CHEMOTHERAPY Volume 8

Cancer Chemotherapy II

CHEMOTHERAPY Volume 1 Clinical Aspects of Infections

Prophylaxis; life·threatening infections; infection in leukaemia; surgical infection; anaerobic infection; respiratory and urinary tract infections; amikacin.

Volume 2 Laboratory Aspects of Infections Sensitivity testing; assay methods; animal models of infection; sisomycin; tobramycin.

Volume 3 Special Problems in Chemotherapy Tuberculosis; genital tract infections; antibiotic resistance and mode of action; topical chemotherapy and antisepsis.

Volume 4 Pharmacology of Antibiotics Tissue concentrations; pharmacokinetics; untoward effects of antibiotics.

Volume 5 Penicillins and Cephalosporins Penicillins and cephalosporins; betalactamases; new agents.

Volume 6 Parasites, Fungi, and Viruses Parasitic infections; fungal infections; chemotherapy of viruses; co·trimoxazole.

Volume 7 Cancer Chemotherapy I Symposia - new drugs and approaches; cell and pharmacokinetics; potentiators of radiotherapy; in vitro screening systems; immunological aspects.

Volume 8 Cancer Chemotherapy II Free papers - new drugs and approaches; cell and pharmacokinetics; mechanisms of action; new analogues; cancer chemotherapy of specific organs.

CHEMOTHERAPY Volume 8

Cancer Chemotherapy II

Edited by K. Hellmann

Westminster Hospital and Imperial Cancer Research Fund

and T. A. Connors

Chester Beatty Research Institute

Plenum Press· New York and London

Library of Congress Cataloging in Publication Data

International Congress of Chemotherapy, 9th, London, 1975. Cancer chemotherapy II.

(Chemotherapy; v. 8) Includes index. 1. Cancer - Chemotherapy - Congresses. I. Hellmann, Kurt. II. Connors, T. A.,

1934- III. Title. IV. Series. RM260.2.C45 vol. 8 [RC271.C5] 1615'.58s 1 [616.9'94'061J 76-1945 ISBN 978-1-4613-4354-7 ISBN 978-1-4613-4352-3 (eBook) DOl 10.1007/978-1-4613-4352-3

Proceedings of the Ninth International Congress of Chemotherapy held in London, July, 1975 have been published in eight volumes,

of which this is volume eight.

© 1976 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1976

A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N. Y. 10011

United Kingdom edition published by Plenum Press, London A Division of Plenum Publishing Company, Ltd.

Davis House (4th Floor), 8 Scrubs Lane, Harlesden, London, NWIO 6SE, England

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming,

recording, or otherwise, without written permission from the Publisher

CHEMOTHERAPY Proceedings of the

9th International Congress of Chemotherapy held in London, July, 1975

Editorial Committee K. Hellmann, Chairman (Anticancer) Imperial Cancer Research Fund, London.

A. M. Geddes (Antimicrobial) East Birmingham Hospital.

J. D. Williams (Antimicrobial) The London Hospital Medical College.

Congress Organising Committee W. Brumfitt K. Hellmann K.D. Bagshawe H. Smith EJ. Stokes F. Wrigley J.D. Williams

I. Phillips M.R.W. Brown D.G.James C. Stuart-Harris R.G.Jacomb D.T.D. Hughes T.Connors

International Society

H.P. Lambert P. Turner A.M. Geddes D. Armitage D. Crowther D.S. Reeves R.E.O. Williams

of Chemotherapy Executive - to July 1975

P. Malek C. Grassi G.H. Werner

H.P. Kuemmerle Z.Modr K.H. Spitzy P. Rentchnick

H. Ericsson G.M. Savage H. Umezawa

Preface

The International Society of Chemotherapy meets every two years to review progress in chemotherapy of infections and of malignant disease. Each meeting gets larger to encompass the extension of chemotherapy into new areas. In some instances, exp~sion has been rapid, for example in cephalosporins, pen­icillins and combination chemotherapy of cancer - in others slow, as in the field of parasitology. New problems of resistance and untoward effects arise; reduction of host toxicity without loss of antitumour activity by new substances occupies wide attention. The improved results with cancer chemotherapy, es­pecially in leukaemias, are leading to a greater prevalence of severe infection in patients so treated, pharmacokinetics of drugs in normal and diseased subjects is rece1v1ng increasing attention along with related problems of bioavailability and interactions between drugs. Meanwhile the attack on some of the major bacterial infections, such as gonorrhoea and tubercu­losis, which were among the first infections to feel the impact of chemotherapy, still continue to be major world problems and are now under attack with new agents and new methods.

From this wide field and the 1,000 papers read at the Congress we have produced Proceedings which reflect the variety and vigour of research in this important field of medicine. It was not possible to include all of the papers presented at the Congress but we have attempted to include most aspects of cur­rent progress in chemotherapy.

We thank the authors of these communications for their cooperation in enabling the Proceedings to be available at the earliest possible date. The method of preparation does not allow for uniformity of typefaces and presentation of the mate­rial and we hope that the blemishes of language and typographical errors do not detract from the understanding of the reader and the importance of the Proceedings.

K. HELLMANN, Imperial Cancer Research Fund A. M. GEDDES, East Birmingham Hospital J. D. WILLIAMS, The London Hospital Medical College

vii

Contents

On the Cytogenetic Criteria of Rational Tumor Chemotherapy • • • • • • • • •

L. S. Evseenko, S. W. Gorkova, E. A. Minenkova, M. M. Fomina, and G. G. Poroshenko

Life Prolongation of Mice Bearing Syngeneic Tumor, Leukemia P388 with the Streptococcal Preparation, OK-432 and Its Mechanism of Action . . . . . . . . . . . . ..

T. Iwaguchi and Y. Sakurai

Breakdown of Non-Immune Metastasis Resistance after Cytostatic Drugs • • • •

J. de Ruiter, Y. Smink, J. Jansen, and L. M. van Putten

Prevention of Lymphoma Growth in Mice by a Covalent Drug-Carrier-Antibody Complex •

G. F. Rowland, G. J. O'Neill, and D. A. L. Davies

Effects of Anti-DNA and Anti-RNA Antibodies Bound to Melphalan and Methotrexate on C3H Mammary Adenocarcinoma and 11210 Leukaemia • • • • •

P. Tran Ba Loc

Immunosuppressive Effects of Some Organic Compounds with Anti-Inflammatory Activity •

I. Barasoain, A. Portoles, J. M. Rojo, and C. Sunkel

Differential Immunosuppressive Effects of Anticancer Agents on Lymphoid Subpopulations • • •

E. Tsubura, K. Yata, G. Hisano, K. Tominaga, H. Sasaki, and S. Sone

ix

1

5

9

11

17

21

27

x

Experiments and Theoretical Considerations on Synchronisation of 11210 Ascites Tumour Cells and Crypt Epithelia of the Mouse with Vincristine • • • • • • • • •

W. Jellinghaus, R. Maidhof, B. Schultze, and W. Maurer

Theoretical Bases for Designing Combination Therapy with Dibromodulcitol (DBD) •••••••

K. Lapis, A. Jeney, L. Kopper, B. Szende, and J. Takacs

Tilorone HYdrochloride: Its Pharmacokinetic Parameters and Its Pharmacodynamic Effects • • • • • • • •

V. Gaur and P. Chandra

Pharmacokinetics of Futraful (FT-207) for Clinical Application • • • • • • • • • • • • • • ••

H. Fujita, M. Sugiyama, and K. Kimura

Effects of Cytotoxic Drugs and/or Corticosteroids on Peripheral Leukocytes • • • • • • • • •

M. Kawano, K. Kohzai, O. Saitoh, and E. Tsubura

The Effectiveness of Sequential Therapy Schedules with Adriamycin and Cyclophosphamide in the P388 Leukemia Model • • • • • • • • • •

I. Wodinsky, J. K. Swiniarski, J. M. Venditti, and R. K. Johnson

Antitumor Activity of Mimosine and Mimosine HYdrochloride Against B16 Melanoma in

CONTENTS

31

37

43

51

59

63

BDF 1 Mice • • • • • • • . • • • • • •• 77 T. A. Khwaja, T. C. Hall, and K. M. A. Sheikh

A New Multipeptide Antitumour Drug A. De Barbieri

Metabolism of the Tumour-Inhibitory 3,3-Dimethyl-l-

87

Phenyl-Triazene and Its 4-Chlorophenyl Analogue 91 G. F. Kolar and J. Schlesiger

Antitumour Activity of Benzofuroxan Derivatives V. C. Barry, J. G. Belton, and M. L. Conalty

97

Antitumour Activity of Tetrazolopyridazines and Tetrazolophthalazines • • • • • • • •• •••••• 103

V. C. Barry, M. L. Conalty, J. F. O'Sullivan, and D. Twomey

CONTENTS

Antineoplastic Effect of Compound 9777-VUFB in Animals with Experimental Tumours; Its Interaction with Some Cutostatics

M. SemonskY, V. Pujman, and H. Vessela

Effects of GP 48 989 Alone and in Combination with Hormones and Chemotherapeutic Agents on DMBA-Induced Mammary Carcinomata II • • • •

K. H. Schmidt-Ruppin and K. Schieweck

R 17934: A New Synthetic Anticancer Drug Interfering with Microtubules • • . • • • . . . . • • • •

M. De Brabander, R. Van de Veire, F. Aerts, G. Geuens, L. Desplenter, J. DeCree, M. Borgers, and P. A. J. Janssen

Antitumour Activity of Carminomycin • • • • • • V. A. Shorin

Variamycin, a New Antitumour Antibiotic • • • • . • S. M. Navashin, T. G. Terentjeva, E. V. Bobikov, L. I. Torbochkina, A. B. Sokolov, Y. O. Sazykin, O. K. IChanykova

and

Inhibition by Caffeine of Post-Replication DNA Repair in Hamster Cells Treated with cis platinum (II) Diammine Dichloride ---. • • • • • •

H. W. van den Berg and J. J. Roberts

The Role of Nuclear Proteins in the Chemotherapeutic

xi

107

115

121

129

133

139

Effect of Dibromodulcitol (DBD) • • • • • • • 145 A. Jeney, E. Dzurillay, K. Lapis, and L. Institoris

The Effect of Dibromodulcitol on the Replication of DNA in Yoshida Sarcoma Cells •••••••••• 153

E. Institoris and L. Holczinger

Clinical Cancer Chemotherapy with Drugs Aimed at Gene Regulators • • • • • •

F. E. Knock, R. M. Galt, Y. T. Oester, and R. Sylvester

159

Characterization of the Bleomycin Action on DNA • • • • • • • 165 H. Umezawa, H. Asakura, and M. Hori

Antitumour Antibiotic Carminomycin: Mechanism of Action 169 G. F. Gause and Y. V. Dudnik

Effect of Combined Chemotherapy with Lysosome Labilizers and Mitomycin-C

T. Taniguchi, H. Niitani, A. Suzuki, N. Saijo, I. Kawase, and K. Kimura

175

xii

Optimal Conditions for Tumor Chemotherapy Chosen on the Basis of Changes in the Lipid Antioxidant Activity • • • •

N. P. Pal'Mina, E. B. Burlakova, V. D. Gaintseva, and N. P. Sezina

An Antitemplate Approach to Develop Selective Inhibitors of Oncornaviral Reverse­Transcriptase . . • • • . • . • .

P. Chandra, T. J. Bardos, U. Ebener, B. Kornhuber, D. Gericke, and A. GDtz

Experimental Approach to Increase the Effects of Cancer Chemotherapy in Tumor-Bearing Rats Pretreated with an Inducer on Microsomal Drug-Metabolizing

CONTENTS

185

191

Enzyme (cytochrome p-450) • . • • . • • • • • • • . . 197 S. Ohira, S. Maezawa, K. Watanabe, K. Kitada, and T. Saito

Effect of the Drug-Metabolizing Enzyme Inducers on the cytostatic Activity of Dibromodulcitol • • • . 203

E. Gati

Studies of N-Methyl-N-Nitrosourea-C1 40 in Mice with Hepatoma 22A

L. B. Gorbacheva, G. V. Kukushkina, A. M. Serbryanyi, and V. S. Tutlyte

Pharmacokinetics

I. S. Sokolova,

Collateral Sensitivity Between an Alkylating Agent and Halogenated Methotrexate • • . • • • •

B. W. Fox

Meso-l,2 bis-(3,5-Dioxopiperazine-l-yl)-1,2-Dimethyl-ethane (ICRF 193): A Potent Antitumour Analogue of ICRF 159 . • .

K. Hellmann

New Derivatives of Nitrosourea with a High Therapeutic

209

215

219

Index for Oncostatism and Immunosuppression 221 J. L. Imbach, M. Hayat, E. Chenu, B. Serrou, and G. Mathe

Effect on 11210 Leukaemia, on Antibody Forming Cells, and on Macrophage cytotoxicity of Ellipticine and Three Derivatives . . . . • • . . . • . • • 229

G. Mathe, M. Hayat, E. Chenu, I. Florentin, M. Bruley-Rosset, M. Janot, P. Potier, N. Dat-Xuong, A. Cave, T. Sevenet, C. Kan-Fan, J. Poisson, J. Miet, J. Le Men, F. Le Goffic, A. Gouyette, A. Ahond, L. Dalton, and T. Connors

CONTENTS

Antitumor Activity of Daunorubicin Derivatives G. Jolles, R. Maral, M. Messer, and G. Ponsinet

New Antitumour Analogues of Cytosine Arabinoside and the Effect Against Mouse Leukemia 11210

M. Aoshima, S. Tsukagoshi, Y. Sakurai, J. Oh-ishi, M. Akiyama, and T. Ishida

Exceptional Responses to Chemotherapy and/or Hormonotherapy of Cases with Generalized Cancer • • • • • • • • • • • • • ••••

D. Razis, M. Constantoulakis, M. Dimitriadis, A. Athanassiou, and T. Messaropoulos

Clinical Considerations in Myelomatosis J. B. Healy

Chronic Gastritis, Atypical Epithelia in Biopsies and Therapeutic Consequences • • • • • •

J. Zangger and M. Taufer

Clinical Studies on Changes in Serum Glycoproteins in Cancer Chemotherapy • • • • • • • • • •

K. Funahashi

Double-Blind Trial with Levamisole in Resectable Lung Cancer • •

W. Amery

Carcino-Embryonic Antigen Determinations and Chemotherapy in Cancer Patients

J. Huys and P. M. Van Vaerenbergh

Aspects of Chemo-Immunotherapy in a Controlled Clinical Study for the Treatment of Bronchogenic Cancer • • • • • • • • • •

Ch. Cerni, o. Kokron, M. Micksche, R. Titscher, and H. Wrba

Pulse-Cytophotometric Monitoring of the Intensive Chemotherapy of Acute Leukaemia • • • • •

S. Pawelski and S. Maj

Treatment of Adenocarcinoma of the Ovary with

xiii

237

243

249

257

275

281

287

295

Combined Immunotherapy and Chemotherapy • • • • 305 P. K. Kalpaktsoglou, A. P. Kondyli, G. B. Ioannidou, K. E. SoulpiMargariti, A. C. Comninos, and G. P. Andritsakis

xiv

Clinical and Experimental Studies on Immuno­chemotherapy Using OK-432, a New Streptococcal Preparation • • • • • • •

T. Hattori, M. Niimoto, S. Yamagata, and T. Tohge

Phase I and Phase II Studies in the Treatment of Cancer Patients by Radiotherapy, Chemo­therapy and Methanol Extraction Residue of an Anti-Tuberculosis Vaccine (MER)

E. Robinson, R. Haasz, A. Bartal, and Y. Cohen

Additional Therapy with Trenimon in Treatment of Carcinoma of the Uterine Cervix . • •

M. Kaether and G. Franz

Interferences of Radiotherapy and Chemotherapy on the Binding of 3H-17a-oestradiol with Its Specific Receptors • • • • • • • • • • •

E. Genazzani, G. L. Sannazzari, G. Conti, and F. DiCarlo

Mechanism of Antitumor Action of Hemolytic Streptococcal Preparation OK-432 (NSC-B1l6209) for Malignant Pleural or Peritoneal Effusion by Intrathoracal

CONTENTS

313

319

327

331

and Intraperitoneal Injection • • • • • • • • • 339 K. Ota and A. Oyama

The Significance of Reduction Surgery in the Treatment of Advanced Cancer Patients

R. Esaki, K. Shibata, and K. Funahashi

Intermittent Long Term Polychemotherapy as an Adjuvant

345

to Surgery of Bronchogenic Carcinoma • • • • • 355 K. Karrer and N. Pridun

Polychemotherapy for Advanced Lung Carcinoma: Results and Further Consequences

J. Kuehboeck, P. Aiginger, and P. Poetzi

Chemotherapy in Conservative Treatment of Lung Cancer Patients • • • • . • • • •

I. V. Kasiananko, A. I. Pozmogov, and E. L. Jerusalimsky

Effective Chemotherapy for Bronchial Carcinoma E. W. Street

Clinical

MUltidisciplinary Curative Assault on Disseminated Carcinoma of the Breast • • • • • • • •

P. Mannes, R. Derriks, R. Moens, C. Laurent, and J. Dalcq

361

375

381

CONTENTS

Potentiation of Drugs Using Sequential Chemotherapy Against Disseminated Breast, Bronchial, and Central Nervous System Solid Tumors • •

P. Pouillart, L. Schwarzenberg, J. L. Amiel, G. Mathe, P. Huguenin, Ph. Morin, A. Baron, Ch. Laparre, and R. Parrot

Animal and Human Studies with Oral Mitomycin c(6): A Preliminary Report • • • • • • • • •

P. D. Boasberg, T. C. Hall, O. Odujinrin, R. S. Benjamin, B. B. Lowitz, H. B. Nevinny, and C. L. Maddock

Chemohormonal Therapy of Breast Cancer - A Pilot Phase I-II Study • • • • • • • • •

O. O. Odujinrin, R. J. Benjamin, R. E. Hardy, P. D. Boasberg, and T. C. Hall

The Results of Cleomycin Treatment in 90 Patients with Malignant Disease • • • • • • • • • • • •

I. Christov and T. Donchev

Cis-Platinum Diaminodichloride in the Treatment of Squamous Cell Carcinoma and Other Malignant Diseases ... . . . . . . . . . . . . . .

E. Loeb, J. M. Hill, A. MacLellan, N. O. Hill, M. D. Khan, J. J. King, R. Speer, and H. Ridway

Anhydro-Arabinosyl-Fluorocytosine HYdrochloride: A Phase I Study • • • ••••••• •

P. Alberto and R. Medenica

Ifosfamide in the Treatment of Lung Cancer and Metastases of Solid Malignant Tumours •

H. Wrba, O. Kokron, and R. Titscher

Preclinical and Phase-I Studies of Ifosfamide for Its Massive Dose Cumulation Schedule

K. Kubo

Clinical Pharmacological Studies with Formyl-Leurosin in Malignant Diseases • • • • • • • • • • • •

S. Eckhardt, I. Hindy, and E. Farkas

Clinical Investigations with F-Leurosine E. Farkas and S. Eckhardt

Clinical Investigations of Dibramodulcitol in the Treatment of Malignant Diseases • • • • •

I. Hindy and J. Szanto

xv

387

405

413

421

425

435

437

445

451

457

463

xvi

Oral Estracyt® (Estramustine phosphate) in the Treatment of Advanced Carcinoma of the Prostate •• • • • . • • . • • . • • •

A. Nillius and I. KOnyves

Treatment of Prostatic Carcinoma with Estracyt® (Estramustine phosphate) .•.•

F. Balogh, Z. Szendroi, L. Kisbenedek, I. ~dnyves, and I. Szendi

CONTENTS

469

475

Clinical Use of DDMP in Cancer Chemotherap,y • . • • • • • •• 481 L. A. Price and B. T. Hill

Combination of Anticoagulants and Antineoplastic Drugs in Cancer Chemotherapy • • . • • • • • • • • • 485

K. Rieche

Adri~cin Cardiotoxicity in Man: Effect of Pretreatment with Beta-Methyldigoxin. A Poligraphic Study . • • • . • • • • • • • 491

F. P. Villani, G. Beretta, A. Pagnoni, and A. Guindani

Combination of Adriamycin and Bleomycin in the Treatment of Advanced Cervical Cancer • . . • . • • . • • • 501

N. Natale, C. Mangioni, and G. Bolis

Treatment of Chemotherap,y Resistant Nonseminomatous Testicular Tumors with DDP (NSC-119875) 507

R. Osieka, U. Bruntsch, W. M. Gallmeier, S. Seeber, and C. G. Schmidt

Combination Chemotherapy of Advanced Hodgkin's Disease wi th Adri~cin, DTIC, CCNU, and Bleomycin 513

R. Osieka, U. Bruntsch, W. M. Gallmeier, S. Seeber, and C. G. Schmidt

Proteolytic Enzymes in the Treatment of Malignant Pleural Effusions and Solid Metastases ••••••• 517

o. Kokron, M. Micksche, C. Cerni, R. Titscher, and H. Wrba

Intra-Arterial Chemotherapy of Head and Neck Squamous Cell Carcinoma . • • •

R. Medenica, P. Alberto, W. Lehmann, and M. A. Hopf

Chemotherapy of Glioblastoma Multiforme: A Statistical Analysis of Its Effect

K. Takeuchi and K. Hoshino

523

529

CONTENTS

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in the Treatment of Primary Central Nervous System Tttm.ors • • • • • • • • • • • • • • • •

G. A. Koutras, N. A. Pavlidid, N. Kordiolis, V. Samaras, and J. Taptas

BCNU and CCNU Chemotherapy of Tttm.ours of the Central Nervous System • • • • •• ••••••••

G. R. Della Cuna and P. Paolette

Combination Chemotherapy and CCNU Treatment of

xvii

535

Glioblastoma: A Comparative Trial • • • • 551 W.-D. Heiss, A. Kroiss, J. KUhbock, and W. Profanter

Combination of Adriamycine, VM26, Cyclophosphamide and Prednisone (AVmCP) in Chemotherapy of LYmPho and Reticulum Cell Sarcoma (Stages and Topographic Forms III and IV) • • • • • • • • • 557

J. L. Misset, P. Pouillart, J. L. Amiel, L. Schwarzenberg, M. Hayat, F. de Vassal, M. Musset, D. Belpomme, C. Jasmin, C. Albahary, R. Depierre, and G. Mathe

Comparison of PDN + VCRO ; PDN + VCRo ~ ADMx; PDN + VCRo ~ ADMx ~ CARx; and PDN + VCR + ASp· in Induction of First Remission of Acute LYmPhoid Leukemia • . • • • • • . • • • • 569

G. Mathe, F. de Vassal, J. L. Amiel, P. Pouillart, L. Schwarzenberg, C. Jasmin, M. Hayat, J. L. Misset, and M. Musset

Clinical Evaluation of Peptichemio in Some Hemoblastoses and Solid Tumours • •

G. Pacilio, L. Annunziato, L. Campanella, and G. Scotti

Chemotherapeutic Management of Non-Hodgkin's Lymphoma:

575

Comparative Study of Various Combinations • • • 581 N. Gad-el-Mawla

Chemotherapy for Gastric and Colorectal Carcinoma by Intra-aortic Infusion • • • • • • • • • • • •

K. Yoshikawa and I. Ito

Effect of Adjuvant Chemotherapy with Mitomycin C on the Recurrence of Gastric Cancer after Radical Surgery • • • • • • • • ••• •

T. Nakajima, T. Kajitani, A. Fukami, and I. Ohashi 591

xviii

Long-Term Cancer Chemotherapy for Stage III to IV Gastric Cancer Following Non-Curative Resection

T. Abe, T. Kajiwara, T. Kamata, and S. Tsuboi

Chemotherapy for Advanced Ovarian Malignancy U. VillaSanta

Changes in Clinical and Histological Patterns Observed in Patients with Advanced Carcinoma of the

CONTENTS

597

605

Ovary Treated with Progesterone • • . • • . • • 611 G. A. Paraskevas, Ph. Angelakis, and H. Deligeorgi-Politi

List of Contributors 615

ON THE CYTOGENETIC CRITERIA OF RATIONAL TUMOR CHEMOTHERAPY

L • S. E v see n k 0, S. lJ. Go r k 0 va, E. A. Min en k 0 va, M.M. Fomina, G.G. Poroshenko Institute of Chemical Physics Academy of Sciences Moscow, U.S.S.R.

SUMMARY

Tumor cells are characterized by high variability and it is possible, as a rule, to isolate from a tumor some stem cell lines with various numbers of chromosomes and with chromosome markers. Studies were carried out on transplantable ascites tumors in mice (NK/Ly, L5178 and Sarcoma 37 strains). Only hypertetraploid cells remained in a nitrosomethylurea-resistant tumor strain. Sarcoma 37 and leukemia L5178 which were also resistant to chemo­therapy, differed from the initial sensitive tumors by the presence of new stem cell lines with a definite number of marker chromosomes. It is suggested that the variety of tumor karyotypes within a strain is a result of cell selection in the course of tumor progression. This fact must be taken into consideration by cancer chemotherapy whether by single agents or by combinations.

Recent achievements of cytogenetic methods permit the establishment of some strictly quantitative criteria for the control of tumor cell populations, both in the course of tumor progression, and in the course of chemotherapy. One of such criteria is the change of tumor cells karyo­type with marker chromosomes.

Tumor cells are characterized by high variability and therefore, tumor cell populations are usually heterogenous. As a rule, it is possible to isolate from a tumor some stem cell lines of different cytogenetic features. These lines differ by the chromosome number and by the presence

2 L.S. EVSEENKO ET AL.

of structurally changed (marker) chromosomes. Since one or several chromosomes could be lost while preparing slides, the presence of marker chromosomes is a more re­liable feature of karyotype than the total chromosome number.

Karyotypes of the ascitic forms of NK/Ly and L5178 leukemias and of Sarcoma 37 have been studied. These tumors are sensitive to alkylating compounds, to anti­metabolites and to supermutagens. We found pronounced aneuploidy and marker chromosomes, namely: a large telo­cehtric chromosome with a secondary constriction almost in the midst of it (A chromosome); a big metacentric chromosome (B chromosome); a very small one, 2 to 3 times smaller than the smallest chromosome of the standard mouse karyotype (C chromosome); a large submetacentric chromosome which is the longest of all the other mouse chromosomes, and it has a marked secondary constriction in the middle of its long arm (0 chromosome).

Combinations of these 4 types of marker chromosomes produce various lines of cells: for instance, there is a cell line with A + B + 2C marker chromosomes, with 0 + B + 2C, A + B + C, A + B + 3C, 0 + B + 3C and A + 2B + 2C chromosomes. Each of these lines is found both in diploid and in tetraploid variants. Single polyploid cells were also found.

Depending on the conditions of tumor growth, one or another cell line prevails. For instance, an increase in number of tetraploid metaphases was observed during the first 4 days after tumor transplantation and also in the final stages of its growth.

The cell line with A + B + 2C marker chromosomes is a model line for tumor growth in the peritoneal cavity of random bred albino mice, whereas the line with 0 + B + 2C marker chromosomes is a characteristic of the development of the same tumor in the peritoneal cavity of BALB mice.

Such increase of the number of tetraploid metaphases in the population of tumor cells during the first 4 days and by the 16th day of its growth suggests that tetra­ploid cells are more resistant to the action of different unfavourable factors. High stability of tetraploid cells to such effects was confirmed by the fact that NK/Ly strain becaomes completely tetraploid and contains some new stem cells after 20 passages with the treatment with N-nitrosomethylurea; the same was observed in Sarcoma 37 after 23 passages and Sarcolysin (Melphalan) and in

CYTOGENETIC CRITERIA FOR TUMOR CHEMOTHERAPY 3

L5178 strain after 20 passages and Bruneomycin or Dipin.

The above mentioned data suggest that the increased survival of tumors after treatment is due to the heter­ogenecity of tumor cell populations. It means that, under different types of treatment, only some tumor cells are killed. The remaining cells, being resistant to such treatments, survive. This results in recorrences which are then more resistant to the action of the same drug.

The problem of acquired resistance of tumors to chemotherapy is of great practical value, since the development of such resistance is able largely to diminish the effectiveness of treatment. The data permits also to investigate the relationship between tumor progression and selective processes in tumor cell population.

The cytogenetic method may be used for the prognosis of resistance and may therefore, be a criterion for the selection of rational combined chemotherapy.

LIFE PROLONGATION OF MICE BEARING SYNGENEIC TUMOR, LEUKEMIA P388 WITH

THE STREPTOCOCCAL PREPARATION, OK-432 AND ITS MECHANISM OF ACTION

Takao Iwaguchi and Yoshio Sakurai

Division of Cancer Chemotherapy, Cancer Institute

Kami-Ikebukuro 1-37-1, Toshima-ku, Tokyo 170, Japan

Intraperitoneal administration of OK-432 prolonged life span of CDF1 mice bearing P388. Cell-mediated immune response of the host was investigated in vitro with the method of electrophoretic mobility of macrophage. At the early stage of tumor development, the ce11-mediated immune response of the host treated with OK-432 was kept stronger than that of the untreated one. However, at the advanced stage of tumor development, there was little difference in the cell mediated immune response between both groups. The host was found to be more immunosuppressed than that at the early stage of tumor development.

It has been reported by Ohashi that the intraperitoneal admin­istration of OK-432 before and after the intraperitoneal inoculation of P388 prolonged the life span (TIC 200%) of CDF1 mice bearing the tumor and this immunological treatment also enhanced the antitumor effect of cyclophosphamide as shown in Table 1 (Ohashi, F. and Tsukagoshi, S., 1974). From these results it seems that the life prolongation is due to the increase in cell-mediated immunity of the host by OK-432. Field reported a new method for checking ce11-mediated immunity in vitro (Field, E. J. et a1., 1973). The prin­ciple is based on determination of the decrease in electrophoretic mobility of normal guinea-pig peritoneal macrophages which were contacted in vitro with the supernatant of a mixture on lymphocytes from cancer patients and the soluble antigen of the cancer. They claimed that the slowing of electrophoretic mobility of macro­phages (MEM) determined by the above-mentioned procedure might be regarded as a parameter of the cell-mediated immunity of the host. We also applied the method with the slight modification for animals resistant to inoculation of the tumor (Iwaguchi, T. and Sakurai, Y., 1974).

5

6

-eo

i~°k:"s"\ 104 x3 x5 106 x2

number of spleen cells per ml

Fig. 1. Percent slowing of ma­crophage and the number of spleen cells from OK-432 treated and un­treated mice on the 5th day after the intraperitoneal inoculation of P388 (106 cells/mouse). The dotted line shows that of the treated mouse, and the solid line shows that of the un­treated one.

T. IWAGUCHI AND Y. SAKURAI

I:t~---'~---L..I -,--:'--&..':J_" ~ 104 105 x3 x5 106 x2

number of spleen cells per ml

Fig. 2. Percent slowing of ma­crophage and the number of spleen cells from OK-432 treated and untreated mice on the 9th day after the intra­per~toneal inoculation of P388 (10 cells/mouse). The dotted line shows that of the treated mouse and the solid line that of the untreated one.

TABLE 1. Combination Therapy of Mice bearing Syngeneic Tumor, Leukemia P388, with OK-432 and Cyclophosphamidea )

Treatment with OK-.32

pre-

pre-+

post-

post-

Interval of pre-treatment and tumor inoculation

2 weeks 4 6 8

2 4 6 8

TIc (%) 100 KEIk9b) -C +c

154 225 113 176 115 183 109 176

173 288 236 27. 202 311 192 282

108 248

a ) Median sur" ivai time of untreated m ice was 10 .• days and that of mice treated with cyclo­phosphamide was 18.3 days ( TIC, 176 % ).

b ) -C and +C indicate without cyclophosphamide and plus cyclophosphamide treatment.

LIFE PROLONGATION OF MICE WITH SYNGENEIC TUMOR 7

The presentation deals with the study on the mechanism of the ac­tion of OK-432 on the life prolongation of mice bearing P388 by the use of the method of electrophoretic mobility of macrophages. CDFI mice (10 mice/group) were pretreated intraperitoneally with OK-432 in a dose of 100 Klinische Einheit (KE)/kg/day, daily for 10 days and a leukemia P388 (106 cells/mouse) was inoculated intra­peritoneally 2 weeks after the last injection of OK-432. From the next day after the tumor inoculation, the same dose of OK-432 was again administered intraperitoneally for 10 days. As the control, the untreated mice were intraperitoneally inoculated with the tumor. The spleen cells of CDFI mice on the 5th and 9th days after the tumor inoculation were used as sensitized lymphocytes of the host. The number of spleen cells were counted microscopically by staining with Turk solution and then adjusted with RPMI-1640 medium to an appropriate number of the cells/ml. The cells were incubated with or without the soluble antigen (protein 200 ug/ful) at 37° for 90 min in C02 incubator. The supernatant obtained by centrifugation at 2500 r.p.m. for 5 min was added to the macrophages (106 cells/ tube) which were collected from the peritoneal cavity of a non­sensitized guinea pig (about 300g body weight), treated by intra­peritoneal injection of sterilized liquid paraffin (20 ml) 6 to 9 days before. The suspended cells were incubated at 37° for 30 min in a C02 incubator and then the medium was changed to MEPM medium by centrifugation at 2500 r.p.m. for 5 min (Zeiller, K. and Hanning, K., 1971). Cell electrophoretic mobility was measured in a constant current of 0.3mA at 25°, checking the velocity of at least 10 cells by cell electrophoretic instruments (Sugiura Lab. Inc. Japan). The level of cell-mediated immunity was determined two times during tumor development. In the group treated with OK-432 on the 5th day after the tumor inoculation, MEM increased to reach a plateau level of 10 % by varying number of spleen cells from 104 to 105/ml and was maintained constantly to 106/ml • In untreated one, MEM increased similarly to that of the treated group to 105/ml, but from this point the value gradually decreased to become nil as shown in Fig. 1. Cell-mediated immune status of the treated group at that time is thought to be similar to that of the mouse resistant to the inoculation of the tumor. Doubled life prolongation of the group treated with OK-432 might be based on the above-mentioned difference in the immune status of them. On the 9th day after the tumor inoculation, there was little difference in the cell-mediated immune response between the untreated and treated groups as shown in Fig. 2. In both groups, maximum of MEM shifted from 105/ml of spleen cells on the 5th day after the tumor inoculation to 106/ml of spleen cells. At the advanced stage of tumor development, the host was found to be about ten times more immunosuppressive than that at the early stage of tumor development. We acknowledges NCI, NIH Bethesda, U.S.A., for the gift of CDFI mice.

8 T. IWAGUCHI AND Y. SAKURAI

References

1) Iwaguchi, T. and Sakurai, Y., (1974), Gann, 65, 561 2) Field, E. J., Caspary, E. A. and Smith, K. S., (1973), Brit.

J. Cancer, 28, Supp1. 1, 208. 3) Ohashi, F. and Tsukagoshi, S., (1974), Gann 65, 563. 4) Zei11er, K. and Hanning, K., (1971), Hoppe-Sey1er's Z. Physio1.

Chern., (1971), 352, 1162.

BREAKDOWN OF NON-IMMUNE METASTASIS RESISTANCE AFTER CYTOSTATIC

DRUGS

J. de Ruiter, T. Smink, J. Jansen and L.M. van Putten

Radiobiological Institute TNO

Lange Kleiweg 151, Rijswijk, The Netherlands

In various animal models the modification of lung metastases after intravenous injection of tumour cells has been described after treatment with local lung irradiation, inflammation promoting agents, anticoagulants or Corynebacteria.

Recently, the effect of pretreatment of mice with cytostatic drugs on the formation of lung metastases after intravenous injection of osteosarcoma cells was described (1). Most cytostatic agents enhanced the formation of lung metastases with factors between 2 and 10. However, administration of Cyclophosphamide resulted in an exceptionally high enhancement factor of more than a hundred. Immunosuppression could not explain these findings since no comparable results could be obtained after intensive immunosuppression with antilymphocyte globulin. Furthermore, evidence was obtained that this tumour was not immunogenic, since immunization with heavily irradiated osteosarcoma cells did not influence the formation of lung nodules significantly. The fact that drugs such as Bleomycin, 5-FU and Methotrexate were active suggested that the effect of these drugs might be mediated by cell killing. Since treatment with Corynebacterium parvum, a known macrophage stimulant, caused a markedly decreased formation of lung metastases in this model, it was investigated whether the macrophage was the cell killed by these cytostatic drugs (2). However, intravenous administration of peritoneal macrophages caused only a minor decrease in the formation of lung nodules and could not diminish the effect of cytostatic drugs. Furthermore, the administration of silica, a known depressant of macrophage function, caused a decrease in formation of lung mestastases. The absence of effect of treatment with anti-macrophage serum provides further evidence against the macrophage as the cell involved in

9

10 J. de RUITER ET AL.

the enhancement of lung metastases after treatment with cyto­static drugs.

Anticoagulant treatment also influenced the formation of lung metastases. A decrease by a factor of 10 to 50 was observed for different anticoagulant treatments. In ordI25to gain insight into the mechanism involved, the retention of IUDR-labelled osteosarcoma cells in the lung was measured (3). These studies indicated that the increased formation of lung metastases was accompanied by an increased retention of cells in the lung, where­as the decreased formation of lung metastases after Coryne­bacterium parvum and Heparin was paralleled by a decreased retention of cells in the lung. These differences were already manifest as early as 1 hour after the first contact with these cells and these early differences cannot be attributed to immuno­logical mechanisms.

In order to investigate whether the early modifications in retention of osteosarcoma cells was associated with the viability of the tumour cells or with some specific characteristics of tumour cells, the retention of heat-killed osteosarcoma cells and of living isogenic embryonic cells in the lung was followed. Cyclophosphamide induced an increased retention of all three cell types. In contrast, Corynebacterium decreased the retention only of living osteosarcoma cells but failed to modify the retention of dead osteosarcoma cells or living embryonic cells. On the other hand, Heparin decreased the retention of living and dead osteosarcoma cells, but did not affect the retention of living embryonic cells. Since different cell types are modified by the different treatments, no evidence is obtained that a common mechanism is involved.

It can be concluded that Cyclophosphamide decreases a non­immunological resistance against the lodging and growth of any type of cell in the lung.

REFERENCES

1. Van Putten, L.M., Kram, L.K.J., Van Dierendonck, H.H.C., Smink, T. and Fuzy, M. Enhancement by drugs of metastatic lung nodule formation after intravenous tumour cell injection. Int. J. Cancer 12, 588 - 595, (1975).

2. Smink, T., Jansen, J. and Van Putten, L.M. Cyclophosphamide and the formation of metastases. I. The role of macrophages. Cancer Chemotherapy Reports, to be published.

3. De Ruiter, J., Smink, T. and Van Putten, L.M. Cyclophosphamide and the formation of metastases. II. Modification in lung retention of various cells. Cancer Chemotherapy Reports, to be published.

PREVENTION OF LYMPHOMA GROWTH IN MICE BY A COVALENT DRUG-CARRIER-

ANTIBODY COMPLEX

G.F. Rowland, G.J. O'Neill, and D.A.L. Davies

G.D.Searle Research Laboratories

Lane End Road, High Wycombe, Bucks, England

SUMMARY

The use of carrier linked drug-antibody conjugates ('carrier­DRAC') in the mouse EL4 lymphoma system is described as a model for selective transport of cytotoxic drugs in cancer therapy. This method of conjugation provides for a high degree of drug substitution without significant loss of antibody activity and with no loss of water solubility. Suppression of tumour growth in vivo, and tests in vitro show greater effectiveness of the drug­carrier-antibody conjugate than of drug-carrier and antibody un­combined.

INTRODUCTION

The explosive growth of tumour immunology in recent years has revived the possibility first proposed by Paul Ehrlich (1906) that antibodies might be used to direct cytotoxic agents to tumour cells, thereby improving selectivity and reducing non-spec­ific toxicity. Several studies have appeared in which agents were linked to antibody either covalently or non-covalently (Mathe et al. 1958, Moolten and Cooperband 1970, Ghose et al. 1972, Flechner 1973, Rubens and Dulbecco 1974). Where non­covalent, attachment is used the possibility of in vivo dissociation exists and results from the use of such mixtures are open to the interpretation of a synergism between free drug and antibody unlinked, a phenomenon (the 'DRAB' effect) now well established (Davies and O'Neill 1973, Davies et al. 1974). The in vitro and in vivo effects of covalent drug-antibody conjugates (DRAC) have also been described using alkylating agents (Linford et al.

11

12 G.F. ROWLAND, G.J. O'NEILL AND DAL. DAVIES

1974, Davies and O'Neill 1974, O'Neill and Davies 1975) and antitumour antibiotics (Hurwitz et al. 1975, Levy et al. 1975). In all these cases the degree of drug substitution is limited by loss of antibody activity or water-solubility of the product. A method of overcoming this limitation using an inert intermediate carrier molecule has been described (Rowland et al. 1975). The present paper gives further results on the use of this drug­carrier-antibody conjugate (carrier-DRAC) showing the effects in vitro and in vivo with different routes and levels of tumour challenge.

MATERIALS AND METHODS

The alkylating agent p-phenylenediamine mustard (PDM) was kindly supplied by the Chemical Defence Establishment, Porton, U.K. The carrier was poly-L-P(-glutamic acid (PGA) molecular weight 35,000, obtained from Miles Seravac (UK) Ltd., and the carbodiimide used for coupling was l-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) , obtained from the Sigma Chemical Co.Ltd.

Antibody was prepared as previously described, (Davies et al. 1974) by immunizing rabbits with mouse lymphoma cells (EL4), absorbing the serum with normal mouse spleen and fractionating to prg1uce immunoglobulin (Ig). Antibody activity was determined by a Cr-release assay (Davies et al. 1974).

EDC was used to prepare the drug-carrier complex (PDM-PGA) and also to couple this to Ig as previously describe4 (Rowland et al. 1969). This gave a conjugate preparation with 90 moles of alkylating PDM per mole Ig, was fully water-soluble and retained 67% of the original antibody activity. In vitro effects were determined using cultured EL4 cells incubated for two days with conjugate. Cytostasis was measured by pulse-labelling cells with tritiated thymidine (Ragiochemical Centre, Amersham) at a concentration of 0.67 pCi/10 cells/ml for the last 4 hours of culture. The cells were harvested, washed to remove unincorpor­ated thymidine and dis&olved in KDH prior to mixing with scintill­ation fluid for counting (Rowland 1969). In vivo effects of the conjugates were tested in groups of C57BL/6 mice inoculated with live EL4 tumour cells and subsequently injected with four daily doses of material commencing 24 hours after tumour challenge. Survival of the mice was noted and used as the criterion of effect­iveness.

RESULTS

Evidence of coupling PDM-PGA to Ig came from three types of physico-chemical study. Treatment of the conjugate with ethanol

PREVENTION OF LYMPHOMA GROWTH 13

to a final concentration of 50% resulted in 80% precipitation of drug with Ig. PDM-PGA alone did not precipitate and PDM-PGA plus Ig unlinked produced only a low level of drug precipitation. Chromatography of the conjugate on ion-exchange resins showed a diminished peak for unmodified Ig and the presence of Ig in the main PDM-PGA elution peak. Immunoelectrophoresis on agar using goat-anti-rabbit Ig antiserum as developing agent showed Ig having anionic characteristics consistent with material coupled to PDM-PGA.

The in vitro behaviour of various preparations is shown in Fig.l in which PDM-PGA is used with either normal rabbit globulin (NRG) or with antibody-containing immunoglobulin (Ig). Thymidine uptake is expressed as a percentage of that in control cells incub­ated without drug conjugates. It can be seen that the drug in the PDM-PGA-Ig conjugate is considerably more cytostatic than when PDM­PGA is coupled to or in the presence of NRG. The conjugate is also somewhat more cytostatic than PDM-PGA plus Ig unlinked.

The in vivo effects are demonstrated by Fig.2 showing a typical survival chart of mice following treatment. Ig alone at the concentration used in the conjugates increases the survival time by approximately 7 days. The drug-carrier PDM-PGA alone gives a slightly greater increase while an additive or superadditive drug­antibody (DRAB) effect is seen with the two unlinked. The greatest increase however is seen by giving the conjugate (carrier-DRAC) with no deaths by 60 days.

The effects obtained with varying doses of conjugate in a mgre severe test are shown in Table 1. Mice were challenged with 10 cells sub-cutaneously and treated as before. Although the complex is less effective under these conditions than with the lower tumour challenge, a significant dose-related increase in survival was obtained. In this test Ig alone had no effect on survival at any dose used and no protection was afforded by PDM-PGA-NRG conjugates.

DISCUSSION

The results described support the notion that cytotoxic drugs can be coupled to antibody through intermediate carrier and that the conjugates obtained can function as effective anti-tumour agents. The results both in vitro and in vivo suggest that the mechanism is not one of drug-carrier and antibody synergism, since linked mat­erials are more effective than the components. In addition the results obtained with the sub-cutaneously implanted tumour show that tumour challenge and treatment can be at separate sites and still remain effective. This demonstration is of obvious import­ance when considering the potential clinical application of 'carrier DRAC' .

14 G.F. ROWLAND, G.J. O'NEILL AND DAL. DAVIES

100 ... __ _

80

I=l 0

• ..! ~ III J.I 0 • Po

'" 0 CJ I=l

• ..! • Q)

I=l 40 • ..! "Cl • ..!

! E-<

~ c

20 0

20 40 60 80 100

Drug cone. (pg/ml)

Fig.l Tritiated thymidine uptake by EL4 cells in culture with drug conjugates : A, PDM-PGA-NRG. B, PDM-PGA plus NRG. C, PDM-PGA plus Ig. D, PDM-PGA-Ig. The relative amounts of drug and protein were the same in all four preparations.

Finally a note of caution; the coupling of drug carrier to Ig is still difficult to control. Some preparations in our hands have given less effective protection and on analysis have shown poor coupling. Different drug-carrier molecules and alternative coupling reactions are under investigation and should soon help to

PREVENTION OF LYMPHOMA GROWTH

100

80

60

40

20 %

Survivors

15

E

~----~10~~--~~--~3~0------~4~0----~5~0~----~60~

Days after tumour challenge

Fig.2 Survival of Mice challenged with 5xl04 EL4 cells on Day 0 and treated with various preparations : A, Saline alone. B, 4mg 19. C, 750pg PDM-PGA. D, 4mg 19 plus 750~g PDM-PGA unlinked. E, PDM-PGA-1g (4mg 19, 750~g PDM-PGA).

Table 1. Survival of Mice challenged with 106 EL4 cells sub­cutaneously and treated with conjugates.

Treatment Dose per injection Mean Survival Significance + (Daily, Drug(alkylating) Globulin Time (-.S.D.) relative to

1-4) (l1g) (mg) (Days) controls

Saline - - 17.B 10 0.4

PDM-PGA-1g 200 1 19.4 ± 1.4 P( 0.05 " 400 2 20.0 j:; 1.0 P<.O.Ol " BOO 4 22.0 t 3.4 P( 0.02

19 alone - 1 17.0 :X 0.9 N.S. " - 2 17.0 :to 0.9 N.S. " - 4 17.6 ± 1.3 l~. S.

IPDM-PGA-NRG 200 1 lB.2 '"'- 3.9 N.S. " 400 2 17.0 ~ 1.8 N.S. " 800 4 16.3~7.0 N.S.

16 G.F. ROWLAND, G.J. O'NEILL AND DAL. DAVIES

resolve this question.

I should like to thank Mrs. S. Mann and Miss R. Goldsmith for coping with much of the technical work involved in preparation and testing of conjugates and Mr. A.J. Manstone and his assistants for the in vivo tests.

REFERENCES

Davies, D.A.L., O'Neill, G.J. (1973), Brit. J. Cancer, 28, Supp.I, 285.

Davies, D.A.L., Manstone, A.J., Buckham, S. (1974), Brit. J. Cancer, 30, 297.

Davies, D.A.L., Buckham, S., Mans tone , A.J. (1974), Brit. J. Cancer, 30, 305.

Davies, D.A.L., O'Neill, G.J. (1974), p.2l8 Proc. XI Int. Cancer Congress, Florence.

Ehrlich, P. (1906), Collected Studies on Innnunity, Vo1.2, p.442, New York: John Wiley.

Flechner, I. (1973), Europ. J. Cancer, 9, 741.

Ghose, T., Norvell, S.T., Guclu, A. et al. (1972), Brit. Med. J. iii 495.

Hurwitz, E. , Levy, R. , Maron R. et al. (1975) , Cancer Res.35, 1175.

Levy, R. , Hurwitz, E. , Maron R. et al. (1975) , Cancer Res. 35, 1182.

Linford, J.H. , Froese, G. , Berczi, 1., Israels, L.G. (1974), J. Nat. Cancer lnst. 52, 1665.

Mathe, G., Loc, T.B., Bernard, J. (1958), Compt. Rend. 244,1626.

Moolten, F.L., Cooperband, S.R. (1970), Science, 169, 68.

O'Neill, G.J., Davies, D.A.L., Proc. 2nd Nat. Congress of Oncology, Bucharest.

Rowland, G.F. (1969), Cancer Res. 29,391.

Rowland, G.F., O'Neill, G.J., Davies, D.A.L. (1975), Nature,255, 487.

Rubens, R.D., Dulbecco, R. (1974), Nature, 248, 81.

EFFECTS OF ANTI-DNA AND ANTI-RNA ANTIBODIES BOUND TO MELPHALAN AND METHOTREXATE ON C3H MAMMARY ADENO­CARCINOMA AND L 1210 LEUKAEMIA

Pierre Tran Ba Loc

Laboratory of Zoology and Parasitology, Div. Experimental Cmcerology and Chemotherapy, Faculty of Medicine and Pharmacy 4, Place Saint Jacques, 25030 Besancon, France

Approaches to immunotherapy of neoplastic diseases can be improved by two kinds of methods: one non-specific, the other has specific aims. Our studies concerned mostly the latter. It is known that the biological sub­strates of neoplastic processes are contained in DNA and RNA molecules of cancer cells. DNA and RNA are involved whatever the etiology of neo­plasms: on the one hand, the possible modification of the genome by direct mutation of DNA or by incorporation of oncogenic viruses; on the other hand, the oncorna viruses and messenger RNA as possible causes of neoplastic trans­fromation of cells. Based on this hypothesis, previous work on animal tumours has shown the cytostatic effect of DNA isolated from the same tumours and chemically modified. It has been postulated that the modified DNA acted as a vaccine in active immunotherapy of the host from which tumour DNA was extracted. With passive immunotherapy, a new approach has also been obtained by getting DNA antiserum from rabbits with the DNA isolated from tumours as antigens and chemically combined with rabbit gamma­globulins; the DNA being considered as a hapten. In another study, RNA extract from tumours was used in the same way and chemically modified for active immunotherapy assays and to produce interferon-like substances.

In the present study, passive immunotherapy is used by means of anti­DNA and anti-RNA antibodies chemically bound to antimitotic substances, Melphalan and Methotrexate which are known to be active on cancer cells but very toxi c to norma I cells. These substances are bound chemi cally to antibodi es produced by our methods descri bed herewi th. The resul ti ng products, antibodies bound to antimitotics, seem to point electively on L 1210 leukaemia cells and C3H mammary adenocarcinoma cells, antibodies used as a specific vehicle.

17

18 P. TRAN BA LOC

MATERIALS AND METHODS

1. Isolati on of DNA and RNA

The tumours used for these experiments are transplanted mammary adeno­carcinoma named TMB ( T=tumour, M=mammary, B=Besancon) arising from spontaneous mammary adenocarcinoma bearing TMV virus in a C3H He female mouse (Orleans Centre National de la Recherche Scientifique Labora tory supply). The TMB tumour was at its 127th transplantation in isogenic C3H mice. Five weeks after the transplantation, the tumours were collected after killing mice by cervical break. The frozen tissue was thawed and homogenized in a Waring Blender for 3 minutes with cooling at a concentration of 109 of wet tissue per lOOml of extraction buffer (O.14M CINa; O.OlM ethylene di­amine-tetra-acetic acid; dodecyl sodium sulphate 1 percent). Centrifugation at 10,000 r per minute during 10 minutes gave a supernatant containing a crude RNA bound to histones (Fracti on 51). The pellet is treated 3 times and all supernatants were added to fraction 51. The pellet remaining after the above treatment was added to 3 volumes of 5M NaCI and homogenized. It was then stored overnight at 4°C. It was then centrifuged at 10,000 r per minute for 20 minutes giving a supernatant, 52, containing a crude DNA bound to histones which was dialysed against a liquid containing 0.001 M Tris + O.OOlM EDTA + 0.05M NaCI 3 times during 24 hours. Crude DNA and RNA obtained were precipitated 3 times by ethanol for purification. After the last centrifugation for eliminating insoluble materials, the DNA and RNA obtained must give absorbance ratio greater than 1.70 (optical density 260:280nm). Every centrifugation was performed at 4°C in a refrigerating ultracentrifuge (Beckman). Ultra-violet spectra were obtained by a UNICAM Spectrophotometer recorder.

2. Preparati on of antisera

Tumour DNA and RNA were previously combined with rabbit gamma­globulins by means of N N'dicyclo-hexyl-carbodiimide and the DNA and RNA considered as hpatens, for hyperimmunization in rabbits. Antisera against DNA and RNA antigens isolated from tumours were prepared by immunizing rabbits with a series of intramuscular injections of the appropriate antigens added to Plasmodium berghei infected red cells as immunizing adjuvant (instead of Freund's complete adjuvant). Immunization was performed by three injections a week during three weeks. Ten days after the last injection, the rabbi ts were bl ed and the serum coli ected was used for gel mi crodi ffusi on analysis.

ANTI-DNA AND ANTI-RNA ANTIBODIES 19

3. Preparation of anti-DNA and anti-RNA gamma-globulin

From the antiserum collected the gamma-globulins are prepared according to the technique of Deustch by ethanol precipitation. The combination of anti-DNA and anti-RNA gamma-globulins with Melphalan is performed according to the following procedure. Binding is made between the acid function of Melphalan and the amine and hydroxyl groups of gamma-globulins by means of N N'dicyclohexyl-carbodiimide. 10mg of Melphalan is dissolved in a solution of water and propyleneglycol and added to a solution of anti­DNA and anti-RNA gamma-globulins (150mg) containing 20mg of N N'di­cyclo-hexyl-carbodiimide. The combination of anti-DNA and anti-RNA gamma-globulins with Methotrexate is performed by binding the acid function of Methotrexate with amine and hydroxyl groups of gamma-globulins by means of N N'dicyclo-hyxyl-carbodiimide. 10mg of Methotrexate is dissolved in a buffer containing sodium carbonate, pH 8.5, and added with stirring to a solution of anti-DNA and anti-RNA gamma-globulins (175mg) containing 25 mg of N N'dicyclo-hyxyl-carbodiimide.

4. Biological assays

a) 120 female C3H He Orl Mice were housed with standard pellets and water supplied ad libitum. A tumour suspension was prepared in normal saline at a finalconcentration of 10 cells per mi. Experimental mice were injected subcutaneously in the flank with 0.2ml of the suspension of cancer cells. On the day after the transplantation, the mice were randomized in 6 groups. Group 1: Controls Group 2: Treated by anti -DNA and anti -RNA antibodies; Group 3: Treated by anti-DNA and anti-RNA antibodies combined with

Melphalan (lOmg/kg) ; Group 4: Treated by anti -DNA and anti -RNA antibodies combined with

Methotrexate (20mg/kg) ; Group 5: Treated by Melphalan alone (lJmg/kg); Group 6: Treated by Methotrexate alone (20mgjkg) . Every group had 20 tumour-bearing mice. Survival times have been compared.

b) 120 DBA 2 mice were inoculated with L 1210 leukaemia. On the day after the transplantation, the mice were randomized into 6 groups as in (a) above t each group containing 20 mice, survival times have been compared.

20 P. TRAN BA LOC

RESULTS

a) L 1210 leukaemia assays: survival time

b) C3H mammary adenocarcinoma assays: survi va I ti me

Group 1: 7 + 1 day Group 2: 7 + 2 days Group 3: 27 + 4 days Group 4: 31 + 5 days Group 5: 12 + 1 day Group 6: 14 + 2 days

Group 1. 42 + 6 days Group 2. 45 + 4 days Group 3: 50 + 7 days Group 4 : 54 + 8 days Group 5 I 22 + 5 days Group 6: 20 + 3 days

CONCLUS IONS

It has been shown that DNA and RNA isolated from tumours of L 1210 leukaemia and C3H mammary adenocarcinoma and chemically bound to rabbit gamma-globulins can produce in rabbits anti-DNA and anti-RNA antisera by the usual hyperimmunization, DNA and RNA having the role of haptens. From these antisera have been jsolated antibodies which were bound to Mel­phalan and Methotrexate by means of N N'dicyclo-hexyl-carbodiimide. The products resulting from anti-DNA and anti-RNA antibodies bound to Melphalan and Methotrexate were used in biological assays on L 1210 leukaemia and C3H transplanted mammary adenocarcinoma. Survival time studies have shown that DNA and RNA antibodies bound to Melphalan and Methotrexate are more active than DNA and RNA antibodies or Melphalan and Methotrexate alone. The hypothesis is that these anti-DNA and anti-RNA antibodies bound to Melphalan and Methotrexate guide the antimitotic substances to the cancer cells rather than to the normal cells. This study is a new approach to speci fi c chemo-immunotherapy.

REFERENCES

Isaacs, A., Li nderman, J. (1957). Proc . Roy. Soc. Bi 01., 147, 258. Mathe, G., Tran Be Loc,~. and Bernard, J. (1958). C.R.Acad.Sc.,246,

1626. -Tran Be Loc, P. and Bernard, J. (1960). C.R.Acad.Sc., 251,477. Tran Ba Loc, P. (1966). C.R. Soc.Biol. 160, 36. -Tran Ba Loc, P. (1970). J.Med.Besancon,6, 143 Tran Be Loc, P. (1972). Adv. Antimicrobial and antineoplastic Chemotherapy,

(Urban and Schwarzenberg Ed. Wi en) p. 183. Tran Be Loc, P. (1972). C. R.Soc. Bi 01., 166, 343.

IMMUNOSUPPRESSIVE EFFECTS OF SOME ORGANIC COMPOUNDS

WITH ANTI-INFLAMMATORY ACTIVITY

I. BARASOAIN, A. PORTOLES, J.M. ROJO, AND C. SUNKEL*

Inst."Jaime Ferran" of Microbiology.C.S.I.C. and

Alter Laboratories* - Madrid - Spain

SUMMARY: Some indomethacin esters have been tested for immunosu­ppressive activity and compared with Bleomycin, Mitomycin C, Ac­tinomycin D and Rifamycin SV. The most active substances were ac­tinomycin and an indomethacin-methylen-benzoic ester (IMB-1). It has been seen that IgM response is the most affected when drugs are administered in a short period of time and previously to the antigen.

INTRODUCTION: Today immunosuppressive therapy is widely applied in transplants as well as in the treatment of autoimmune and neoplas­tic diseases. In addition, immunosuppresants are often administe­red in association with other drugs which further hampers a proper evaluation of the treatment. As it is known, several clinicopatho­logical side effects can be originated under indiscriminate con­ditions of immunosuppression. An enhancement and generalization of tuberculosis, brucellosis, typho-bacillosis and other infectious diseases has been pointed out (Krueger, 1972). On this basis it appears to be necessary to screen the newly developed anti-inflam­matory compounds for their immunodeppressive effects by comparing them with antibiotics interfering with the nucleic acid metabolism since they may affect the immune response.

In this work some indomethacin derivative molecules have been tested for immunosuppressive activity and compared with the effects produced by antibiotics such as Bleomycin, Mitomycin C, Actinomy­cin D and Rifamycin SV on primary immune responses.

MATERIALS AND METHODS: Drugs.Actinomycin D (AD) and Mitomycin C (MC) (Calbiochem), Blemycin (BL) (Almirall) and Rifamycin SV (Le­petit) were used.

21

22 I. BARASOAI N ET AL.

Indomethacin (Merck) and three Indomethacin esters (patent pending.Spa,numbers 432544 and 432545) synthesized by Alter, S.A. Laboratories were employed.

Animals. Male Ham/ICR Swiss mice, weighing 18-20 were emplo­yed through out all the experiments.

Anti-inflammatory activity. Carrageenin test was performed according to the Winter et al. (1962) method.

Immunization pattern and drug dosage. Sheep red blood cells (SRBC) were washed three times in saline phosphate buffer pH 7.2 before immunization and 2.5 x 108 SRBC in 0.1 ml were injected intraperitoneally (i.p.). AD (0.4 mg/Kg) and BL (34 mg/Kg) were administered in a single i.p. injection simultaneously to the an­tigen while 1.25 mg/Kg/day of MC was injected during two days star­ting simultaneously with immunization and 10 mg/Kg/day of RF was given i.p. along all the immune response curve, beginning on the day of immunization. Anti-inflammatory drugs were given orally at a dose of 11.15 ~,moles/Kg/day during three days before, and simul­taneously with the antigen.

Determination of antibody levels. The hemagglutination test was performed as described by Yamaki et al. (1969) with slight modifications. The immunohemolysis test was carried out with 51Cr labeled SRBC following Rojo et al., (1975). The direct hemolytic plaque assay was performed following Jerne et al. (1963) with the modification introduced by Bullock and Moller (1972).

RESULTS AND DISCUSSION: The molecular structures and activities of the anti-inflammatory molecules are shown in Table I and in Fig. 1 a primary immune response curve to SRBC is depicted. The hemolytic plaque assay, which detects IgM forming cells (Jerne et al. 1974), showed a single peak with a maximum around the 4th day after immunization. This maximum was also detected in serum anti­body tests. A second broad peak presumably due to IgG was only de­tected by the hemagglutination test. Consequently, although the immunohemolysis test is far more sensitive than the hemagglutina­tion, we employed mainly this second assay in order to have more complete information of the antibody synthesis.

Table II shows the immunosuppressive activity of antibiotics acting on nucleic acid metabolism, and the results can be summariz­ed as follows: (i) AD strongly inhibits the activity of the anti­body producing cells as well as the levels of circulating antibo­dies; (ii) MC produces a reduction in both assays without affect­ing the IgG synthesis, while a shift of 2 days was observed in the IgM peak; (iii) RF produces a very strong inhibition on the anti­body serum levels and also a shift in the IgM peak as well as a

IMMUNOSUPPRESSIVE EFFECTS OF SOME ORGANIC COMPOUNDS 23

TABLE 1. Molecular Structure and Antiinflammatory Activity of the Three Indomethacin Esters Tested

CH3°"l§:r]::.CH2-COO(~

Compounds ~ CH3 CI-@- CO Antiinfl~~ R octlvlty

Indomethacin H 100

1MB-I -c~-ooc-ill 100

IMT-3 -C~-OOC~ 87.0

IF-31 -Ctt-C§} CH3 13.6 CH3

reduction in the plaque forming activity; and (iv) Bleomycin was found to be the least active drug. Although Yamaki et al., (1969) did not find any effect of BL on the immune response we found a shift of two days in the peak of IgM serum antibodies.

In Table III the results obtained after treatment with anti­inflammatory drugs are summarized. With IF-31 no effect at all was found while with indomethacin a reduction of the IgM peak, follow­ed by a slight reduction of the IgG,is observed and a shift of one day was produced on the activity of the antibody forming cells. IMT-3 and IMB-1 compounds were the most immunodeppressive anti-in­flammatories. In the former the IgM synthesis was strongly reduced while in the latter no separate IgM peak was detected. The plaque forming cells activity was delayed until day 6 in both cases,being very low in comparison with the control experiment.

Icr 104 I T 6 Z 0& ~ ~

I E ~\ ~ Q. ~ 104 J \,'A 1-'\ ! 15 t= /7 \..\ . u- (I)

... z .! .,...--_ 0- :::I ~ in I \ -"']i:"... 0.......0 ~

! i 103 fl \1\':' \ .................. °'0 lif~ i o! ~....... -.... I&.

c!' "....... II. 2 2 A-'-~~'-A ~! 102 101

3 5 7 9 II 13 DAYS AFTER ANTIGEN INJECTION

Fig. 1. Immune Response Patterns to Sheep 1rythrocytes in Male Ham/ICR Swiss Mice.

24 I. BARASOAI N ET AL.

TABLE II. Effect of Several Antibiotics on a Primary Immune Response to SRBC, in Mice

DRUG Antibody level variations after the antiQen injection DATA GIVEN

ASSAYED 3 4 5 6 7 8 9 10 II 12 13 dayS AS:

None 200 3200 380 800 1100 2500 - 4000 3200 2100 1300 ~ 0

Actinomycin-D (10 (10 <10 150 500 2000 - 3350 3200 1600 650 ~ ~ IX: W I-

Mitomycin-C <10 30 200 1100 300 2600 - 2250 I~ 550 310 '" ;r; t: Rifamycin-SV <10 <10 300 600 300 800 - 1600 600 180 110 ~

;:) u

Bleomycin <10 100 1300 3200 1600 530 - 2530 800 290 100 g:; u

None 4 407 657 218 16 4 2 '" ~ - - - - Z (3

~ctinamycin-O ;:)

~ 4 9 50 17 13 0 - - - - - 0

ffen Z w Mitomycin-C 2 144 396 30 2 0

Q...J ~ - - - - - 1...1 >- w Q. 8 u en

~ifamycin- SV 2 2 300 36 6. 0 - - - - - aI ~ - ........

Bleomycin 2 62 720 94 9 0 ~ fr - - - - - ct ~

DATA EXPRESSED WITH (-) WERE NOT DETERMINED

TABLE III. Effect of Several Antiinflammatory Drugs on a Primary Immune Response to SRBC, in Mice

DRUG Antibody level variations after the antiQen injection DATA GIVEN

ASSAYED 3 4 5 6 7 8 9 10 II 12 13daY5 AS:

None 140 1810 160 480 1426 3840 4260 3520 2500 1840 en

1340 !!:! 0 0

Indomethacin 5 320 160 120 580 950 980 1100 2000 1100 990 aI -i IX:

11.1 t-

1MB-I 15 87 108 320 660 1200 907 670 520 350 220 '" Z I-

~ ~

IMT-3 5 290 130 210 1070 1450 2990 1420 1120 1060 940 ...I ;:) u

IF- 31 107 1840 115 187 187 2600 2700 2400 2000 1410 1070 g:; u

None 52 463 189 52 3 - - 2 - - - ! ~ u ;:) w

Indomethaci~ 6 165 270 33 15 0 u - - 5 - - -ff en z

..Jw Q. ..J~ 1MB-I 28 48 93 94 13 - - I - - - I >- wQ. 0 uen

19 0 IOQ IMT-3 46 67 129 19 - - 4 - - - aI i= ~ IF-31 20 450 93 58 12 3 Z - - - - - ct Q. -

IMMUNOSUPPRESSIVE EFFECTS OF SOME ORGANIC COMPOUNDS 25

Since these two anti-inflammatory drugs have a similar chemi­cal structure i1 may explain their parallel activity and consequen­tly a similar mode of action.

In Fig. 2 the effect of the treatments with the two most im­munosuppressive drugs used in this work is compared. As can be seen IMB-1 immunosuppressive effect is comparable although not so dras­tic with that of AD. In both cases the IgM and IgG responses are overlapped. IgM response is the most affected when immunosuppressi­ve drugs are administered in a short period of time and previously to the antigen.

During recent years some studies have been made on the inhibi­tory action of non steroid anti-inflammatory drugs, such as indo­methacin and aspirin.on the synthesis of prostaglandins (Ferreira and Vane, 1974). This action may explain not only their known effect on inflammation and associated phenomena such as oedema, pain and fever but on the immunocompetent cell proliferation and antibody release.

10~ 10" \' -,~~ 102 v~/ H-". 103

.... • • 0' ...

o .; 0 - 101 0

102 i !J \PFC

iii ° Co) c ___ crc z 0 1&1 1&1 -I AFTER TREATMENT It. AD .......... --., II) 1MB-I ~

102 I H~ ~ Icr

" 0"'~'-Co) I&. It.

101 I . HMG rJ2

{! "".PFe rJl

II 13 3 5 7 9 II 13

TIME (DAYS)

Fig. 2. Immune Response in Mice After Actinomycin D and IMB-l Treatments.

~ ~

2 ~ I: 1&1 II)

I ~ c

26 I. BARASOAI N ET AL.

Although indomethacin is commonly referred as not deeply affecting immune response (Levy, 1974 and Grinwich et al. 1974), these results suggest that an immunosuppressive effect occurs at clinical doses with the drug and some of its derivatives.

Acknowledgements: This work has been carried out with the assis­tance of a Grant for Basic Research from Alter, S.A. Laboratories (Spain) .

REFERENCES

Bullock, W.W. and Moller, E. (1972) Eur. J. Immunol. 2: 517-522 Ferreira, S.H. and Vane, J.R. (1974) Ann Rev. Pharmacal. 14: 57-

73 Grinwich, K., Skelly, R., Sheridan, J. and Plescia, O.J. (1974)

Can. Fed. BioI. Sci. 17: 350 Jerne, N.K., Nordin, A.A. and Henry, C. (1963) In cell-bound

Antibodies, ed. Amos, B. and Koprowski, H. p. 109 Wi star Institute Press.

Jerne, N.K., Henry, C., Nordin A.A., Fuji, H., Koros, A.M.C. and Lefkovits, I. (1974) Transplant Rev. 18, 130-191

Krueger, G.R. (1972) Adv. Pharmacol. Chemother. 10, 1-90 Levy, L. (1974) Arch. Int. Pharmacodyn 211, 8-1~ Rojo, J.M., Barasoain, I. and Rubio, N. (1975) Comm. V. Congres.

Nac. Microbiol. Salamanca. Spain Winter, C.A., Risley, E.A. and Nuss, G.W. (1962) Proc. Soc. Exp.

Med. 111, 544 Yamaki, H.:-Tanaka, M. and Umezawa, H. (1969) J. Antibiotics 22

315-321

DIFFERENTIAL IMMUNOSUPPRESSIVE EFFECTS OF ANTICANCER

AGENTS ON LYMPHOID SUBPOPULATIONS

E. Tsubura, K. Yata, G. Hisano, K. Tominaga, H. Sasaki and S. Sone 3rd Department of Medicine, Tokushina University School of Medicine, Tokushina, Japan

SUMMARY

The immunosuppressive effects of Cyclophosphamide, Mitomycin C, FT-207, Hydrocortisone and Azathioprine were investigated by studying their effects on the proportions of T cells and B cells in mouse spleen and on the hemoly­tic plaque forming cells to sheep red blood cells (SRBC) and lipopolysaccharide (LPS). These compounds all caused a marked reduction in the total number of thymus cells in mice. Five consectuve daily intraperitoneal injections of Cyclophosphamide caused a decrease in the percentage of B lymphocytes and increase in that of T lymphocytes in the spleen and lymph nodes. Hydrocortisone had the same effect as Cyclophosphamide on the spleen, but Mitomycin C, FT-207 and Azathioprine had no significant effects on the pro­portions of Band T lymphocytes in the spleen. Cyclophos­phamide had more marked efpects on activated spleen cells than on normal spleen cells. Cyclophosphamide treatment also caused marked suppression of antibody production to SRBC or LPS, whereas treatment with Azathioprine, Hydrocor­tisone, Mitomycin C or FT-207 caused only moderate sup­pression of LPS-PFC. Treatment with Cyclophosphamide or Azathioprine increased the blastogenic response to PHA. ~hereas treatment with Cyclophosphamide or Mitomycin C de­creased the response to LPS and Mitomycin C also decreased the response to PHA.

INTRODUCTION

The immune deficiency state in patients with cancer or

27

28 E. TSUBURA ET AL.

autoimmune disease is due both to the disease itself and to administration of various immunosuppressive agents. Thus treatment of patients with immunosuppressive agents places them at a great risk to infection from a wide variety of organisms. This is a serious drawback to this therapy but at present it is unavoidable because the mechanism of deve­lopment of secondary immune deficiency are not clear. In future a combination of cancer chemotherapy and immunopot­entiat ion of the host, so called immunochemotherapy, will probably become widely used in clinical fields; it is important therefore to study the basic problem of the effects of anticancer agents on the immune system of the host. Accordingly, to elucidate the effects of various immunosuppressants on the host defence mechanism, we measured the effects of anticancer agents on the number of thymocytes, the subpopulation of spleen lymphocytes and antibody production in C3 H/He mice and the proliferative response of lymphocytes in vitro to mitogens.

MATERIALS AND METHODS

Animals. Female C3 H/He strain mice of 6-8 weeks old were used.

Immunosuppressive treatments. Mice were injected intra­peritoneally for 5 consecutive days with doses of one fifth of 43.6% of the LD~O of the test compounds. In mg/kg these doses were; Cyclopnosphamide 40, Azathioprine 90, Hydro­cortisone 116, Mitomycin C 0.48 and Futraful (FT-207) 83.

Immunization. Sheep red blood cells (SRBC) were obtained from Nihon Biotest Kenkyusho (Tokyo). Lipopolysaccharide (LPS) of Escherichia coli 055:B5 was purchased from Difco Laboratories (Detroit, USA) and was boiled for 60 min to abolish its toxicity. Animals were immunized by intr~ venous injection of either 0.25 ml of 20% SRBC suspension or 30 mg of detoxicated LPS in physiological saline on exp­erimental day 2 (5 days before essay of plaque formation).

Preparation of cell suspensions. On day 7 mice were killed and suspensions of thymus and spleen cells were prepared by squashing the organs between glass slides in Eagle's MEM (Nakarai Chemical Co. Ltd) and then passing the homogenate through a No. 150 platinum mesh (Ikemoto Rikagaku, Tokyo).

Detection of T and B lymphocytes. Anti 9 C3 H/AKR antibody was prepared by the method of Gorcznskii (1972) and the Trypan blue dye exclusion cytotoxicity test (Terasaki 1964) was used for detection of T cells.

The direct immunofluorescent method (Rabellino et aI,

DIFFERENTIAL IMMUNOSUPPRESSIVE EFFECTS 29

1971) was used for detection of B cells.

Measurement of blastogenic response (to PHA. PWM. LPS) Spleen lymphocytes were suspended at a concentration of Ixl06 cells/2ml in MEM supplemented with 20% fetal calf serum and duplicate 2 ml samples were incubated with l~l of phytohema~-glutinin-P (PHA-P)(Difco Lab), pock week mi~ogen (PWM) or lipopolysaccharide (LPS)(Difco Lab) at 37 C for 72 hours under an atmosphere of 5% CO 2 end 95% air. Then 24 hours before harvesting, 1 ~Ci of tritiated thymidine (3H-TdR)(Daiichi Pure Chemicals Co. Ltd) was added. Radioactivity was counted in an Aloka, Model LSC-601, liquid scintillation counter. Activity is expressed in counts per minute (cpm).

Detection of Antibody. Cells producing antibody to SRBC five days after immunization were detected by the hemoly­tic plaque technique, as described by Jern and modified by Cunningham (1968)(SRBC-PFC). The ratio of splenic plaque forming cells (PFC) to LPS (LPS-PFC) was determined by a slight modification of the method of Britton (1969). The total number of splenic PFC and the PFC per 106 spleen cells were then determined.

RESULTS

Number of thymocytes. The number of thymocytes in mice treated with Cyclophosphamide or Hydrocortisone decreased markedly to 3.6% and 9.8% respectively of the control value. On treatment with FT-207 the number decreased mod­erately to 21.3% of the control and on treatment with Azathioprine or Mitomycin C to 60%-52% of the control.

Number of Spleen cells. Cyclophosphamide caused the greatest decpease in the number of spleen cells in mice. Mitomycin C, Azathioprine and Hydrocortisone also caused some decrease, but FT-207 did not.

Proportion of T lymphocytes. The proportion of T cells in untreated mice, estimated by our method varied from 19% to 28.0% in the spleen and 62.3 to 73.8% in the lymph nodes. Treatment with Cyclophosphamide or Hydrocortisone increased the proportion of T cells in the spleen. Treat­ment with cyclophosphamide also mcreased the proportion of T cells in the lymph nodes but Hydrocortisone did not. Treatment with Mitomycin C, FT-207 or Azathioprine did not cause any significant change in the T cell population.

Proportion of B lymphocytes. The proportion of B lympho­cytes in the spleen of untreated mice varied from 30.3% to

30 E. TSUBURA ET AL.

40.0%. The proporti~ decreased markedly on treatment with Cyclophosphamide or Hydrocortisone, and these changes cor­responded to increase in the proportion of T cells. Treat­ment with Mitomycin C, FT-207 or Azathioprine caused no significant change in the proportion.

Proportions of T and B cells in spleen activated with SRBC

or LPS. Similar but more marked changes were observed in activated spleen cells. On treatment with Cyclophospha­mide plus LPS, there was a more significant decrease ot Ig-bearing cells and increase in the proportion of Q bearing cells. Treatment with Hydrocortisone caused no significant change of ig-bearing cells. Thus, activated B lymphocytes seem to be resistant to steroid.

Blasto enic res onse to mito ens. Cyclophosphamide (24mg! kg day was injected i.p. for 7 consecutive days and the blastogenic response observed was expressed as a percent­age of that in control mice. It was found that Cycloph­osphamide increased the blastogenic response to PHA. Similar treatment with Azathioprine also increased the response to PHA. However, a remarkable decrease in the responses to LPS were observed in Cyclophosphamide and to PHA in Mitomycin C treated mice.

Antibody production. SRBC-PFC. Treatment with Cyclophos­phamide greatly suppressed antibody production to SRBC in mice to below 25% of the control value. Whereas Azathio­prine, Hydrocortisone, Mitomycin C and FT-207 suppressed antibody production to about 50% of the control value.

Anti~ody production. LPS-PFC. Specific antibody production to LPS was measured. Significant suppression of its pro­duction was observed in Cyclophosphamide or Azathioprine treated mice. Mitomycin C treated mice showed moderate suppression of antibody production to LPS as well as to SRBC. Hydrocortisone and FT-207 did not affect antibody production to LPS.

REFERENCES

Gorcznskii, R.M. (1972) J. Immunol., 108, 547. Terasaki, P.E. (1964) Nature, 204, 998.

Rabellino, E. et al (1971) J. Exp. Med. 133, 156.

Cunningham, A.J. & Szenberg, A. (1968) Immunol. 14, 559.

Britton, S. (1969) Immunol., 16, 513.

EXPERIMENTS AND THEORETICAL CONSIDERATIONS ON SYNCHRONISATION OF L 1210 ASCITES TUMOUR CELLS AND CRYPT EPITHELIA OF THE MOUSE WITH VINCRISTINE

W.Jellinghaus, R.Maidhof, B.Schultze, W.Maurer

Institut fur Medizinische Strahlenkunde der Universitat Wurzburg, 87 Wurzburg, West-Germany

The chemotherapy of malignant tumours is limited as many drugs are most effective against cells in one specific phase of the cell cycle. If by synchronisa­tion the number of cells in the drug sensitive phase could be increased the chances of successful therapy would be improved. Such a cycle phase specific therapy after synchronisation is often practiced clinically using Vincristine in combination with cyclophosphamide. With this form of therapy the most important question is if it is possible to achieve in vivo synchronisa­tion of cells.

The results of only a few experimental studies have been reported in the literature and the data ob­tained so far with Vincristine are contradictory (3,4, 5,7,8). For this reason experiments to investigate the possibility of in vivo synchronisation with Vincris­tine, using the L 1210 leukemia and the crypt epithelia of the normal mouse, have been carried out.

Mitotic indices of the L 1210 cells after Vincristine On the 5th day after transplantation of 105 cells

the mice were injected with 0,1 ~g Vincristine per ani­mal at time zero. From time zero to 48 hours the ani­mals were killed at intervals of two hours. The experi­mental period of 48 hours is three to four times the cycle time of the L 1210 cells. The mitotic indices after Vincristine are given in figure 1; the crossed points are the average of 4 to 7 animals, and each of the other points represents one animal. From the native

31

32 w. JELLINGHAUS ET AL.

Mitotic Index

30

% L1210 ASCItes tumor

~ 20

J \ 10

o 10 20 30 40 50 hours after VInCrlstl ne

Fig. 1 Mitotic index of the L 1210 cells after injec­tion of 0,1 ~g Vincristine per animal. The points are measured mitotic indices. (For details see text)

mitotic index of 2,2% the percentage of arrested mi­toses increases rapidly to reach a maximum of 26% at 4 hours. The cell flux is 7%/hr and this means that almost all the cells entering mitosis are arrested in the 4 hour period. This fact is supported by the fail­ure to see any anaphases during this time. Between 4 and 12 hours the percentage of arrested mitoses falls to the native value. We have called this 8 hour period the "releasing time". From 12 hours on the mitotic in­dices lie within the normal range and there is no ev­idence of a second peak of mitoses. Thus, in this ex­periment no synchronising effect could be seen.

Calculation of the mitotic indices of the L 1210 cells from a theoretical model

Having failed experimentally to find any evidence of synchronisation we decided to calculate theoreti­cally whether any synchronisation could be expected. Figure 2 explains the method of these calculations which is based on the changes in the frequency distri­bution of cells throughout the cycle. To make the mod­el easier to understand, we made idealised assumptions. We assumed that the cycle time remains constant even

L 1210 ASCITES TUMOUR CELLS AND CRYPT EPITHELIA 33

10 20 30 hours

Fig. 2 Theoretical model to calculate the mitotic and labelling indices at any time after Vincristine injec­tion. (For details see text)

under the influence of Vincristine, that the duration of mitosis after the release of the Vincristine bloc is not changed and that the cells have a constant fre­quency distribution along the cycle. Fig.2 A shows the normal distribution of the L 1210 cells throughout the cell cycle (T=14 hrs.,Gl=4 hrs., 5=8 hrs.,G2+M=2 hrs.). The native mitotic index is 2%. Fig.2 B shows the distribution of cells four hours af­ter Vincristine injection. As shown in fig.l, by this time 26% of all cells are arrested in mitosis. This is indicated by the column at mitosis MI. During this time no cells leave mitosis and thus the four hour period of the cell cycle after mitosis is empty of cells. This is indicated by the broken line. During the next 8 hours in the so called II r eleasing time ll the arrested cells are assumed to pass into the following cycle. 12 hours after Vincristine injection an 8 hour long subpopulation of formerly arrested mitoses has been formed. We have called this subpopulation the Vincris­tine (IIVCRII)-population. At the end of the Vincristine

34 w. JELLINGHAUS ET AL.

bloc those cells not affected by Vincristine are as­sumed to pass through the cycle and 12 hours after Vincristine they form the "Rest"-population. Fig.2 C demonstrates that, according to this theory, the effect of Vincristine can be considered formally as a splitting of the original cell population into two subpopulations, which we have called the VCR-popu­lation and the Rest-population. These two subpopula­tions then pass jointly through following mitoses Ml, M2 and M3. The theoretically expected mitotic index at any time can then easily be calculated. The solid line in the lower part of fig.2 gives the results of these calculations. The points C-G on the solid line corre­spond to the frequency distributions C-G shown above.

This model can also be used to calculate the time course of the labelling index to be expected at any time after Vincristine arrest. For this purpose the positions of the S-phases are marked on the diagram. The labelling index at any time is the ratio of cells in the S-phase to the total number of cells.

A comparison of the theoretical and experimental mito­tic indices of the L 1210 cells

In fig.l the experimental results are represented by the points and the theoretical indices by the solid line. The dotted line represents the theoretical results calculated assuming exponential growth. Even with the idealized assumptions made, the expected maxima are flat and too small to be detected experimen­tally. Thus, the theoretical model and the experimen­tal results are in good agreement and show that there is no synchronisation after Vincristine.

Labelling indices of the L 1210 cells after Vincristine Fig.3 shows the time course of the labelling in­

dex after Vincristine injection at time zero. Each point represents one animal. At first there is a fall in labelling index, which is followed by a rise to just above the native value of 52%. From 12 hours on the values lie within the normal range and there is no further peak. Therefore once again there is no evidence of synchro­nisation. Assuming a completely constant cycle time even under the influence of Vincristine the theoreti­cally expected pattern of labelling indices is given by the short-dashed line. However, from our own and published data (1) it is known that the cycle time of the L 1210 cells shows considerable variation. For this reason the calculations were repeated assuming a varia-

L 1210 ASCITES TUMOUR CELLS AND CRYPT EPITHELIA

Labeling Index

100

% L 1210 Ascites tumor

(' ,"'-"\ I \ I \

I \ f \ I \ I \

_. f \ f \ -. -. ~ \ { \ '~\ 1\

• '/ '\ -.. I \

• .. ~ ' .. '~~. , - \ •• '-. --I I \ '-.... • 50 •• ~\ _. _'I •••• .,. • \ •

\ fe· \ •• ---' ' • .. ~ .' . \ I.·. \-.~ __ • :,,:/i' \./ J .\~J .. ,: ....

•

•

o 10 20 30 40 50 hours after VI ncrist I n e

Fig. 3 Labelling indices of the L 1210 cells after

35

0,1 ~g Vincristine per animal. The points are measured labelling indices. (For details see text)

tion in cycle time of 11 to 17 hours with a mean value of 14 hours. The result of this calculation is given by the solid line in fig.3. Taking into consideration the real variation in cycle time found within the L 1210 cells, good agreement can be seen between theory and experiment.

Similar experiments were carried out on the je­junal crypt epithelia of the normal mouse. The Vincris­tine dose used was 0,2 ~g/g body weight. Once again no evidence of synchronisation was found (4,7).

The results show that there is no synchrony after Vincristine treatment in either L 1210 ascites cells or in the crypt epithelia of the normal mouse. The the­oretical model demonstrates that none could be expected. There are two main reasons for the failure to achieve synchrony. One is the long releasing time and the other is the big variation of the cycle time. Other workers (2,3,4,5,6) have found the releasing time after Vin­cristine to be even longer.

36 w. JELLINGHAUS ET AL.

If a long releasing time is a general property of Vincristine it is unlikely that Vincristine will be a suitable agent for synchronisation in vivo.

Literature

1. Dombernowsky,P., Hartmann,N.R.: Cancer Res. 32: 2452-2458 (972)

2. Frei,E.III, Whang,J., Scoggins,R.B., Van Scott,E.J., Rall,D.P., Ben,M.: Cancer Res. 24: 1918-1925 (1964)

3. Hartenstein,R., Ehrhart,H., Hoffman,K.: Z.Krebs­forsch. 79: 213-220 (1973)

4. Jellinghaus,W., Maidhof,R., Schultze,B., Maurer,W.: Z.Krebsforsch. in press (1975)

5. Klein,H.O., Lennartz,K.J., Gross,R., Eder,M., Fi­scher,R.: Dtsch.med.Wschr. 97: 1273/1282 (1972)

6. Klener,P., Donner,L., Houskova,J.: Proc. 7th Int. Congr. Chemother. 2: 353-355 (1972)

7. Maidhof,R., Jellinghaus,W., Schultze,B., Maurer,W.: Dtsch.med.Wschr. 100: 54-56 (1975)

8. Pouillart,P., Hoang Thy Huong,T., Lheritier,J.: Bull. du Cancer 61: 509-526 (1974)

THEORETICAL BASES FOR DESIGNING COMBINATION THERAPY

WITH DIBROMODULCITOL (DBD)

Lapis,K.,Jeney,A.,Kopper,L.,Szende,B.andTakar.s,J.

1st Institute of Pathology,Semmelweis Medical

University, Budapest, Ulloi ut 26, Hungary

From the results of clinical and experimental tumour chemotherapy it seems that no single agent seems to be able to deal effectively with most of the human tumours and reasonable therapeutic results can only be reached by combinations of drugs. At present, however, selection of drugs and treatment schedules for combina­tion treatment is mostly empirical. Experiments should therefore be done to help the selection of the appropr­iate drugs for combinations as well as to help to deter­mine the correct timing of treatments. We have chosen two different considerations to tackle this problen.

In selecting drugs for the combination, we took into consideration firstly the dependency of their effect on the rate of proliferation of the tumour to be treated and secondly the data on the mode of their action at the subcellular level.

In the following experiments a cytostatic hexitol, dibromodulcitol (DBD) has been given in various combina­tions, chiefly because we have a great deal of data on the mechanism of action of this agent.

In previous years, we worked out an assay method to study the relationship between growth rate and drug sensitivity of our test object the NK/Ly ascites lyphoma (Fig. 1). The tumour is transplanted by giving 10 7 cells per animal i.p. Treatment was given for 60 minutes on the fourth or on the tenth day following transplantation.

37

38

i 1

GROWTH CURVE OF NK/l¥ ASCITES TUNOUR AND SCHENE OF EXPERINENTS ®----/~.-~Vvi :.,a,JD.,., l ; ,*".. I

111;1, II-I I TREATNENT

&t ~

RETRAI{SPLANTATION 70't»III ,

EVAWATION(tota/ cell number) on ""day 1 "/0"" •••

AFTER TRANSPt..ANTATION

Figure 1

K. LAPIS ET AL.

Then the cells were retransplanted into healthy animals and the drug effect was evaluted by measuring the total cell number on the seventh day.

In this system we found that DBD acts more effici­ently on young, relatively rapidly proliferating tumour cell populations than on old, slowly profliferating ones. Cyclophosphamide on the other hand has an equal inhibit­ing effect on the two different cell populations while BCNU proved to have a more pronounced effect on the slowly growing cell population (Fig 2).

Our further aim was to study the effect of these agents in combination-therapy. Simultaneous adminstra­tion of DBD and BCND, resulted in an additive effect while in the case of combined administration of DBD and cyclophosphamide a more than addi ti v.e effect was observed (Fig. J).

There is another theoretical possibility to get a better therapeutic response and this is the treatment of a synchronized cell population with a cell cycle (phase) specific drug. We presumed that DBD was such a drug. Vinblastine (1 gamma/animal) was used as a syn­chronizing agent which produced a partial syn­chronization of NK/Ly ascites tumour (Fig.4). DBD treatment was carried out for one hour at

COMBINATION THERAPY WITH DIBROMODULCITOL

Age of treated Dibromodulcltol Cyclophosphamide tumour 350 mg/kg 140 mg Ikg 80 mg/kg

4 day 72,2 +

45,9 80, 5 10 day 36. 1 5, 7 69,0

Age factor++ 0,5 0, 12 0,85

+ The values illustrate the rate of growth inhibition in 0/0

Growth inhibition (G.!.) Nc of treated on 7th day

100 - .-:.------.,--­N of cont rol on 7 th day

c

++ Age factor G.!. on lOth day

G.!. on 4th day

40 mg/kg

27.0 31, 5

1, 16

BCNll 40 mg/kg

50.0 92,5

1,59

Figure 2. The effect of cytostatic agents in different stages of growth of NK/Ly ascites tumour.

Growth inhibition (01.)+

L>rugs Dosage Single treatment ++ Combination ++

mg/kg Drug DR/) Calculated Obs .. r\'t'd alone 140 mg/kg

Cyclophosphamide 100 20 18 38 84

BC\T 10 59 19 78 78

5-Fluorouracil 100 H 17 48 64

V inc ristine 1 45 18 61 58

Bleomycin 10 10 19 49 42

Dianhydrodulcitol .1 51 17 70 55

+ Growth inhibition (%)

Evaluation on lOth day following transplantation

t+ Treatment on 4th day (ollowin, transplantation

Schedule: simultaneous treatment

Figure 3. Combination therapy with Dibromodulcitol (DBD)

39

40

TREAT"'ENT OF PARTIALLY SYNCHlKJNIZED NK Ly

ASCITES TIJIo/()()R WITH DIBR()Iof(){)(.(CITOL

early

S ·S

CONTRCI.

tI 10 JO '0 50 10 10 HOURS

I I DIBROfoIODULCITOL

(100",,1,,) fO

RETRANSPLANTATION IrI'CELLS)

AFTER VINBLASTINE AND JH - THYMIDINE

INJECTION

EVALUATION on 711>ctay

I I

I GROWTH 0" '00 " INHJ8mON!%)

Figure 4

K. LAPIS ET AL.

various intervals after Vinblastine administration, and then, 106 tumour cells were transplanted into new hosts to determine the tumour-inhibiting effect. DBD was most effective when given 42 hours after Vinblastine treatment. At this time, according to the mitotic rate and thymidine index, the majority of the cell population was found in the early S phase (Fig. 4). It seems that the length of the interval between the administ­ration of Vinblastine and DBD is rather critical.

Studying the mode of action of DBD, we found that though the cells on the boundary of the Gl / S phase, or in the early S phase were the most sensitive, they still entered and proceeded in the S phase and their DNA content doubled, and the cells accumulated in the second half of the S phase suggesting a block at the S / G2 boundary (Fig. 5). On the basis of this observation, we concluded that the DBD does not inhibit the initiation of DNA synthesis. So, it can be assumed, that 5-FU by influencing DNA synthesis at a different way may enhance the tumour-inhibiting effect of DBD.

We observed that after simultaneous administration of DBD and 5-FU, the tumour growth-inhibiting effect was found to be more than additive (Fig. 3).

Other conbinations were also tried. DBD, which heavily

COMBINATION THERAPY WITH DIBROMODULCITOL

Figure 5

5cl~eJlCtie preaentvtlon of data conCl'rn~r.£ ccr.-.:':u;etlon

tnerllPY with Dibromodulcitol IOBDI Bccordln£ to Cnrter'£ proposal +

CJ Growth lnhlbitlon as B

unlt for either drug

drug

DBD ... CycloiltlOsrhclLide

DED + 5-FU

D3D ... "elm I

DED ... VCR I

OBD+BLEC I

DBD+DilD

Effect of combination

more than "dClitive

core than edJltive

audItive

lese than nduttive

less than additIve

less than adctlhv~

+ Flanning combined therapy. The! Interaction of J:.1tperimentsl and Clinical Stud1",,,,. St.K.Cfll'ter.

Cancer CheClotherspy Reports. Pert 2. Vol •. '. He.l.

) - l?pp.

Figure 6

41

42 K. LAPIS ET AL.

alkylates the components of the chromatin, differs from Bleomycin in its chemical activity, and the two drugs belong to two groups with different mechanisms of action. Our studies concerning the combination of these two drugs, however, point to the fact, that the effect observed after simultaneous adminstration was less than additive (Fig. 3).

Dianhydrodulcitol (DAD) is one of the metabolic products of DBD. Despite chemical relationship, these two agents have different pharmacological activities. Evaluating the tumour growth-inhibiting effect observed after simultaneous administration of these two agents, we observed an example of combination therapy, when the tumour growth-inhibition was only as great as if one of the drugs would have been administered.

In the following experiments, we wanted to give some examples of how various agents could be selected on a rational base for combination treatment, on consid­ering the available data concerning their mechanism of action.

Based on such considerations, DBD as an alkylating agent, which acts preferentially on fast proliferating cells, and does not cause DNA damage directly has been combined with cyclophosphamide and 5-FU, and a more than additive tumour growth-inhibing effect was observed in each case.

Combined treatment with DBD in our experiments can be summarized on the basis of the classifacation introd­uced by Carter into 3 groups (Fig.6) : a) where the eff­ect of the combinations was just additive, b) where the effect was more than additive, c) where the effect was less than additive. This classification, however, only concerns tumour growth-inhibition and it is not clear therefore how the toxicity of the drugs is modified in such combinations.

TILORONE HYDROCHLORIDE: ITS PHARMACOKINETIC PARAMETERS AND ITS

pHARMACODYNAMIC EFFECTS

Virendra Gaur and Prakash Chandra

Dept. Molecular Biology, Zentrum der Biologischen Chemie Universitgtskliniken 6 Frankfurt am Main, West Germany

SUMMARY

Tilorone's pharmacokinetics and tissue distribution dynamics 1, 2, 4, 6, 8, 12, 16, 24, 48, 72 and 144 hours after intraperitoneal injections of 20 mg/Kg in male AKR mice disclosed the following features: the drug's biological half-life was about 72h with major excretory route as kidney. The spleen and liver showed, in order, the highest specific activities which were found to be reversed with relation to each other at higher doses. The drugs distribution 24h after injection was liver 25%, spleen 2.5%, kidney 2.3%, lungs and pancreas about 1.5% each and less than 0.5% of the administered dose in the remaining tissues. Drug-levels in thymus and testes were lower but more persistent. Tilorone stimulated general growth­rates of post-natal mice; induced transient involutions of thymus, pancreas and glomerular tissue; showed specific interactions with zinc-sensitive biochemical reactions; and exerted selective muta­genic pressures on post-natal tissue growth patterns of mice treated chronically with tilorone solutions at concentrations of 20~e/ml.

INTRODUCTION

Tilorone hydrochloride, the orange water soluble dihydrochloride salt of 2,7-bis(2-(diethylamino)ethoxy)fluoren-9-one is a synthetic broad-spectrum antiviral agent (Krueger and Mayer, 1970), an orally active interferon inductor (Mayer and Krueger, 1970), with anti­tumour (Adamson et al., 1971; Munson et al., 1971, 1972; Morahan et al., 1974), antileukaemic (Rheins et al., 1971; Barker et al., 1971) and uniquely selective immunostimulatory (Rohovsky et al.,

43

44 V. GAUR AND P. CHANDRA

1970; Munson et al., 1970; Morahan et al., 1971; Diamant stein , 1973; Megel et al., 1974) and immunosuppressive properties (Mobraaten et al., 1973; Megel et al., 1974; Friedlaender et al., 1974). In revelance to its mode of action, the drug has been found to inhibit replicative and reverse-transcriptive enzymes of E.coli and RNA-tumour viruses in vitro (Chandra et al., 1972; Waalkes et al., 1974) with paradoxically stimulatory effects in vivo on the similar enzymes of neoplasms (Rhoads et al., 1971; Gazdar, 1972a) and metaplasms (Gazdar et al., 1972b) as evidenced by apparent stimulation of tumour growth rates and enhancement of viral oncogenesis. In continuation of our investigations concern­ing the drug's mode of action (Gaur and Wacker, 1972; Gaur and Chandra,1973; Wacker et al., 1972; Chandra et al., 1972), we wish to report some of the drug's pharmacokinetic and physiological properties which appear to us to be of relevance for the drug's pharmacodynamic effects.

MATERIALS AND METHODS

Labelled Tilorone hydrochloride-914C was synthesized in our laboratory as described elsewhere (Gaur and Wacker, 1972). The crude product was subjected to chromatographic purification on columns of Sephadex LH-20 (Pharmacia) in methanol to obtain a final product whose chemical and radiochemical purity was above 99.5% as estimated by tlc-analysis.

The animals used were male AKR-mice, 8-10 weeks old, weighing 30±2g. which were kept on food and water ad libidum till the time of sacrifice.

Drug-dosage in 24 experiments designed to investigate the pharmacokinetics and the tissue distribution dynamics was 20mg/Kg body weight administered i.p. To study the dose-dependent relative changes in the tissue distribution patterns at the time of maximum interferon titres (De Clercq and Merigan, 1971) four single experi­ments at 40, 70, 80 and 90 mg/Kg body weight were also carried out (the detailed data of experiments at higher dosages will be reported elsewhere).

Radioactivity counts in the tissues were carried out by dis­solving aliquots of the wet tissues in soluene-100 (Packard) in ratios of 100 mg wet tissue/ml soluene at 700 C for 24h and counting the CPM in toluene-based scintillators. Alternatively, for tissues, faeces and urinary excretions absorbed on filter pads, 95-99% of their total radioactivity could be extracted by dounce-homogenization of the tissues in methanol and by subsequent centrifugation at 10,000 rpm. The radioactivity in methanol extracts was determined using dioxan­based scintillators, the residual CPM of the unextractable sediments with soluene-100 as above. All values were corrected for quench

TILORONE HYDROCHLORIDE

60

Q) CIl 50 0 rd

rd 40 Q)

+> () Q)

'IJ 30 I=l • .-1

'+-I 0 20 ~

10

24h

Diagram lao

% 100

80

60

40

20

excretion

48h 72h

total excretion

55%

120h 144h

Cumulative excretion of radioactivity from 14C­Tilorone-HCl (2Omg/Kg) i.p. as % of injected dose during the first 144 hours after a single dose

\ .... . . ........-. '.- ..... ·-·-~---_._t - "--.---.

f~ ",-. -..:r-' -~--v'--..-:-:..,::- ~.--,.,-. -=---.~-:::-:-::~::::- .:, - - _.'

7

24h 48h

45

Diagram lb. Changes in the mode of excretion as a function of time after injection. Upper curve (% of total excreted in urine); lower corresponding value for faeces.

60

50

40

30

20

10

~

<lJ

>-<

<lJ

<lJ

ri

:> P

I .,

-i

(/) ri

Dia

gra

m l

c.

>-<

(j)

'd

'd

'd

oj

>-<

ri

0 ri

P

~

..c:

m

Ul

S (/

) ()

<lJ

<lJ

'd

oj

:>,

ri

(j)

m

..c:

~ ~

<lJ

:>,

.,-i

(j

) >-<

>:l

(/

)

S S

mri

>-<

()

ri

o m

(j

) U

l (/

) (j

)

Ul

(j)

0 >-<

m

<l

J

~ +>

>:l

>:l

()

..c:

m

m

+>

>-<

ri

.,-i

0

;j

tlD

>:l

>-<

()

>:l

'd

+>

()

>-<

.,-i

.,

-i

.,-i

P

I ~

>:l

.,-i

P

()

+>

PlU

l

>:l

'd

:>,

i01

.,-i

0

()

<lJ

m

m

~

Ul

m

0 .,

-i

>-< ~

Ul

Ul

.,-i

(/

)

;j

.,-i

..c:

o

j >-<

;j

..c:

(j

) m

(j

) >-<

(j

) (j

) .,

-i

+>

p.,

<lJ

<lJ

§ (j

) 'd

.,-

i

ri

..'<!

+>

p.,

;j

'd

+>

>-<

()

..c:

p U

l :>

'd

Ul

Ul

p.,~

+>

m +

>

Tis

sue:

pla

sma

rati

os

(pg

Til

oro

ne/g

w

et ti

ssu

e:

fg/m

l p

lasm

a)

of

the m

ou

se ti

ssu

es

(ab

scis

sal

Ih,4

h,8

h,1

6h

an

d

24

h aft

er

i.p

. in

jecti

on

. D

rug

co

ncen

trati

on

s

in sp

leen

li

ver,

lu

ng

s,

kid

ney

, th

yro

id

an

d p

an

cre

ati

c ti

ssu

e

wer

e alm

ost

2

0 ti

mes

mo

re th

an

th

ose

in

pla

sma.

Th

e co

ncen

trati

on

s

in th

e cir

cu

lati

ng

blo

od

cell

s ~g

Til

oro

ne/g

o

f se

dim

en

ted

b

loo

d cell

s

wer

e o

n th

e

av

era

ge

3:1

w

hen

co

mp

ared

w

ith

rg

/ml

pla

sma.

-I>--

0- :< G>

» c :D » z o ;"t

J ()

:J: » z o :D »

tJ I-' I-' I-' 1-" [\) +:-0'\ 00 0 [\) +:-0'\ 00 0 I\) ">I.\l. III 0 0 0 0 0 0 0 0 0 0 0

Oll 1-'1 liver '1

~ [\)

[\) w th us +:-lungs

[\)tJ Vl kldne +:-+:- +:-f-'. 00'\ urinary bladder 00 ::Y ::YUl 'i ::Y u c+ p.---..l semina], vesicles po

+:-"'1 f-'. po H) 00 f-'. ::100 pancreas H) urlnary c+ ::Yo' po c+ (I)

~ c+\o testls (I) rect~ 'i

III c+ (I) 'i ::s f-'. •• I-' rectum th us

I~ p.o 0

I~ ::s Ul r:::: duodenum ~cum I-'

+:-">d '"d '-' +:-"Ill

(I) [\)

::Yc+ () 1-'

c+ f-'.w

~ ~i~ ;~;~..; co~u C 0

~ III (I) ~.~ brain H)'i () I-'

diaphragm c+::s Vl heart (I) Ul ~~ m scle muscle '1

0 C+'-' III H) f-'.---..l

Ul '1 <: ~-, 1-" 00

1-"III C+I-' ::s p. '<:\0

Oll 1-" o e5 1-'0

(I) III H) ()

f-'. c+ I-' f-'. f-'.

>d <: <: I-' I-' I-' f-'. (I) [\) +:-0'\ 00 0 [\) +:-0'\ 00 0 [\) ~ c+ 'i 0 0 o. 0 0 0 0 0 0 0 0

f-'. '<: ~ I-' ::s

c.....H) c: (I) '1 Oll [\) () 0

8 C+S f-'. f-'.w 0 I-' ~+:- ::s +:-

0 6Vl .n....Lu.uc::.y [\) 0 I H)t-3 ~ CJ\

I-' thymus +:-+:-::Y f-'.

----+:- [\)1-' Oll---..l ::Y pancreas III 00

urln ry bladder H)

'1 ~ 00 po c+ 13 0 (I) H)

semi al vesicles (I)

Oll ::I c+\O c+ 'i '-.(1)

c+b (I)

duodenum ~I 'i

I~ (lq ::r:: ~. ~

----f 0 I-' caecum

0'1-' ~ I\)

0 +-........... ..J-~ ,....

0 ~ f-'.

~tJ ~

• ;:s I-'

caecum brain \0 f-'·S III +:-Vl

::I 0 Ul I-' ur'nary bla-der

I-' diaphragm .~ Vl ---..l c: 13 Ul t;b'-, b ain 7tomach

Q III (I) c:

---- I-' ~~ stomach ----

sedimentary blood (I) c+ f-'. tp peritonium

~~ '-' \0 sinal chord

(I) ~ serum i3 Ul f-'. rsuinal chord () +:-" (I) ::Y

L'f' 301!:lOlH:lO!:lOAH 3NO!:lOlll

48 V. GAUR AND P. CHANDRA

errors by recounting after addition of comparable amounts of internal standards.

RESULTS AND DISCUSSION

The drug's cumulative excretion via kidney and through bile into faeces as a percentage of injected dose is shown in Diagram lao The percentages in the unshaded columns represent percentage of total excretions eliminated renally whereas the shaded portions of the columns are the corresponding amounts of excretions eliminated through bile into faeces. The significance of glomerual filtra­tion as the major route of the drug's elimination during the first 48 hours is evident. However, diagram lb demonstrates the change in the mode of excretion as a function of time after injection. From the upper curve of the diagram , which represents the percent­age of total CPM excreted renally, it can be seen that the relatively high efficiency of the renal function decreases with the time whereas the efficiency of the biliar elimination rises. Diagram lc demon­strates the very high drug-concentrations in spleen, liver, lungs, kidney, thyroid and pancreas during the first 24 hours with relation to the drug concentrations in plasma. The drug levels in the above mentioned tissues are more than 20 times higher than those in plasma. The plasma clearance of Tilorone is very rapid. Within an hour after the i.p. injection the plasma levels drop from 4pg per ml to an average level of about 1 fg/ml. The drug concentrations in the sedimented blood cells (rg Tilorone/g sedimented blood cells) also show a rather rapid drop in the specific activity which during the first two hours drops down from the initial high value of 12 pg/g to about 3-4 yg/g blood cells. These values when compared to the 24 hour average values of liver, spleen, lungs, kidney, thyroid and pancreas as being 95, 100, 85, 68, 43 and 32 microgramms of Tilorone/g wet tissue respectively do bring home the remarkable differences among the tissues to bind Tilorone. In addition to these differences the tissues also show a considerable diversifi-­cation in regard to the drug's tissue distribution dynamics and persistance of binding. Spleen and liver bind Tilorone much more persistently than the lung and kidney. The drug's tissue distri­bution 4, 24, 48 and 144 hours after drug administration is shown in Diagram 2. The most remarkable feature of these findings is indeed the unique behaviour of thymus- seminal vesicles and testic­ular tissue. These tissues bind very little Tilorone during the beginning but 48 and 144 hours afterwards the drug levels in these tissues are very high, indicating a tendency of these tissues to retain Tilorone. These tissues also share with each other the common property of having the highest mitotic index. We feel inclined to interpret this finding as a supporting evidence for Chandra et al. 's contention (Chandra et al. 1972) that Tilorone may be binding to the double-stranded DNA by intercalation. This should also explain the drug's high antitumour activity since

TILORONE HYDROCHLORIDE 49

tumour cells must divide more frequently than other tissues.

Unique behaviour of thymus tissues, also evidenced by the drug­induced involution, appears to us to be of relevance towards the suppressive effect of Tilorone on the cell-mediated immune response. After all the T-cells are the main carriers of the cell-mediated immunity. Their specific vulnerability to the drug's toxic effects should be expected to impair the cell-mediated immune responses.

CONCLUSION

Initially very high specific activities of lung and kidney with relation to Tilorone and a relatively sharper drop in the same during the first 4 hours after injection recommends itself very well for acute infections of these tissues. Tilorone's pharmacodynamic effects on Thymomas, and pancreatic tumours as well as its interactions with testicular and thyroid hormones should yield fruitful insights in correlations between Tilorone's pharma­cokinetics and its pharmacodynamic effects.

REFERENCES

Adamson, R.H. (1971). J. Natl. Canc. Inst., 46, 431. Barker, A.D., Rheins, M.S. and Wilson, H.E. (1971). Proc. Soc.

Exp. Biol. Med., 137, 981. Chandra, P., Zunino, F. and GBtz, A. (1972). FEBS LETTERS, 22,101. Chandra, P., Zunino, F., Gaur, V., GBtz, A., Zaccara, A.,

Woltersdorf, M. and Luoni, G. (1972). FEBS LETTERS, 28,161. De Clercq, E. and Merigan, T. (1971). J. Inf.Dis., 123, 190. Diamantstein, T. (1973). Immunology, 24,711. ---Friedlaender, G.E., Mosher, M.B., Mitchell, M.S. (1974). Cancer

Res., 34, 304. Gaur, V. and Wacker, A. (1972). J. Lab. Comp., IX, 281. Gaur, V. and Chandra, P. (1973). Nature, 60, 263. Gazdar, A.F., Steinberg, A.D., Spahn, G.F. and Baron, S. (1972).

Proc. Soc. Exp. Biol. Med., 139, 1132. Krueger, R.F. and Mayer, G.D. (1970-)-.- Science, 169, 1213 Mayer, G.D. and Krueger, R.F. (1970). Science, 169, 1214 Megel, H., Raychaudhar, A., Goldstein, S., Kinsolving, C.R.,

Shimano, I.and Michael, J.G. (1974). Proc. Soc. Exp. Biol. Med., 145, 513.

Mobraaten, L.E., De Maeyer, E. and De Maeyer-~ignard, J. (1973). Transplantation, 16, 415.

Morahan, P.S. and Hamilton, L.D. (1971). J. Reticuloendothelial Soc., .2., 645.

Morahan, P.S., Munson, J.A., Baird, L.G., Kaplan, A.M. and Regelson, W. (1974). Cancer Res., 34, 506.

50 v. GAUR AND P. CHANDRA

Munson, A.E., Munson, J.A.,and Regelson, W. (1971). Int. Coll. on Interferon and Interferon inducers, Leuven, Belgium, Abs.27.

Munson, A.E., Munson, J.A., Regelson, W. and Wampler, G.L. (1972). Cancer Res., 32,1397.

Rheins, M.S., Barker-,-A.D. and Wilson, H.E. (1971). Canad. J. Microbiol., 17, 1259.

Rohovsky, M.W., Newberne, J.W. and Gibson, J.P. (1970). Toxicol. Appl. Pharmacol., 17, 556.

Waalkes, T.P., Sander, K., Smith, R.G. and Adamson, R.H. (1974). Cancer Res., 34, 385.

Wacker, A., Lodemann, E., Gaur, V. and Diederich, J. (1972). Nature, 22, 520.

PHARMACOKINETICS OF FUTRAFUL (FT-207) FOR CLINICAL

APPLICATION

* ** *** H.Fujita, M.Sugiyama and K.Kimura

* ** Tsurumi University; Yokohama City University and National Cancer Centre Hospital*** Tsurumi 2-1-3, Tsurumi-Ku, Yokohama City, JAPAN

Futraful, (FT-207) is a fur an conjugated 5-FU which was synthesized by Hiller, et.al. From coopera­tive studies between Japan and USSR, it has become clear that FT-207 is a masked or transport form. There­fore although FT-207 itself shows a very week direct activity against tumor cells, after entering the body, FT-207 is activated to 5-FU and thus becomes a potent carcinostatic substance.

EXPERIMENTAL METHODS

Drug concentration of FT-207 and the active substance, 5-FU was separately measured by a bioassay method using Staphylococcus aureus, 209P as the test organism. A Chloroform extraction method was employed for dividing FT-207 from the materials which contain its active metabolites.

In some experiments, spectrophotometric assay, paper chromatography and a method using radioactive ~H-FT-207 were also employed.

RESULTS

1) Persistency of Blood and Tissue Level: Fig. 1 shows the blood level of FT-207 after intravenous injection in rabbits. The result shows that FT-207 remains for a long time in the blood and it is gradually

51

52

0.60

H. FUJITA, M. SUGIYAMA, AND K. KIMURA

Blood L.Ils of FT-207 and It I Active Substancn after Intravenous Administration in Rabbits

15 Fu.etc.1

0.78 0.80

0.52

3 4 5

17.6

0.47

6

Dose : 100mg/kg Average of 2 Rabbits

3.8 3.8

0.40 0.20

8 10 TIme after Administration, Hours

Figure 1

converted to the active substance 5-FU.

Cytocidal effects of FT-207, 5-FU, 6-MP etc. against cultured Yoshida Sarcoma cells were studied. The antitumor activities of these drugs were time dependent, rather than concentration dependent. The long time persistency of FT-207 and its active metabol­ites in the body seems to satisfy this time-dependency.

2) Oral or Rectal Administration: The second characteristic of FT-207 is able to admister by simple methods. The absorption of the drug from gastrointes­tinal tract is good and shows high and continued blood level after oral adminstration in rabbits or in cancer patients.

The blood level of FT-207 and its active substances after intrarectal administration was studied. As a result, relative high and long continued blood level was obtained in experimental animals, such as rats and rabbits. However, the absorption of the drug through human rectum was not so good, and the blood level was somewhat low and varied among individual cases.

Direct injurious action of FT-207 to gastrointest­inal or rectal mucous membrane is mild, because of mask­ed form. Thus, we can give the drug orally or rectally not only to in-patients but also to out-patients.

PHARMACOKINETICS OF FUTRAFUL

Concentration of FT-207 and Its Active Substances in the Blood and the Cerebro­spinal Fluid in Cancer Patient

Dose and Method : 1600 mg, ~

-20

! :!' E

0.01

Blood

IActive Substances I

L------2----3------------~~4 Time after Administration 'Hours)

Figure 2

Table 1

The Brain LeveL of Radioactive Metabolites in Rats foLLowing Intravenous Administration of 3H-FT-207 and 3H- 5Fu

Dose: 90mg/kg (FT-207); 30mg/kg (SFu)

Time after Metabolites

F T -207 5 F u Adm.(hrsJ mcg Iml : Percentage mcg/mll Percentage

F T-2 Q7 41.85 j 97.68 - -

1 5 Fu 0.16 0.56 0.1 B 7.93 FUPA 0.43 [ 1. 43 1.77 72.3 2 F - alanine 0.04 O. 16 0.32 14.92

F T -207 32.28l 97.80 - -4

5 Fu 0.07 i 0.34 0.01 i 2.21 FUPA O. 10 i 0.43 0.35 i 54.88 F -alanine 0.25 ! 1. 40 0.18 i 37.45

53

3) Penetrability through Blood-Brain Barrier: The third characteristic of FT-207 is the penetrability through blood-brain barrier because it is lipophilic. As shown in Table 1, the brain level of FT-207 is remarkably higher than that of 5-FU following injection of 3H-labelled FT-207 or 3H-5-FU in rats. Furthermore, FT-207 and the active metabolites tend to persist for longer time in the brain. When 5-FU was administered, the catabolic products, such as FUPA or F-~-alanine

54 H. FUJITA, M. SUGIYAMA, AND K. KIMURA

were mostly found in the brain, and 5-FU itself was detected only as a few parts.

A clinical study as to the penetrability of FT-207 to the human cerebrospinal fluid is indicated in Fig. 2. 3 hours after p. o. administration, about the same level of FT-207 or its active metabolites was observed between the cerebrospinal fluid and the blood. Thereafter, mod­erately high level continued for several hours in both

body fluids.

The clinical applications of FT-207 to brain tumors, especially to brain metastasized adenocarcinomas are under study in our hospital.

4) Activation (in vitro Experiments): In vitro experiments concerning the activation of FT-207 were carried out by means of paperchromatography. Purified FT-207 and various tissue emulsions were mixed and incubated at 37°C for 2 hours, and followed by paperchro­matography, and the spots having antibacterial activities were checked on a large agar plats. After incubation, 2 kinds of spots appeared, the upper bacterial growth­inhibitory spots were in accord with Rf of FT-207 and newly formed lower spots were in accord with Rf of 5-FU. From the experiments, it is clear that the main active substance of FT-207 is 5-FU.

In Fig.3, quantitative study on the in vitro act­ivation of FT-207 among various human tissue-emulsions

is presented. After incubation of the drug and the emulsions, the amount of produced 5-FU was estimated. The result shows clearly that FT-207 is activated most remarkably by the hepatic homogenates, while less by other tissues. However, it must be noted that FT-207 is heat-labile and a few parts of it are spontaneously converted to 5-FU at 37°C, and so we can expect partly this natural activation in orther tissues including cancer tissues.

5) Activation (Hepatic Clearances): In order to confirm the activation of FT-207 by the liver, in vivo experiments were carried out by using dogs. Table 2 shows the simultaneous clearances of FT-207 and its active metabolites by the liver. In detail, the blood level of FT-207 and produced 5-FU and the blood circula­tion volume were measured in the blood vessels entering the liver, that is, the hepatic artery and the portal

PHARMACOKINETICS OF FUTRAFUL

FT-207 : SOOmcg/ml TIssue Homogenates : 10·,.

30 60 120 Incubation Time (Minutes)

55

Fig. 3. Activation of FT-207 by Various Tissue Homogenates from Man

vein and the vessel oomming out the liver, that is, the hepatio vein.

As a result, the concentration of FT-207 itself did not show large differenoe in amount among these bloods, but the liver. On the other hand, the amount of active substanoe, S-FU in the hepatic vein showed marked increase in any moment after administration, and the hepatio clearances calculated were 117% on the average.

A similar experiment where FT-207 was injected into the inferior mesenteric artery was also oarried out. In this method, the drug will flow into the portal vein. The result also shows that 5-FU level in the hepatic venous blood increases remarkably. The hepatic clear­anoe rates were 123% on the average. These in vivo data also show that FT-207 is activated to 5-FU by the liver.

6) Factors Affeoting Activation: Aocording to Fujii, Saitch and Hiller, the biochemical meohanism of the aotivation of FT-207 in the liver has been shown to correlate with microsomal drug metabolizing enzyme, P 450, which is enchanced by NADPH.

56 H. FUJITA, M. SUGIYAMA, AND K. KIMURA

From the viewpoint of this, we carried out some experiments as to the factors influencing the activation of it by the liver.

Fig. 4 shows that in vivo pretreatment with phenobarbital of mice resulted in great increase in the decomposition of FT-207 and the production of S-FU by the liver homogenates. Furthermore, the livers from mice which were administered in combination with pheno­barbital snd glutathione gave more pronounced result. The addition of NADPH was essential for this in vitro reaction.

In vivo experiment of the blood level of FT-207 and its active metabolites was carried out in rabbits which were pretreated by several drugs.

Pretreatment with carbon tetrachloride resulted great inhibition of the metabolism of FT-207 and gave high FT-207-levels and very low S-FU levels in the blood. The inhibition by carbon tetachloride was slight­ly prevented by simultaneous treatment with glutathione. On the other hand, pretreatment of phenobarbital or phenobarbital and glutathione in combination resulted rapid decrease of FT-207 and increase of S-FU in the blood.

Table 2

Hepatic CLearance of F T-207 and Its Active Substance ( 5 Fu )

Experimental Animal : Dog

Time after Adminis. (Minutes)

1 5 C' 60 0 N 90 I I-

150 LI..

180

;f 15 1.1'1

:g 60 CI) 90 ~ 150 :;; u

180 «

Dose of FT-207 : 100mg/kg Administration Method : One Shot Injection into the

FemoraL Vein

Drug Concentration (mcg/ml ) Portal ! Hepatic Hepatic Pheripheral Hepatic

Vein 1 Artery Vein Vein Clearance

1 1 4 100 11 4 84 98.80 ("!o)

85 84 88 100 99.08 72 64 73 94 99.43 68 62 68 74 99.15 57 57 62 68 98.05

0.24 0.27 0.30 0.18 115.65 0.1 8 0.24 0.28 0.2 1 125,08 0.1 7 0.18 0.22 0.22 122.26 0.17 0.1 7 0.21 0.19 118.94 0.18 0.15 0.18 0.1 9 101. 84

PHARMACOKINETICS OF FUTRAFUL

Influence of Phenobal and Glutathione on the Activation of FT-207 by Mouse Liver

_ Homogenates

(

FT_207 : 200mcgl ml NAOPH : SmM

37·C.4~ Live' Hom. : 20 '1. ~'00 ... o N 0!­IL. '15

.6 ~50 i o '0 GI <ii II:: T

~ 1 -[10

If 20 Ll>

."

2:30 " ." fa

Q.. 40

1.15 '1. KCL - 0.01101 Na/K-70.3 Phosphat. Buffer

31.0

10.8

L--;;p-;-,,-.Jfnob~al~-;!-G ~s H;-;--;;P~BILIl. G~SH:::-;-""""'''---- !!,;~ :.t_ i.p. tomie.3timos

80 rngJIog SOOmg~ Control --+t..flnth •• >q>Ofi -r--".,.-- --Y.,.-- -mr--.,.,.--- mont.

13.2 16.8

24.6

34.'

Fi gure 4

57

From these studies, it is assumed that these drugs influence the activation of FT-207 by inducing or inhibiting NADPH-dependent drug metabolizing enzyme, P 450. Analogous mechanism has been recognized in the activation of Endoxan.

EFFECl'S OF CYTO'IOXIC mUGS AND/OR CDRl'ICDSTEROIDS ON PERIPHERAL

Surmary

Michiaki Kawaoo, Katsuhito Kohzai, Osamu Saitoh, Eiro Tsurura Depart:nent of Intemal Medicine (III rd), Tokushima University School of Medicine Tokushima City, Japan

The myeloperoxidase (MPO) activity of patients treated with cytotoxic dru:Js and/or cortioosteroids was lower than that of healthy controls. Cyclopmsphanide decreases the nl.nlber of leukocytes witb:>ut affecting the MPO activity per cell. At the same dose of anti-inflanmatory potency, dexamethasone caused IOOre decrease than other cortioosteroids in the MPO level, nitro-blue tetrazolium (NBl') arx1 nl.nlber of polynorphonuclear leukocytes (Ro1N). lbwever, on successive daily trea1lnent with dexamethasone for 7 days, the MPO level and NBl' returned to nomal, althou:Jh phagocytosis of Candida by Ro1N CWninished· to about 60 % of the nomal level. Pretreatment of rats with heat-killed Candida albicans protected the functions of Ro1N against dexamethasone.

It is suggested fran these results that one cause of increased susceptibility to certain infections during imnunosuppressive therapy may be damage of the functions of R-1N. The results also suggest that the actions of cytotoxic drugs and corticosteroids may be different.

Introduction

The catplication of infection is an iIrp:>rtant problem when using newer nethods of therapy of neoplastiC and auto:irrmune diseases. There are many reports of semndary :inmunologic deficiencies in patients with neoplastic diseases, such as Hodgkin I s disease (Schier, W W et al. 1956; Lamb, 0 et al. 1962) and lympOOcytic leukemia (Ragab, AlI et al. 1970). Many cytotoxic dru:Js and glucooorticoids are available for treatment of cancer or hanatologic

59

60 M. KAWANO ET AL.

neoplasms, but they IIOstly cause increased susceptiblli ty to certain infections which may prove fatal. One reason for the increased susceptibility of these patients is myelosuppression (Meyers, RE, 1973~ Anderson, RJ et al. 1973~ Vietti, TJ, 1975). Granulocytes have many fmctions related to the defence mechanisn of the body, inclu:li.ng charotaxis, phagocytosis and a bactericidal action. Therefore, we studied the effect of cytotoxic drugs and corticos­teroids on peripheral polymorphonuclear leukoyctes (EMN).

Materials and Methods

1) leukocytes (EMN) ~ A mixture of 6 rnl of heparinized blood fran the test subject and an equal volune of 6 % dextran in nomal saline was stood for 45 min to allow sedimentation of red cells. Then the leukocyte-rich plasna was centrifuged at 800 rpn for 10 minutes and the precipitated white cells were ·resuspended in nonnal saline at a concentration of about 1 x 107 leukocytes/rnl. Rat leukocytes were collected by a similar method.

2) Myeloperoxidase activity (MPO) ~ 1. 2xlO 7 leukocytes were resuspended in 3 rnl of acetate buffer (pH 3.7). The preparation was sonicated for 1 minute and then centrifuged at 20,000 x g for 30 minutes and the supernatant was used for enzyme assay with o-dianisidine as substrate (Klebanoff, SJ, 1965 ~ Alexander, JW and Meakins, JL, 1972). MPO activity is expressed as lmits per 106 PMN.

3) NBI' test: This test was perfonned by Gifford' s method.

4) Phagocytosis: Rats were treated with corticosteroids and then their leukocytes were collected and allowed to becane attached to glass slides' in a tissue culture chamber (Lab-Tex Products Division, Miles Lab Inc. USA). Then a heat-killed suspension of Candida albicans was added to give the same nU11bers of Candida and leukocyte cells. The nunber of phagocytic PMN was counted and phagacytic activity was expressed as the percentage of EMN which had ingested Candida albicans.

5) Corticosteroid treatrrent: Ik:>ses of 100 mgjk.g of hydrocortisone, 25 ITW3/kg of prednisolone and 2.5 mgjk.g of dexamethasone were injected intra peritoneally into Wistar-king strain rats once or on 3 or 7 successive days and leukocytes-were obtained 24 oours after the last treatrrent.

6) Pretreatrrent with Candida: Candida albicans B-1445 was cultured at 37'C in Sabouraud's glucose broth for 5 days. Then the cells were washed 3 times with nonnal saline and heated. The heat-killed cells were suspenged in nonnal saline at a concentration of about 5 x 108 cells/rnl and injected into rats intravenously at a dose of 1 x 108 cells/rat.

CYTOTOXIC DRUGS/CORTICOSTEROIDS AND PERIPHERAL LEUKOCYTES 61

Results

1) MPO activity of human peripheral leukocytes: Patients with collagen disease had a normal MPO level (178±68.9 U) before therapy with cytotoxic drugs or corticosteroid. However, therapy reduced their MI?O level (83.6±56.0 U) •

The data suggested that corticosteroid decreased the Mro level of the peripheral leukocytes more than other cytotoxic drugs.

2) MPO activities of peripheral leukocytes of rats treated with cyclophosphamide, azathioprine or prednisolone: When 50 ng/kg of cyclopmsphamide, 80 ng/kg of azathioprine and 100 ng/kg of prednisolone were injected intraperitoneally into rats, the numbers of peripheral leukocytes decreased to about 10 %, 75% and 55 %, respectively of the control number. Alth:>ugh cyclopmsphamide caused the greatest decrease in the number of leukocytes, it did not change the MPO level per cell, whereas prednisolone caused leukocytopenia and decrease in the MPO level.

3) Effect of corticosteroids on MPO activity and the NET score of rat peripheral leukocytes: The effects of doses, 25 mg/kg of prednisolone, 100 ng/kg of hydrocortisone and 2.5 mg/kg of dexamethasone, which had the same anti-inflarrmatory potencies, were canpared. Leukocytepenia was observed after treatment with dexamethasone or prednisolone. Dexamethasone caused the most decrease in the MPO level of leukocytes, to 66 % of the control and it also reduced the number of NET positive cells to 32 % of the control level.

4) Changes of MJ:(, activity and NBT during successive treatments of rats with dexamethasone: After ,injection of dexamethasone once and on 3 successive days, the MPO level decreased to 66 % and 76 % of the control level and the NET score to 32 % and 14 % of the control values, respectively. However, on treatment for 7 successive days, the MPO level and NBT returned to the nonnal values.

5) Effect of successive treatments with dexamethasone on Candida phagocytosis by PMN: Treatment with dexamethasone for 3 or 7 successive days reduced the phagocytic activity in 30 minutes to about 60 % of the control level.

6) :Protective effect of Candida albicans on NBT and the MPO activity of rat leukocytes treated with dexamethasone: When rats were injected with killed Candida albicans before 1, 3 or 8 days and then treated with dexamethasone, their MPO level and NET did not decrease. After one or 3 pretreatment with Candida albicans, the levels were actually raised. This shows that Candida albicans

62 M. KAWANO ET AL.

protected the leukocytes against dexamethasone treatment. Pretreatment with candida 3 or 8 days before, did not change the phagocytic activity.

References

1. Alexander, J.W.; and Meakins, J.L. (1972) Ann. SUrg. 176, 273

2. Anderson, R.J.; Shafer, L.A.; Olin, D.B. and Eickhoff, T.C. (1973) Am. J. Med. 54, 453

3. Klebanoff, S.J. (1965) Endocrinology, 76, 301

4. Lamb, D.; Pilney, F; Kelly, W.O. and Good, R.A. (1962) J. Immuno1. 89, 555

5. Meyers, R.D.; Young, L.S.; Armstrong, D.; and Yu,B (1973) Am. J. Med. 54, 6

6. Regab, A.H.; Lindgvist, K.J.; Vietti, T.J.; Clx:>i, S.C. and Oster1and, C.K. (1970) Cancer 26, 890

7. Shier, W.; Roth, A, Ostroff, G and Schrift, MH (1956) Am. J. Med. 20, 94

8. Vietti, T.J. and Regab, A.H. (1975) Cancer 35,1007

THE EFFECTIVENESS OF SEQUENTIAL THERAPY SCHEDULES WITH ADRIAMYCIN

AND CYCLOPHOSPHAMIDE IN THE P388 LEUKEMIA MODEL

I. Wodinsky*, J. K. Swiniarski*, J. M. Venditti**, and R. K. Johnson** *Arthur D. Little, Inc., Acorn Park, Cambridge, Ma. 02140 **National Cancer Institute, Silver Spring, Md. 20910

SUMMARY

Treatment with adriamycin (AD) followed sequentially with cyclophosphamide (CP) produced a synergistic effect with no increase in toxicity to the L12l0 tumor-bearing hosts. Synergism was observed only when AD was administered early in the course of the disease (Proc XI Int Cancer Congress, ~:737, October 1974, Florence, Italy). In the current studies, delayed therapy on the more sensitive P388 leukemia model was used. One million ascites cells were inoculated i.p. on day O. AD was administered as a single dose i.p. on day 5 only, and CP was given Ix only on day 5 or on subsequent days (days 6-10). In an obverse series of experiments, CP was given on day 5 only and AD given on subsequent days. Administration of AD alone produced significant increases in life span of tumor-bearing mice through day 7 and CP alone was effective through day 8. Concomitant therapy on day 5 or sequential therapy through day 8 resulted in significant therapeutic poten­tiation for both series. There was marked synergism in 31/32 dose combinations with a significant number of long-term survivors when AD preceded CP treatment by 4 or 5 days but not in the obverse experiment. The P388 leukemia studies using delayed sequential therapy con­firms the earlier L12l0 experiment that AD administered prior to CP was the more effective therapeutic regimen.

Last year we reported that combination chemotherapy with AD and CP was highly synergistic in the L12l0 leukemia system1 (Fig. 1). It was also noted that this combination produced the enhanced effect

63

64

. .., ... c: ... . ~ c ...... <'" ~ ......

'" E

I. WODINSKY ET AL.

o 25

o 0 13

2.5 42

5 55

10 50 107

(2/10)

-25

cp* mg/kg/inj.

50 I 100

% ILS

42 53

>256

(5/10)

>226 214

(5110) (4/10)

119 189

(2/10) (4/10)

200

23

*CP administered i.p. on Day 1 only 30 mins. prior to AD lxDay **AD administered i.p. on Day 1 only lxDay

400

-9

Figure 1. Combination chemotherapy studies with cyclophosphamide and adriamycin in CnFl d mice bearing I.P. inoculated leukemia L12l0

ADRIAMYCIN AND CYCLOPHOSPHAMIDE IN LEUKEMIA MODEL

525 0

• 500 t::.

~ 0

• 400

300

200

100

o

-96h -72h -48h -24h Oh CP alone Oh

50 100 Cyclophosphamide

mg/kg

AD - 4 mq/kg administered i. p. at times indicated prior to CYT CP - administered i. p. on Day 5 only at doses indicated

200 300

Figure 2. The increase in life span of L12l0 tumor-bearing mice to in vivo sequential chemotherapy with adriamycin and cyclophos­phadmide.

65

0-

EXPE

R I M

ENTA

L

DES

I GN

0-

Da~s P

ost T

umor

Im~

lant

5

5 51

12

6 7

8 9

10

AD

AD

AD

AD

AD

AD

CP

CP

AD

CP

AD

CP

AD

CP

AD

CP

AD

CP

AD

CP

AD

Ser

ies

A

6 :-

P388

-10

ce

lls i

n 0.

1 m

l i.

p. ::E

0

CP

-Da

y 5

only

Ixd

ay i.

p.

0 z AD

-

As s

ched

uled

Ixd

ay i.

p.

en '" -< m

Fig

ure

3

-l » r

EXPE

RIM

ENTA

L DE

SIG

N » 0 :x

l l> 3:

-< Da~s P

ost T

umor

Im~lant

Q z

5 5

51/2

6

7 8

9 10

» z 0

CP

CP

CP

CP

CP

CP

n -< n AD

r 0 "'

0 ::t:

AD

CP

0 en

"'0 ::t:

AD

CP

» 3:

AD

CP

0 m

z AD

CP

r m

C

AD

CP

~

m

3:

AD

CP

l> 3:

0

AD

CP

0 m

r

Serie

s B

P3

88 -

106

cells

in

0.1

ml

i. p.

AD

-Da

y 5

only

Ixd

ay i.

p.

CP

-As

sch

edu

led

lxda

y i.

p.

Fig

ure

4

0- ....,

68 I. WODlNSKY ET AL.

only when administered early after tumor implantation and when the tumor burden was small (Fig. 2). It therapy was initiated on day 5 (when systemic disease was already present) there was no chemo­therapeutic advantage over that obtained with CP alone.

During the past year, Tobias et al. 2 has confirmed the syner­gism of this combination in the L12l0 tumor system and also reported that the AD/CP combination essentially inhibited the DNA synthesis in L12l0 cells whereas each agent alone allowed significant DNA synthesis to continue and that the combined treatment also produced a greater tumor cell kill than the single agent. Recently Corbett et al. 3 have demonstrated the enhanced therapy of this combination over single agent therapy in mice bearing C3H mammary tumor, B16 melanoma, Ridgway osteogenic sarcoma, and P388 leukemia. Prelimi­nary results in human studies4 ,S'6,7 indicate that combinations of AD and CP are promising in the treatment of solid tumors.

The experiments described herein are a continuation of these combination studies and were designed to determine what is the temporal relationship between the administration of AD relative to CP and vice versa.

Because the L12l0 tumor becomes systemic early in the course of the disease and AD is not too effective against the systemic disease, the L12l0 tumor does not lend itself to study the temporal relationship question. Thus for the present studies we used the P388 lymphocytic leukemia which has been maintained in our labora­tory since August, 1962. The neo~lasm is serially transplanted by intraperitoneal inoculation of 10 ascites tumor cells into DBA/2 and CDFI hosts at weekly intervals. The P388 leukemia is not prone to becoming systemic early and the initiation of therapy could be delayed allowing the following experiments to be carried out.

Aliquots of tumor cell suspensions were counted in a hemacy­tometer. The suspensions were then diluted with cold Earle's balanced saline solution so that 106 cells could be injected intra­peritoneally into each mouse in a volume of 0.1 mI. Groups of CDFl (BALB/c ~ x DBA/2 d) d and ~ mice weighing 20-25 g were used and each experimental group consisted of 280 mice. There were 10 mice in each drug treated group and 40 mice in the control group.

In the first series of experiments, the tumor-bearing mice were treated on day 5 only with CP and AD was administered sequen­tially on the days shown in Fig. 3. In the obverse experiment (Series B), tumor-bearing mice were treated on day 5 only with AD and then CP was administered sequentially (Fig. 4).

All drug solutions were prepared in physiological saline and administered in a volume of 0.5 mI.

Chemotherapeutic synergism for these experiments was defined as the combined chemotherapy producing an increase in life span (ILS) of greater than 25% above the ILS produced by either drug alone at their respective dosage levels.

Cyc

loph

osph

amid

e (C

P)

mgl

kglin

j. C

yclo

phos

pham

ide

(CP)

m

glkg

linj.

Seri

es I

-A

[T-l-

sO 1-

100-[~I3ooJ

Seri

es 1

-6

I--0

1-5

0 I 1

00 J

200 I

300 I

.... "0

ex

:

0 4

18

145

22

159

45

172

54

200

• lO

v ce

lls i

n 0.

1 m

l i.

p. CP

-Da

y 5

only

lxd

ay i.

p.

AD -

Day

5 on

ly l

xday

i. p

. % IL

S

81

150

177

100

172

3/10

68

145

268

209

?l1

n

>45(J

86

5

/H

363

90

4/1

0

3/10

13

59

Exp.

P300

3

.... "0

ex

:

% I

lS

0 63

18

72

27

86

54

127

100

~72

• 10

6 ce

lls i

n 0.

1 m

l i.

p.

AD -

Day

5 on

ly l

xday

i. p

. CP

-Da

y 5

only

lxd

ay i.

p.

90

122

150

163

1/l

0

236

4110

154

181

186

213

268

>400

3

/l0

5

/l0

227

313

4/10

3/

10

>363

>4

45

5110

81

10

Exp.

P304

1

Fig

ure

5

. C

om

bin

ati

on

C

hem

oth

erap

y S

tud

ies w

ith

C

ycl

op

ho

sph

amid

e an

d }~riamycin

on

C

DF

l M

ice

Beari

ng

P3

88

L

ym

ph

ocy

tic

Leu

kem

ia*

:t:­ o :0

~ s: ~ ('

) z :t:­ z o (') ~

(')

r o '"\J ::r:

o en

'"\J ::r:

:t:­ s: o m

Z r m

C

A

m s: ~ s: o o m

r 0-

-0

CP

mgl

kglin

j.

Seri

es 3

-A

1 0

1 50

110

0 1

200 I

300

I 0

4

0 72

9 95

27

136

31

59

CP -

Day

5 on

ly lx

day

i. p.

AD -

Day

6 on

ly lx

day

i. p.

% IL

S

81

168

177

222

131

21

10

68

145

240

263

1/10

1/

10

318

>368

3/

10

5/10

>259

33

6 5

/10

41

1Q

18

0

Exp.

P300

3

CP

mgl

kglin

j.

Serie

s 3-

B

01

5OIH

~I20

0130

01

0 18

18

63

27

81

54

140

109

159

AD -

Day

5 on

ly lx

day

i. p.

CP -

Day

6 on

ly lx

day

i. p.

" IL

S

54

109

150

177

268

21

10

122

159

154

240

1/10

1/

10

186

227

204

177

-5

54

1/10

1/

10

Exp.

P304

1

Fig

ure

6.

C

ombi

nati

on C

hem

othe

rapy

S

tud

ies

wit

h C

ycl

op

ho

sph

amid

e an

d A

dri

amy

cin

on

CD

F 1 M

ice

Bea

rin

g P

388

Ly

mp

ho

cyti

c L

euke

mia

~ ~ 52

z en ~ !!l » r

CP

mg/

kglin

j.

Serie

s 4-

A [ -o

r 50110

0 I 20

0 I 30

0 I

0 4

0 77

0 12

7

18

159

90

195

CP -

Day

5 on

ly I

xday

i. p

; AD

-Da

y 7

only

lxd

ay i.

p.

% IL

S

81

172

163

118

1/10

118

68

145

300

277

4/10

2/

1C

259

381

2/10

3

/H

300

340

3/10

4

/H

281

136

'Uln

4

/lr

Exp.

P3OO

3

CP

mg/

kgli n

j.

Serie

s 4-

8 o

I SO

] lOOT

200 1

300]

0 18

18

68

27

90

54

100

100

163

AD -

Day

5 on

ly l

xday

i. p

. CP

-Da

y 7

only

Ixd

ay i.

p.

" IL

S

18

100

145

154

200

1110

4 9

159

200

200

231

181

240

2110

272

40

2110

Exp.

P3

041

Fig

ure

7

. C

om

bin

atio

n

Ch

emo

ther

apy

S

tud

ies

wit

h

Cy

clo

ph

osp

ham

ide

and

Ad

riam

yci

n

on

C

DFI

M

ice

Bea

rin

g P

388

Ly

mp

ho

cyti

c L

euk

emia

l>

o :D

5> :s::

-<

(') z l>

z o ~

(') r o -c

::I: o C

J)

-c

::I:

l>

s: o m

Z r m

C

A

m :s:: 5> s: o o m

r ::::!

CP

mgl

kg/in

j.

Serie

s 5-

A 1

01

50

11

00

12

00

13

00

1

0 30

-10

35

-10

40

-5

45

-10

55

CP -

Day

5 on

ly lx

day

i. P.

AD -

Day

8 on

ly lx

day

i. P.

% IL

S

80

80

80

95

115

1110

130

145

195

175

2/10

l/1

C

160

210

1110

225

85

2/10

2/

10

20

20

Exp.

P3

004

CP

mgl

kglin

j.

Serie

s 5-

8 1

0 1

50 Il

(~ 1

20013

00)

0 22

36

100

45

145

54

145

86

240

11l1o

AD -

Day

5 on

ly lx

day

i. p.

CP -

Day

8 on

ly lx

day

i. p.

% IL

S

18

150

195

259

1/10

290

311f

t

136

150

218

250

259

145

3/H

1/

10

304

68

3111

1

104

18

2/10

Exp.

P3

042

Fig

ure

8.

Co

mb

inat

ion

Che

mot

hera

py S

tud

ies

wit

h

Cy

clo

ph

osp

ham

ide

and

Ad

riam

yci

n

on

CDF I

M

ice

Bea

rin

g P

388

Lym

phoc

ytic

L

euke

mia

"-I ~ ~ o Z

en

~ m

-I

» r

CP

mg/

kglin

j.

Seri

es 6

-A

[0 I

50 I 10

0 I 2

00 I 3

00 I

0 0

30

2 .~.

0 40

c

o:a,

<c

..x:

: I

4 0

-0

40

E

-10

45

-5

50

CP -

Day

5 on

ly lx

day

i. p.

AD -

Day

9 on

ly lx

day

i. p.

% IL

S

80

130

145

80

265

110

1110

80

70

200

1/10

80

55

175

60

35

140

Exp.

P300

4

CP

mgl

kgli n

j.

Seri

es 6

-B I 0

I 50

I 1

00 I 2

00u

I 300

I

0 18

36

86

45

127

54

168

86

177 2/10

AD -

Day

5 on

ly lx

day

i. p.

CP -

Day

9 on

ly lx

day

i. p.

% IL

S

36

154

200

240

290

4/10

54

45

254

100

300

259

2/10

2/

10

272

100

l/1

n

136

45

4110

Exp.

P3

042

Fig

ure

9

. C

om

bin

atio

n

Ch

emo

ther

apy

S

tud

ies

wit

h C

ycl

op

ho

sph

amid

e an

d

Arl

riam

yci

n

on

C

DFI

M

ice

Bea

rin

g P

388

Ly

mp

ho

cyti

c L

euk

emia

» o :c » s: -< n z » z o n -< n r o " ::I: o en " ::I: » s: o m z r m

C

A

m

s: » s: o o m

r ~

CP

mgl

kglin

j. CP

m

g/kg

linj.

Ser

ies

7-A

I 0

I 50

1

100[2

00 I

300J

S

erie

s 7-

6 o I 5

0 I 1

00 I

200 I

300]

c:

0 30

0 0

0 35

-10

40

-5

45

"------

CP -

Day

5 on

ly lx

day

i. p.

AD -

Day

10 o

nly

lxda

y i.

p.

% IL

S

80

130

145

80

130

40

75

90

95

80

55

120

85

55

35

Exp.

P300

4

0 0

36

90

45

127

54

154

1/10

86

231

21

10

AD -

Day

5 on

ly lx

day

i. p.

CP -

Day

10 o

nly

lxda

y i.

p.

% IL

S 0

140

200

231

2/10

300

31

10

Fig

ure

10

. C

om

bin

atio

n

Ch

emo

ther

apy

S

tud

ies

wit

h

Cy

clo

ph

osp

ham

ide

and

A

dri

amy

cin

on

CD

F 1

Mic

e B

eari

ng

P38

8 L

ym

ph

ocy

tic

Leu

kem

ia

0 0

245

222

1/10

254

227

1/10

2/

10

245

336

4111

1

190

100

Exp.

P304

2

;: ~

r s: ~ m ~

m ~ » r

ADRIAMYCIN AND CYCLOPHOSPHAMIDE IN LEUKEMIA MODEL

Figure 5 demonstrates the synergistic effect of the combined therapy when both drugs were administered on day 5. When either drug was administered as a single agent, there were no 60 day survivors in both series and both AD and CP were highly active. The combined therapy produced a large number of drug combinations which were synergistic and which produced 60 day survivors.

75

Similar results were obtained in both series when AD or CP was administered on day 6 and day 7 (Fig. 6 and Fig. 7) demonstrating no schedule dependency. Both AD and CP were still effective as single agents administered i.p. on day 7.

The administration of CP on day 5 only and AD on day 8 showed that AD was no longer effective as a single agent and only 6/16 drug combinations demonstrated potentiation of the chemotherapy over single agents alone with few long-term survivors. When AD was administered on day 5 and CP on day 8, there was no decrease in the therapeutic efficacy of CP as a single agent and there were 12/16 drug combinations which were synergistic (Fig. 8).

By day 9, Series A had only three drug combinations which were synergistic whereas in Series B (where AD was given on day 5 only and CP on day 9 only) there were 15/16 drug combinations which were still synergistic. CP alone administered on day 9 only showed only slight activity (Fig. 9).

AD was no longer effective on mice bearing P388 when injected i.p. on day 10 and there were no combinations which showed thera­peutic potentiation with this therapy schedule (Fig. 10).

In the obverse experiment, CP administered one day before the median survival time of the control mice showed no effect but administration of AD on day 5 and of CP on day 10 to these mice resulted in 15/16 drug combinations showing synergism and 50% of the combinations producing one or more 60 day survivors (Fig. 10).

In summary, we have shown in our experiments that there were no treatment schedule differences up through day 7 for the AD/CP combinations in the P388 system. However, the treatment schedule was more effective when AD was administered on day 5 and CP on day 8 or day 9 or day 10 rather than the obverse.

Further investigations are in progress to determine if a greater cell kill is produced by the optimal treatment AD/CP schedule and if the inhibition of DNA synthesis at the later intervals are sustained for longer periods.

Supported by HEW, NCI, DCT, Contracts NOI-CM-33727 & NOl-CM-53765

76 I. WOOl NSKY ET AL.

REFERENCES

1. Wodinsky, I., J. K. Swiniarski, and J. M. Venditti. An Evalu­ation of Adriamycin (NSC 123127) and Cyclophosphamide (NSC 26271) Used Alone in Sequential Combination for the Therapy of Murine Leukemia L12l0. Proc. XI Inter Cancer Congress ~:737, 1974, Florence, Italy.

2. Tobias, J. S., L. M. Parker, M.H.N. Tattersall, and E. Frei, III. Adriamycin/Cyclophosphamide and Adriamycin/Melphalan in Advanced L12l0 Leukemia. Br. J. Cancer (in press).

3. Corbett, T. H., D. P. Griswald, J. G. Mayo, W. R. Laster, and F. M. Schabel, Jr. Cyclophosphamide-Adriamycin in Combination Chemotherapy of Transplantable Murine Tumors. Cancer Res. 35: 1568-1573, 1975.

4. Muggia, F. M., M. Perloff, G. A. Chia, L. J. Reed, and G. C. Escher. Adriamycin (NSC 123127) in Combination with Cyclophosphamide (NSC 26271): A Phase I and II Evaluation. Cancer Chemoth. Rep. ~:9l9-926, 1974.

5. Salmon, S. E. and S. E. Jones. Chemotherapy of Advanced Breast Cancer with a Combination of Adriamycin and Cyclophosphamide. Proc. Amer. Assoc. Cancer Res. 15:90, 1974.

6. Parker, L. M., J. J. Lokich, C. T. Griffiths, and E. Frei, III. Adriamycin-Cyclophosphamide Therapy in Ovarian Cancer. Proc. Amer. Assoc. Cancer Res. 16:263, 1975.

7. Lloyd, R. E., S. E. Jones, S. E. Salmon, and Southwest Oncology Group Members. Phase II Trial of Adriamycin and Cyclophospha­mide: A Southwest Oncology Group Pilot Study. Proc. Amer. Assoc. Cancer Res. 16:265, 1975.

ANTITUMOR ACTIVITY OF MIMOSINE AND MIMOSINE HYDROCHLORIDE AGAINST

B16 MELANOMA IN BDFl MICEl

T.A. Khwaja, T.C. Hall* and K.M.A. Sheikh

LAC/USC Cancer Center, Los Angeles, California, U.S.A.

* American Cancer Society Research Professor PRP 47

SUMMARY

Mimosine? p-N-(3-hydro~-~-~yrido~e)~~~inopropionic acid, and its hydrochlorlde produced slgnlflcant lnhlbltlon of subcutaneously transplanted murine B16 melanoma in BDFl female mice. In such mice during mimosine treatment differences developed in the reactivity of both surface membrane and cytoplasmic antigens as compared to un­treated cells.

INTRODUCTION

Mimosine, ~-N-(3-hydroxy-4-pyridone)~ -aminopropionic acid, is present in high amounts in the seeds of mimosa and leucaena species (1). Animals feeding on these plants and their pods show signs of toxicity such as weight loss, skin irritation, loss of hair and infertility (2). Mimosine, the active principle, also completely inhibits the growth of Escherichi coli (3) and of mung bean seedlings (4). Rats fed on mimosine showed reduction in weight of reproduc­tive organs and reversible infertility (5). DeWys and Hall studied the effect of mimosine against a number of transplantable animal tumors (6.7). Mimosine was effective against leukemia L1210 and Lewis Lung carcinoma systems in vivo, however, more significant activity was observed against-Walker carcinosarcoma 256 (7).

L-Mimosine is a structural analog of L-tyrosine and L-DOPA. Mimosine is substrate for phenylalanine aminoacyl synthetase and not for the corresponding tyrosine enzyme (8). It does not inhibit the transfer of phenylalanine tyrosine into proteins and there is no evidence of incorporation of mimosine into proteins (8). Crouse et al.

77

78 T.A. KHWAJA, T.C. HALL, AND K.MA SHEIKH

have reported the inhibition of tyrosine decarboxylase and tyrosinase by mimosine (9). On the basis of its structural similarity to tyrosine it was felt that mimosine should be of interest in the treatment of melanomas in which tyrosine metabolism may be related to tumor growth. In this paper we report the activity of mimosine and its more soluble derivative, mimosine hydrochloride, against transplantable melanatic Melanoma B16 in BDFI female mice. Because immune effects have been implicated in melanoma therapy, the effects of mimosine on cellular reactions in the tumor cells were also studied.

METHODS AND MATERIALS

B16 melanoma was obtained from Dr. G.R. Laster Jr., of Southern Research Institute, Birmingham, Alabama. The tumors were maintained by serial transplantation into the axillary region of C57 Blk/6 female mice every 10-14 days. Animal model experiments were carried out in BDFI female mice according to the methods outlined along with the tabIe of results. The tumor weight was calculated from the volume measurements on days 9, 12 and 14. On the 14th day the animals were sacrificed, the tumors were excised and weighed.

Mimosine hydrochloride was synthesized by dissolving mimosine in 2N HCl by gentle warming. The acid solution was filtered and mimosine hydrochloride was precipatated by addition of two volumes of ethanol and excess of ether. The precipitated product was filtered and washed with ether and dried under reduced pressure at 600 c for 4 hours to provide a quantitative yield, m.p. 175-6°c. Ultraviolet absorption:

"H+ 277 and 250 (sh),A,H: 231 nm;~OH- 306,\ O~- 250 nm;A H20 284, max mln max '\ ml n max

)\H20 242 nm. On thin layer chromatography mimosine hydrochloride mln had Rf values identical to the parent compound in three different systems.

Immunofluorescent antibody studies to detect tumor associated surface membrance and cytoplasmic antigens were carried out according to methods outlined by Lewis et al (10).

RESULTS

Table 1 shows the effect of oral administration of mimosine against subcutaneous transplants of B16 melanoma in C57 blk/6 mice. Mimosine at a dosage of 350 mg/kg/day, induced a 45% inhibition of tumor growth. There were no dose related effects seen: at 700 mg/kg there was a 32% inhibition in weight, but no greater weight loss than at half this dose.

Table II shows the effects of intraperitoneal administration of mimosine hydrochloride against subcutaneously transplanted B16

ANTITUMOR ACTIVITY OF MIMOSINE 79

Treatment (oral) Tumour Wt. A Wt. T/C % Inhibitioll (day 14) (5 days)

(grams)

Controls, Saline 0.715 - 0.15 - -~imosine

35Omg/kg,qd (1-5) 0.39 - 2.25 0.545 45.5 700mg/kg,qd (1-5) 0.48 - 2.2 0.671 32.9

5-Fluorouracil (i.p.)2Omg/kg,qd (1-7) 0.52 - 1.6 0.716 28.4

TABLE I Effect of Oral Administration of Mimosine against Subcutaneous B16 Melanoma in BDFI Female Mice

melanoma. Mimosine hydrochloride produced a dose dependent re­gression: at 300 mg/kg there was 84% inhibition of the tumor weight. The drug at this dose was toxic and produced considerable weight loss and 2/6 animals died. However, the remaining animals made a remark­able weight recovery at the cessation of the treatment (see Charts I and II). The effect of mimosine on humoral melanoma antibodies was measured by indirect immunofluorescence studies, on viable cells for surface membrane antigens and on fixed cell smears for cytoplasmic components on specimens obtained from treated and untreated animals obtained on day 14. These results (Table III) suggest that the serum of untreated B16 melanoma bearing mice may contain a low titer of antibodies to both surface membrance and cytoplasmic antigens of the tumours. Serum from mimosine-treated animals with or without B16 melanoma does not show such reactivity with membrance components of the tumour cells. However, serum from tumor bearing mimosine treated

Treatment (i.p.) Tumor Wt. /}. Wt. T/C % Inhibition (Gram) (5 day)

(Day 14)

Control, Saline 0.496 + 1.17 - -Mimosine HCL

10Omg/kg,qd (1-7) 0.38 - 0.83 0.766 23.4 200mg/kg,qd (1-7) 0.32 - 2.3 0.496 35.48 300mg/kg,qd (1-6) 0.08 - 4.3 0.161 83.87

5-Fluorouracil 2Omg/kg, qd (1-7) 0.16 - 2.0 0.322 67.74

TABLE II: Effect of Mimosine Hydrochloride Intraperitoneally against Murine B16 Melanoma in BDFI Female Mice

80 T.A. KHWAJA, T.C. HALL, AND K.MA SHEIKH

an~mals show a comparatively higher titer of fluorescence than in the untreated mice. Mimosine treated and untreated groups of BDFl mice were sacrificed on day 14 and average weights of spleen and tnymus recorded in each case (Table IV). A significant increase in these average weights of mimosine treated B16 tumor bearing animals we observed at 100 mg/kg/day and 200 mg/kg/day doses as compared to un­treated controls. Both spleen and thymus weights were reduced after 300 mg/kg/day treatment, indicated possible immune suppression at this dose. These results are in agreement with results obtained from our antitumor antibody studies (Table III). It is interesting to observe that while mimosine hydrochloride at 250 mg/kg/day administer­ed by the intraperitoneal route decreased spleen size, the same dose administered by subcutaneously enhanced the kidney and spleen weights of the tumor bearing animals.

SERA B16 MELANOMA CELLS

C.I.F. M.I.F.

B16- Saline Controls + + B16- Mimosine HCl, 100 mg/kg ++ -B16- Mimosine HCl, 200 mg/kg ++ -B16- Mimosine HCl, 300 mg/kg + -

~imosine HCl, 100 mg/kg - non-tumor mlce - -~imosine HCl, 200 mg/kg - non-tumor mice - -~imosine HCl, 300 mg/kg - non-tumor mice - -

TABLE III: Anti-melanoma Antibody Study in BDFI Mice on Chemotherapy with mimosine hydrochloride.

TREATMENT MEAN MTS. IN MG.

SPLEEN THYMUS

pontrols, Saline 53.4 5.3

~imosine ~mosine l1imosine

l1imosine l1imosine

TABLE IV

HCl, 200 mg/kg, lop. 111.4 11.0 HCl, 250 mg/kg, i.p. 45.0 7.2 HCl, 300 mg/kg, i.p. 49.4 4.2

HCl, 100 mg/kg, s.c. n6.7 n.S HCl, 250 mg/kg, s. c. 91.0 6.5

Effect of Mimosine HCl on the weights of Spleen and Thymus of BDFI Mice bearing B16 Melanoma.

ANTITUMOR ACTIVITY OF MIMOSINE

SOURCE:

STRUCTURE:

LEGUMINOSAE PLANTS

(LEUCAENA GLAUCA~ MIMOSA PUDICA)

/J -N- (3-HYDROXy-4-PYR IDONE) - ct­AMINOPROPIONIC ACID

MELTING POINT: 227-2280C

SOLUBILITY: 2GM/LITRE (2S0C) IN WATER

Figure 1 Structure and Properties of Mimosine

81

82

Figure 2

T.A. KHWAJA, T.C. HALL, AND K.M.A. SHEIKH

eoe H e

o := -c-c-coo - HI

NH2 MIMOSINE DIAN ION

OH O HH 0= -C-C-COOH

H I

NH2-HCL

MIMOSINE HYDROCHLORIDE

ULTRAVIOLET ABSORPTION:

H2° A 284 J ');20 242 NM

MAX MIN

H+ 'AH+ ~ 277 J 250(sH)J

MAX MIN

~;I ~H 306 J 250 NM MAX MIN

231 NM

Structure of Mimosine Dianion, Mimosine Hydrochloride and Ultraviolet Properties of Mimosine

ANTITUMOR ACTIVITY OF MIMOSINE 83

OJ

0.&-

0.5-

.,; E

'" 0.4 ~

~

'" 0.3-! ~

0.2-

0.1-

~HH HO~ ~-C-COOH

NH2

L~TYROSINE

CHART I

INHIBITION OF B16 MELANOMA BY

MIHOSINE HYDROCHLORIDE

INTRAPERITONEALLY

X CONTROLS, SALINE

MIHOSINE HYDROCHLORIDE (% INHIBITION)

o l00HG/KG X 7 «(.P.)--<23.4) o 201lHG/KG X 7 «(.p.)--(35.Q) o 300HG/KG X 6 «(.p.)--(84.0)

5-ELUOROURACIL

EU 25HG/KG X 7 (l.p.)--(68.0)

DAYS POST TRANSPLANT

OH C HH 0- N-C-C-COOH

r H COH HH

HO C-C-COOH H I

NH2 NH2

L-MIMOSINE L-DOPA

Figure 3 Similarity in Structures of L-Tyrosine, L-Mimosine and L-DOPA

84

A WT.

(GMS)

T.A. KHWAJA, T.C. HALL, AND K.M.A. SHEIKH

CHART II

EFFECT OF MIMOSINE HYDROCHLORIDE INTRAPERITONEALLY o

ON THE WEIGHT OF BDF1 + MICE (BEARING B16 MELANOMA)

+2.0

+1.0

0

-1.0

-2.0

-3.0

-4.0

-5.0

-6.0

-7.0

(CONTROL. SALINE)

5

100MG/KG+ 8___..

8

9

DAYS POST TRANSPLANT

12 14

FU /~ __ FU

200MG/KG+ .~./ FU

FJ

ANTITUMOR ACTIVITY OF MIMOSINE 85

DISCUSSION

Our studies show that mimosine produces a significant inhibition of the growth of B16 melanoma in vivo. The use of more soluble mimosine hydrochloride seems to give a definite advantage in drug administration. Although the highest dose of mimosine hydrochloride used in our experiments produced weight loss as well as possible immunosuppressive effects to the host, tumor inhibition at lower, relatively non-toxic dosage, was consistently observed with two different routes of administration. The immunological studies pre­sented here suggest that mimosine hydrochloride may produce changes in surface membrane components of B16 melanoma as exhibited by loss of reactivity of serum obtained from mimosine treated animals. On the other hand, mimosine at a dosage of 100 mg/kg and 200 mg/kg caused a change in cytoplasmic reactivity which could be due to circulating mimosine or enhancement of antibody production. These results are interesting in view of recent interest in the immunotherapy of human melanomas and suggest a possible application of mimosine in treatment of human disease.

We thank Miss Janet Varven for excellent technical assistance.

REFERENCES

1. J.L. Brewbaker & J.W. Hylin. Crop Sci. 2, 348 (1965).

2. M.P. Hegarty, P.G. Schinckel & R.D. Court. Australian J. Agri. Res. 15, 153 (1964).

3. S. Suda. Bot. Mag., Tokyo 73,142. (1960).

4. I.K. Smith & L. Fowden. J. Exp. Botany 17, 750 (1966).

J.W. Hylin & LJ. Lichton. Biochem. Pharmacol., 14, 1167 (1965). 5. 6. 7. 8.

W.D. DeWys & T.C. Hall, Cancer Chemothr. Rep., 57,41 (1973).

W.D. DeWys & T.e. Hall. Europ. J. Cancer, 2, 281 (1973). LK. Smith & L. Fowden. Photochemistry, I, 1075 (1968).

9. R.G. Crouse, J.D. Maxwell & H. Blank. Nature 194, 694 (1962).

10. M.G. Lewis, P.J.G. Avis, T.M. Phillips & K.M.A. Sheikh. Yale J. Biol. Med., 46, 661 (1973).

A NEW MULTIPEPTIDE ANTI TUMOUR DRUG

A. De Barbieri

Istituto Sieroterapico Milanese S. Belfanti

Milan - Italy

Summary: Investigations are reported concerning a new antiblastic drug, Peptichemio, a complex of 6 peptides of m-[di-(2-chloroethyl~ amino]-L-phenylalanine, with amino acids, physiologic or antagoni­stic of L-configuration. Chemical formula, pharmacological, bioche­mical , immunological and biological investigations are briefly out­lined.

In the ten year period 1960-70, in the framework of a far-reaching programme of investigations in the field of antitumour peptides, se­veral peptides (more than 300) of m-[di-(2-chloroethyl)-aminQ]-L­phenylalanine (m-SL) with amino acids physiologic or antagonisti~

of L-configuration, were synthesized by De Barbieri et al. After a careful biochemical and pharmacological screening, 6 pepti­des among those which showed a higher activity in comparison with m-SL were selected and compounded to yield a new chemotherapeutic antiblastic drug called Peptichemio. Peptichemio contains four tripeptides, one tetrapeptide and one pentapeptide: all amino acids must belong to the L-configurationin order to show chemotherapeutic activity. Acute toxicity was studied in mice and rats by i.m., i.p. and i.v. routes; chronic toxicity was investigated in rats and dogs. No ill effects were observed on the growth of the test animal, nor did histological examination detect any alteration at the level of lung, liver, kidney, intestine at the two-dose level tested, ex­cept for an evident hypotrophy of the splenic tissue. Blood pic­ture, serum enzyme activities, plasma electrophoretic pattern were

87

88 A. De BARBIERI

not appreciably modified but white cells decreased at the higher dose used. No fetal toxicity was detected in rats, while it was ob­served only at the higher dose tested in rabbits. Fertility was slightly affected in rats. The effects on the cardiovascular system and the isolated heart we re studied: neither did the infusion or the injection of Peptiche­mio bring about any evident alteration of blood pressure, respira­tion rate, pulse, EKG, nor did it modify heart rate. The screening of the chemotherapeutic activity was done on sarcoma 180 (mice), Yoshida ascites sarcoma (rats) and Leukemia L 1210 (DBA/2 mice). In the first experimental model a clear drop of the weight of the tumour in the treated animals was observed in compa­rison with the controls. In the other two experimental models, a doubling of mean survival time was recorded in the treated animals in comparison with the controls. The chemotherapeutic index eva­luated on mice bearing sarcoma 180 was 3.S, if calculated as the ratio between LDSO/ EDSO' while it turned out 1.S8 as the ratio between LD10/ED90 • Besides the chemotherapeutic effect, an antispastic effect was re­corded. Morphological examinations of the antipro1iferative effect of Pep­tichemio, carried out on sarcoma 180 (mice),showed that Peptichemio affects cells undergoing mitosis, which almost completely disap­pear after treatment, or mitoses are highly atypical. The antiprO­liferative effect Was also studied on tumour surgical specimens Jf intrarteria11y treated patients and showed an analogous picture. Electron microscopy investigations pointed out contemporaneus le­sions at the level of nucleus and cytoplasm in the tumours of the treated animals. From a theoretical viewpoint, as Peptichemio bears alkylating and antimetabolic groups, an alkylating and antimetabolic mech-anism of action might have been expected. This assumption was substantiated by the biochemical investigations carried out. The tests were performed comparing the effects of Peptichemio and m-SL on some biochemical events connected with nucleic acid and protein synthesis. Some points deserve special attention: RNA po­lymerase and RNA are unaffected both by Peptichemio and m-SL; DNA synthesis and DNA polymerase are affected by both compounds, but to a different extent, the inhibition being higher for Peptichemio and lower for m-SL. DNA polymerase RNA dependent (reverse transcrip­tase) is inhibited by Peptichemio, not by m-SL: this finding was substantiated in repeated tests. Protein synthesis studied, based on the inhibition of 14C L-1eucine incorporation in tumour systems,

A NEW MUL TIPEPTIDE ANTITUMOR DRUG 89

cellular and subcellular, and in normal cell systems, is inhibit~ ed by Peptichemio, more in neoplastic than in normal cell systems. To get a deeper insight in the mechanism of protein synthesis inhi­bition the effect of Peptichemio on the incorporation into Yoshida ascites tumour mitochondria of the amino acids contained in the peptides sequences of Peptichemio was studied. A selective inhibi­tion of this incorporation was observed and this selectivity of the inhibitory effect was observed also at the level of the of aminoa­cyl-t-RNA synthetases and of specific t-RNA's of the amino acids belonging to the peptide structure of Peptichemio; m-SL was devoid of this action. The effect of Peptichemio was investigated also on the immune re­sponse: antibody formation, anaphylactic reaction, immunoglobulin serum levels are unaffected by Peptichemio. Lymphocyte blastic transformation was carefully studied using different mitogens:PHA, PWM and Con-A. Peptichemio slightly inhibits lymphocyte blastogene­sis: the inhibitory effect seems more evident on B in comparison with T lymphocytes in the range of doses tested. This finding was substantiated by other tests (rosette E, EA and EAC). Besides, and this is quite important from a discriminative point of view, the effect of m-SL on the above mentioned tests is different as it is unable to discriminate among the different lymphocyte populations. Studies are now in progress on the effect of Peptichemio at the level of the cell cycle. These experiments were performed on human heteroploid cell line (E.U .E.: human embryonic epithelium), on Col-­cemid synchronized cells. Two biological parameters were chosen for the evaluation of Peptichemio effect: percent inhibition of 1) the plating efficiency and 2) of colony growth. Peptichemio inhibits the early 8 - phase: this result was substantiated by stu­dies performed on the effect of the drug on ~ thymidine incorp~­ration in the synchronized cells. Research is now in progress to study the effect of Peptichemio on other macromolecular biosynthe­ses (proteins,RNA) to obtain a more comprehensive picture of the action of Peptichemio on cell cycle.

References

A. De Barbieri et al.: Atti del Simposio sui Peptichemio, Milano, 18 Novembre 1972. Ed. I.8.M., Milano 1974, p. 13.

METABOLISM OF THE TUMOUR-INHIBITORY 3.3-DIMETHYL-l-PHENYLTRIAZENE

AND ITS 4-CHLOROPHENYL ANALOGUE

G.F. Kolar and J. Schlesiger

Institute for Toxicology and Chemotherapy German Cancer Research Centre Heidelberg, F.R.G.

It is generally accepted that the inhibition of neoplasia by triazenes depends on their conversion into reactive intermediates. The triazenes are dealkylated by the hepatic microsomal enzymes (1) and it has been suggested that degradation of the drugs to alkylating agents is an essential step in their activation (2). Since altern­ative modes of activation cannot be excluded and metabolism of this class has been little investigated, urinary metabolites of 3.3-dimethyl-l-phenyltriazene and its para chlorinated congener, 1-(4-chlorophenyl)-3.3-dimethyltriazene, were identified. Both compounds are known to increase the life-span of mice bearing the TLX lymphoma by about 60% at their optimal doses (3, 4).

Metabolic experiments showed that rats injected with ring­labelled DMPT excreted about 80% of the radioactivity in two days urine and that the most abundant class of metabolites were hydro­xyaniline conjugates. In addition, the urine was found to contain triazene metabolites which could be cleaved to arenediazonium cations with cold acid. Since the triazenes are labile compounds which are difficult to handle, the acid-catalysed fission and subsequent coupling of the released diazonium cations with N-ethyl-l-naphthyl­amine was utilised for the identification of ring hydroxylations which Here introduced into the triazene molecule in vivo (Figure 1).

The acidic reagent protonates the tertiary nitrogen in the metabolite after which the labilised N-2/N-3 bond is cleaved, releasing the modified diazonium cation which reacts with the added N-ethyl-l-naphthylamine to produce a coloured azo dye.

The urine of the treated animals was collected directly in an acidified solution of N-ethyl-l-naphthylamine in vessels protected

91

92

3.3-Dimethyl-l- phenyl­triazene

1 2 3 ...... CH3

I 'CH ~ 3

G.F. KOLAR AND J. SCHLESIGER

ON~N ...... N

1 Biotransformation of the phenyl ring

...... CH3 -oN~N ...... N

b) Conjugation '"

a) y ~ I 1 ....... CH3

HVI Protonation at N3

&> / CH3 ~N--N ....... N-H

H0-V ....... CH3

1-HN ...... CH3 Cleavage of the '" CH3 N2 - N3 bond

-O=~N-S""tJ-CH2-CH3 H

0 lib. I j \ -I ~ '''W;'''' of tho "" •• ..,

arentdiazonium cations with N-ethyl-1- naphthylamine

S' N-CH2-CH3 -(f N-N - I HO I H

~

Figure 1

Conversion of triazene metabolites into modified 4-benzeneazo-N­ethyl-l-naphthylamines.

from light. The coloured azo derivatives which formed were extracted from the neutralised aqueous phase, separated by column and thin­layer chromatography and identified on the basis of their chromato­graphic and spectral properties. The excreted aniline metabolites were extracted from neutralised urine hydrolysates with ethyl acetate separated by thin-layer chromatography and identified by the same methods.

METABOLISM OF A TUMOR-INHIBITORY TRIAZENE 93

The metabolism of the parent 3.3-dimethyl-l-phenyltriazene was investigated first. All three isomeric hydroxyphenyl azo dyes were synthesized, and the chromatographic and spectral properties of these model compounds were determined. The protonated form of the azo derivatives was either red in case of no hydroxylation or meta hydroxy­lation or it was blue when either an ortho hydroxyl or para hydroxyl had been introduced into the phenyl ring. The colour of each deri­vative is therefore of diagnostic value.

Male Sprague-Dawley rats which had been injected with 2 mmoles (298.4 mg/kg, 96% LD50 ) of DMPT excreted 0.9-1.1% of the dose as triazene metabolites capable of diazo coupling and 38-46% as modified anilines (Fig 2). The principal coloured derivative was 4-benzeneazo­N-ethyl-l-naphthylamine which was obtained in a 0.6-0.7% yield, followed by 0.3-0.4% of 4-(4-hydroxybenzeneazo)- and by 0.02% of 4-(2-hydroxybenzep-eazo)-derivative. The most abundant metabolite was 4-hydroxyaniline which accounted for 31-36% of the injected radio­activity. Further metabolites of DMPT which could be identified were aniline, 2-hydroxyaniline and 3-hydroxyaniline.

/H

ONH2 1 -2 f(5'C'"' ON""N :1

/H

Q-NH2 5-7 Q -%'C'"5 N""N ~ - 1

OH OH ~

P-NH2 ",1 OH

/H

HO-o-NH2 31-36 -0- f(5'C'"5 HO 'I _ ~ N""N : 1

Figure 2

Urine metabolites of 3.3-dimethyl-l-phenyltriazene. Percent of injected dose.

0.6-0.7

0.02

0.3-0.4

94 G.F. KOLAR AND J. SCHLESIGER

Since para hydroxyaniline is a sensitive compound which deteriorates rapidly during isolation and chromatography, its concentration was also determined by the indophenol reaction. The direct assay showed that the amount of excreted 4-hydroxyaniline ranged between 56-61% of the injected dose.

In a similar way the metabolism of 4-CI-PDMT was investigated. The ring-substituted triazene was selected for metabolic studies because of its higher st~bility to hydrolysis at physiological conditions (5)(2.42 x 10 min, 40.33 h against 210 min, 3.5 h). Additional factors Which35avoure~7this choice were the natural isotopic ratio (3:1) or Cl to Cl which allowed convenient detection of chlorine-containing metabolites by mass spectrometry. The metabolism of 4-CI-PDMT was found to be similar and the yields of both classes of metabolites were about the same as those isolated from biotransformed DMPT. However, some significant differences in the structure and distribution of individual compounds were observed (Fig 3).

The principal coloured derivative was 4-(4-chlorobenzeneazo)-N­ethyl-l-naphthyl~ine which was isolated in a 1.0-1.1% yield, followed by a 0.2-0.3% yield of the 4-(4-chloro-2-hydroxybenzeneazo)­analogue. When the para position of the phenyl ring in the applied drug is blocked by the chlorine atom, hydroxylation occurs pre­dominatly at the ortho position. Therefore, the most abundant metabolite was 4-chloro-2-hydroxyaniline which made up between 15 to 20% of the applied dose, followed by 5-6% of 4-chloroaniline and low yield of 4-chloro-3-hydroxyaniline and 4-hydroxyaniline.

In addition, the chromatograms contained a spot at R 0.53 (toluene, acetone, acetic acid, 60:39:1 v/v) which could ~e dia­zotised and coupled with N-ethyl-l-naphthylamine to yield a lilac-coloured dye with an absorption maximum at 565 Dm. The mass spectrum of the metabolite showed a molecular ion at m/e 143/145 in the ratio 3:1, indicating the presence of one chlorine atom and of one hydroxyl group in the molecule. This interesting metabolite, obtained in 8-10% yield, was therefore isomeric with the hydroxylated 4-chloroanilines. A comparison with an authentic sample confirmed the identity of this metabolite as 3-chloro-4-hydroxyaniline which arose by hydroxylation-induced chlorine migration from para to meta postion of the phenyl ring (6).

The findings can be summarised as follows:- Subcutaneous injecton of DMPT or 4-CI-PDMT to rats resulted in the excretion of 0.9-1.4% of metabolites capable of diazo coupling after cold acid

METABOLISM OF A TUMOR-INHIBITORY TRIAZENE 95

... H

C1-oNH2 5-6 I 2 5 ~-C" CI-Q-N'=>N I 1.0 -1.1

... H

CI~NH2 15 -20 .n:o-c2" , 0.2-0.3 CI-QN'=>N I OH OH

CI-Q-NH 2 ,..,1 OH

H09-NH2 8-10 CI

H0-Q-NH2 < 1

Figure 3.

Urine metabolites of 1-(4-chlorophenyl)-3.3-diamethyltrizene. Percent of injected dose.

treatment and of 30-46% of modified anilines. Since the principal azo derivatives were not hydroxylated on the phenyl ring, the lip­ophilic triazenes had to be rendered water-soluble by hydroxylation and subsequent conjugation on the methyl group(s) at N-3, in agreement with the proposed activation of triazenes by DC-hydroxylation (1). The attack of the hydroxylating enzymes occurred mainly at the para position of the phenyl ring; however, when the para position was substituted with chlorine, hydroxylation occurred predominately at the ortho postions. Catabolic degradation of 1-(4-chlorophenyl)-3.3-dimethyltriazene, accompanied by an hydroxylation-induced migration of a substituent, the so-called N.r.H. shift, has been detected for the first time in a cytoRtatic compound.

96 G. F. KOLAR AND J. SCHLESIGER

REFERENCES

1. R. Preussmann, A. von Rodenberg & R. Hengy. Mechanism of carcinogenesis with l-aryl-3.3-dialkyltriazenes. Enzymatic dealkylation by rat liver microsomal fraction in vitro. Biochem. Pharmacol. 18 (1969), 1-13.

2. R. Preussmann & A. von Rodenberg. Mechanism of carcinogenesis with l-aryl-3.3-dialkyltriazenes II. In vitro alkylation of guanosine, RNA and DNA with aryl-mono­alkyltriazenes to form 7-alkylguanine. Biochem. Pharmacol., 19 (1970) 1505-1508.

F.W. KrUger, R. Preussmann & N. Niepelt. Mechanism of carcinogenesis with l-aryl-3.3-dialkyltri~~enes III. In vivo methylation of RNA and DNA with l-phenyl-3.3-( C) dimethyltriazene. Biochem. Pharmacol., 20 (1970) 529-533.

3. R.C. Stanley Audette et al. Studies on the mechanism of action of the tumour inhibitory triazenes. Biochem. Pharmacol., 22 (1973) 1855-1864.

4. G.F. Kolar, unpublished results.

5. G.F. Kolar & R. Preussmann. Validity of a linear Rammett plot for the stability of some carcinogenic l-aryl-3.3-dimethyltriazenes in an aqueous system. Z. Naturforsch., 26b (1971) 950-953.

6. G.F. Kolar & J. Schlesiger. Biotransformation of 1-(4-chlorophenyl)3.3-dimethyltriazene into 3-chloro-4-hydroxyaniline. Cancer Letters, in the press.

ANTI TUMOUR ACTIVITY OF BENZOFUROXAN DERIVATIVES

V.C.BARRY, J.G. BELTON and M.L. CONALTY

LABORATORIES, MEDICAL RESEARCH COUNCIL OF

IRELAND, TRINITY COLLEGE, DUBLIN, IRELAND

In a series of 4-Amino-7-Nitrobenzofuroxans, those with 4-piperazinYl sUbstituents showed significant antitumour activity in mice.

In a number of papers Ghosh and his colleagues (Ghosh 1968, Ghosh and Whitehouse 1968, Whitehouse and Ghosh 1968, Ghosh and Whitehouse 1969. Ghosh et al.1972) have reported that 4-nitrobenzofuroxans and 4-nitro­benzofurazans were active in vitro in inhibiting nucleic acid and protein synthesis in sheep lymphocytes. We have synthesized a variety of 4(7)­aminosubstituted-7(4)-nitrobenzofuroxans (I) and 4(7)-aminosubstituted-7(4)-nitrobenzofurazans (II) and have examined them for antitumour activity in a spectrum of transplantable tumours in mice.

I IT

97

98 V.C. BARRY, J.G. BELTON, AND M.L. CONALTY

BIOLOGICAL ACTIVITY

Methods

In evaluating anti tumour activity use was principally made of the P388 lymphatic leukaemia, the Ehrlich ascites tumour and, to a lesser extent, the LandschUtz and sarcoma 180 ascites tumours and the solid sarcoma 180. A preliminary in vitro evaluation for growth inhibition activity against He La cells was also made.

The evaluation procedures used have been published previously (Barry et al. 1966) and are based on those of the National Service Centre of the United States National Cancer Institute (Geran et al. 1972) although with some differences in details. In the case of the P388 leukaemia the compounds were given intraperitoneally once daily on days one, five and nine following the day of implantation. The criterion of activity was increase in mean survival time to 125 per cent that of the controls, or greater. With the other tumours treatment was given intraperitoneally once daily for 7 days following the day of implantation. Activity against the ascites tumours was assessed on increase in survival time as for the P388 leukaemia. In addition, the degree of fluid inhibition at day 7 was taken into account. Assessment of activity against the sarcoma 180 was based on tumour weight.

Results

On comparing the antitumour results obtained with the various 4-amino-7-nitrobenzofuroxan analogues it was found that only the compounds most active against the Ehrlich tumour were active against the P388 leukaemia. The tables show only these P388-active compounds for reasons of space.

The compounds Ghosh used, 4-nitrobenzofuroxan and 4-nitrobenzofurazan, were inactive in the Ehrlich and P388 screens. 7-Nitrobenzofuroxans with alkylamino and arylamino sUbstituents in the 4-position were also inactive. Of the dialkylamino compounds examined, the dimethylamino derivative showed activity against the P388 leukaemia (Table I). Some activity against the Ehrlich tumour but not against the leukaemia was also observed with the dibutylamino compound. However, the

ANTITUMOUR ACTIVITY OF BENZOFUROXAN DERIVATIVES

TABLE 1. Activity of 4-Amino-7-Nitrobenzofuroxans against P388 Leukaemia in B6D2F1 Mice.

-N(Me)2

-N(CH2 CH 20H)2

-(1

Treated LP. Once Daily on Days 1,5,9

Mg/~g

64-16

64-16

64-16

Mean Survival Time Test/Control

Percent.

128

151-125

130-126

126-134

Control MST = 11.9-12.2 days.

99

TABLE 2. Activity of 4-piperazinyl-7-Nitrobenzofuroxans against P38R Leukaemia in R6D2F 1 Mice.

R= CH 3

C3H7 n

C4H9 n

CH 2 CH 2 OH

CHO

COOEt

Treated LP. Once Daily on Days 1,5,9.

Mg/!(g

64-8

32-4

64-8

64-8

64-8

128-32

Control MST =

Mean Survival Time Test/Control

Percent.

174-125

158-132

142-126

143-126

132-137

138-128

10.6-12.2 days.

100 V.C. BARRY, J.G. BELTON, AND M.L. CONAL TY

most active compound of this type proved to be the 4-dihydroxyethy1amino-7-nitrobenzofuroxan (Table 1).

With the various k-po1ymethylenimino compounds activity was found to be confined to 4-ethy1enimino-7-nitrobenzofuroxan which showed marked activity against the Ehrlich tumour and moderate activity against the P388 leukaemia. The morpho1ino analogue gave similar activity (Table 1). 4-Amino compounds with both alkyl and aryl sUbstituents on the amino nitrogen had limited activity against the Ehrlich tumour and no activity against the leukaemia.

From the point of antitumour activity the most interesting of the 7-nitrobenzofuroxans examined were those with a1kylpiperaziny1 substituents in the 4-position. These are set out in Table 2 where it may be seen that several gave significantly increased survival times over eight-fold dose ranges. There was no marked difference in activity between the more active compounds although the best result was obtained with the methyl-piperazinyl derivative. Replacement of the alkyl substituent on the piperaziny1 moiet~ by aryl or aralkyl substituents gave com~ounds Wh1Ch were inactive. All the P388-active piperazinyl compounds were also highly active against the Ehrlich tumour, and many were able to inhibit HeLa cell growth to 50%8 of that in the controls at concentrations of 10-7-10-g/m1.

Benzofuroxans active against the Ehrlich ascites tumour were similarly active against the Landschutz and sarcoma 180 ascites tumours. However, against solid sarcoma 180 no significant reduction in tumour weight was observed with compounds which had shown substantial activity against the P388 and Ehrlich tumours.

4-Substituted-7-nitrobenzofurazan analogues of representative members of the more active benzofuroxans were found to be inactive against all our tumours. The synthesis and evaluation of these and other benzofuroxans are being continued.

REFERENCES

Barry. V.C., Conalty, M.L. McCormick, J.E.,McElhinney, R.S. and O'Sullivan, J.F.(1966), Proceedings of the Royal Irish Academy, 64B. 335.

ANTITUMOUR ACTIVITY OF BENZOFUROXAN DERIVATIVES

Ceran, R.I., Greenberg, N.R., Macdonald, M.M. Schumacher, A.M., and Abbott, B.J. (1972), Cancer Chemotherapy Reports, 3,9.

101

Ghosh, P.B. (1968), Journal of the Chemical Society (B) 334.

Ghosh, P.B., Ternai, B. and Whitehouse, M.W. (1972) Journal of Medicinal Chemistry, 15, 255.

Ghosh, P.B. and Whitehouse, M.W. (1968), ibid. 11,

Ghosh, P.B. and Whitehouse, M.W. (1969), ibid. 12,

305 .

505.

Whitehouse, M.W. and Ghosh, P.B. (1968), Biochemical Ph armacology, 17, 158.

AClCNOWLEDGEMENTS

We thank Dr.J.F.O'Sullivan for the ReLa cell evaluations and Mr.E.Hickey, B.Sc. for assistance with the experiments in mice. We acknowledge with thanks financial support from the Irish Cancer Society. We also wish to acknowledge the helpfulness of Mr. C. B. Reeder, Drug Research and Development, National Cancer Institute, Bethesda, in the maintenance of our mouse breeding colonies and our apprec­iation of the interest of Dr. A. Goldin and of Dr. O. Yoder in our work.

ANTITUMOUR ACTIVITY OF TETRAZOLOPYRIDAZINES AND TETRA­

ZOLOPHTHALAZINES

V.c. Barry, M.L. Conalty, J.F.O'Sullivan, D. Twomey

Laboratories, Medical Research Council of Ireland Trinity College Dublin 2

Derivatives of pyridazine and benzodiazines have been found to possess growth inhibitory properties against HeLa cells in vitro and experimental tumours in mice.

A variety of nitrogen containing heterocyclic compounds has marked activity against experimental tumours in animals and some of them have proven useful in the clinic (6MP, 5FU etc.).

We have been synthesising and evaluating heterocyclic materials for activity against HeLa cells in vitro and transplantable tumours in mice, in an attempt to detect new types of compounds having anticancer effects. Uniquely among the pyridine derivatives examined 6-nitrotetrazolo( l,5-b) pyridine (I) showed activity against the HeLa cell in vitro but was inactive in vivo. This led to the examination of heterocycles with a tetrazole ring

Tfnked to a ring containing two nitrogen atoms.

The 6-chlorotetrazolo( l,5-b) pyridazine II (R=CI) was active in vitro and had activity against a number of tumours in mice. Replacement of the chlorine atom led to reduction of activity. Thus the corresponding Ome, OEt, OPr, OPri and a number of nitrogen derivatives, morpholino, N4-methyl piperazinyl and substituted hydrazines had no activity while some sulphur derivatives showed activity against the Ehrlich but were ineffective against the P .388 Leukaemia. Replacement of the tetrazolo ring by a triazole as in VI also resulted in loss of activity (figure 1) .

The activity was not reduced if a benzene ring was fused to the pyridazine

103

104

TI-fl C N

HC/ 'N/

" I

HC, N c'l N02

1

N-N Ii Ii

HC/c'N~

" I

HC, /.N c~

R

II

N==1T H i i C N N

HC'l 'c'" "c'l I II I

HC C ~N ~/ '·c'/

H R

Figure

v.c. BARRY ET AL.

N-·N H Ii II C C N

HC9 '0/ 'f/ I II

HC C )f ~/'C

H C1

m

N-N

" II 1 ,C, /CR HC'" N

" I He, /.N 'C ..... R

as in I". The 6-chlorotetrazolo (1 ,5-b) phthalazine again proved the most active of the compounds. Substitution of a pyridine ring instead of the benzene ring caused loss of activity.

The nitrogens in the diazine ring in these compounds have been in a 1,2 orientation but we have also looked at the isomeric 1,4 quinoxaline (IV) and the qunazoline (V). The quinoxaline (IV R=CI) is active but the quina­zoline has no activity.

The evaluation of these compounds was carried out in a Hela cell system in vitro and in mice against the Ehrlich Ascites Tumour and the P.388 leukaemia.

Simple pyridazines had no growth inhibitory activity against the Hela cell in culture or against the Ehrlich Ascites Tumour or the P.388 leukaemia in vivo. However the tetrazolopyridazine (II R=CI) had activity. It inhibited growth of the Hela cells at a concentrati on of 10-6 to 10-7 g/ml

ANTITUMOUR ACTIVITY OF TETRAZOLOPYRIDAZINES 105

TABLE 1. ACTIVITY V. EHRLICH ASCITES TUMOUR IN MICE

HeLa cell Dose Survivors 1:% Inhibition mg/kg i.p. at day 35 C

Compound 10-xS/1II1

II R= x

Cl 6-7 135 0/7 125 90 1/7 138

SOPh 6-7 8 0/7 162

S02 Ph < 6 18 3/7 207 8 2/7 162 5.4 3/7 199

SoQ 6-7 12 0/7 234 H- 8 0/7 133

5.4 1/1 142

III R=

Cl 8-9 60 4/7 185 40 5/7 222 27 6/7 245 18 7/7 273 12 5/7 209

8 3/7 207

S02cycloHexyl 90 0/7 154 40 1/7 169 18 2/7 157

S02 CH 2Ph <6 27 4/7 169 18 2/7 149 12 3/7 137

8 2/7 124

IV R=

Cl <6 18 1/7 175 8 6/7 260 3.6 0/7 137

S02 Ph 135 2/7 277 90 3/7 259 60 0/7 136 40 0/7 150

106 V.C. BARRY ET AL.

TABLE 2 ACTIVITY AGAINST P388 LEU~AEMIA IN MICE

Compound Dose Mean Survival Survival mg/kg i.p. Time Time days 1,5,9. days 1% Eost imE1ant C

II R=C1 256 14.4 143 128 13.6 134

64 13.3 132 32 13.3 132 16 12.7 126

III R=C1 128 15.0 124 64 15.7 132 32 15.9 134 16 15.4 121

IV R=C1 32 14.3 123 16 16.1 139

8 14.0 121

ACKNOWLEGEMENTS

We thank Mr. E. Hickey, B.Sc. for assistance with the experiments in mice and the Irish Cancer Society for financial support. We also wish to acknowledge the helpfulness of Mr. C.B. Reeder, Drug Research and Development, National Cancer Institute, Bethesda, in the maintenance of our mouse breeding colonies and our appreciation of the interest of Dr. A. Goldin and of Dr. O. Yoder in our work.

The financial support of the Irish Cancer Society is gratefully acknowledged.

and also showed activity against the experimental tumours in mice (see tables) •

Work is still continuing on further variations of the structures.

However when we look at the results obtained with the P.3A8 leukaemia in mice, we find that only the chloroderivatives are effective in extending the survival time. The tetrazolopyridazine is active over a sixteen fold range and the tetrazolophthalizine is active over an eight fold range. The tetrazoloquinoxaline with the nitrogens in 1,4 alignment is active over a four fold range (see Table 2) •

The compounds have yet to be tested against the L1210 leukaemia.

ANTIN'1!:OPLASTIC H:FFli:CT CF COMPOUND 9777-VUFB IN ANIMALS

WITH EXPERIMENTAL TUMOURS: ITS IN'RRACTION WITH SOME CY'l'OSTATICS

M. Semonsky, V. Pujman, and H. Vesela

Research Institute for Pharmacy and Biochemistry

17, Kourimska, 130 00 Praha J, Czechoslovakia

We wish to briefly report our experiences with the antineo­plastic activity of the compound 9777-VUFB in animals with trans­plantable tumours, and the influence of this compound, provisionally named Damvar, on the activities of cyclophosphamide and 5-fluoro­uracil. On the basis of spectrometric studies, Damvar in solid sta­te was allotted the structure of delta-/2-amino-6-hydroxy-3,4-di­hydro-4-oxo-5-pyrimidinyl/valeric acid /Fig. 1/. Damvar, together wi th a series of other analogous 5-pyrimidinylalkanecarboxylic acids and a series of their derivatiTee, have been synthesized at our Institute both as potential cytostatics - pyrimidine-type anti­metabolites - and as compounds which might serve as detoxicants in combinations with some of known cytostatics.

Damvar, a colourless crystalline substance, is stable when stored under usual conditions. It is poorly soluble in water, but, for example, in the form of an alkali metal salt it is considerably better soluble. An aqueous solution of Damvar is practically stable, whereas aqueous solutions of its salts, for example, of its sodium salt, are unstable owing to a relatively rapid cleavage of the compound by atmospheric oxygen. This also applies to the adduct of D8DWar with diethanolamine. The poor stability of aqueous solu­tions of salts of Damvar was one of reasons why microsuspensions of free acid were used for parenteral administration.

For tests for antineoplastic activity of Damvar and for its possible role in combination with selected cytostatics, the follow­ing transplantable tumours were chosen: S ISO, solid; S 180 and S 37, ascitic, all implanted into H-strain mice, and leukaemia LA implanted into mice C 57 Bl. With the first three tumours, the changes in the tumour or in the ascitic fluid weights, respectively,

107

108 M. SEMONSKY, V. PUJMAN, AND H. VESELA

LD 50 (mtt I

IICEIHI I AT S I liST II I

p.D. =- 10 ,I kl p.l. =- 11 ,Ik, s.c.=- 65011ika s.c.=- 2 I'ka

Rltl 28 _"I 20. 100. 500. II' kl '.1. III Illie

Figure 1. DAMVAR, 9777-vUFB

checked 24 hours after the last delee, and with the leukaemia LA, the change in the survival time served as criteria of drug activity.

Damvar in the form of aqueous microsuspension, and the cyto­statics cyclophosphamide or 5-fluorouracil dissolved in 0.90 % so­lution of sodium chloride, were administered in volumes of 0.2 ml per 20 g of body weight. The routes of administration and dosage patterns are shown in Figures 2. The absolute values found in the treated animals, namely, the tumour weights, and the survival times in the leukaemic mice, were compared with the corresponding findings in the control animals. The findings were statistically processed by Wilcoxon's stochastic test at thA significance level p 0.05. For the sake of clarity, the leukaemia graphs show relative values, the appropriate control findings being accepted to represent 100 per cent.

ANTINEOPLASTIC EFFECT OF COMPOUND 9777-VUFB 109

TUMOUR CY-DAYS OF APPLICATION O-DAYS OF APPLICATION OAY OF EXAM. AFTER TRANSPL.

S lao SOLIO

5 lao ASC.

S 37 ASC

7 + 11 •••• 60.g /kg I.e. 8 - 10 and 12 - 16 •••• 100.g/kg •• c.

1 + 4 •••• 30mg/kg 3.C. 2 + 3 and 5 - 8 •••• 100.g/kg •• c.

1 + 4 •••• 60.g/kg 5.C. 2 + 3 and 5 - 8 •••• 100.g/kg •• c.

Figure 2. Dosage Schedule.

17

9

9

From Fig. 3 it is evident that when the administration of Damvar alone was finished, the tumour weights exhibited either marked or aat least slight decreases. After a combination of Damvar with cyclophosphamide, the decrease in the weight of the tumour S 180 was significant against the findings in the controls as well as those in the groups receiving Damvar alone and cyclophosphamide alone. In the case of the ascitic sarcoma S 180, after the combina­tion of Damvar with cyclophosphamide the weight of ascitic fluid Was significantly reduced against the findings in both the controls and the group receiving cyclophosphamide alone. Damvar alone also reduced the ascitic fluid weight, but not Significantly. In the case of S 37, after the combination of both &Rugs the ascitic fluid weight was significantly lesser than in the controls as well as in the groups treated with Damvar alone or cyclophosphamide alone. The weight decreases of ascitic nuids in both ascitic tumours after the combination of Damvar with cyclophosphamide were accom­panied by significant decrease in the cellularity. After single or repetitive subcutaneous or oral administrations of Damvar alone in various doses, no effect on the survival time in the leukaemic mice w&ssobserved. On the other hand, as apparent from Fig. 4, when Damwar was administered orally /100, 200, or )00 mg/kg/ either on days 1, 2, J, or on day J only after transplantation of LA leukaemia, and cyclophosphamide dosed 28 mg/kg was injected subcutaneously 60 minutes after the last dose of Damvar, evidently the mean survival time /MST/ was significantly prolonged after the optimal doses of Damvar in combination with cyclophosphamide. The MST was prolonged more markedly and significantly, after Damvar-cyclophosphamide com­bination, when Damvar was injected subcutaneously on the days 1, 2, and 3 /Fig. 4./.

110

I 6

S 180 solid.

M. SEMONSKY, V. PUJMAN, AND H. VESELA.

S 180 asc.

_ r--

S 31 asc.

4 -

2

. I--

CDC,

1., 60

CDC,

1., 30

-

-r--

CDC,

100 60 la/ka

1:Q1, -~

~ C, C, 8 :, C, D , C, o D

60100 60 30 100 30 60 180 ... lkl 100 100 100

• , - 0.05 vera. C 0 NT RO L C 4 , c 0.05 '.fl. DUVAl 0 * P - 0.05 reno CYCLOPHOSPHAMIDE C,

Figure 3

o C

, 1

lmll

k,

s.c.

D 1

. o

Dam

10

0 m

Ilk

, s.

c. D

1,1

,1.

() D

am

x D

am

!>D

am o

C,

MST

°10 .. SO

40

10

10

I •

10

tOO

,.0. D 1

. 6

0' b

efo

re C

,

p.o.

D U

,lt

o D

am n

o m

'lk

, I.

C·

D 1

,1,1

,

o C

, I.

e. D

1,

:)D

am

oC

,; C

, ,r

aft

er

D.m

C

100

\,1

8

da,.

..p

< O

,OS

Wil

e L

a V

UF

B

+

;./~

o :if

: 0

100

100

lOG

D

am

m'l

II,

Fig

ure

4

MS

T

010

I + &J

7G

60

SO

40

JO

10

10

100

100 ----

-;';

-;--

::-=

----

11

21

lS

C

, m

'lk

,

» ~ Z m

o ~ ~ C

')

m

"11

"11 m

C')

-I

o "1

1 8 s:: ~

o c z c co

......

......

...... <: c "11

IJI - -

II

h",,

,a,

20

0-.

,11

... e

.D 1

21

• S

F"

-.,1

1.

,-,0·

D

l

8 D

e",

. S

F.

,S F

.W .,

ter

De"

"

n D

e",

20

0 ..

.. '"

... e

.Dl

5'"

-.,1

1 ..

.. o.D

l

8 D

a ...

5..

. ,5

" .0

' aft

er 0

.""

o D

e ...

200l

1li,

11,

'.e.

D J

. 5'"

.... 1"'

• .e-D

J

(j

0..

... s

F. ,S

F..

o' .,t

.r Da"

"

C

10

00

1 0 =.

clay

., ..

. C

0

,05

Wil

e L

. Y

UF

B

MS

T

01

0

/@

/;~:e/

~-·

fj"

35

4

2

SO

75

1

00

• r

lOl II

50

1

00

Fi g

ure

5

• it ./ to)

. ;;.

/ 'V

0 i 25

5

0

75

5

Fu 1

119

1 k

9

~

s: en

m s: 0 Z

en '" :<- <

"'C

c '- s: » z » z 0 :J

: <

m

en

m

r »-

ANTINEOPLASTIC EFFECT OF COMPOUND 9777·VUFB 113

The Fig. 5. shows that after one dose of Damvar 200 mg/kg, injected subcutaneously on day 3 sixty minutes before oral or sub­cutaneous administration of the optimal dose of 5-fluorouracil, the effect of 5-fluorouracil was enhanced significantly. When Damvar was injected subcutaneously on the days 1, 2, and J, the effect of 5-fluorouracil administered orally was not enhanced.

The results show that when optimal aoses of Damvar and of cyclophosphamide were combined in suitable proportions, then the antineoplastic effect of cyclophosphamide was enhanced.

The MST in leukaemic mice was increased after both combina­tions, Damvar + cyclophosphamide and Damvar + 5-fluorouracil.

Studies in animals with other transplantable tumours have been continued of the antineoplastic activities of Damvar and its combi­nations with other cytostatics belonging to various effector types. Studies of the mechanisms of the antineoplastic action of Damvar and its effect in combination with the cytostatics tested, with due regard to its immunobiological activity and its effeet on the tumour cell membrane, have been continued as well.

EFFECTS OF GP 48 989 ALONE AND IN COMBINATION WITH HORMONES AND

CHEMOTHERAPEUTIC AGENTS ON DMBA-INDUCED MAMMARY CARCINOMATA II

K.H. Schmidt-Ruppin and K. Schieweck

Research Department, Pharmaceuticals

Division, CIBA-GEIGY Ltd., Basle, Switzerland

GP 48 989, a thiazolidinone derivative, causes regression of DMBA­induced mammary carcinomata in female Sprague-Dawley rats and of DMBA-induced skin carcinomata. It inhibdts the growth of some transplantable carcinomata, but was inactive against certain transplantable sarcomata and leukaemias in mice and rats (1, 2).

In rats with 1-2 mammary tumours of comparable size (8-12 mm dia­meter) it was found that long-term oral treatment with GP 48 989 (e.g. in a total dose of 750 mg/kg administered as 30 x 25 mg/kg in 6 weeks) was relatively more effective than its administration in higher doses over a shorter period (e.g. a total dose of 1500 mg/kg administered as 15 x 100 mg/kg in 3 weeks). The com­pound was at least equally active when injected intramuscularly and subcutaneously (e.g. 10 mg/kg i.m. once per week).

The activity of the substance proved to be independent of the size and number of tumours. Multiple tumours varying in size up to 50 mm diameter regressed just as well as smaller, single tumours. Regression was found, for instance, in all of 21 tumours in 5 rats treated with a total dose of 756 mg/kg s.c. (18 injections of 42 mg/kg in 6 weeks).

A comparable degree of activity was seen in rats with tumours that had become refractory to daily treatment with oestradiol. The re­gression rate was 84 % after a total dose of 90 mg/kg i.m. (9 in­jections of 10 mg/kg in 3 weeks). GP 48 989 also caused regression of carcinomata showing progressive growth after bilateral ovariec­tomy. In 11 out of 100 rats, ovariectomy failed to arrest the car­cinomatous growth. Eight of these animals were treated with

115

116 K.H. SCHMIDT·RUPPIN AND K. SCHIEWECK

Effect of GP 48 989 (18x42 mg/kg s.c.) in rats with multiple DMBA-induced mammary carcinomaIB of different sizes.

M ~ ~ ~ ti Start of treatment \ I \ I

M ~ M hi M After 3 weeks \ I \ I

xx M xx xx xx 'After? weeks \ I \ I \ I \ I \ I

-One week after end of treatment

FIGURE 1

GP 48 989, and 7 of the 8 tumours responded to treatment with at least 9 intramuscular injections of 10 mg/kg given over a period of three weeks. The eighth refractory tumour was a fibroadenoma. The control animals' tumours c8ntinued to grow.

An increased regression rate in response to treatment with GP 48 989 (30 x 25 mg/kg p.o. in 6 weeks) was seen after preceding treatment with oestradiol (15 x 0.1 mg/kg s.c. in 3 weeks). In this experiment, treatment of 40 tumour-bearing rats with oestradiol (0.1 mg/kg s.c.) for 3 weeks resulted in a regression rate of 23 %. Treatment was continued for a further 6 weeks with the same dose of oestradiol in 20 of these animals and with GP 48 989 (25 mg/kg p.o.) in the remainder. Tumour regression in the group treated with GP 48 989 was significantly greater (95 %) than in the oestradiol-treated group (49 %).

In another experiment it was found that testosterone (5 mg/kg s.c. in 3 weeks) caused a further increase in tumour regression in ani­mals previously treated with GP 48 989 (100 mg/kg p.o. in 3 weeks). Relapses and the appearance of new tumours were observed 5 weeks after the withdrawal of testosterone. In response to a further period of treatment with GP 48 989 (9 x 10 mg/kg i.m. in 3 weeks) all these tumours regressed completely.

EFFECTS OF GP 48 989 117

T AS LE 1

Oestradiol dipropionate@ and GP 48989 in sequential treatment.

% Tumour regression after

3 weeks 9 weeks

40 rats treated with oestradiol 15xO.l mg/kg s.c. 23

20 subsequently treated with oestradiol 30 xO.l mg/kg s. c. 49

20 subsequently treated with GP 48989 30x25 mg/kg p.o. 95

Ovocyciin@

T-size (x%)

70 60

GP48989 Testosterone GP48989 £; 50 3: 15x100 9x5 9x10 Q 40 mg/kg po mg/kg sc ••••• mg/kg Lm. (!J 30 .. .. .. .. 20

+10 ", , 6 9 • -10 , ,

~----~----~----~--+-~~--~----~18~--~2~1----weeks

20 • , 30 • • • t: 40 • .Q ,

U) 50 • U) , ~ 60

, Ol , ... 4~

, (I) • 70

, a: ~~ ,

80 ~~~ , •

90 ~.. : ........ 100

Effect of sequential treatment courses with GP 48 989 and testosterone isobutyrate' on the 9rowth of mammary tumours in rats. 1 Perandren®

FIGURE 2

118

~ ~ 0 % E .a 100 "C

~ 90

I!:! 80

~ 70

t 60 50

E 40 0 0

"0 30 Q) 20

f 10

~

K.H. SCHMIDT·RUPPIN AND K. SCHIEWECK

- GP 48 989 10 mg/kg i.m. •••••• DESD 0.25 mg/kg s. c.

{GP 48 989 10 mg/kg i. m. } _.- DESD 0.25 mg/kg s.c.

" . . ....-._._._. \ ." '\ ." r . . \.._._._._ ..

I i i

j l· .... ,. ,# .. _ •• __ ..... ., .. "

/ ' -----.. , , • , 'Treatrnent~ 4111 •••••••••••••••••• ,

1 5 10 15 Weeks Complete regl88llon of DMBA-Induced mammary carcinomata in rats tntaI8d with GP 48 989, Dlethylatllbeatrol diphoephate$ and a combination of these.

Honvan®

FIGURE 3

The results indicate that the compound is remarkably active after treatment with oestrogens as well as androgens and is fully com­patible with these hormones.

In view of the responses to GP 48 989 observed in some advanced cases of mammary and prostatic cancer (3), the activity of GP 48 989 in combination with diethyldihydroxystilbene diphosphate (Honvan~) appeared worth investigating. Because of the side effects it provokes in humans Honvan was given in a low dose (15 x 0.25 mg/kg s.c. in 3 weeks, followed by 3 injections spread over a further 3 weeks) together with GP 48 989 (6 x 10 mg/kg i.m. in 6 weeks). Compared with the effects of each compound alone, the resultant tumour regression was of earlier onset and longer duration.

It appears possible that the side effects caused by Honvan at the usually higher doses administered in man might be diminished if the compound were given in a lower dosage in combination with GP 48 989.

A similar, but less pronounced effect was observed with a combi­nation of GP 48 989 (12 x 10 mg/kg i.m. in l~ weeks) and Fluoro­uracil~ (12 x 2.5 mglkg i.v. 12 weeks.

EFFECTS OF GP 48 989

I!! ::J 0

% E -= 100 "C

~ 90

~ 80 Cl ~ 70

f 80 a 50 E

40 0 (J

a 30 OJ 20 j c: 10 OJ ~ cf

- GP48989 •••••• 5-FU

-'-'-'-'-"""--""C"'""\ // \ , \

10mg/kg Lm. 2.5 mg/kg Lv . 10 mg/kg i.m.} 2.5 mg/kg Lv.

i \. .. ---.. , I ",-",-- \ "" ,. -_ .. -_......... \ , , '\ .. ", I. , / \ "

" I ~

.. " ~, Treatment • '"

5 10 15 20 22 Weeks

Complete regression of DMBA-induced mammary carcinomata in rats treated with GP 48989, 5-FU® and a combination of these.

Fluoro-uracil' FI GURE 4

119

No leucopenia was detected in the rats treated-with either FU or the combination, despite the relatively long duration of treatment.

To sum up, there are a number of noteworthy features emerging from these results: First of all, GP 48 989 causes regression of OMBA-induced mammary carcinomata irrespective of their size and number. Secondly, it causes comparable regression of carcinomata-that have become re­fractory to continuous creacment with oestradiol or continue to grow after ovariectomy and are therefore independent of ovarian steroids. Thirdly, the compound is remarkably active after pre­ceding treatment with oestradiol or testosterone. Finally, GP 48 989 is fully compatible with both Honvan® and Fluorouracil® when given in combination with either of these drugs.

References 1. K.H.Schmidt-Ruppin, A.Meisels, E.Schott, A.Storni and K.Schie­weck, Experientia 29, 823-825 (1973). 2. K.H.Schmidt-Ruppin and K. Schieweck, "Progress in Chemotherapy" Proceeding of the 8th International Congress of Chemotherapy, Athens 1973. Vol 3, 815-818 (1974). 3. M.S.Zedek, B.Oorsk, M.Teller, H.W.J.Marquardt, F.S.Philips, S.S.Sternberg, C.C.Stock and I.Krakoff, "Progress in Chemotherapy" Proceeding of the 8th International Congress of Chemotherapy, Athens 1973. Vol 3, 430-433 (1974).

R 17934 : A NEW SYNTHETIC ANTI-CANCER DRUG

INTERFERING WITH MICROTUBULES *

M. De Brabander, R. Van de Veire, F. Aerts, G. Geuens, L. Desplenter, J. De Cree, M. Borgers and P.A.J. Janssen

Janssen Pharmaceutica, Research Laboratories 2340-Beerse, Belgium

As has been shown by Atassi (1) R 17934 is active against lTlany experilTlental tUlTlors and leukelTlias. PrelilTlinary clinical observa­tions have already established its activity against hUlTlan neoplastic cells in vivo.

We have investigated the effects of this cOlTlpound on neoplas­tic cells in tissue culture in order to gain inforlTlation on the site of action of R 17934 at the subcellular level.

A rapid (within 10 lTlinutes) loss of the norlTlal bipolar sYlTl­lTletry, which is the result of active directionallTlovelTlents, follows treatlTlent of tissue cultured cells with R 17934, at doses higher than 0.04 P-g/lTll. The undulating lTlelTlbrane activity, norlTlally confined to the leading pole of the cell is distributed all over the cell peri­phery giving the cells a rounded forlTl with a radial sYlTllTletry. As a consequence the cells cease to show net translationallTligrations.

The saltatory lTlovelTlents of intracellular particles and endo­cytic vacuoles disappear also and are replaced by periodic bursts of cytoplaslTlic lTlass -streaming. This results in a randolTlisation of the location of these structures which norlTlally forlTl a perinu­clear rirrl.

At the ultrastructural level, R 17934 induces a cOlTlplete loss of lTlicrotubules within 10-20 lTlinutes, in a lTlanner identical to col­chicine or the vinca alkaloids. Here again a rapid loss of the norlTlal cOlTlpartlTlentalisation and organisation becolTles apparent. This is lTlost obviously visible in the "explosion" of the Golgi area, norlTlal­ly concentrated in a perinuclear area around the centriolar cOlTlplex. Within 20 lTlinutes the individual Golgi organelles are dispersed over

·Supported by a grant frOlTl the 1. W. O. N. L. (Brussels, BelgiulTl)

121

122 M. De BRABANDER ET AL.

the entire cytoplasm and the centrioles too often assume a periphe­ral location near the plasma membrane. Lysosomal structures, normally forming a perinuclear rim, assume also a randomised distribution.

In cultures treated with R 17934 the mitotic cells round up in a normal fashion. However, the ordered chromosome distribu­tion and movement is completely lost. Two major types of final outcome occur depending on the cell line used. Some cultured cells (e. g. HeLa, melanoma B16) rapidly become necrotic during the abortive mitotic phase resulting in the extermination of the culture as a whole. Other cells remain in the rounded pseudomitotic phase for a very prolonged time (± 1Z hrs.) but finally readhere onto the substratum, without previous cell division. These cells show up either as mononucleated polyploid cells (e. g. human and mouse fibroblasts) or as multinucleated cells containing a large number of micronuclei each provided with one or several nucleoli (e. g. MO 4 cells, virally transformed mouse embryonal cells).

At the ultrastructural level, treated mitotic cells are shown to be completely devoid of spindle mic rotubules. The unseparated double chromosomes are scattered throughout the cytoplasm inter­mingled with other organelles. The HeLa cells show a pycnotic degeneration of the chromatin, and accumulation of lamellar mem­brane structures and lipid droplets, before becoming completely necrotic (Z).

The endopolyploidisation is the re sult of reenveloping of the chromatin material in a new nuclear membrane before readhesion of the cells. This results in the formation of 1 large nucleus (such as in human or mouse embryonal fibroblasts) or in the formation of multiple micronuclei (such as in M04 cells) where the chroma­tin masses are separated in such a way that they are wrapped indi­vidually or in small groups into new nuclear membranes (Z).

The foregoing shows that the antineoplastic effect of R 17934 resides most probably in its ability to disintegrate the microtubular apparatus of the cell. In order to see whether this compound showed a similar effect on neoplastic cells in vivo we have studied the ultra­structural appearance of L1Z10 cells in vivo. For this purpose mice were inoculated with L1Z10 cells intraperitoneallv or intravenously.

After 4-5 days the animals were treated with 160, 80 or 40 mg/kg of a micronised suspension (mean diameter Z u) of R 17934, either intraperitoneally or intravenously. After 5, 10 and 30 hrs. samples were fixed and embedded for ultrastructural observation. These samples included the ascitic fluid, the peritoneal serosal membrane and gastrointestinal tissue of the mice injected intraperitoneally and the liver, spleen and gastrointestinal tissue of the mice injected intravenously.

One human patient suffering from a carcinoma of the gastric parietal cells with accompanying malignant ascitic effusion, was

ANTICANCER DRUG INTERFERING WITH MICROTUBULES 123

Figure 1a : A malignant ascitic cell derived from a gastric parietal cell carcinoma, 24 hrs. after treatment with 3 mg/ kg of R 17934 as a micronised suspension. A large phagosome contains the phagocy­tosed crystals of R 17934. In this cell and all the other malignant ascitic cells, (both mitotic and non-dividing) microtubules were completely absent.

124 M. De BRABANDER ET Al.

Figure lb : Mitotic ascitic cell of the same patient 24 hrs. after in­traperitoneal treatment with 3 mg/ kg of a micronised suspension of R 17934. Chromosomes (chr) are scattered in the cytoplasm which is completely devoid of spindle microtubules .

ANTICANCER DRUG INTERFERING WITH MICROTUBULES 125

treated intraperitoneally with 3 mg/kg of R 17934 in the micronised form. A sample of the ascitic fluid was withdrawn l4 hrs. after treatment and processed for ultrastructural observation.

The micronised particles of R 17934 were avidly phagocytosed by both the murine and human neoplastic cells and normal phago­cytes (polymorphonuclear leukocytes, monocytes and Kl1pffer cells (fig. la, la). Microtubules were completely absent, with all doses used, in mitotic and non-mitotic LIllO cells up to 30 hrs. after the treatment, and in the human neoplastic cells l4 hrs. after treat­ment (fig. la, Ib). This resulted in phenomena identical to those observed in tissue cultured cells. However, the non-malignant cells residing in the same compartment (leukocytes and the mesothelial cells lining the peritoneal cavity, respectively liver cells, vascular endothelial cells and Kl1pffer cells) showed normal amounts of mi­crotubules and a normal cellular organisation both in the experi­mental and in the clinical situation (fig. la, lb). In the LIllO mice this was true even at the highest dose tested (160 mg/kg). Non-divi­ding intestinal epithelial cells showed normal microtubules too. The mitotic crypt cells, however, were often seriously affected with the higher doses (160-80 mg/kg). Although microtubules were present, centriolar migration had often failed, resulting in a mitotic block.

Apparently the antitumoral effect in vivo of R 17934 is also related to its microtubule dissoluting properties, an effect similar to that of the vinca alkaloids. It is worth stressing that these com­pounds, which are known in an oversimplified way as "spindle poisons", have profound effects on non-dividing cells. The total dis­organisation of both the cell interior and the cell surface should strongly affect all the vital processes which depend on ordered sub­cellular movements. The interference with directional cell migration could have important consequences for the invasive and metastatic properties of tumoral cells. At last, both the alteration of the fluid mosaic structure of the cell membrane (3) and the altered turnover of the cell coat (4) could seriously change the interaction with immune and non-immune host defense mechanisms. In view of this, the appa­rent difference in sensitivity between interphase malignant and non­malignant cells towards R 17934 could be a favorable factor in deter­mining its therapeutic efficacy.

The use of a micronised suspension as the formulation for in­travenous or local injections makes this compound a new "phagoso­motropic" drug (5). This property may favor an accumulation of the compound in actively phagocytosing cancer cells and the formation of a slow release depot which ensures prolonged active blood levels. Preliminary clinical observations have shown that this can be favor­ably exploited in the local treatment of ascitic or pleural effusions where regressions can be obtained without any systemic toxicity.

126 M. De BRABANDER ET Al.

Figure 2a : A znononuclear phagocyte in the ascitic fluid of the sazne patient as in figure 1 after intraperitoneal treatznent with 3 zng/kg of a znicronised suspension of R 17934. The vacuoles (v) are filled with R 17934 crystals. However, znicrotubules are present around the centriole (arrow) and the cellular organisation is norznal. Indeed the Golgi organelles (arrowheads) are fixed in a pericentriolar position.

ANTICANCER DRUG INTERFERING WITH MICROTUBULES 127

Figure 2b : An eosinophylic leukocyte in a Ll2l0 ascites 5 hrs. after intraperitoneal treatment with 160 mg/kg of a micronised suspension of R 17934. Note the abundant presence of microtubules in the vici­nity of the centriolar satelites (arrowhead) and the normal pericen­triolar location of the Golgi organelles (arrows).

128 M. De BRABANDER ET AL.

References

1. Atassi, G. and Tagnon, H. 9th International Congress of Chemotherapy, London, July 1975.

2. De Brabander, M. J., Van de Veire, R. M. L., Aerts, F. E. M. , Borgers, M.J.M. and Janssen, P.A.J. Submitted

3. Berlin, R.D., Oliver, J.M., Ukena, T.E. and Yin, H.H. Nature, 247, 45 -46 (1974).

4. Ginsel, L. A., Daems, W. Th. and Debets, W. Proceedings of the International Symposium on Microtubules and Microtubular Inhibitors. Beerse (Belgium), September 1975 (in press).

5. Trouet, A., Deprez-De Campeneere, D., De Duve, C. Nature, '239, 110-111 (1972).

ANTI TUMOUR ACTIVITY OF CARMINOMYCIN

V.A. Shorin

Institute of New Antibiotics, Academy of Medical Sciences

Moscow, U.S.S.R.

SUMMARY

Antitumour action of carminomycin was studied in mice with 8 different transplantable tumours. Carminomycin was found to be effective in the treatment of leukaemia L1210 and P388, bronchogenic cancer of the lung, cancer of the lower part of the oesophagus, lymphosarcoma Lyo-l and lymphoadenoma NkLy. It is less effective in Sarcoma 180 and not effective at all in the Ehrlich carcinoma. Carminomycin is effective both intravenously and orally. The death of dogs from the toxic doses of carminomycin results from aplasia of the bone marrow. The histopathologic study of organs of dogs killed after administration of carminomycin in toxic doses has not shown any irreversible toxic effect.

Antitumour activity of carminomycin was studied ln mlce with 4 transplantable solid tumours and 4 ascitic tumours.

Lymphosarcoma, strain Lyo-l

This strain was most sensitive to the effect of carminomycin. Two intravenous injections of carminomycin, each equal to 0.5 of LD50, inhibit tumour by 95-100%. Similar results were obtained by oral application of carminomycin in doses of equivalent toxicity.

129

130 V.A. SHORIN

Cancer of the Lower Part of the Oesophagus, Strain OZ-5

Three intravenous injections of carminomycin, in the single dose of 0.5 of LD50, inhibit this tumour by 80%, in the average of three experiments.

Bronchogenic Cancer of the Lung

Three intravenous injections of carminomycin, in the single dose of 0.5 of LD50, inhibit this tumour by 95%, in the average of two experiment s .

Sarcoma 180

This is less sensitive to carminomycin as compared to the three previous tumours. Two intravenous injections of carminomycin In the single dose of 0.5 of LD50 inhibit this tumour by 50%.

Leukaemia L12l0

In this system carminomycin was found to be more effective than daunorubicin and methotrexate. Daunorubicin prolonged the lives of mice by 29%, and carminomycin by 44%, after two intravenous injections in the single dose of 0.5 of LD50. After two injections of carminomycin in the single dose of 0.75 of LD50, the effect of this antibiotic exceeds that of methotrexate by 160%. It should be noted that 20-30% of animals treated by carminomycin are perm­anently cured.

Leukaemia P-388

The superiority of carminomycin over daunorubicin and metho­trexate in this system is even more pronounced than in the studies with leukaemia L12l0.

Lymphoadenoma NkLy

Daily subcutaneous injections of carminomycin in the single dose of 0.3-0.4 of LD50 inhibit this tumour by 60-75%. Daily oral applications of carminomycin in the doses of equivalent toxicity inhibit tumours by 90%. In this system carminomycin is more active by oral application than by intr.avenous or subcutaneous injections.

ANTITUMOR ACTIVITY OF CARMINOMYCIN 131

Ehrlich Carcinoma

In this system carminomycin is not effective.

Carminomycin differs from daunorubicin by its greater activity in leukaemia L1210 and P-388, and by the absence of effect upon Ehrlich carcinoma (Shorin et al. 1973). The single intraperitoneal, intravenous, subcutaneous and oral LD50s Of carminomycin given to white mice are 1.3, 3.7, 3.8 and 7.3 mg/kg respectively. When compared to daunorubicin, carminomycin is four times more toxic when given i.p., seven times more toxic when given i.v., 11 times more toxic when given s.c., and 27 times more toxic when given orally. Such strong differences in toxicity between carminomycin and dauno­rubicin given by the oral route are due to much better absorption of carminomycin from the gastrointestinal tract as compared to dauno­rubicin.

In studies with dogs it was observed that single and multiple i.v. injections of carminomycin, even in toxic and lethal doses, do not alter the functioning of the liver and kidney, the levels of sugar and electrolytes in the blood or cause changes in electrocardio­graphic study of organs in dogs killed after administration of car­minomycin in toxic doses has not shown any irreversible toxic effects. Carminomycin possesses lymphotrophic action and affects lymphoid tissue in the spleen and lymphatic nodes. The death of the dogs from the toxic doses of carminomycin results from aplasia of the bone marrow (Vertogradova et al. 1974).

Irreversible inhibition of haematopoiesis appears after a single i.v. injection of the antibiotic at a dose of lmg/kg and also after ten daily i.v. injections of carminomycin each at a dose of O.15mg/kg. Severe but reversible inhibition of haematopoiesis in dogs was recorded when carminomycin was injected daily for 5 days at a dose of 0.15mg/kg. The course can be repeated after an interval of 1 month. Similar severe reversible inhibition of haematopoiesis is observed after five daily oral administrations of carminomycin each at a dose O.30mg/kg. If carminomycin is injected i.v. twice weekly in a single dose of O.15mg/kg for 5 weeks, alterations in the blood are only moderate.

The study of the pharmacokinetics of carminomycin in rabbits has shown that after i.v. injection at a dose of 1 mg/kg and after oral. administration at a dose of 2mg/kg the durg can be found in the blood for 3-5 hours (Goldberg et al. 1974). Carminomycin penetrates practically all tissues and organs and can be detected in the spleen, kidney, lungs and liver for 3-5 hours after administration. However, the largest concentration is in the spleen, where it can be detected 24 hours after administration. Carminomycin is excreted

132 VA SHORIN

with the bile (up to 11% of the injected dose) and with the urine (2.5%-4.0%).

Intravenous injections of carminomycin given to cats narcotized by urethane in doses from 1 to 5 mg/kg, produced no alterations in the blood pressure or in the frequency and amplitude of the resp­iratory movements. No alterations were noted on their cardiograms.

REFERENCES

Goldberg, L.E., Filiposianz, S.T. and Kunrat, I.A. (1974). Antibiotiki, 19, 57.

Shorin, V.A., Bazhanov, V.S. and Averbuch, L.A. (1973). Antibiotiki, 18, 681.

Vertogradova, T.P., Goldberg, L.E. and Filiposianz, S.T. (1974). Antibiotiki, 19, 50.

VARIAMYCIN, A NEW ANTITUMOUR ANTIBIOTIC

S.M. Navashin, T.G. Terentjeva, E.V. Bobikov, L.I. Torboch­kina, A.B.Sokolov, Y.O.Sazykin and O •. K. Khanykova National Research Institute of Antibiotics,

Moscaw, U.S.S .R.

SUMMARY

The new anti tumour antibiotic, variamycin, was active against a number of mouse and rat tumours. There were differences in the anti tumour spectrum and pharmacological activity between variamycin and mithramycin. The patterns of distribution and excretion of variamycin and mithramycin were in general similar, but the levels of variamycin in the spleen and kidneys were higher. In experiments on rats with gl ioblastoma multiforme c14-variamycin penetrated into the normal and tumour tissues of the brain; the radioactivity of the tumour tissue was higher than that of the normal brain tissue at any period of the experiment. The mechanism of action of variamycin was studied.

Variamycin, a new antibiotic of the group of aureolic acid, was isolated at the National Research Institute of Antibiotics in 1969 (Yu. V .Zhdanovitch et al., 1971). The organism producing variamycin is an actinomycete classified as a new species: Act. olivovariabilis sp. nov. The structure of variamycin, CS2H7602 is shawn in figure 1.

In addition to chromomycinone, variamycin has in its mol~cule the residues of three desoxysugars t two of which are identical to oliose and olivose and one is variose, a new 2,6-dideoxysugar. Variamycin significantly differs· from aureolic acid (which is close to it) by the presence of the variose residue in the antibiotic structure (Lokshin, G.B. et aI., 1975). Therefore, varia­mycin is a new antibiotic of the group of aureolic acid not described before.

133

134

HO

He

OHt

S.M. NAVASHIN ET AL.

O H Me

o OH

He Y0 \Y.H

/O~He

~

Q Figure I. Structure of variamycin

Variamycin is active in vitro against many grampositive and gram­negative bacteria and has a cytotoxic effect on cultures of HeLa cells. Variamycin was found to be active against mouse tumours: carcinoma 755; sarcoma 180; lymphosarcoma LlO-I; Harding-Passey melanoma; lymphadenosis NKLl; sarcoma 37. The activity was evident after repeated intrareritoneal and intravenous administrations of the drug at doses of 0.15-0.5 mg/kg. No activity of variamycin with respect to cancer of the stomach of mice OJ-5 and Ehrlich carcinoma was seen.

The following rat tumours were sensitive to variamycin: Walker carcinoma; sarcoma 45; Gueri n carci noma and sarcoma M- I • Pronouncecl"therapeuti c effect was found with respect to hemocytoblastosis La. The control animals died by the 7th day, while the use of variamycin prolonged the survival of the animals by 60 per cent. The number of blastic cells decreased to one­tenth. The therapeutic efficiency of variamycin in mouse leukaemia was lower. The average survival rate of the animals in all the test groups increased by 37-50 per cent as compared to the control.

Administration of the maximum tolerated dose of variamycin to mice resulted in a short-term decrease, up to 60 per cent in the total number of nucl ear el ements of the bone marrow duri ng the first 24 hours. By the 3rd day the number of myelokaryocytes returned to normal.

VARIAMYCIN, A NEW ANTITUMOUR ANTIBIOTIC

fumout utalj/,i l.n"l~lt.lon

180 ,sozcomo CO'l.cLnomo 'lLmOut O:t-

'/5 -5

Coac.Lnomo d :li.mJtJ$Dl(OItIO

l'ne.l.anom IIQZ c/l.n9-Pa

'I J.

1:10-1 Q

ss)j

.so'ZC!oma 3"1-J,J.

S 'i}/m~obucDSi. COic.lnomQ iA ,i'

gO'lCOmO t/l/lzln ~02tU.n SO~COmQm '2J.Jct.fJe ~

c.o'lt!.t"~D2t:.O

it5 Dmo

-1

mo

~ lilt .

~

~ 0\ ~

0-~

Utbot SIa

t '" 'c..I /::)

"

e e CI\ ~ ~

Figure 2. Variamycin antitumour spectrum.

135

Administration of a toxic dose of variamycin to rats resulted in decreased numbers of myelokaryocytes of up to 63 per cent in 24 hours. A decrease in the number of nuclear elements during the first 3 days was due to decreased numbers of non-differentiated and slightly differentiated cell forms, as well as immature forms of the granulocytes. By the 7th day the morphological picture of the bone marrow was almost normal. The erythrocyte counts and haemoglobin levels in the peripheral blood practically did not change. Some decrease in the number of the reticulocytes and thrombocytes was noted during the first 3 days. later the number of thrombocytes rapidly came back to the normal level

We obtained some preliminary data on the mode of action of variamycin. Variamycin proved to be an inhibitor of RNA synthesis in the experiments with intact cells of Staph.aureus and Hela tumour cells. The antibiotic had a marked inhibitory effect on the DNA-dependent synthesis of RNA in

136 S.M. NAVASHIN ET AL.

the cell-free system containing isolated cell nuclei. The inhibitory effect of variamycin in synthesis of RNA in the nuclei of sarcoma M-1 cells of rats being more pronounced than that in the nuclei of the normal rat liver. With the method of equilibrium dialysis and spectrophotometry it was shown that the sites of DNA bound with variamycin and mithramycin were common. In this connection it is possible to assume that variamycin inhibits the synthesis of the ribosomal RNA.

Table 1 presents the results of the study on distribution of Cl4-variamycin in the organs of albino mice. During the first hours after administration of the antibiotic its maximum levels were found in the blood after which the concentration of the label in the blood markedly decreased, just as it did with the very high initial levels of the label in the kidneys. High levels of the label were found in the liver and spleen. The antibiotic was determined in the spleen for 24 hours but showed no tendency for the level to decrease.

Table 1

Organs and Time after drug administration* (hours) tissues 0.5 I 2 3 6 24 48

Blood 513 315 247 135 91 44 Kidneys 212 140 75 60 35 49.5 29 Liver 187 167 180 152 82 27.2 8 Spleen 143 132 122 127 115 108.5 47 Brain 6 7 8 6 6 3.1 3.1

* Cl4-variamycin (1.5mg/kg), impuises/IOO sec/g

The fraction of variamycin in the total amount of the labelled products of the urine was 30 to 40 per cent at any peri od of the experiment. In 48 hours the radioactivity decreased to one - tenth compared to the level registered 24 hours after the antibiotic administration. The dynamics and levels of the radioactivity in the normal and tumour tissues·of the rat brain are evidence of penetration of Cl4-variamycin through the blood-brain barrier and its predominant accumulation in the tumour tissue. The maximum anti­biotic levels in the glioblastoma multiforme of rats were determined 2 hours after drug administration (4.3 times higher than that of the normal brain tissue) .

Experimental studies on variamycin showed that it significantly differed from other antibiotics of the group of aureolic acid with respect to the toxic effect on the animals and peculiar properties of the specific activity. Thus, variamycin had a broader anti tumour spectrum than mithramycin (Table 2) .

VARIAMYCIN, A NEW ANTITUMOUR ANTIBIOTIC 137

Table 2. Comparative biological characteristics of variamycin and mithra­mycin.

Experimental chemotherapy

Carcinoma 755 Sarcoma 180 Lymphosarcoma L. J . 0 . -1 Melanoma Harding-Passy Tumour OJ-5 Sarcoma 37 Sarcoma 45 Sarcoma M-l Walker carcinosarcoma Guerin carcinoma Haemocytoblastosis La Leukaemia P-388

Pharmacology

Delaying of blood coagulation Increased permeability of vessel walls Changes of blood cell composition

(anemia, trombocytopenia, lympho­penia)

Changes of biochemical parameters of urine (level of chloride, protein etc)

Variamycin

+++ ++ +++ ++

++ +++ ++ +++ +++ ++ +

+

+

Mi thramyc in

+++ ++

++

++

++ +

+ +

+

+

With respect to the pharmacological properties variamycin differs from the other representatives of the group of aureolic acid in the following:

- absence of the negative effect on thrombocytopoesis, erythropoesis and leucopoesis,

- absence of the effect on the content of calcium in the blood, - absence of the effect on haemoglobin levels, - acceleration of the blood coagulation.

Accumulation of variamycin in the tissues of the liver and spleen in sig-n i fi cant amounts, its penetrati on through the bl ood-brai n barri er and accumu­lation in brain tumours are peculiar properties of the antibiotic pharmaco­kinetics.

Variamycin is now in the first stage of its clinical studies.

138 S.M. NAVASHIN ET AL.

REFERENCES

1. Zhadanovitch, Yu.V., Lokshin, G.B., Kuzovkov, A.D. and Rudaya, S.M. (1971). Chimija prirodnich soedineniji, 4,646.

2. Lokshin, G.B., Zhdanovitch, Yu.B., Kuzovkov, A.D. (1975). 1, 4, 79.

3. Phmip-: J.B., Schenck,J.R. (1953). Antib. Chemotherapy, 3, 1218. 4. Fatsuoka, S. (1958). Cancer (Suppl.), 49, 23. -5. Gause, G.F., Ukolina, R.S. and Sweshnikova, M.L. (1962). Anti­

biotiki, 7,3, 34-39. 6. Eselevitch, M.M., Sazykin, Yu.O., and Torbotchkina, L.1. (1971).

Antibiotiki, 5, 400-404. 7. Eselevitch, M.M., Torbotchkina, L.1. and Sazykin, Yu.O. (1972).

Antibiotiki, 11 i 975-977. 8. Bobikov, E.V.,Terentieva, T.G., Torbotchkina, L.I., Sokolov, A.B.,

Sazykin, Yu.O. and Navashin, S.M. (1975). Antibiotiki,8, 705.

INHIBITION BY CAFFEINE OF POST-REPLICATION DNA REPAIR IN HAMSTER

CELLS TREATED WITH CIS PLATINUM (II) DIAMINE DICHLORIDE

H.W. van den Berg and J.J. Roberts

Institute of Cancer Research, Pollards Wood Research

Station, Chalfont St. Giles, Bucks., U.K.

There is considerable evidence to suggest that DNA is a critical cellular target for the anti-tumour agent cis platinum (II) diammine dichloride (cis Pt(II))l. An important factor in determining the susceptibility of a cell to the cytotoxic action of this agent will be, therefore, the ability of the cell to re­pair cis Pt(II)-induced damage to its DNA. The results of earlier studies 2 using Chinese hamster cells in culture led us to believe that the majority of DNA-platinum products - those in­volving only one strand of the double helix - are chemically stable and refractory to enzymatic removal. This suggested that these persistent lesions may be circumvented by another DNA re­p~ir mechanism, namely post-replication repair. This is a pro­cess which enables the cell to tolerate a certain amount of un­excised damage to its DNA by leaving, during DNA synthesis, a gap in the newly synthesised DNA strand opposite the damage in the template strand. This gap is subsequently filled - probably by de novo synthesis in mammalian cells 3 - and this gap-filling process is inhibited by the trimethyl xanthine, caffeine. Con­sequently many cell lines competent in post-replication repair are rendered extremely sensitive to the lethal effects of UV irradiation~ and chemical damageS by post-treatment incubation ln the presence of non-toxic concentrations of caffeine.

In this paper we report that caffeine greatly enhances the lethal and chromosome-damaging effects of cis Pt(II) in Chinese hamster lung V79-379A cells. These effec~ are accompanied by a dose-dependent reduction in the molecular weight of DNA syn­thesised in cis Pt(II)-treated cells in the presence of caffeine. Furthermore, the results of separate birlding studies suggest that at several levels of reaction, the new DNA is synthesised up to a size approximately equal to the distance between DNA-platinum

139

140

c 0·1 o -u o <-u.. 0\ C

.:;: 0·01 > <­:::l

If)

0·001

H,W. van den BERG AND J.J. ROBERTS

10 20 30 40 50

Concn. of cis Pt [II) (}JM)

Fig. 1 Survival curves for treatment of V79-379A cells in suspension culture with cis Pt(II) alone (0) or cis pt(II) followed by growth in the presence of 0.75 roM caffeine (~). Cells were exposed to cis pt(II) for 2 hrs. at 37°C.

products in the template strand. The effect of a 2 hr. treatment with C1S pt(II) on V79-379A

cell survival and the potentiation of this effect by caffeine is shown in Fig. 1. (Survival has been estimated from the ability of treated cells to divide and form COlonies). It can. be seen that the shoulder on the cis pt(II) survival curve (characterised by the '~uasi-threshold' dose, DQ) is completely abolished if the treated cells are subse~uently grown in the presence of 0.75 roM caffeine. (This dose of caffeine alone is non-toxic to these cells). The DO, or dose increment re~uired to reduce the surviving fraction from f to 0.37 f on the exponential part of the curve, is also reduced by caffeine.

For cytological studies, samples were taken from suspension cultures of cells treated with 15 ~M cis pt(II), which reduces cell survival to 50%, or 4% if the cells are grown in the pre­sence of caffeine (Fig. 1). 4 hrs. after treatment with 15 ~M cis Pt(II) the number of metaphases containing visible chromo-

POST-REPLICATION DNA REPAIR INHIBITION BY CAFFEINE

III c .Q -E ... QI

~ 01 C

100

c 50 .!2 c o u ~ ;§

15J.lM cis Pt[IIl

+0·75mM Caffeine

o~ ~iSPtllIl •

~O O~~---r------r------r------~~

10 20 30 40 45 Hours after Treatment

Fig. 2 Relationship between appearance of chromosomal aber-

141

rations and time after treatment with cis pt(II). (0), 15 ~M cis pt(II); (~), 15 ~M cis pt(II) followed by growth in the pre­~ce of 0.75 mM caffein~ Cells were exposed to cis pt(II) for 2 hrs. at 37°C. ---

somal aberrations was not significantly above control level, and this proportion was unaffected by caffeine (Fig. 2). By 14 hrs. after treatment, 60% of platinum-only treated cells contained chromosome aberrations, with only 4% of cells post-incubated in medium containing 0.75 mM caffeine being classified as normal at this time. Caffeine not only increased the number of cis pt(II}­treated cells containing chromosomal aberrations, but it also enhanced the severity of the damage observed. The most dramatic effect was the sharp increase in the number of cells containing 'shattered' chromosomes, and those with numerous chromatid de­letions and exchanges (see ref. 6).

The delayed appearance of chromosomal abnormalities after cis pt(II) treatment suggests that DNA replication is necessary for their formation, and in this respect cis pt(II) resembles UV irradiation and alkylating agents rather than X-irradiation (see ref. 7).

Fig. 3 shows that the potentiation by caffeine of cis pt(II)­induced lethality and chromosomal aberrations is associated with a reduction in the molecular weight of DNA synthesised immediately after treatment. In these experiments, cis pt(II)-treated cells

142 H.W. van den BERG AND J.J. ROBERTS

SEDIMENTATION OF DNA SYNTHESISED IN THE PRESEt«:E OF 0·75l1li4 CAFfEINE 10 • 10 10

\ CONTRa.

\ 15~M 8!PtIllI

-'

\ 5 g 5

'\ /"' ~

I \/-W--.. ~ . J "\ \ I \

, .. \ . "'J . \~

~91Pt(1I1

~---,---.----,---~0L---.----.----,---,0L---.----.----,---~ 10 20 30 40 10 20 30 40

~ FRACTION MJMBER ~ 12

20

10 10 1\ J \

50~M8!Pt (II)

I \ . i \ l j \

.. -.----' 10 20 30 40 0 10 20 30

<-- FRACTION NlMlER ~

30

Fig. 3 Alkaline sucrose gradient sedimentation profiles of DNA synthesised during a 2 hr. period in the presence of 0.75 mM caffeine in V79-379A cells treated for 2 hrs. with cis Pt(II). Sedimentation (5,000 r.p.m./16 hrs./200 C) is from right to left.

were resuspended in medium containing 0.75 mM caffeine and 0.5 ~C/ml [3 HJ-thymidine for 2 hrs. Cells were then lysed on top of 5-25% alkaline sucrose gradients which were then centrifuged at 5,000 r.p.m. for 16 hrs. at 20°C prior to fractionation and radioactivity determination (see ref. 8 for experimental details). cis Pt(II) treatment led to a dose-dependent reduction in the molecular weight of DNA synthesised during a 2 hr. period in the presence of caffeine. The number average molecular weight of DNA synthesised in treated cells ranged from 4.35 x 10' daltons (15 ~M cis Pt(II)) to 0.95 x 10' daltons (100 ~M cis Ft(II)).

Fi~4 attempts to explain these and other data6 ,8 by way of a model. In the absence of caffeine, DNA synthesis is in­hibited in cis Pt(II)-treated cells and discontinuities in daughter DNA are not detectable if the length of the radioactive labelling period is extended to compensate for the slowing in DNA synthetic rate 8 • This we interpret as the successful op­eration of a post-replication repair system which allows the syn­thesis of a continuous daughter DNA molecule on a template con-

POST-REPLICATION DNA REPAIR INHIBITION BY CAFFEINE

WITH CAFFEItE ~ +4 ~ I I

I I I I I I

I I I

T I t::. t::. I t::. 6 6 t::. I

A WITHOUT CAFFEINE

~ + I I I I I :

T A A I t::. t::. t::. t::.

D-o 0

D------~ I I I I I I I I I

DNA Synthesos Inhibited as a result of delay at site of lesions

compenste for delay

I I I I I 0

I I 0

I nitlatlon of DNA Synthesos beyond

lesions • leaving gaps. No reduchon In rate 01 DNA SynthesIs

Remove ~ Caffeine

D D _____ -:--___ -:-_

No gaps detectable Gaps filled

Key T = Templote DNA D = Daug,ter DNA 6 = DNA-Pt product a = DNA SynthesiS

InrtlQtlon site

Fig. 4 DNA strand elongation in Chinese hamster V79-379A cells treated with cis Ft(II): the effect of caffeine. For explanation, see text.

143

taining unexcised damage. Caffeine inhibits this repair process, and consequently newly synthesised DNA contains discontinuities. Separate binding experiments have demonstrated a close correlation between the size of the DNA synthesised in the presence of caf­feine and the distance between DNA-platinum products in the tem­plate strand. We believe, therefore, that, as shown in Fig. 4, DNA is synthesised in the presence of caffeine up to a size approximately equal to the inter-platinum distance on the tem­plate strand. Whilst caffeine remains in the growth medium, these discontinuities persist and act as focal points for the pro­duotion of damage which is visualised at mitosis as chromosomal aberrations. Inhibition of post-replication repair by caffeine is further associated with the complete abolition of the shoulder on the cis Pt(II) survival curve (Fig. 1), suggesting that the cells are no longer capable of tolerating low levels of damage to their DNA. The effect of caffeine at the molecular level is re­versible, since on its removal from the medium, the newly syn­thesised DNA in cis Ft(II)-treated cells rapidly reaches the high molecular weight observed in control cells.

It has been suggested that the ability of caffeine to potentiate the anti-tumour activity of cyclophosphamide 9 or nitro­gen mustard and X-rayslO may be in part attributable to eXC1Slon repair inhibition. We suggest that a post-replication repair

144 H.W. van den BERG AND J.J. ROBERTS

system may operate in tumour cells in vivo and it is this repair system that is inhibited by caffeine, since low doses of caffeine do not inhibit excision repair in mammalian cells ll .

References

1. J.J. Roberts, Recent Results in Cancer Research: Platinum coordination complexes in cancer chemotherapy, Springer­Verlag 1974, pp. 79-97

2. H.W. van den Berg and J.J. Roberts, Chem.-Biol. Interactions, in the press

3. A.R. Lehmann, J. Mol. BioI., 66 (1972) 319-337

4. A.M. Rauth, Radiation Res., 31 (1967) 121-138

5. A.M. Rauth, B. Barton and C.P.Y. Lee, Cancer Res., 30 (1970) 2724-2729

6. H.W. van den Berg and J.J. Roberts, Mutation Res., In the press

7. M.A. Bender, H. Gaston-Griggs and J.S. Bedford, Mutation Res., 23 (1974) 197-212

8. H.W. van den Berg and J.J. Roberts, Chem.-Biol. Interactions, submitted for publication

9. D. Gaudin, K.L. Yielding, A. Stabler and J. Brown, Proc. Soc. Expl. BioI. Med., 137 (1971) 202-205

10. D. Gaudin and K.L. Yielding, Proe. Soc. Expl. BioI. Med., 131 (1969) 1413-1416

11. J.E. Cleaver, Radiation Res., 37 (1969) 334-348.

The role of nuclear proteins in the chemotherapeutic effect of Dibromodulcitol (DBD)

Jeney, A., Dzurillay, E., Lapis, K. and Institoris, L. 1. Institute of Pathology, Semmelweis Medical University Budapest, 1085. Hungary

We have previously reported that DBD induces not only inhibition but also enhancement of RNA labeling. (Valyi-Nagy et al. 1969) Later it was observed that the growth stage of the cell population determines which type of alteration in nucleic acid labeling will be manifested upon DBD treatment. (Jeney et al. 1971) Table 1. demonstrates an example for the growth rate dependent effect of DBD on nucleic acid labeling of Yoshida cells in suspension culture. Yoshida tumour cells were treated with 10 or 100 }lg x mC1 DBD for one hour 2 or 5 days after culturing, that is in the iogarithmic and in the stationary stages respectively. Measuring incorporation of 3H- Uridine into hot PCA soluble fraction an enhanced labeling was found only in the 2 days old culture at the case of 10 J.lg x x ml-1 which produces a 30-40 % inhibition in the growth of the cell culture. This type of alterations are not confined to Yoshida cells since in the P388F lymphoma cells DBD induced an enhanced RNA labeling in the early logarithmic stage and a decreased one in later stages.

Evaluating these experiments we felt entitled to presume that DBD in­fluences the regulation of nucleic acid synthesis by binding to chromatin proteins. (Jeney et al. 1970, Szab6 et al. 1973). To obtain further data for supporting our presumption the binding of 3H- DBD to nuclear components comparing to other cellular constituents was studied. The distribution of 3H-DBD among macromolecular fractions extracted according to the procedure of Scott et al. (1956) was followed in Yoshida tumour cells treated in vivo for 1 and 24 hours with 100 mg/kg 3H- DBD. Table ll. shows that at 1 hour 80 % while at 24 hours after drug treat­ment 60 % of the total radioactivity is located in the PCA soluble

145

146 A. JENEY ET AL.

Table 1

{ffeet at O,,,"of'nCJdulcdOI "" tn" ,ncorporat,on of 5· T· L/r.dm"

Lnto YQ$h,da sorCDn7a c«is m 2 or 5 c:kJY. old .su.p.n.,on

cuitlJrfI

CP'" • OO~O Prot.,n contt!nt mg ".,. cult"Jr.

I=ract~O'" Cold peA Hoi ACA

.. of cult""., 2 days .5 '*'!,IS 2~Sqs 2dO'1S 5do':/!J

Control o317! .J6 M!.5 2S&!.52 1O!¥J &!1!J 211! 10

15! 12

YCBhlt:Ja SOf"COI7JO c~lI. cultt/lrlld U1 FlkMr mftiu.lnt , I}~ I'tor.ea!rum

Tr#otment ..,tin DBO for 2 hour$

l.ol»bng wlih IO.,.,C'I.1,", 5·/- L/r,dm. (SC,lm 1"1)

PCA .:s= Pr!rchlor, c aCId

Table 2

D'stnbui!on of .)H-OBO among c~Uulor

froctton.s of YoshIda sarcoma ,n VIVO

dol a rtNol.d to 0 8 c"lI.. Froct.ons

fhour 21; hour!J '"9 V- cpn ,.

'"9 ~ cp-n

Cald PCA 506,8 81 0.726 (Solub/,,;

Alcohol· <11"-(lipIds)

001 17 0097

NoOH-J.lCI 2,9 10,1, (RlVA)

/,6 3,6 0,021;

PeA -we (ONA)

2.2 alS Q02 2,1 QOI<9

No 01-1 12.2 1,1;9 (pot"",!J)

0,2 12,6 a28S

7r~otrnl!"nt tXl'"9/lrg .31-1-DBO (188 ",e,lm /'f) 'P FrocflonciiCYJ Scott .t 01 1956

%

61

<12

2,0

/;,0

21;

Eoeh pomt re~~~nt rn.an of four o.t",.,.,.,.nal,on. (PIS %)

0° cpn - '/'9 .3/-1-080

NUCLEAR PROTEINS AND DIBROMODULCITOL 147

fraction, i. e. not bound to macromolecules. During the observation period the radioactivity decreased very substantially in the cold PCA fraction, in the alcohol-ether, and in the dilute alkali soluble fractions, at the same time in the hot PCA and in the residual fractions (containing protein solubilized in alkali) the radioactivity showed a much slower decrease, therefore drug relatively accumulated in these later two frac­tions for 24 hours. On Table III. the binding of 3H- DBD to various nuclear components is demonstrated. These data confirm the results of E. Institoris et al. (1974) by indicating that more radioactivity is as­sociated to histones and especially to chromatin acidic proteins than to DNA. Our present data however point out that at 24 hours this type of distribution among chromatin components changed since DNA contained substantially more radioactivity than the chromatin proteins. Further­more the partition of the radioactivity between histones and acidic proteins also changed. At 1 hour the acidic proteins while at 24 hours after drug treatment the histone fractions contained more radioactivity. For determining whether radioactivity in these fractions represents bound molecules chromatin proteins isolated from Yoshida tumour and for comparison also from rat bone-marrow were separated on SDS­-polyacrylamide gel-electrophoresis. Table IV. shows that in the 0.35 M NaCI extracted fractions radioactivity was detected as a distinct peak on the gels, indicating that drug has a different access to the various proteins present in this fraction. Studies on the acidic proteins suggested a differential binding of DBD or its metabolite in Yoshida tumour and in bone-marrow. Radioactivity was mainly bound to the slowly migrating component in the case of the Yoshida tumour, while acidic proteins from bone-marrow contained the majority of the radioactivity in the fast migrating components. Among the histones mainly the F1 and to a lesser extent the F3 and F2al contained radioactivity. In many experiments radi~activity was observed in front of the histones where Coomassie Brilliant Blue shows no staining on the gel. Recently it was verified that at this position the radioactivity is associated with a band stained with periodic acid-Schiff reagent (PAS) suggesting the presence of glyco­proteins. The question has arisen as to whether the binding of DBD to chromatin proteins brings about alterations in their functions. In the case of the histones it was found feasible to estimate the interaction of this molecule to DNA by measuring the melting profile of DNA in the presence of histones. Table V. shows the increase of Tm point of DNA after the complex formation with F 1 histone. However if the F1 histone had been isolated from Yoshida sarcoma treated with 50 mg/kg DBD for one hour no increase in Tm point was observed. This result seems to us as a strong evidence that DBD prevent the interaction of histones and DNA by binding to the former, and could be considered as an explanation for the unique effect of DBD on nucleic acid synthesis. The compilation of these data offer a possibility to set up a model for the consecutive

148

Table 3 O'str"butlon of .JH-080 among nuc'~or

fracttons of Yoshida sarcoma

data r'lC1t~d to 0 1 c,.lIs Fracttons

I hour rotlo IO'dpm rat,·o 24 ':?/irs

10 clpm

ql5 tf iliaCI lI.1troct

0,35 "1.5 0,22 8.11

0,35 tf /Voel 'Itract

a25 31,7 412 10,9

J.IIston ... 1,2 74,7 aliI} 14,4

ACidic residual a57 123,0 0.7 5,5 prot~ln.

DNA 7,00 28,0 UJO 20,1

rr.otrntN1t .3W -OBO 700 mg/kg (18~ mCi/mH)

NuClear fracilons St"tn and Barl.K1 (1971)

IhdloaclL vdy TO' dpm ~ Ing 311-080

Table 4 BlNOING OF " · OIBROitIODUILI7OL fO ~/N PROrC/~

-.

~----~~~~~+ ~-~--==~------~ - - -OCN$HONETER TRACING

~--~nV'TY PIiIOrEJ~ S£PCRATEo ON SOS · POI.'(AC~yI:;;tAHIOE GEL- cf.E' ~IKJPr-HJRES/ S

A. JENEY ET AL.

NUCLEAR PROTEINS AND DIBROMODULCITOL

E 'h ...

Table 5

Effect eX DBD (T/ the FarootJon eX 0NA-H5tr:ne [ Carpbx

1m pOints .

I j i ,

/

.. •

Table 6

....... /

r i

i /

lit

-'"

THE EFFECT OF O/SINlNOlXJLCITOL (080) ON THE

FUNCTIONS OF CH/iOMATIN

...-- .-.. • I

i

~ TRANSCRIPTION·· .RHA

I'! nuClf-O/u$ .. ,1'IIli1J,IkI

In Cltl"Ol'hOllh . .nllone H

149

150 A. JENEY ET AL.

alterations in the chromatin after DBD treatment. Table VI. shows our present view on the mode of action of DBD at the nuclear level. At an early time after treatment if one molecule DBD is associated to DNA threetimes and fourtimes as much are bound to histones and to acidic proteins respectively. As a consequence of the drug binding the inter­action of histones to DNA is prevented which causes a deficient re­pression on the genome followed by the production of undesirable transcripts. The acidic proteins are more heterogenous than histones not only in molecular weight but in function as well. It is unknown whether DBD binds preferentially to those components of this fraction with regulatory or with enzymatic function, if any distinction may be involved at all. However alkylation on the acidic proteins which has been implicated in the specific regulation of the genome probably induces damages in their function resulting alterations in the replicative and transcriptional function of the genome. On the same ground it is also conceivable that DBD by binding to the chromatin proteins upset the balance between repression and derepression, furthermore the intimate connections between replication and transcription get confused. It seems to us that the present model helps to understand certain pharmacobio­chemical features of DBD. So the fact that DBD not only inhibits but also enhances RNA labeling can be explained by the possibility thaL in certain cases DBD preferentially effects the repressor and in other cases the derepressor component of gene regulation. The growth rate dependent tumour inhibitory effect of DBD is also in harmony with this model since fast proliferating cells require high level of chromatin protein synthesis. However in attempting to establish the relevance of these data to the chemotherapeutic mechanism of DBD one must certainly be aware of the complex meaning of drug binding to chromatin proteins. In this respect four possibilities should be taken into consideration: 1. No consequence at all on cell viability and no influence on the chemothera­peutic effect. 2. No consequence on cell viability but it represents a de­toxication reaction. 3. Contribute to the chemotherapeutic action as a second or adjuvant target. 4. It represents a critical target. As far as DBD is concerned the question must remain open as to whether the chromatin protein does not contribute to the chemotherapuetic action. For our part it is hard to accept that covalent binding to chro­matin proteins has no contribution ot the chemotherapeutic effect, since they are not just a defender of DNA but play an important role in the tissue specific function of the genome.

NUCLEAR PROTEINS AND DIBROMODULCITOL

REFERENCES

Institoris, Ethel, Ho1czinger, L., Banfi, D. and Institoris, L. (1971) Proc. Vllth Internat. Congress of Chemotherapy, Prague.

Jeney, A. Szabo, T. Va1yi-Nagy, T., Institoris, L. and Szabo, J. (1970) Europ.J. of Cancer 6. 297.

Jeney, A. Szabo, I. Fox, M., Fox, B. Garrad, J. and Ho11and,J. (1971) Proc. Vllth Internat. Congress of Chemotherapy, Prague.

Va1yi-Nagy, T., Jeney, A., Szabo, J., Szabo, I. and Institoris, L. (1969) Europ. J. of Cancer 5. 403.

151

THE EFFECT OF DIBROMODULCITOL ON THE REPLICATION OF

DNA IN YOSHIDA SARCOMA CELLS

Etel INSTITORIS and L. HOLCZINGER

Research Institute of Oncopathology

1112 Budapest, Rath Gy. u. 7, Hungary

SUMMARY

Doses of dibromodulcitol which significantly suppressed the growth rate of Yoshida sarcoma cells ~n culture produce an alkylation level of 2.3 - 4.3 xlO nucleotides per molecule of dibromodulcitol in DNA. This extent of alkylation does not lead to single strand breaks in the parental DNA, whereas it causes postrepli­cative lesions in the newly synthesized DNA strand, the repair of which is dose dependent.

INTRODUCTION

1,6-Dibromo-l,6-dideoxy-dulcitol (DBD, Elobromol, Mitolacto~ is a widely used alkylating agent in cancer chemotherapy. Previous studies of its mode of action revealed that DBD alkylates mainly the chromo­somal proteins and to a less extent DNA after in vivo treatment Institoris et ale 1974. Under these circum­stances noticeable lesions were not detectable in the DNA molecule. In the present work we made a more detail­ed study of DBD effect on DNA using in vitro conditions.

MATERIALS AND METHODS

Yoshida sarcoma cells were routinely maintained in suspension culture (Fox and Fox 1971). The cells were exposed to various doses of D8Dfor one hour. After re-

153

154 E. INSTITORIS AND L. HOLCZINGER

moval of drug the cells were washed, resuspended in fresh medium and the growth rate of treated cells were compared to that of the control over four days. For binding exper­iments 100 ml suspension of cells (3 x 105 cells per ml) were exposed to 3H-DBD (spec. act. 518 ~Ci/mg) for one h and the drug continaing medium was removed by centrifuga­tion and after washing DNA was isolated (Kirby 1957). The pronase treated D~A was dialysed before the measurement of radioactivity.

For template labellin~ the cells were uniformly label­led with ~-(Me)-thymidine (spec. act. 15 Ci/mmol) for 18 h. The isotope-containing medium was removed and after washing the cells were resuspended in fresh medium before the treatment.

For assay of newly synthesized DNA the cells after a 1 h treatment were collected by centrifugation and after washing pulse labelled with 3H-(Me)-thymidine in fresh medium for 30 min. In the chase experiments the cells were incubated in medium containing 2 ~g/ml thymidine for various time. Sedimentation analysis was performed by the method of McGrath and Williams (1966) in isokinetic alkaline sucrose gradient (Noll 1967).

RESULTS AND DISCUSSION

The effect of DBD on the growth rate of Yoshida sarcoma cells growing in suspension culture is shown in Fig. 1. Significant supression of cell growth already occured at DBD concentration of 50 ~g/ml. 200 ~g/ml DBD the highest drug concentration used in this study, proved to be very toxic.

Using the above drug concentrations the cells were exposed to 3H-DBD for 1 h and the radioactivity was measur­ed in DNA samples isolated from these cells. (Table 1). The extent of binding is expressed in pmoles DBD bound to 1 mg DNA as well as in the number of nucleotides containing 1 molecule of DBD bound.

Doses ~g/ml

50 100 200

TABLE 1 : Binding of 3H-D8D to DNA

DBD bound to DNA pmoles/mg Nucleotides x 105 per 1 molecule of DBD

7.7 4.3 9.1 3.6

14.7 2.3

DIBROMODULCITOL AND REPLICATION OF DNA 155

control

$0"u5 Im[

fOO flg/"'[

da!Jl

FIGURE I : Growth curves of cells exposed to DBD for I h.

In order to investigate the kind of lesion in DNA caused by these alkylation levels, cells were exposed to the most toxic dose of DBD (200 ~g/ml) after template labelling and the cells were submitted to the alkaline sucrose gradient centrifugation. Fig. 2. shows the sedimentation patterns of DNA released from treated and untreated cells. As it can be seen even 200 ~g/ml DBD did not lead to single strand breaks in the parental DNA. Furthermore, the same sedimentation profile was obtained for template DNA from cells which had been incubated in the absence of the drug for 5 h after treatment.

(/)

..... Z ::::l e u

10

~ 5 ..... e .....

~ UNTREATED _ TREATED

10 20 ~

• SEDIMENTATION

FIGURE 2 : Alkaline sucrose gradient sedimentation profile of template DNA after I h treatment with 200 ~g/ml DBD.

156 E. INSTITORIS AND L. HOLCZINGER

For study of DNA synthesis on the template DNA conta­ining alkylated parts, the cells were pulse-labelled with 3H-thymidine after treatment and then incubated with cold thymidine for times of various length. During the in­creasing chase time the cells were submitted to alkaline sucrose gradient centrifugation every hour. The differ­ence between the sedimentation profiles of newly synth­esized DNA from treated and untreated cells could be observed after 5 h chase time (Fig. 3). At 50 ~g/ml DBD concentration, the difference is uncertain but at 100 ~g/ ml - which caused a marked supression of cell growth and the alkylation level was 3.6 x 105 nucleotides per 1 molecule of DBD - the peak of treated DNA definately shifted towards the higher region of gradient compared to that of the control. The most marked difference between the sedimentation profiles of DNA synthesized in treated and untreated cells were found at 200 ~g/ml DBD con­centration where the alkylation level was 2.3 x 105 nucle­otides per one molecule of DBD for parental DNA. These data revealed that the DNA synthesized normally on the alkylated template DNA until 5 h after treatment, but increasing the chase time the DNA synthesis becomes abnormal.

Subsequent incubation of the 100 ~g/ml DBD-treated cells in cold thymidine led to a shift of DNA peak to-

~UNTREATED

_TREATED

FIGURE 3: Alkaline sucrose gradient sedimentation profiles of DNA synthesized in cells after a 1 h treatment, 30 min pulse and 5 h chase prior to centrifugation.

DIBROMODULCITOL AND REPLICATION OF DNA 157

wards the heaview region and 12 h after treatment it sedimented in the same position as the control. Whereas the DNA synthesized in 200 ~g/ml DBD-treated cells does not show any sign of the repair process by this time (Fig. 4).

In order to test whether the decrease of DNA mole­cular weight may simply be due to a slower rate of DNA synthesis in treated cells, we determined the incorporat­ion of 3H-thymidine into the cells 3 and 5 h after treat­ment (Table 2).

TABLE 2 Incorporated 3H- TdR (dpm x 10-2 /10 5 cells)

After Control DBD treated treatment 50 ~g/ml 100 ~h/ml 200 ~g/ml

3 h 275 269 280 270 5 h 340 335 355 192

As can be seen, both the untreated and treated cells incorporated about the same amount of 3H-thymidine except the cells treated with 200 ~g/ml DBD. In this case the incorporation is about 60% of the control value.

On the basis of these data presented here the effect of DBD on DNA could be explained in the following way:-

Lower doses of DBD alkylate the parental DNA, result­ing in a post replicative lesion in the newly synthesized DNA t.i. gaps are formed in the daughter strand opposite

IJ)

I­

Z ~ o U

10

....I 5 « I-o I-

10 20

c-.CONTROL

_'00llg/ml _200llg/ml

30 II SEDIMENTATION

FIGURE 4: Alkaline sucrose gradient sedimentation profiles of DNA synthesized in cells after a 1 h treatment, 30 min pulse and 12 h chase prior to centrifugation.

158 E. INSTITORIS AND L. HOLCZINGER

to the alkylated nucleotides of parental DNA. These alk­ylated sites in the parental DNA are not so frequent, so during sufficiently long time the cells are able to repair them. In the case of higher doses the occurrence of alk­ylated sites in DNA are more frequent which already leads to a depression of DNA synthesis.

REFERENCES

1. Fox, M. & Fox, B.W. (1971/72), Chemico-Biol. Interactions, i, 363.

2. Institoris, E., Holczinger, L. & Banfi, D. (1974) Zeitschrift f~r Krebsforschung, ~, 101.

3. Kirby, K.S. (1957), Biochemical Journal, &£,495.

4. McGrath, R.A. & Williams, R.W. (1966), Nature, 212, 534.

5. Noll, H. (1967), Nature, 215, 360.

CLINICAL CANCER CHEMOTHERAPY WITH DRUGS AIMED AT GENE REGULATORS

F.E. Knock,R.M. Galt, Y.T. Oester, R. Sylvester

Augustana Hospital,University of Illinois,Loyola Univ.

411 w. Dickens ,Chicago ,Illinois 60614, U.S.A.

Clinically useful sulfhydryl (SH) inhibitors appear to show preferential toxicity against a variety of animal and human cancers, relative to normal tissues. Such preferential effecrs9have been seen both in vivo and by in vitro sensitivity tests. -

Black, Kleiner and Bolker reported that patients whose solid tumors were regressed by the SH inhibitor iodoacetate plus adjuncts malonate and fluoride showed no change in blood components at the 10 same doses clearing blasts from the circulation of leukemic patients. For human cancers capable of objective measure, the SH inhibitor2 oxophenarsine (J-amino-4-hydroxyarsenosobenzene) and iodoacetate -9 plus adjuncts have regressed the majority of cancers sensitive to the drugs by in vitro sensitivity tests on each patient's own cancer.

In histochemical studies. clinically useful SH inhibitors were found to react mainly ~i~h protein-SH groups of chromosomes, cell and nuclear membranes. - From the clinical and histochemical data, the main clinical target of the SH inhibitors, the SH-bearing non­histone chromosomal prote~n~ (NHP),were predicted to playa major role in regulating genes. - Recent studies in multiple laboratories now strongly suggest that1rhr6 NHP may indeed playa major role in regulating gene readout. -

Kinetic studies now in progress appear to confirm the usefulness of the in vitro sensitivity tests instrumental in finding the rela­tively preferential effects of the clinically useful SH inhibitors against a variety of human and animal cancers. The sensitivity tests rule out inactive drugs and permit select~o~ from among drugs active against each patient's own cancer cells. - The most useful tests monitor drug effects on isotope incorporation into DNA, RNA and

159

160 F.E. KNOCK ET AL.

protein and on DNA polymerase activity of intact cancer cells rela­tive to normal cells. Preliminary studies suggest the possibility of further refinement of the tests by monitoring drug effects on tracer incorporation into fractions of the chromosomal NHP separated by polyacrylamide- SDS gel electrophoresis.

MA TERIALS AND ME'IHODS The methods used to follow tracer incor~o9ation into DNA.RNA

and proteins have been previously described. - • For the data in Table 1. S-180 cells were treated in M-199 medium supplemented with 10~ calf serum. Treatment times ranged from 2-48 hours before admixture with tracer doses of tritiated thymidine for 20 hours. all at J70 C. The data. expressed as % of control values. are the averages of triplicate runs and did not vary over 5 %. For the drugs listed in Table 1. tracer incorporation to DNA is the most significant parameter of drug activity. 50 only data for DNA are included.

For DNA polymerase activity. the double isotope method of Chae et al was used. Control cells were incubated with thymidine (TdR)14 labelled with triti~ and experimental cells with inhibitor and C labelled thymidine. To equalize losses in thin layer chromatogra-phy. control and treated cells were pooled. DNA an§ ~ntermey!ate~ were isolated and counted as previously described. • The C: H ratios of intermediates proximal to the inhibited site should be high­er and those distal to the block lower,than the ratios obtained in the absence of effective inhibitor.

For the data in Table J, Hela cells were grown in Eaglets Basal Medium supplemented with calf serum. Drug treated cells were exposed to drugs at levels proportional to clinically permissible d?ses, and inversely proportional to toxicity, as set down in Table 1. -, Syn­thesis of nonhistone chromosomal proteins in control and drug treated cells was assayed by incorporation into chromosomal proteins of I-try­ptophane-J H which labels only NHP. since histones contain no trypt­ophane. Chromatin was isolated by the mer80d of Stein and Thrall and fractionated on polyacrylamide-SDS gels.

RESULTS AND DISCUSSION

Kinetic studies appear to confirm the value of the sensitivity tests in current use to rule out inactive drugs and to permit select­ion from among active drugs for treatment of a given tumor. As a compromise between prolongation of drug effects and maintainence of viability of human cancer cells, we routinely have used 6 hours of drug expos~r8' followed by admixture with tracers for an additional 20 hours. -,

The relative order of effects for various antitumor agents in these tests and for assays of DNA polymerase activity in mice cancers has not been altered significantly from results of the kinetic

DRUGS AIMED AT GENE REGULATORS 161

Table 1. Drug Effects on In Vitro Metabolism of S-180 Carcinoma and Normal Mouse Liver Cells

Tissue Drug mg/ml JH-Thymidine Incorporation to DNA at 2 hrs. 4 hrs 6 hrs 12 hrs. 24 hrs. 48 hrs.

S-180 SHI-l 0.4 43.1 42.1 40.6 39.3 36.1 34.6 SHI-2 0.4 51.6 44.6 40.5 31.7 30.3 28.9 5-FU 0.5 61.7 52.7 45.1 36.9 ~.9 39.0 Cyt 0.5 60.8 56.1 50.1 39.4 .8 32.0

Liver SHI-l 0.4 68.0 67.4 59.7 49.5 48.1 53.8 (House) SHI-2 0.4 69.2 65.3 59.0 50.7 50.7 52.3

5-FU 0.5 67.3 60.1 50.6 46.8 43.8 36.4 Cyt 0.5 63.7 59.1 45.9 44.2 40.5 39.2

The numbers represent ~ of control value. SHI-l = iodoacetate plus adjuncts menad~ol,malonate, fluoride and heparin in the ratios used clinically; SHI-2 = oxophenarsine plus adjuncts; 5-F'U = 5-Fluorouracil; Cyt = cytoxan.

studies.

Even more importantly, the preferential toxicity of clinically useful SH inhibitors against various cancer cells relative to nor­mal liver and wound healing tissues, can be seen from both the kin­etic studies and from the routine sensitivity tests used to rule out inactive drugs and to select active drugs for treating each patient. J-9 The data are given in Tables 1 and 2.

Thus, from Table 1, 5-fluorouracil and cytoxan are approximate­ly as toxic to normal liver as to 3-180 carcinoma cells from mice. B,y contrast, the two SH inhibitors at all time intervals maintain greater toxicity to cancer than normal cells,as seen also in Table 2.

From clinical studies and histochemical data, it was predicted that the main clinical target of the SH inhibitors, the SH-bearing nonhistone chromosomal prQteins , would be found to play a major role in regulating genes.)-S The preliminary studies outlined in Table 3 show that tracer incorporation into non histone chromosomal proteins of Hela cells was more affected by the clinically useful SH inhibitors than was tracer incorporation into total celtalar pro­teins monitored by assay of incorporation of dl-leucine-l- C. Thus, iodoacetate plus adjuncts depressed tracer incorporation into total cellular protein to only 68.S % of control, but depressed tracer incorporation into nonhistone chromosomal proteins, their main clinical target, to less than 1 %. Concomitantly,tracer incorporation to DNA was reduced to 34.9 %,indicating marked sensi­tivity. In general, tracer incorporation to DNA down to 50% or less of control is correlated with good clinical regression from the

162 F.E. KNOCK ET AL.

Table 2. Drug Effects on Conversion of ]dR_2_14C to TdR Nucleotides and DNA in intact Carcinoma and Normal Cells

Average experimental values of 14C:JH % of control ratios

'fissue Drug Time(Hrs.l Thl2!!ine ]dR TMP TDP TIP DNA

S-180 SHI-l 0.5 130 120 340 262 149 32 6 126 119 413 311 129 10 12 135 122 452 300 241 0 48 123 120 361 283 191 0

Hydrea 0.5 123 117 357 298 191 22 6 181 213 569 437 181 0 12 202 233 613 465 231 0 48 179 206 549 334 217 0

Cytoxan 0.5 U8 156 274 199 144 39 6 104 142 299 168 131 14 12 123 153 314 211 144 0 48 119 135 346 233 156 0

Liver SHI-l 0.5 113 110 169 121 103 67 Normal 6 l26 U4 20~ 16~ 104 a~ Mouse 12 121 118 21 17 U

48 118 109 267 188 124 40

Hydrea 0.5 131 U6 286 224 180 44 6 184 200 650 511 312 0 12 175 165 639 501 326 0 48 162 183 506 422 239 0

Cytoxan 0.5 109 101 154 134 100 89 6 104 122 344 219 186 51 12 100 135 413 326 212 0 48 100 156 402 319 200 0

Drugs were used at 1 mM concentration. SHI-l = iodoacetat~ plus adjuncts in the same concentration ratios used clinically. Hydrea = hydroxyurea. Data are the averages of three experiments with each agent, and did not vary over 5 ~.

given antitumor agent. 8•9

The preferential toxicity of clinically useful SH inhibitors is illustrated in Table 3. for isotope incorporation to all fractions of cancer cells relative to normal cells, here fibroblasts from normally healing 5 day old wounds of the anterior abdominal wall of rats. Enhanced toxicity against cancer cells relative to normal

DRUGS AIMED AT GENE REGULATORS 163

Table ). Drugs Effects on TraQer Ip~orpOration to Hela Cells and Rat Fibroblasts

Tissue Fraction

Hela DNA Cells

RNA

Protein (total)

NHP

Normal DNA Wound, Rat, 5 RNA Day Old

Protein (total)

NHP

SHI-l SHI-2 (Adj) (Adj)

Adj. Only

)4.9

52.2

68.8

0.2

66.3

75.2

86.9

66.8

37.4

60.0

73.3

78.5

94.5

97.8

53.1

61.8 91.2

73.5 100.0

82.5 100.0

5-FU

40.4

68.7

82.1

43.0

57.)

69.8

Pyr.

86.2

70.9

46.5

Dry! effects on incorporati~n of thymidine-'H to DNA; uri­dine-2- C to RNA; dl-leucine-l- C to total cellular protein; and l-tryptophane-3H to chromosomal nonhistone protein were moni­tored. Figures represent ~ of control value. SHI.l(Adj) = iodo­acetate plus adjuncts menadiol diphosphate, malonate, fluoride and heparin in concentration ratios used clinically8; SHI-2(Adj)= oxophenarsine plus adjuncts; 5-FU = 5-fluorouracil; Pyr=pyruval­dehyde. Data are the averages of triplicate runs and did not vary over 5'1>.

cells for the clinically useful SH inhibitor is seen especially for the SH-bearing chromosomal nonhistone protein fraction. Whereas tracer incorporation into chromosomal nonhistone proteins of Hela cells is reduced to less than 1%, that for the fibroblasts is reduced only to 66.8%.

Since DNA polymerase (s) are part of the nonhistone proteins, DNA polymerase activity would be expected to be markedly depressed for the Hela cells, relative to normal fibroblasts, by clinically useful SH inhibitors. This has indeed been found to be true. Data on drug effects on DNA polymerase activity of Hela cells and fibroblasts essentially duplicate that shown in Table 3 for chromosomal nonhistone proteins, with marked effects of iodoacetate plus adjuncts against SH-bearing DNA polymerase (s) of Hela cells, and little against DNA polymerase activity of normal fibroblasts.

164 F.E. KNOCK ET AL.

The data to date suggest that subfractions of chromosomal nonhistone proteins showing high reactivity towards drugs reacting preferentially with a broad spectrum of human cancers should be further defined. Special interest may attach, for example, to the reaction of clinically useful SH inhibitors with nuclear deoxy­ribonuc1eases playing a putative role in DNA replication and repair as well as to their reaction with proteins which bind to,recognize and may regulate selected small portions of DNA. Such delineation of chromosomal proteins reacting with antitumor agents showing preferential toxicity to a wide spectrum of human cancers may not only improve target selection for future sensitivity tests for clinical use, but may also improve understanding of mechanisms of cell regulation and carcinogenesis.

REFERENCES 1. Szent-Gyorgyi,A., Bioelectronics, Academic Press, New York,

1968, pp. 75-89. 2. Knock,F.E.,Arch. Surg., 86 (1963) 489. 3. Knock,F.E.,J. Am. Med.Assoc,191 (1966) 151. 4. Knock,F.E.,J. Am. Geriatrics Soc. ,14 (1966)883;15(1967),882. 5 •• Knock,F.E.,Perspectives Biol. Med. 10 (1967) 310. o Knock,F.i.,Anticancer Agents,Char1es C. Thomas ,Springfield ,1967 ,

pp. 93,122,166,183,213. 7. Knock,F.E., Nat1.Cancer Inst.Mono.,)4 (1972)247. 8. Knock,F.E., Galt, R.M., Oester,Y.T., Sylvester,R.,Oncology,

26 (1972) ,515. 9. Knock,F.E., Galt, R.M. Oester,Y.T., Sylvester,R.,Oncology,

,0 (1974) 1. 10. Black,M.M., Kleiner, I.S. and Bo1ker, H., cancer Res.,7(1947)8l8. 11. Ste11wagon,~., and Cole ,H. , Annu.Rev. Biochem,,8 (1969) 951. 12. Gilmour,R.S. and Pau1,J., FEBS Letters 9 (1970) 242. 13. Teng,C., Teng,C. and Allfrey,V.G., J. Biol.Chem,246(1971),597. 14. Spe1sberg,T.C. and Hnilica,L., Biochem. J., 120 (1970) 435. 15. Stein,G.S. and Farber,J., Proc. Mat1. Acad. Sci. 69 (1972)2918. 16. Stein,G.S., Spe1sberg,T.C. and K1einsmith,L.J., 5cienoe,183,

(1974)817. 17. Chae, C., et a1, Cancer Res., 30 (1970) 2652. 18. Stein,G.S. and Thrall, C.L., FEBS Letters, )4 (1973) 35.

CHARACTERIZATION OF THE BLEOMYCIN ACTION ON DNA

Hamao Umezawa, Hideki Asakura & Makoto Hori

Institute of Microbial Chemistry

Kamiosaki 3-14-23, Shinagawa-ku, Tokyo, Japan

The activity of bleomycin in causing a strand scission in a superhe1ica1 form of SV40 DNA was utilized for quantitative assay. The bleomycin action was counteracted by simultaneously added native or denatured DNA of other species, poly dG-C.po1ydG-C, poly dA-T· poly dA-T and poly dA-T (denatured) while not by tRNA of E. coli, apurinic acid, poly dA, poly dT and various deoxyriboo1igonuc1eo­tides. The results suggested that the least structural requirement of DNA in binding with bleomycin would be a single stranded deoxy­ribopo1ynuc1eotide chain with both purine and pyrimidine bases. Various b1eomycins and their derivatives were tested for possible activity of cleaving SV40 DNA and the results indicated the require­ment of the following groups for the activity: (1) lone-pair electrons of a-NH2 of S-aminoa1anine moiety, (2) C-termina1 amide or ester group of b1eomycinic acid, and (3) the location of the carbamoyl group on 3-hydroxy1 of mannose moiety. A derivative with a hydrogenated thiazole ring (phleomycin) was as active as bleomycin. Actinomycin D stimulated the bleomycin activity while ethidium bromide counteracted it.

Blm causes strand scission of DNA in vitro as well as in vivo. We have reported an in vitro assay system in which 3H-SV40 DNA was used as the substrate and the reaction time was strictly controlled by terminating the reaction with alkali (Umezawa et a1., 1973). This method enabled us to perform a quantitative determination of the bleomycin activity. The reaction was apparently inhibited by simultaneous addition of DNA, irrespective of its source, since the additional DNA also could bind with bleomycin, thus competed with 3H-SV40 DNA. This effect is referred to as "protection by competi­tive binding." RNA had no protective effect. Various nucleic acids, natural or synthetic, were tested for possible "protection

165

166 H. UMEZAWA, H. ASAKURA, AND M. HORI

Table I Protection of SV40 DNA from the bleomycin action by

simultaneous addition of various nucleic acids. (Protection by competitive binding)

( Rate of strand scission ~n a test run -)XlOO(%) Rate of protection= I-Rate of strand scission 1.n a control rurl

Additional nucleic acid

none: control DNA (calf-thymus) heat denatured DNA tRNA (E. coli) apurinic acid poly dA-T

alkaline denatured poly dA-T

poly dG-C

poly dA poly dT d(pApT)3 d(pGpT) 2 d(pCpA)2

amount added (xlO- 3 A260 units)

1.2 1.3

100 20 0.5 2

{ ~.5

0.5 2

100 100

37.4 33.6 39.0

Rate of protection

0* 69.7 61.0

8.9 o

45.2 75.5 32.5 62.6 62.2 88.5

7.3 12.6 o o o

* Rate of conversion from superhelical form to nicked open circular form (% conversion) by 0.39 \.1M bleomycin B2 was 60%.

by competitive binding1l and the results are shown in Table 1. These results suggested that the least required structure of DNA to bind with bleomycin would be a single stranded deoxyribopolynucleotide chain with both purine and pyridine bases.

To find an active group(s) in the structure of bleomycin, various members of bleomycins, their derivatives and moieties were tested for possible activity of cleaving SV40 DNA and the results are summarized in Table II. For comparison, the antimicrobial activity of each compound is also indicated. The enzymically in­activated bleomycin also showed much reduced activity in our assay system. It is believed that an aminopeptidase in tissues hydrolyzes the amide group of the S-aminoalanine moiety. This modification causes increase in basicity of the neighboring a-NH2 from pKa 7.3 to 9.4. Therefore, under the assay conditions, the a-NH2 of this derivative is mostly protonated. The a-NH2, in native Blm, is

BLEOMYCIN ACTION ON DNA 167

Table II Structure-activity relationship of bleomycin

The activity of each compound is expressed as relative value to the activity of bleomycin B2 (100%). The antimicrobial activities were determined with Mycobacterium 607 as a test organism.

Compounds tested

-NH-(CH ) -NH-C-NH 2 4 II 2

NH (Terminal amine of Blm B2)

Bleomycin derivatives

Enzymically inactived Blm B2 ++ Blm B2 (Cu -chelated)

++ Blm B2 + Blm B2 (Cu -chelated)

Phleomycin A2 ++ Phleomycin A2 (Cu -chelated)

Epi-Blm B2

Iso-Blm A2 (2-0-carbannyl)

Strand scission of DNA

100

5

<2

~100

87

2

~100

5

Bleomycins with various terminal amine parts I

-NH2 (Blm-Bl ) ~100

-O-(CH ) -NH 2 3 2 (B1m ester) ~100

-OH (B1eomycinic acid) 5 +

-NH2-(CH2)3-S-(CH3)2 (B1m A2) ~100

r\ (MOP-Blm) -NH-(CH ) -V 113 2 3

( ..... CH3 -NH- CH2)3-N'CH3 (DMP-Blm) 100

-NH-CH -CH-CH 2 I 3

(DAP-Blm) 83

NH2

Antimicrobial activity

3,385 u/mg

<300

3,480

595

595

584

305

685

1,335

159

938

592

810

2,178

168 H. UMEZAWA, H. ASAKURA, AND M. HORI

Table III Effect of DNA-binding compounds on the bleomycin action

In a reaction mixture dissolving SV40 DNA, actinomycin D or ethidium bromide was added 10 min before initiation of reaction.

Compounds tested Rate of strand scission(%)

Ac tinomycin D 10 llg/ml 0 Blm B2 0.2 37.2

+ Ac tinomycin D 1 66.6 2 81.1

Ethidium bromide 20 llg/ml 2.8 Blm B2 0.6 95.7

+ Ethidium bromide 1 77.7 2 53.0 5 25.1

involved in chelation with Cu++. The lack of activity in both the enzymically inactivated Blm and the Cu++-chelated Blm, together with other observations, strongly suggested that a nucleophilic attack by the a-NH2 of the S-aminoalanine moiety plays an important role in the bleomycin action. Phleomycin differs from bleomycin only in partial hydrogenation of the thiazole ring and was as active as bleomycin and also lost activity upon chelation with Cu++. As far as the in vitro activity is concerned, the terminal amines of complexed structures can be replaced ~y NH3 or some alcohols, as indicated respectively by bleomycin Bl and bleomycin ester. In contrast, bleomycinic acid, in which the terminal carboxyl group is free, was inactive. The negative charge on the free carboxyl group may interfere with the function of an active group(s) of the same molecule. Chemical fragments of bleomycin, such as tripeptide S, tetrapeptide A, heptapeptide (Muraoka et a1., 1972), were inac­tive in our assay system at a concentration of 6 11M.

Ethidium bromide inhibited the bleomycin action while the reverse was true with actinomycin D, as shown in Table III. The inhibitory effect of ethidium bromide on the bleomycin action suggests that some planar structure of bleomycin is first inter­calated between adjacent bases of one strand of DNA. On the other hand, actinomycin D may cause a conformational distortion in the superhelical DNA in such a way that bleomycin could act more readily.

Muraoka et al., (1972), J. Antibiot., 25, 185 Umezawa et al., (1973), J. Antibiot., 26, 521

ANTITUMOR ANTIBIOTIC CARMINOMYCIN: MECHANISM OF

ACTION

G.F. Gause and Y.V. Dudnik

Institute of New Antibiotics, Academy of Medical Sciences Moscow, USSR

Carminomycin selectively inhibits synthesis of nucleic acids in cells of microorganisms and malignant tumors. Carminomycin complexes with DNA in vitro and considerably increases the melting temperature of DNA. The antibiotic inhibits the template activity of DNA in the system of DNA-dependent RNA polymerase. Carminomycin inhibits repair in bacterial cells injured by radiation and alkylating agents.

Antitumor antibiotic carminomycin in produced by Actinomadura carminata (Gause et al. 1973). The struc­ture of carminomycin (Brazhnikova et al. 1974) is shown on Fig. 1.

Fig. 2 shows the effect of carminomycin on the synthesis of DNA, RNA and protein in the culture of Micrococcus lysodeikticus 53-5. Carminomycin selectiv­ely inhibits the synthesis of DNA. In the concentration of 10 mcg/ml this antibiotic immediately and completely stops the synthesis of DNA; at the same time the rate of synthesis of RNA does not differ from that of the control culture for 8 minutes. The inhibition of RNA synthesis appears later, and is followed by the inhibi­tion of protein synthesis. It should be mentioned that in these experiments the growth of bacteria was inhibit­ed slightly, and appeared only after 30 minutes at the concentration of carminomycin attaining 10 mcg/ml. It is clear that in this case the primary effect of the antibiotic is the inhibition of synthesis of DNA.

169

170 G.F. GAUSE AND V.V. DUDNIK

o OH

Fig.l. Structure of carminomycin.

In the action of carminomycin upon ascites tumor cells of lymphadenoma of mice (strain NkLy) in vitro, the incorporation of labeled precursors into DNA and RNA is inhibited to the same extent. In concentrations strongly inhibiting the synthesis of nucleic acids in asci tes tumor cells, carminomycin for 2 hours practically does not affect protein synthesis. To conclude, carmin­omycin in common with other anthracyclines selectively inhibits synthesis of nucleic acids in bacterial as well as in tumor cells.

Carminomycin was found to be an inducer of phage production in lysogenic bacteria. Titre of the phage in the lysogenic strain of Micrococcus lysodeikticus 53-40 (N-S phage production) is increased 100 times under effect of carminomycin in the concentration 10 mcg/ml.

Carminomycin complexes with DNA in vitro. The shift in the absorption spectrum of carminomycin in the presence of native DNA indicates the formation of a complex between antibiotic and DNA. A similar shift in the spectrum of carminomycin is observed upon the addi­tion of DNA denatured by heat. Carminomycin interacts not only with DNA, but also with ribosomal RNA of ~. coli, as well as with purine nucleotides, nucleosides and with purine bases. It is not bound by pyrimidine nucleotides, nucleosides and bases. Synthetic polyri-

ANTIMOUR ANTIBIOTIC CARMINOMYCIN 171

0 PROTEIN DNA

o 1.5 10 3

0

b 1.0 2 _ ..... _~10

:; 5 0-U 10

0.5

10

048 16 o 4 8 16 o 4 8 16 32

MINUTES

Fig. 2. Effect of carminomycin on the incorporation 2-C14-urac il into RNA and DNA, and C14_ phenylalanine into protein of Micrococcus lysodeikticis 53-5. Incor­poration into DNA was assayed after hydrolysis by 0.5 NKOH for 3 hours at 37°C. Concentrations of carminomy­cin are 1 and 10 mcg/ml.

bonucleotides poly-A and poly-UA induce a shift in the spectrum of carminomycin similar to that induced by RNA. Although carminomycin is not bound by uracil, uri­dine and UMP, the addition of poly-U induces a character­istic shift in the spectrum of this antibiotic.

The complexing of carminomycin with DNA has a stabilizing action upon the secondary structure of DNA to the effect of heat (Fig. 3). By adding carminomycin in the molar relation antibiotic/nucleotide DNA equal to 0.03, the increase in the melting temperature of DNA attains 7°. At higher concentrations of the antibiotic the melting temperature increases by 30-35°C, and also hyperchromic effect is increased.

Carminomycin considerably increases the viscosity of DNA in solutions. Carminomycin inhibits the template activity of DNA in the system of DNA-dependent RNA poly­merase of~. coli. It inhibits the synthesis of RNA in experiments with both native and denatured DNA used as templates. It is of interest that low concentrations of carminomycin inhibit the template activity of denatured DNA stronger than that of native DNA.

172 G.F. GAUSE AND V.V. DUDNIK

1.8

DNA + CARMINOMYCIN

1.6

1.4

DNA

1.2

50 60 70 80 90 100

Fig. 3. Effect of carminomycin on the melting tempera­ture on DNA from ~. coli. Molar relation antibiotic/ nucleotide DNA attains 0.2.

Carminomycin inhibits repair in bacterial cells injured by radiation and alkylating agents. In these studies carminomycin was added to nutrient agar in the concentration 0.6 mcg/ml. At this concentration it does not affect colony formation in the mutant of E. coli. 19-8 with the increased permeability of the c;ll~b­rane (this mutant is sensitive to actinomycin, daunoru­bicin and carminomycin). By plating upon this agar suspensions of~. coli 19-8 treated by ultraviolet radiation one can observe considerable decrease in the number of survivors (Fig.4). A similar decrease in the number of survivors is observed after plating upon the nutrient agar containing carminomycin the suspensioqs of E. coli 19-8 treated by mitomycin C.

ANTIMOUR ANTIBIOTIC CARMINOMYCIN 173

"1 0

i z 0 i= ~ 2 a: u. Cl z :> :> a: 3 :I en

o 10 20 30 40

UV, sec

Fig. 4. Effect of carminomycin on bactericidal action of ultraviolet radiation upon~. coli 19-8.

To conclude, the biological effect of carminomycin can probably be explained by distortion of DNA-dependent biosynthetic reactions, resulting from complexing of the antibiotic with DNA-templates.

References

Brazhnikova, M.G., Zbarsky, V.B., Ponomarenko, V.I. and Potapova, N.P. (1974) Journal of Antibiotics, 27, 254

Gause, G.F., Sveshnikova, M.A., Ukholina, R.S., Gavrilina, GV., Filicheva, V.A. and Gladkikh, E.G. (1973) Antibiotiki, 18, 675

EFFECT OF COMBINED CHEMOTHERAPY ~ITH LYSOSOME LABILIZERS

AND MITOMYCIN-C

T. Taniguchi**, H. Niitani*, A. Suzuki*, N. Saijo*, I. Kawase* and K. Kimura *National Cancer Center Hospital, Toyko, Japan

** Yokosuka Kyosai Hospital, Yokosuka, Japan

Our experimental studies showed that both plasmin and lipoproteinlipase are lysosome labilizers capable of en­hancing the cytocidal effect of Mitomycin-C (MMC) through increased release of lysosomal enzymes of tumor cells, and that lysosomes have differences in their membrane stabili­ties against labilizers, that is, the lysosomal membranes of tumor cells are less atable than those of liver cells. In clinical studies of lysosome labilizers, urokinase which activates plasminogen to plasmin and/or dextran sulfate, which activates lipoproteinpase in blood were used in com­bination with MMC. Regressions of nodular pulmonary met­astases was seen in 46% of patients treated with this combined chemotherapy, compared with only 25% in patients treated with MMC. Side effects caused by the combined chemotherapy were not increased compared with those pro­duced by MMC alone. Mean survival period of the responding patients treated with the combined chemotherapy was greater than that treated with MMC alone.

Attention has been focused on the lysosome because of its important role in the autolysis of the cells. It has already been demonstrated in several studies that the lysosomal enzyme activities are increased in the tumor cells after treatment with antitumor agents (1). This fact indicates that the lysosome plays an important role in the self-destruction of tumor cells in cancer chemother­apy, suggesting the possibility that the cytocidal effect of antitumor agents might be enhanced by additional treat­ment with lysosome labilizers which further increase the lysosomal enzyme activities. Our experimental studies

175

176 T. TANIGUCHI ET Al.

indicate that both plasmin (2) and lipoproteinlipase (3) are lysosome labilizers, and that the cytocidal effect of Mitomycin-C (MMC) is enhanced through the acceleration of the release of free-form lysosomal enzyme in combination with these labilizers. When lysosome labilizers are used concomitantly with antitumor agents, it becames indispen­sable to study how lysosome labilizers influence the lysosomes of non-tumor cells for understanding the side effects caused by this combined treatment. Differences of lysosomal membranes stabilities against labilizers were therefore studies in lysosomes of Yoshida ascites tumor cells and liver cells of tumor bearing rats.

The lysosomal fraction of tumor cells and that of liver cells were separated according to the fractionation scheme. On Table 1 and then, suspending these fractions in medium, the tumor cell lysosomal preparation and the liver cell lysosomal preparation were made. As lysosome labili­zers, we selected four kinds of labilizers; phospholipase­A, TritonX-lDD, lipoproteinlipase and lysozyme. By dis­solving these in different media, labilizer solutions of gradually varying concentration were prepared and were allowed to act on the tumor cell lysosomal preparation and the liver cell lysosomal preparation. After incuba­tion of the mexture consisting of the labilizer solution and the lysosomal preparation, free activites of ~-glucuronidase, a kind of lysosomal enzyme released from lysosomes into each of the media, were measured and the percentages of their activities to~ -glucuronidase bound activities of the control were calculated for lysosomal membrane stabilities against labilizers.

Figure 1 shows the difference between lysosomal membr­ane stabilities of the lysosome of tumor cells and that of liver cells against phospholipase-A. Comparing the stabi­lities of the two types of cells against phospholipase-A, it can be said that the lysosomal membrane of the tumor cell is less stable than that of the liver cell at the low concentration.

Figure 2 shows the difference between lysosomal mem­brane stabilities of both lysosomes against TritonX-lDD. With increase in concentration of TritonX-lDD, the bound form of the tumor cell lysosome was converted to the free form more rapidly than that of the liver cell lysosome. Comparing the stabilities of the two against TritonX-lDD, it is possible to say that the lysosomal membrane of the tumor cell is unstable while that of the liver cell is stable at the low concentration.

TABL

E 1

PR

EP

AR

AT

ION

O

F

LY

SO

SO

MA

L

FR

AC

TIO

N

Yos

hida

A

scit

es T

umor

C

ells

hom

ogen

eize

(E

MA

NU

EL

-CH

IKO

FF

' s

)

hom

ogen

eize

r cl

eara

nce

IO

}l

6000

rpm

10

'

(130

00g-

min

) I

r I

Su

p

I 13

000

rpm

20'

(260

000

g-m

in)

Pp

t

(Lig

ht

Mit

ocho

ndri

al F

ract i

on)

Rat

L

iver

hom

ogen

eize

(T

eflo

n

ho

mo

gen

eize

r)

4000

rpm

10'

(860

0g- m

in)

I I

Su

p

I 12

000

rpm

20

'

(240

000g

- min

)

I .----

----I

Pp

t (L

igh

t M

ito

cho

nd

rial

F

ract

ion

)

~ ~

o :s::

m

r » IX!

r N m

::tJ

C/l » Z o :s:: ~ :s:: ~ z h ::::

178 T. TANIGUCHI ET Al.

Differences of Lysosomal Membranes Stabilities against

Phospholipase-A between the Lysosome of Yoshida Ascites

Tumor Cell s and That of Liver Cells of Tumor bearing Rats

"lo 100 A

50

I I I I I

AI )( ,

x f , , A'

X XTumor eell

A--- ---A Liver

",-=A_r--_~ __ -""T"""------r---;J"""-"'i 1 00 200 300 500

Conen. of phospholipase A }lg

Lysosomal preparation was incubated for 30 min at 30·C in 2SmM Tris-HCI buffer (PH7.S) in the presence of 0.2SM sucrose and phospholipase A. Control was treated in identical manner, without adding phospholipase A in the incubation mixture.

FIGURE 1

LYSOSOME LABILIZERS AND MITOMYCIN-C

Differences of Lysosomal Membranes Stabilities against

TritonX- 100 between the Lysosome of Yoshida Ascites Tumor

Cells and That of Liver Cells of Tumor bearing Rats

Q) I/) to

"'0 c ....... o >. L.. .~ ~ .~ o .... ~ u

- 1\1 'l'I'O ~5 .... .B 0 >,0 .~ -= > c:

0 '';:; u 0 0 to .... Q)IiI-Q)---L.. lL

'" 100 x

.. Xl( ,"AA

50

)( I::.. " X " X , I::.. "

x It" I::.. , I::.. I::..~' I::.. 4,'

0.5

I I ,

I I

I I

I I

I I

I I

I )( I

I I

I I::.. I

I

I

1::../ I

I I

I I

II::.. I

x

2

)( , 1::..' I

~ I

I I::.. I

I

3

, ,

I ,

! ...... ", A

, .. ' )(

" , I::..

"

~~---X Tumor cell

1::..-----1::.. Liver

4 5 Xl0-' Concn. of Triton X-IOO (V/V,,)

Lysosomal preparation was incubated for 30 min at 30"C

in 25mM Tris-HCI buffer(PH7.5} in the presence of 0.25M

sucrose and TritonX-1 00. Control was treated in identical

manner. without adding Triton X-IOO in the incubation mixture.

FIGURE 2

179

180 T. TANIGUCHI ET AL.

Differences of Lysosomal Membranes Stabilities against

Lipoproteinlipase between the Lysosome of Yoshida Ascites

Tumor Cells and That of Liver Cells of Tumor bearing Rats

100

x

t::.

L\.

25

~ I I

I I

t::.

50

I

I I

I , I

I , ,

;'

... , ;" x

" t::.

100

X--X Tumor cell

L\.----L\. Liver

150

X t::.

--:'X

x

200

Concn. of lipoproteinlipase (pg/mI)

Lysosomal preparation was incubated for 30 min at 30·C in 25mM Tris- HCI buffer (PH 7. 5) in the oresence of 0.25M

sucrose and lipoproteinlipase. Control was treated in identical manner, without adding lipoproteinlipase in the incubation mixture.

FIGURE 3

Figure 3 shows the difference between lysosomal membrane stabilities of both lysosomes against lipoproteinlipase. With increase in concentration of lipoproteinlipase, the bound form of the tumor cell lysosome was converted to the free form more rapidly than that of the liver cell lyso­some. Comparing the stabilities of the two against lip­oproteinlipase, it is possible to say that the lysosomal membrance of the tumor cell is unstable while that of the liver cell is stable at the low concentration.

LYSOSOME LABI LlZERS AND MITOMYCIN-C

Differences of Lysosomal Membranes Stabilities against

Lysozyme between the Lysosome of Yoshida Ascites Tumor

Cells and That of Liver Cells of Tumor bearing Rats "to

50

X---l)( Tumor eell

40 .6._ - - - -.6. Liver

0) I/)

'" ~>. 5 .~ L.. .-::J .... o CJ 30 ::J to --0 ~c: ~:l

.8 '+--o 0 .... >, .... .... 5 20 > CJ .~ 0 o .... lOse.. 0)------0)

)(. .6..6. .6.)(

~ )(

)( ~

L.. )( )(

IJ.. 10

" x .. ~f .. .. .... .6..6. .6.

~OOO 500

X .6. _------.6. __ .&.- - --- - - -- -4;..6..6.- ---

50 1 00 200 300 400

Conen. of Iysozym e !' g

Lysosomal preparation was incubated for 30 min at 3Q'C in 25mM Tris-HCI buffer (PH6.a)in the presence of 0.25M sucrose and lysozyme. Control was treated in identical manner, without adding lysozyme in the incubation mixture.

FIGURE 4

181

Figure 4 shows the difference between lysosomal membrane stabilities of both lysosomes against lysozyme. With inc­rease in concentration of lysozyme, the bound form of the tumor cell lysosome was converted to the free form more rapidly than that of the liver cell lysosome. Comparing the stabilities of the two against lysozyme, it is possi­ble to say that the lysosomal membrane of the tumor cell is unstable while that of the liver cell is stable at the low concentration.

182 T. TANIGUCHI ET AL.

Experimental studies on the lysosome particles proved that lysosomes have differences in their membrane stabili­ties against labilizers. That is, the tumor cell lysosome is less stable than the liver cell lysosome. These exper­imental results suggest the possibility of clinical appli­cation for enhancing the cytocidal effect of antitumor agents combined with lysosome labilizers without increas­ing side effects caused by the combined treatment.

In clinical studies, we used as lysosome labilizers, urokinase (4) which activated plasminogen to plasmin and/or dextran sulfate (5) which promoted the activity of lip­oproteinlipase in blood in combination with MMC to the selected cancer patients with nodular pulmonary metastases. The effects were evaluated by the change in diameter of

Primary Site

H •• d • Neck

Lung

Breast

Uterus

Others

Total

TABLE 2

Effects of chemotherapy on the nodular pulmonary metastasis from various organs

MMC Only MMC+LL*

Strong Slight Effect Effect Effect Effect Non

Rate Strong Effect Slight Effect Effect

Non

1 1 3 2/5 2 2 7

1 1 4 2/6 3 5 2 11

1 3 114 1 1 1

1 1 6 2/8 5 13 3 7

1 1 12 2/14 1 1 19

3 3 3 28 9/37 11 19 9 45

25" * Dextran sulfate and/or Urokinase

TABLE 3

Effect Total Effect

Rate Rate

4/11 6/16

10/21 12/27

2/3 3/7

21/28 23/36

2/21 4/35

39/84 48/121

46"

Incidence of Toxic Effect after 5 Consecutive Administrations of Mitomycin-C and the Combined Therapy

Treatment Total No. of

patients

Mitomvcin-C 37 Mitom;'·cin-C

+ dextran sulfate 84 and/or urokinase

No. of patients with toxic effcct Leukopenia Thrombocytopenia

( < 3,000 cells/mm3) ( < 70,000 pIa:telets/mml )

17 (46%) 8 (22%)

28 (33%) 19 (23%)

Hemorrhagic diathesis

4 (11%)

10 (12%)

LYSOSOME LABILIZERS AND MITOMYCIN-C

TABLE 4

Mean survival*of patients** with nodular pulmonary metastasis treated with MMC or MMC + lysosome labilizer (LL)***

Treatment No. of Mean Survival (Months)

Patients

Non- tre ated 14 4.4 MMC 23 8.0

! Effective 11. 7

Non-effective 16 6.4

MMC+LL 31 17.3 ! Effective 16 19.8

Non- effective 15 14.6

* I. from the time of detection of pulmonary metastasis 2. without prior chemotherapy

** I. primary tumor had been resected 2. including uterin cervical cancer radically treated with radiation 3. excluding cases of breast cancer and thyroid cancer

*** dextran sulfate and/or urokinase

183

nodular density in the chest films. According to the per­centage of reduction in total length of diameters of mea­surable tumor, an objective response was classified as follows:- strongly effective over 90%, effective over 50%, slightly effective over 25%. non-effective less than 25%.

Table 2 shows the effects of chemotherapy on the nodular pulmonary metastases from various organs. The effective rate was 46% in the patients treated with the combined chemotherapy, whereas MMC alone produced a res­ponse in only 25% of patients.

Table 3 shows the incidence of side effects caused by the combined chemotherapy did not tend to increase as com­pared with those caused by MMC alone.

Table 4 shows the difference of mean survival periods of the patients with nodular pulmonary metastas~s treated with the combined chemotherapy or treated with MMC alone. Mean survival period of the effectively treated patients with the combined chemotherapy was longer than that of patients treated with MMC alone.

In the chemotherapy of cancer aiming at the total kill of cancer cells, the significance of the lysosome and its close connection with the mechanism of cell-autolysis will become gradually important.

OPTIMAL CONDITIONS FOR TUMOR CHEMOTHERAPY CHOSEN ON THE

BASIS OF CHANGES IN THE LIPID ANTIOXIDANT ACTIVITY

N.P. Pal'Mina, E.B. Burlakova, V.D. Gaintseva, N.P. Sezina Institute of Chemical Physics, Academy of Science, Moscow, U.S.S.R.

SUMMARY

Changes in antioxidation activity of lipids (ADA) from tumor and tissues of tumor host have been studied in the course of tumor growth, by action of various chemo­therapeutic drugs on intact animals and tumor hosts. It was found that development of a number of inoculated tumors ran parallel with increasing ADA of tumor host organs, and such ADA changes were important and charact­eristic of tumor growth. Different antineoplastic drugs suppressed the tumor and normal cell division more strongly with decreasing ADA value. Compounds increasing ADA accelerated tumor growth. Study of ADA changes in course of tumor growth gave the possibility of following changes in the sensitivity of tumors and organs of tumor­hosts relative to the action of antineoplastic drugs, and to choose the optimal conditions of rational therapy. Experimental data permit the conclusion that the use of drugs of the same class for action on a tumor at any stage of its growth is absolutely ineffective and the use of intermittent administration of alkylating agents and antioxidants might be promising.

A suggestion made by a number of authors (Haddow, 1947; Emanuel, 1958; Jensen, 1950) about the intensifica­tion of oxidative processes in organism is one of the hypothesis attempting to explain the causes responsible for the appearance and growth of malignant neoplasms. One of the most important physico-chemical properties of lipids is their antioxidative activity, which is likely to

185

186 N.P. PAL'MINA ET AL.

provide information as to the extent of oxidative reaction development and which might be, in a certain way, connected with free radical level in lipids. This value character­ises the ability of a particular substance or a set of substances to inhibit oxidation and is due to the presence of natural antioxidants in animal organs and tissues.

This paper is devoted to the study of ADA change in the development of various inoculated tumors and the effect of different drugs. Antioxidative activity of lipids was determined on the methyloleate oxidative model as the relationship of the induction periods difference on the curves of methyloleate oxidation with added lipid extract and pure methyloleate to the lipid concentration dissolved in methyloleate.

~e have established that the development of inoculated tumors: leukosis (La), Ehrlich ascites carcinoma (ACE) and hepatoma 22a occurs against the background of extreme ADA increase in host organs and tissues, which is essential for tumor growth. Any inhibition of the development of cancer caused by decreased doses of inoculum or chemo­therapeutic agent administration brings about a shift of the maximum along the axis of time (Fig. 1.)

t,day5

FIGURE 1: Changes of ADA liver in course of different transplanted tumors

OPTIMAL CONDITIONS FOR TUMOR CHEMOTHERAPY

.,0

o,s

~ ADA.

1.0

o '---~_-,-_ ... 1.0 4 t . dQs~

A LK~I.AT II<(, AGtWT~

o '--,..----,----.--6 t.~S

187

FIGURE 2: Changes of ADA after injection of antitumor agents

Chemotherapeutics of different classes: alkylating agents, hormones, antimetabolites, and other cytostatics or antioxidants, when administered to intact animals in therapeutic doses brin~ about considerable ADA decrease in organs and tissues (Figure 2). Such nonspecific effects on tumor growth as that produced by various doses of x-ray irradiation and stress result in decreases ADA as well . Experiments on the transplanted leukosis La have shown the leukosis La coefficient to be proportional to the ability of a particular substance to decrease ADA (Palmina & Burlakova, 1973). Important implications for tumor therapy can be deduced from the regularities of ADA change in tumor growth and when injecting chemotherapeu­tics. First, on the basis of the direct linear dependence obtained one can estimate the advantages of using new drugs as compared with those being used at present. Second, knowing the predictability of ADA changes in tumor growth processes it is possi hle to arrive at the correct treatment taking into account specific individual features of a particular preparation to establish the individual time of drug administration optimal dose, time interval between injections (which is the period when ADA is at the lowered level after one injection). The antioxidative activity model may be of use for the choice of the mode of administration of a particular preparation. Is the best result obtained when a single large dose is given or

188 N.P. PAL'MINA ET AL.

when smaller doses are given continuously. If continuous administration loweres ADA to the same level as with single dose then single administration seems to be pre­ferable. In fact, in a number of cases when alkylating agents and radiomimetics were used, single administration of a large dose produced a better effect than continuous dose. As a rule these are preparations of the type which when injected lead to ADA decrease. They are, however, antitumor agents which may be used only when given contin­uously, because they induce temporary ADA decrease. These are hormone preparations. Then single injection may even lead to an increased tumor growth as a short phase of ADA decrease is followed by a long phase of ADA increase. Thus the ADA model enables one to take into account both general and individual properties of chemotherapeutic substances and thus allow them to be used as a test for choosing optimal conditions of tumor chemotherapy. All the above refers to the attack on tumor growth in early stages. When the tumor is of a considerable size the organism has a lowered ADA. It seems correct at such stages to use other drugs and another treatment strategy. This conclusion is based on the results obtained earlier which showed that ADA levels determine the sensitivity of both the organs of tumor-host animal and the tumor to damaging effects. When tumors rrow ADA of both organ and tumors is lowered (hepatoma 22a or tumor ADA is higher than that of animal organs (ACE - (Figure 3) In this case the administration of "classical" antitumor agents exerts more damaging effects on the organism than on the tumor. We believe that at such a stage of tumor growth it is expedient to introduce substances increasing ADA in the organism, which allows it to fight the tumor with its own recources. The experiments on ACE demonstrated that

AOA/AOAo 2.0

1.0

~t OAo

1.0

AOA/AOAo

ACE

2.0

1.0

o l----.---r--..------.---r----Jo 0 '----.---.-----..--.----,,.........0 2 4 6 8 10 t. days 2 4 6 8 10 t. days

FIGURE 3: Changes of ADA liver (left axis) and tumor (rt)

OPTIMAL CONDITIONS FOR TUMOR CHEMOTHERAPY 189

the administration of inhibitors-antioxidants (e. g. 4-methyl~2,6-ditretbutylphenol in a dose of 30 and 100 mg/kg) on the 8th day after inoculation increased the average lifespan of animals by 30-50%, compared with the control animals, while the injection of Thio-TEPA(5mg/kg) at the same time accelerated the death of the animals. On the basis of the ADA changes in the presence of tumor growth we checked the scheme of successive introduction of Thio-TEPA(lO mg/kg) at the early stages of tumor growth and antioxidant (4-methyl-2,6-ditretbutylphenol 30 mg/kg-BHT) at later stages. Thio-TEPA was introduced on the second day after hepatoma 22a transplantation, BHT-on the 8th day. ~e obtained an average increase of animal lifespan of 40-45% compared with control and of 20% compared with a group of animals to which only Thio­TEPA was given. (Table 1). Injection of antioxidant on the 8th day did not influence either the death of animals or tumor growth. Tumor growth in the animals having received both Thio-TEPA and BHT was inhibited to a greater extent than when only Thio-TEPA was injected.

Taking into account the fact that when ADA is incr­easing at later stages of tumor growth we can then again introduce "classical" antitumor agents it may be hoped that the scheme of intermittent administration of alkylating agents and antioxidants might be promising.

A conclusion may be reached on the basis of the data presented here that the change of such physico-chemical value as ADA may serve as a test for the development of techniques for rational tumor therapy which take into account the interrelationship of tumor and organism and the change of stability to damaging agents as the tumor progresses.

TABLE 1

DRUG DOSE TIME OF DRUG LIFESPAN INJECTION DAYS

Control - 21. 5 ~hio-TEPA 10mg/kg 2 days after tumor

transplantation 27.2

Thio-TEPA 10mg/kg 6 24.1 Irhio-TEPA 10mg/kg 11 15.3 BHT 30mg/kg 6 25.8 BHT 100mg/kg 6 11.8 BHT 30mg/kg 11 21. 5 ~hio-TEPA 10mg/kg 2 29.2 & BHT 30mg/kg 11

190 N.P. PAL'MINA ET Al.

REFEREN CES

1. Haddow, A. (1947), Growth, 11.., 339.

2. Emanuel, N.M. et ale (1958). Dokl. Acad. Nauk. 121, 1, 14. -- --

3. Jensen, E.W. (1950) in Book of Abstracts of the Fifth Conference on Biological Antioxidants Transactions i, 159.

4. Palmina, N.P., Burlacova, E.B. (1973). in "Actualnie Vooprosi Sovremennoi Oncologee", 1, 86.

5 . Bagley, E. A • ( 19 70). In" F i z i c 0 - chi m i c h e ski e mechanizmi slocachestvennogo rosta". 16.

AN ANTITEMPLATE APPROACH TO DEVELOP SELECTIVE

INHIBITORS OF ONCORNAVIRAL REVERSE-TRANSCRIPTASE

* Po Chandra, T.J. Bardos, U. Ebener, Bo Kornhuber, D. Gericke and A. Gijtz Gustav-Embden-Zentrum der Biologischen Chemie, University of Frankfurt; and Dept. of medicinal Chemistry, Buffalo, U.S.A.

Single-stranded polyribonucleotides are known to act as efficient templates for the viral DNA-polymerases in the presence of the complimentary oligo-deoxyribonucleotide primer. Chemical modification of such template would be expected to alter the interaction between the template and the viral enzyme (1-5). This appears to be a very useful approach for designing specific inhibitors of viral DNA polymerases, which might find application in the chemotherapy of cancer (6).

The inhibition of DNA polymerases from RNA tumor viruses by partially thiolated polynucleotides has been described by us earlier (1-3,6,7). Partially thiolated polycytidylic acids MPC I-III (containing 1.7%, 3.5% and 8.6% 5-mercapto­cytidylote units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly rA.p(dT)14 or poly (dA-dT) templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing approx. one 5-mercaptopyrimidine nucleotide per 50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the

* Correspondence should be sent to: Professor Prakash Chandra, Head of Molecular Biology, University Medical School, Theodor-Stern-Kai 7, Frankfurt-70, W. Germany

191

192 P. CHANDRA ET AL.

endogenic reaction, and also in the presence of synthetic templates. The thiolated DNA was the most active, but thio­lated tRNA also showed substantial inhibitory effects, while the thiolated rRNA was less effective. In a bacterial DNA­polymerase (E. coli K12, using denatured DNA as template), MPC I-III showed no inhibitory effect. By contrast, MPC III (8.6% 5-mercaptocytidylate units) and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase I system; however 8-10 times higher concentrations were needed to achieve the in­hibitory response compared to that of viral system. Kinetic analysis of the inhibitory action of a thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template with a Ki/Km ratio of 0.5.

To measure the effect of mercapto polyC (MPC III) on the production of splenomegaly by FLY, we first incubated the cell-free extracts of spleen from mice (Groppel strain) with 100 ~g/ml of MPC at 37°C for 1 h. In the control group, where no compound was used, the cell-free spleen suspension was pre­incubated with Tris-buffer (O.OlM, pH 7.4), the solvent for MPC. The aliquots of this suspension (0.2ml, LD90) were in­jected into each group, consisting of 10 animals. The spleen weights were analyzed on the 8th or the 12th day after in­jection. There was a 60% reduction of spleen weights (arithmatic mean of five individual values) in the MPC treated group, measured on the 8th day. However, no differences in spleen weights in the treated and the control group were ob­served on the 12th day. This is probably due to the fact that at this MPC concentration the whole of virus is not inacti­vated, so that the residual active virus particles lead to potentiation of leukemogenesis.

To ensure that the splenomegaly is an actual indicator of leukemogenesis, and the effects obtained on the 8th day were due to MPC inhibition of the viral activity, cell-free spleen extracts from mice injected with FLY pretreated at higher MPC doses (200-400 ~g/ml) were injected to normal mice, and their leukemogenic potential was indexed by the spleen size. As follows from Table 1, all the animals inoculated with cell-

INHIBITION OF ONCORNAVIRAl REVERSE-TRANSCRIPTASE 193

TABLE I. ASSAY FOR LEUKEMOGENIC POTENTIAL OF CELL-FREE SPLEEN EXTRACTS FROM MICE INFECTED WITH FLV PRETREATED WITH THE PARTIALLY THIOLATED POLYCYTIDYLIC ACID (MPC III)

*

Treatment of donor mice*

Untreated

MPC 200 \-Lg/ml MPC 400 \-Lg/ml

Leukemogenesis in Recipient Mice 10 Days After Injection

** with Spleen Extract

No. of POSe Mice/No. of Neg. Mice

10 / 10

6 / 10 4/10

These mice were obtained from the groups inoculated 12 days previously with FLV, or FLV preincubated with MPC at 370 C for 30 min. ** Cell-free spleen extracts were prepared from spleens of 5 mice in each group pooled together.

free spleen extracts from control mice (donor) developed leukemia (positive); whereas, inocula from donors pretreated with MPC-containing viral suspensions have partially lost their leukemogenic activity. It is interesting to note the dose dependency in the activity of MPC in donor mice. Our preliminary experiments have shown that the in-vitro effect of MPC can be increased by the in-vivo application of MPC to animals, after their challenge with the preincubated viral suspensions o This indicates that though MPC is a very potent inhibitor of reverse-transcriptase activity, it may undergo degradation under the in-vivo conditions o In order to study the effect of nucleases on MPC degradation, 35s-labelled MPC (10.1% 5-mercaptocytidylate units) was subjected to exhaustive digestion with micrococcal nuclease (Worthington Biochemical Corporation), followed by column chromatography of the hydro­lysate on DEAE-cellulose using a linear gradient of NaCI (pH 7.5 buffer, O.OOlM)9ME).

194 P. CHANDRA ET AL.

The results of the degradation of MPC showed that about 63% of the MPC was hydrolyzed to mono- and di- nucleotides, but they contained only trace amounts of the 35s-labelled activity. Essentially, all of the 35s-label was present in tri-, tetra- and higher oligonucleotide fractions. These results are an indication of the random distribution of 5-mercaptodytidylate bases in the modified homopolymer, and show that the thiolation of cytosine bases renders the phos­phodiester bonds of the trinucleotide resistent to hydrolysis by micrococcal nuclease.

TABLE 2. TEMPLATE ACTIVITIES OF OLIGO (dG).POLY (rC) AND OLIGO (dG).(MPC) IN DNA-POLYMERASE SYSTEM FROM FRIEND LEUKEMIA VIRIONS (FLV)*

(3H)dGMP Incorporation Concentration into DNA

Template (lJg/ c.p.m./ Percent

used react. mixt.) react. mixt. of Control

None (endogenic) 856 100

Oligo (dG). poly (re) 1 3157 369

2 5791 676 4 7637 891

Oligo (dG). (MPC) 1 953 111

2 1228 143 4 1363 159

* Reactions were performed under standard conditions as described elsewhere o

INHIBITION OF ONCORNAVIRAL REVERSE-TRANSCRIPTASE 195

Viral DNA polymerases require a primer molecule, with a free 3-OH end for copying of single-stranded regions of tem­plates to form the double-stranded product. The two synthetic template-primers that appear to be relatively specific for oncornaviral reverse transcriptases are oligo (dG).poly (rC) and oligo (dT).poly (rA), see review by Sarin and Gallo (8). The template activities of oligo (dG)·poly (rC) and oligo (dG) .(MPC), i.e. 1:1 hybrid of oligo (dG) and mercapto-polycyti­dylic acid(MPC), for FLV DNA-polymerase system are shown in Table 2.

As follows from Table 2 the incorporation of (3H)dGMP into DNA by the viral enzyme is stimulated 3 to 9-fold in the presence of oligo (dG)·poly (rC). However, under similar ex­perimental conditions oligo (dG).(MPC) fails to stimulate the (3H)dGMP incorporation into DNA. The small response, observed at higher concentrations may be due to the fact, that the thiolation of cytosine bases in the polymer is a random pro­cess, and some long stretches of unmodified cytosine bases may be used up by .the enzyme.

TABLE 3. OLIGO (dG).POLY (rC) - DIRECTED ACTIVITY OF DNA POLYMERASE FROM FLV IN THE PRESENCE OF OLIGO (dG).(MPC)*

Oligo (dG). Oligo (dG). (3H)dGMP Incorporation

poly (rC) (MPC) into DNA

(\Jog/reaction (4 \Jog/reaction c.p.m./ Percent mixt.) mixt.) react. mixt. of Control

None (endogenic) 903 100

None + 1270 141 1 + 1310 146 2 + 1450 161 4 + 1670 185

* Reactions were performed under conditions described else-where. MPC contains 15% 5-mercaptocytidylic acid units.

The effect of oligo (dG).(MPC) on the kinetics of the enzyme reaction at various concentrations of oligo (dG). poly (rC) is shown in Table 3. The addition of 1 to 4 \Jog of

196 P. CHANDRA ET AL.

of oligo (dG)opoly (re) in the reaction mixture leads to a slight increase of (3H)dGMP incorporation, however the mag­nitude of stimulation exhibited by oligo (dG).poly (rC) in the absence of oligo (dG).(MPC), as shown in Table 2, is not seen here. This indicates that the enzyme may have a higher binding affinity to the primer-template oligo (dG).(MPC).

Further studies showed that the effect of oligo (dG). (MPC) on the template functions of unmodified duplex oligo (dG)opoly (rC), can be influenced at higher enzyme concen­trations o Thus, these data show that MPC acts by interacting directly with the enzyme. This has been confirmed by the ultracentrifugation studies in which the binding of 35S­labelled MPC to a purified enzyme fraction was investigated (manuscript in prep.).

REFERENCES

1. Chandra, Pa, and Bardos, T.J.: (1972) Res. Commun. Chem. Path. and Pharmacol. i, 615.

2. Chandra, P.: (1974) In: Modern Trends in Human Leukemia, (Neth, R., Gallo, R.C., Spiegelman, S. and Stohlman, F. (eds.) J.F. Lehmanns Verlag, Munich, p. 2110

3. Chandra, P., Ebener, U., Bardos, T.J., Chakrabarti, P., Ho, Y.K., Mikulski, A.J., and Zsindely, A.: (1975) Ann. N.Y. Acad. Sci., (in press).

4. Pitha, P.M., Teich, N.M., Lowy, D.R., and Pitha, J.: (1973) In: DNA synthesis in vitro (Wells and Inman, Eds), MTP-Lancaster, p. 369.

5. Erickson, R.J., Grosch, J.C.: (1974) Biochem. ~ 1987.

60 Chandra, P., Kornhuber, B., Gericke, D., Gijtz, A., and Ebener, U.: (1974) Z. Krebsforschg. Klin. Onkol., (in pressl

70 Chandra, Po, Bardos, T.J. and Ebener, U.: (1974) in: Prog o

in Chemotherapy, Helenic Soc. Chemotherapy, (Edt. G.Ka Daikos), !!I, 182.

8. Sarin, p.So and Gallo, R.C.: (1975) in: Int. Rev. of Science (K. Burton, ed.) Butterworth and MTP, Oxford, Vol. 6, chapter 8, (in press). -

EXPERIMENTAL APPROACH TO INCREASE THE EFFECTS OF CANCER CHEMOTHERAPY IN TUMOR-BEARING RATS PRETREATED WITH AN INDUCER ON MICROSOMAL DRUG­METABOLIZING ENZYME (CYTOCHROME p-450)

Sadao OHlRA, Sakae MAEZAWA, Kazuhiro WATANABE, Kazuhiro KITADA, Tatuo SAITO Department of Clinical Cancer Chemotherapy, The Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University, Sendai, Japan

In this study, we have observed for the first time that 1-(2-tetrahydrofuryl)-5-fluorouracil (FT) when mixed with cytochrome p-450 from the microsomal fraction of rat liver produces a differ­ence spectrum classified as type II (Ks=26.3mM). The appearance of this spectrum suggested that FT was bound to cytochrome p-450 and became an active metabolite (5-fluorouracil; 5FU). In normal rats pretreated with phenobarbital, we demonstrated a significant increase in quantity of cytochrome p-450 in liver microsome, and at the same time, a remarkable elevation of the blood level of 5FU. It is con­verted in vivo from FT which is administered intraperitoneally.

In the next experiment which was carried out based on above data, the tumor weight in the rat (AH130, inoculated subcutaneously) treated with FT in combination with phenobarbital was much lighter than that in the rats treated with FT alone.

We have a1ready reported that the eurviva1 period in tumor­bearing rats became prolonged when treated with cyclophosphamide (one of the masked compounds) in combination with inducer (pheno­barbital) of microsomal drug-metabolizing enzyme in the liver (Ohira et ale 1974, 1975).

In the present paper experiments were made on the inducer of liver microsomal drug-metabolizing enzymes in order to enhance the effects of FT. In the electron transfer chain related to drug metabolism, NADPH, which is an electron donor, is found in associ­ation with NADPH-cytochrome C reductase and cytochrome p-450 attached to the membrane of endoplasmic reticulum of the liver. As known already FT itself has no anticancer activity, because it is one of the masked compounds. Thus it is likely that 5FU, which is a main active metabolite in vivo produced from FT, exerts anticancer

197

198

.. u c .. D.

0 ., D. .. ...

S. OHIRA ET AL.

... ~ 70 -- 30mM ~

0.02 -~ .0 50

~ D.

0.01 .. ...

KS=26.3mM

0

0.01 0 .05 01 1

370 420 490 FT(mM) wave length (nm)

Fig. 1. 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) difference spectrum with microsomes from rat liver. (protein concentration: 2.9 mglml, cyt. p-450: 2.3 ~ mole/mg protein)

activity. Therefore, an attempt was made to enhance the conversion of FT

to 5FU by the induction of drug-metabolizing enzymes in the liver.

RESULTS AND DISCUSSION

It has been reported that the microsomal fraction of the liver shows a unique difference spectrum after the addition of substrates. The patterns of the spectra are different depending on the kinds of substrates added. Therefore, the difference spectrum results from the interaction of the drug with liver microsomal cytochrome p-450. schenkman, J.B. et ale (1967) classified the difference spectra into 3 groups, namely type I, type II and modified type II.

As shown in Fig. 1 the difference spectrum of FT-p-450 obtained from the liver microsome of the normal rats has a peak at 420nm and a trough at 385nm. Thus the difference spectrum of FT belongs to the type II.

The figure on the right in Fig. 1 is a double reciprocal plot­ting of the concentrations of FT ranging from 10mM to 3DmM and the amount of oxidized microsomal cytochrome p-450 determined by the pattern of absorbancy. The Ks value of FT calculated from this graph was 26.3mM. The evidence obtained suggested that cytochrome p-450 in liver microsome participates mainly in oxidation of FT.

The levels of cytochrome p-450 and cytochrome b5 in the micro­somal fractions of the liver determined by the method of Omura, T. and Sato, R. (1964) are shown in Fig. 2. This figure shows the levels of the microsomal enzymes in the liver of the normal rats

INCREASE OF EFFECTS OF CANCER CHEMOTHERAPY

normal contrds

PB 5Omg/kgx 3days

12 hr after

24 "

48"

eM 1OOrrgJkgx3days

12hr after

24 "

48 "

~ cyt.P-450

:::::::::::::: cytbs Q88!0D9

0.39!0D1

::;:;»"~~~M~ 2.14

:::::::::::::::::: 0A8

:::::s:~@~::.~~~~ 2,06

.::::::::::::::::::: 054

o 1.0 2.0

0.61 Q35

0.64

0.38

0.62

043

m}Jmole/mg protein

Fig. 2. Effect of phenobarbital (PB) and chloramphenicol (eM) on rat liver microsomal constituents.

199

which received either phenobarbital at a daily dose of 5Qmg/kg body weight or chloramphenicol at a daily dose of 100mg/kg body weight for three days. The rats thus treated were sacrificed every twelve hours after the final injection and the enzyme levels were de­termined. The level of cytochrome p-450 of the rats treated with phenobarbital is 2 to 3 times as high as that of the normal rat. On the other hand, the level of cytochrome p-450 of the rats treated with chloramphenicol is 70 to SQ% of that in the control.

The results indicate that phenobarbital stimulates and, on the contrary, chloramphenicol supresses the induction of cytochrome p-450. The changes in the level of cytochrome b5, which has a less stimu­lative effect on drug metabolism as compared with cytochrome p-450, was not remarkable.

Fig. 3 shows the blood level of FT and its active metabolite, 5FU in normal rats which received a sing~intraperitoneal injection of FT at a dose of 500mg/kg body weight. The blood levels of both FT and 5FU were determined by bioassay using the cup method. In this method, very thin agar plates were used, in which staphylococcus

200

5

4

3

2

5·FU ()---o Pretreated with Phenobarbital

150mg/kg/day for 3 consecutive days,lpl

~ pretreated with chloramphenicol 1100mg/kg/day for 3 consecutive days,l P I

o 2 4 6 8 10 12 hrs LIIIIL dlllel lilt! alJ~lltlJf1eallnlectlon Of 50Umg/kg Of FT,n single dObe

S. OHIRA ET AL.

Fig. 3. Blood levels of FT-207 and its active metabolite (5-FU) in rats pretreated with various drugs.

aureus 209P was mixed as a test organism. As compared with the normal control, a remarkable elevation of

the level of 5FU was observed in the rats which had received an intraperitoneal administration of phenobarbital at a daily dose of 5Omg/kg body weight for 3 consecutive days prior to the injection of FT. Until the 2nd hour the level of 5FU was somewhat higher in the rats treated with chloramphenicol at a daily dose of 100mg/kg body weight for 3 days, but after the 4th hour, the level became lower than that of the control. On the contrary, the blood level of FT in the rats treated with phenobarbital prior to the injection of FT was lower than that of the control rat. Thus the blood level of 5FU became elevated remarkably when the rats were treated with pheno­barbital. As mentioned previously, the level of cytochrome p-450 in the microsomal fraction of the liver also was increased remarkably by phenobarbital. Moreover, the degree of elevation of the blood level of FT was almost in parallel with an increase of the level of cytochrome p-450 in the microsomal fraction of the liver.

Therefore, it is likely that cytochrome p-450 plays a very

INCREASE OF EFFECTS OF CANCER CHEMOTHERAPY

Fig. 4.

~g, ........ 01 ...

_ c:onIraI

+±~ 5.8:1:1.5 10

FT IOOma/kg 5.0:1:1.7" 10 PB+FTIIII 2.1:1:US"· 10

FT 300 rng/kg 2.9:1:1.1 10 PB+FT 3111 ~ 1.9:1:0.9 10

FT 500mglkg 01 0.5:1:0.3 9 PB+FT !iOO • 0.1 :1:0.1 7

5-Rl !iIIrngjkg 5.5:1:1.4 10 PB+5-FU 5.3:1:1.3 10

5-Rl~ 2.6±1.2 10 PB+5-FU 2.4±1.3 10

5-RlI!illrnakl 0 0.1:1:0.1 10 PB+5-FU .. 0.4%0.3 10

I ( •• pcO.O!i)

4 tumor weIght(lI)

. subcut.-us _ weIghI 01 10 days afIer Inoculation

::~ :J~T'end"1'""J:r.""'~l~'o~~) befCre " •• 1isInIIlon 01 FT or 5'FU

FT.5.FU: FT or 5·FU .clmlnlolreted inlraperitoneally In a single delle 3.5 days afIer 1nocu18llan 01 AH130 cell.

Effect of FT-207 or S FU on subcutaneous tumor weight in AH130 bearing rats.

201

important role in the conversion of FT to SFU. Presum­ably the effects of FT on cancer cells is enchanced by the administration of an inducer for cytochrome p-450 which, in turn, stimulates the conversion of FT to SFU.

On the other hand, on electron micrograph of a section of the liver of the rat treated with phenobarb­ital, more remarkable proliferation of S-ER was observ­ed, as compared with the untreated control. Thus it was clearlydemonstrated that the phenobarbitalinduced activation of FT is accompanied by proliferation of the SER of the liver cells.

Then, experiments were carred out in order to deter -mine the survival period of the tumor-bearing rats, which had received an intraperitoneal injection of AH130 or Yoshida sarcoma. The survival period remained un­changed in the rats treated with FT ( a single dose of 100, 300 or SOO mg/kg intraperitoneally, respectively) alone or with FT in combination with phenobarbital (SOmg/kg for 3 days).

202 s. OHIRA ET AL.

Fig. 4 shows the weight of tumor of AH130 cells transplanted subcutaneously to rats. As shown in this figure, the tumor in the rats treated with FT in combination with phenobarbital was signifi­cantly lighter than that in the rats treated with FT alone. It is of interest that no difference was observed in weight of tumor between the rat treated with 5FU alone and those treated with 5FU in combination with phenobarbital.

CONCLUSION

In the present studies, it was found that phenobarbital causes the proliferation of S-ER in liver cells and stimulates the in­duction of liver microsomal drug-metabolizing enzyme, namely, cyto­chrome p-450, which enhances the rate of conversion of FT to 5FU, the active substance. Therefore, when FT is used in combination with phenobarbital for tumor bearers, the inhibitory effect of FT on tumor growth could be markedly increased.

In summary, it must be emphasized that the fluctuation in level of the drug-metabolizing enzyme, cytochrome P-450 should be taken into consideration, when a masked compound is administered to tumor­bearing hosts.

References

1 ) Ohira, S., Maezawa, s. et al. (1974) , Tohoku J. expo Med. , 114, 55.

2) Ohira, S., Maezawa, s. et ale (1975), Tohoku J. expo Med. , 115, in press.

3) Omura, T. and sato, R. (1964), J. bioI. Chem., 239, 2370. 4) schenkman, J.B. et ale (1967), Mol. Pharmacol., 3, 113.

EFFECT OF THE DRUG-METABOLIZING ENZYME I1IDUCERS ON

THE CYTOSTATIC ACTIVITY OF DIBROMODULCITOL

tVA GATI

RESEARCH INSTITUTE OF ONCOPATHOLOGY

1122. BUDAPEST, RATH Gy. STIle 7.

Sm,1IAARY

Intact Swiss albino mice and Wistar rats bearing Yoshida ascites sarcoma have been pretreated with 3-methy1cholanthrene /Mch/ or with 1,2-5,6 dimethyl­benzanthracene /D1IDA/ prior to dibromodulci~ol /DBD/. The drug combination increased the benz/a/pyrene hyd­roxylase /BaPH/ level in the skin, liver and tumour cells of int~ct mice and rats bearing Yoshida ascites sarcoma sensitive to DBD, respectively. The growth rate of the Yoshida tumour cells was unaffected by treatment with Llch. The total number of the sensitive tumour cells decreased more rapidly with the Mch+DBD combination than with DBD treatment alone. This drug combination was without effect on the tumour cells resistant to DBD.

It was suggested, that the cytostatic action of dibromohexito1s may be partly mediated by epoxide intermediates. /Elson et a1./ The formation of 1,2--5,6 dianhydrodulcito1 from dibromodulcitol in rats bearing Yoshida ttunours could be demonstrated both in vitro and in vivo /Gati et a1./. As the microsomal epoxide hydrase is known to catalyze the conversion of both K- region and non K- region epoxides to the corresponding dihydrodiols, /Sims/ we were interested in studying whe-ther substances inducing this enzyme system could influence the cytostatic activity of DBD.

203

204 E. GATI

In order to determine the possible alteration in the drug- metabolizing enzyme level upon the combined effect of DBD and the enzyme inducers, BaPH activity was measured by the method reported previously IGati et al.l.

liver

i'tD Mch treated

C] !v1ch- D8D treated

1§3 DBD freated

o untreated

skin

Figure 1. Effect of the combined treatment of Mch/10mg/kg i.p. I+DBD/ 500 mg/kg i.p./ upon BaPH in the liver and skin of mice. Each value represents the mean of 6 experiments.

DRUG-METABOLIZING ENZYME INDUCERS AND DIBROMODULCITOL

80

70

60

liver

C DMBA trflaifld

Em ~A·DBDtrealed CJ DBD Ireatro

~ untreated

skin

205

Figure 2. Effect of topical application of DMBA (lOOO~g) combined with OBD (500mg/kg/i.p.) on BaPH in the skin and liver of mice. Each value represents the mean of 6 experiments.

The BaPH activity reached a nearly three-fold level in the liver and skin at 48 hours following the Mch injec­tion. The DBD in a dose equivalent to LDSO/2 exhibited a moderate enxyme inducing effect in the sk1n. Injecting the Mch 16 hours prior to DBD administration the BaPH reached a higher level in the liver and skin than in those treated with Mch alone. Applying the DMBA topically 16 hours prior to the intraperitoneal administration of DBD, the BaPH activity was nearly double in the skin at 48 hours compared to those treated with DMBA alone. This combination caused a significant rise of DaPH activity in the liver also.

206

DBD-sensitive

6() I/)

50 30

~,o 10 :~ gl:)

t fi'(". 10

10

'0 . . 30

20

10

..

I/) DBD-resistart

30

_ Hdt treatod

t· .. Hch'a;o tr..,tod o a;o troatod ~ untreatod

E. GATI

Figure 3. Effect of the combined treatment of Mch/IO mg/kg i.p. / +DBD /25Omg/kg i.p. / upon BaPR in the liver and tumour cells of Wistar rats bearing Yoshida ascites tumour sensitive or resistant to DBD. Each value represents the mean of 4 experiments.

Mch increased the enzyme activity in the liver of rats bearing Yoshida tumour sensitive to DBD from 20 to 41 units whereas the Mch+DBD combination caused a rise to 57 units at 48 hours. At the same time a nearly three­fold increase in the low constituent enzyme level of the sensitive tumour cells could be demonstrated upon the effect of this combination.

In the liver of rats bearing Yoshida turnour re­sistant to DBD the Mch caused a rise of BaPH activity from 20 to 31, Mch+DBD to 40 units. No enzyme inducing effect of Mch or of Mch+DBD combination could be re­corded in the tumour cells resistant to DBD.

In order to determine the influence of enzyme inducer Mch on the cytostatic action of DBD, the change in the total cell number Yoshida tumours was followed up_

DRUG-METABOLIZING ENZYME INDUCERS AND DIBROMODULCITOL 207

, I' I

/ / 9 I

10 / /

I 9 ~r'

10 __ unfreatpd /1 '>-<JMe"" treatpd 1'1 ~~ OBo treated ~ I I

.Q I I .... _Me'" oBD

E CIt :;, I /' .Q c: E treated ~ I / 2 v II

::::: 1; 1/ .. v ~ 0-"", unfreafed <; ~ OBO treated ~ __ Mch treated

8 lrJ

-Mch·OBO

10 treated

5 5 7 11 .: 5 6 7

Tumour agP,days Tumour age,days

Figure 4. The grovnh curves of the DBD-sensitive Yoshida cells following the combined treatment of hlch/lOmg/kg i.p. / +DBD 25Omg/kg i.p./. Each point represents the mean of 4 experiments.

Figure 5. The growth curves of the DBD-resistant Yoshida cells following the combined treatment of Mch/lOmg/kg i.p. /+DBD/ 250mg/kg i.p.

The growth rate of the tumour cells was unaffec­ted by treatment with Mch. T:i.e total cell number decreases more rapidly following the pretreatment vlith !I1ch 16 hours prior to the DBD in dose equivalent to LD50/2 compared with the DBD treatment alone.

The l£ch+DBD combination produced no significant change in the growth rate of DBD- resistant tumour cells, as compared with those caused by the 1.Ich or DBD alone.

DBD is thought to be a direct alkylating agent which does not require microsomal metabolism for action. The

208 E.GATI

ability of DBD to induce BaPH activity in the skin of intact mice could be explained by the fact that the epoxide hydrase may be involved in the metabolism of the epoxide intermediates of DBD.

The increased cytostatic effect of the Mch+DBD combination may be due rather to the enhanced cytotoxic action of the Mch as the extent of the induction of the oxidase activity is generally much higher than that of the hydrase activity.

Corresponding to the data of Hill at ale concerning the chlorambucil-resistant Yoshida ascites sarcoma, the tumour cells resistant to DBD proved to be also unable to respond to microsomal inducers.

REFERENCES

Elson I.L.A.; JarIrlf}n M.; Ross ,{{.C.: Europ. J. Cancer 4. 617 /1968/. Gati E.; Horvath I.P.; Kralovanszky J.; Toperczer J.: Advences in Antimicrobiol. and Antineo­plastic Chemotherapy Ed. Urban and Sqhwarzenberg, MUnchen, 1972. Vol. II.p. 33. Gati E.; Calop J.Y.; Lafaverges F. Ann Histochim. 18. 311. /1974/. Hill B.T.; Douglas J.R.C.; Grover P.L.: Biochem. Pharmacol. 22. 1083. /1973/. Sims P.: Biochem. J. 125. 130 27. /1972/.

14 STUDIES OF N-METHYL-N-NITROSOUREA-C 0 PHARMACOKINETICS IN

MICE WITH HEPATOMA 22A

L.B. Gorbacheva, G. V. Kukushkina, loS. Sokolova, A.M. Serebryanyi, V.S. Tutlyte

Institute of Chemical Physics, Academy of Science, Moscow, U.S.S .R.

SUMMARY

The appearance of the carbonyl label in target molecules of liver, spleen, kidney, <}I mucosa and hepatama 22a was studied after a single injection of MNU-d 0 in a therapeutic dose (80 mg/kg). A high proportion of radio­activity was found in the protein and lipid fractions. The carbonyl label was found in RNA of all organs only 1-5 hours after MNU-CI4 administration. The radioactivity detected in proteins and lipids after MNu-c14o administration is retained in hepatoma cells for longer periods of time (72 hours) while it is rapidly eliminated from normal cells.

N-nitrosourea alkyl derivatives are active therapeutic agents for the treat­ment of a number of experimental and clinical neoplasms. BCNU, CCNU and MeCCNU have been shown to be active against a wide spectrum of animal tumours and in subsequent clinical trials have been demonstrated to have anti­tumour effect against Hodgkin1s disease and non-Hodgkins lymphoma as well as a wide variety of solid tumours in man. N-methyl-N-nitrosourea (MNU) was recommended for the palliative treatment of patients with undifferentiated carcinoma of the lung and Hodgkin1s disease (Emanuel et al., 1974). These compounds decompose at physiological conditions to yield alkylating and carbomoylating moieties. MNU decomposes to give a methyl carbonium ion alkylating DNA, RNA, proteins and lipids and isocyanic acid carbomoylating of proteins. The reasons for the preferential toxicity of nitrosoureas for neo­plastic tissues with a resulting therapeutic effect are not known. One of the possible reasons for the differential sensivity of cells to these agents might

209

210 L.B. GORBACHEVA ET AL.

result from their differing ability to degrade damaged RNA, proteins and lipids. It has been found that murine hepatoma 22a cells repair DNA and replace alkylated RNA, protein and lipid molecules at a much slower rate than the corresponding host cells. This differential effect was accounted for by different rates of the turnover processes in the mechanisms responsible for selective toxicity of nitrosoureas towards tumour cells (Lerman, et al., 1974). It has been shown recently (Rosenoff et al., 1974; Wheeler and Alexander, 1974) that DNA synthesis in the host tissues recovers from the damage by nitro­soalkylureas more readily than in cells of plasmacytoma, Lewis lung carcinoma and leukaemia L121O. In this work the appearance of carbonyl label in target molecules of some or~ans and solid hepatoma 22a was studied after a single injection of MNU-C 40 in a dose of 80 mg/kg. Studies required the use of tumourous mice and were initiated at day 7 of tumour growth (at time zero). After different intervals of time (1,5,24,96,120 and 144 hours) mice were sacrificed and liver, kidney, spleen, brain, GI mucosa and hepatoma were rapidly removed and homogenized. Data on the radioactivity determinations were usually given in cpm mg DNA of organs. After the injection of MNU­C140 radioactivity appeared in the total acid-insoluble fraction (table 1) . The greatest values were obtained 5 hours after MNU administration. The radioactivity of all cellular macromolecules was rapidly eliminated from brain, liver, kidney, spleen, GI mucosa. 24 hours after MNu-c140 administration the radioactivity in brain was 21%, in liver 40%, in kidney 31%, in spleen 40%, in GI mucosa 38% of the maximum appearance of carbonyl label which was 5 hours after MNU administration. A different phenomenon was observed for hepatoma. The greatest va I ue of radi oacti vi ty for hepatoma was obta i ned in 24 hours after MNU-C140 administration (160% to % hour). By 72 hours the radi oacti vi ty in hepatoma decreased to 107%. However practi cally all radioactivity was eliminated from the hepatoma at 120 hours. The level of radioactivity in dividing cells (hepatoma, GI mucosa and part of the spleen) was markedly lower originally than in non-dividing cells (brain and liver); kidney occupied an intermediate position in this respect. The radioactivity in mouse plasma is retained at high level 72 hours after MNU administration. Analysis of distribution of the radioactivity in fractions is largely in good agreement with the data on kinetics of the appearance of total radioactivity (figures 1 and 2). A high proportion of radioactivity was found in the fraction of lipids. 5 hours after MNU administration it was in liver and hepatoma 55% of the total radioactivity; spleen and brain 65%; kidney and GI mucosa 40% This phenomenon might possibly have been due to high solubility of MNU in lipids. One may therefore suggest that nitrosoureas might rapidly damage cellular and intracellular membranes and this process depneded on carbamoy­lation of lipids. The proteins of different organs and especially of hepatoma were carbamoylated to a considerable extent; the radioactivity in hepatoma was 60%, in spleen 40%, in liver 21%, in GI mucosa 22%, in kidney 30%, in brain 12%. Earlier it was shown that in vivo and in vitro C14-cyclohexyl

f400

0

~ ~,ooa

o ~ wo

o 15

2+

/(ill

etlc

S

0/

Lf1t

etin

a 0/

Mac

'l.om

otec

u/es

f1

no'

I.lp

ufs

01

L£lI'

et,

Spte

en

and

Hep

atom

a 01

Mi.c

e -1

3eau

na

Hep

atom

a ZZ

a af

ter.

SL

nate

In

;ect

ion

0/ M

NtI

-CI'

fO

Hep

atom

a

I.ic;

ez

4000

21

( 1(

8

2J]oa

... 72

96

fra

fq~

Fig

ure

o Th

e ta

ta!

acid

-ins

ott/

tel'

!tac

tion

6

'lJN

A

D l.i

pi.d

S

~ P

tct.

eL

ns

f2

96

120

1+'1(

Spee

en

if

1(8

12

96

(20

(4+

iLm

e (l

it)

~

Z c h :;: o '"C

:z: » ::

ll ~ » ("

) o A

Z

m

-I

(")

CIl

t-.>

Ki"

eucs

01

La

6eu.

"9

of M

aC'to

fTTo

tecu

teJ

am

i Iv

lpl l

is

01 6

I M

vcos

o,

~,li

ne!l

an

ti B

tcn

n o

f M

,ce

-Bea

un

s /-I

epato

/OO

2?a

alte

? S'

''9te

l1

1ect

ion

01 M

Nt}

-C"O

6.l.

Mvc

o 5

(1

1500

0

48

72

96

laJ

Iltl(

IODDO

Kidn

ey

---~

9:=

-t"

15

i.

..,

12

S6

dO

14'f

Fig

ure

2

o Th

e to

tae

(feW

· inJ

Qlu

tte

Itad.

iolT

t.1

>N

A

D l

-ip

tds

K P

tote

ins

t.:I

t.:I

! !'J

C) o :D

a:I » (")

:J: m

< » m

-I

» r

MNU-C140 PHARMACOKINETICS 213

and Cl4-carbonyl derivatives had a particularly high affinity for protein (Cheng et 01., 1972). There was negligible binding of these labels to nucleic acids. In our experiments the carbonyl label was found in RNA of liver and spleen only 1 hour ofter MNU administration. The radioactivity detected in RNA of hepatoma, kidney, brain and GI mucosa was retained for 5 hours. The radioactivity associated with DNA and RNA was mainly the direct result of carbamoylation. This follows from chemical analysis of carbamoylated bases of DNA and RNA obtained from liver and hepatoma at 1-5 hours after MNU-c'40 administration.

Table 1. Distribution of the radioactivity of total acid-insoluble fraction in mice with hepatoma 22a after i .v. administration MNU-CI40.

Organs Time after administrati on of MNU-d4o (hours)

5 24 72 96 120

Plasma 30250 11800 11650 3350 6860 3890

Brain 15390 18800 4100 7410 4780 1315

Liver 8010 13600 5440 4930 2470 2185

Kidney 3725 7520 2400 4350 1880 1280

Spleen 3230 6220 1940 2720 580

GI mucosa 2920 4570 1590 445 150 130

Hepatoma 3110 3150 5000 3770 570 265

REFERENCES

1. Chun Jui Cheng, Shinjimura, Dezider Grunberger and I. Bernard Wein­stei n (1972). Cancer Research, 32, 22.

2. Emanuel, N.M., Vermel, E.M., Ostrovskaya, L.A. and Korman, N.P. (1974). Cancer Chemotherapy Reports, Part 1, 58, 135.

3. Lerman, M.I., Abakumova, O.Yu., Kucenco, N.G.-;-Gorbacheva, L.B., Kukushkina, G. V. and Serebryanyi, A.M. (1974). Cancer Research, 34, 1536.

4. Rosen off , S .H.-;-Bostic, F. and Young, R.C. (1974). Biochem. Pharm. 23, 3097.

5. Wheeler, G.P., Alexander, J.A. (1974). Cancer Research, 34, 1957

COLLATERAL SENSITIVITY BE~EN AN ALKYLATING AGENT AND

HALOGENATED METHOTREXATE

Brian W. Fox

Paterson Laborator~es

Manchester U.K.

o \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \

o 10 20 30 40 pg/ml

Fig.l. Survival curves of three resistant and the original sensit­ive lines towards MOMS.

Mutation Res. 19 (1973) 119-128) that

215

We have established four cell lines designated YMDR6 to YMDR9 by in vivo sub­threshold treatment of Yoshida lymphosarcoma with methylene dimethanesulph­onate (MOMS). These have been transferred to cell culture and grown contin­uously. Only YMDR8 was cloned.

Tumour growth of each tum­our in Wistar rats is resistant to lOmg/ml MJ)MS whereas the sensitive, wild type tumour is com­pletely sensitive and the animals are all cured with this dose level.

All cell lines in culture derived from solid resist­ant tumours are resistant to MOMS, three of which are compared to the sens­itive lines in Fig.l. Furthermore, we have shown (M. Fox and B.W. Fox,

the YMDR8 line differs from

216

Fig.2. Formulae of halogenated methotrexates.

B.W. FOX

the original sensitive (S) line in being able to under­take repair replication following damage to the DNA by the drug. Cross­sensitivity studies with YMDR8 has shown (M. Fox and B.W. Fox, Chem-Biol. Interactions, 4 (1971/72) 363-375) has shown that this line is cross-resist­ant to nitrogen mustard and UV light but the two lines are equally sensity to X-rays and methyl methane­sulphonate. In vivo, it has now been shown that cross-sensitivity towards cyclophosphamide Busulphan E.O.R.T.C. 1502 hexamethyl­melamine and propylene di­methane sulphonate also occurs.

During these in vivo studies, three halogenated methotrexates, viz NSC 98580 (3'-bromo methotrex­ate) NSC 29630 (3',5'di-

chloromethotrexate) and NSC 98579 (3'bromo 5'-chloromethotrexate) (Fig.2) were shown to have a slightly greater effectiveness to­wards a YMDR8 implant than against the sensitive line. However, from in vitro work, it was readily confirmed that a strong coll­ateral sensitivity had been established (Fig.3). It was also shown that similar collateral sensitivity patterns had developed against NSC98579 and NSC29630. Furthermore, two of the other resistant cell lines YMDR7 and YMDR9 also showed collateral sensitivity to NSC98580.

It would thus appear from this data that the factors selected for resistance towards MDMS are also linked with the increased sens­itivity towards the halogenated methotrexates. Preliminary data (D.J. Pillinger and B.W. Fox) has suggested that no significant difference in transport of the labelled non-metabolisable amino­iso-butyric acid occurs between the cell lines, either in treated or untreated cells. An alternative explanation is proposed, that of differential clonal selection by different drugs. It is pro­posed that a tumour mass consists of a large number of tumour clones, which differ in their relative sensitivity towards a drug A. These could have arisen as a result of genetic insta-

ALKYLATING AGENTS WITH HALOGENATED METHOTREXATE 217

10'

-I 10

I­u. «10 a: u.

o~ o~

~-----.----------. 0.5

MICRO Gm/ml

YMDR

1·0

Fig.3. Survival curve of YMDS and YMDR8 following 3'-bromo MTX (NSC 98580) •

bility characteristic of tumour tissue during growth. Such a spectrum of sens­itivity would also be a product of the special biochemical changes induced by that drug which causes the death of some cells but nQt others. A different spectrum of clones within the same population would be expected to be sensitive to Drug B. If these two spectra overlap considerably one would expect cross­resistance. If they over­lap very little, collateral sensitivity would be expected to result. If the first drug can be used to select for resistance and after growth of resistant tumour tissue (allowing host imm­unity also to increase) a tumour clonal structure would be achieved which would now be more sensitive than the original to Drug B, with which collateral sens­

itivity has been demonstrated (Fig.4). In view of the fact that, NSC 98580, unlike MDMS, can cause regression but not cure of the Yoshida tumour, the tumour was first made resistant to NSC 98580 and challenged with a sub-effective dose of MDMS. A cure was achieved, which was not achieved in untreated animals at this dose level (Fig.5). This differential is still very small but a more detailed study of the population structural changes following treatment and regrowth could provide a more rational basis for two drug selection and application in tumour chemotherapy on this basis with other more widely used antitumour agents in which collateral sensitivity can be demonstrated in vitro •

• - EORTC Screening and Pharmacology Group and supported by the Medical Research Council and Cancer Research Campaign.

s ~ I

RL,r<

l S

R

-:

Q-

" A

S

R

:

I

d~B

~ I rt

ldps

t t

S

R+ "

S R

+'

S S

~

umcx

.w:-,

now

~

stn

s,tlY

e

to

dru

g A

rnR:.~

: R

R

::;

R

R'

I

~dr~g~

if'

;t.:

R

R+.~

I rt

lop

st

J R

R+

R

R+

R

+

R

"

+

R

Non

le

tha

l dam

age

by d

rug

B t

o

dru

gA

re

sis

tan

t cl

one.

m

Host

imm

unol

ogic

al s

urve

illan

ce

Fig

.4.

Mod

el

of

the

po

ssib

le u

se

of

co

llate

ral

sen

siti

vit

y t

o

incre

ase

ch

emo

ther

apeu

tic

eff

icie

ncy

an

d

cho

ice

of

dru

g p

air

s.

~ ~ ~

EFF

EC

T (F

5m

g/1(

g I"

'l»IS

0,

'ORI

ofAL

& 9

8580

R

ES -

YOSH

IDA

StR

CO

'ofA

"., 1

'10'

0.

~ ~

.., 2

i;

6 ~

if ~

t Ji 5

10

15

20

25

3

0

Oo)

's o

ft.r

1st

tro

nsp

lon

t

Fig

.5.

The

eff

ect

of

MOM

S on

N

SC

9858

0 p

retr

eate

d a

nd

un

treate

d

Yo

shid

a tu

mo

urs

in

viv

o.

t-,)

00

o:J ~ "'T1 o X

MESO-l,2-BIS-(3,5-DIOXOPIPERAZIN-l-YL) -1,2-DIMETHYL­

ETHANE (ICRF 193); A POTENT ANTITUMOUR ANALOGUE OF ICRF 159

K. Hellmann

Cancer Chemotherapy Department

Imperial Cancer Research Fund, London, U.K.

ICRF 193 was amongst a group of bisdioxopiperazines tested against the Sarcoma S 180, the leukaemia L1210 and the Lewis lung carcinoma (3LL) (Creighton, A.M., Hellmann, K., and Whitecross, Susa~ 1969). It was some ten times as active as ICRF 159 «-)1,2-bis(3,5-dioxopiperazin-l-yl) propane), on Sarcoma S 180, though reproducibility within one experiment and between experiments was erratic. This was to some extent overcome by making suspensions in corn oil rather than CMC, the customary suspending medium in this laboratory (see Table 1).

Although the activity increased some tenfold compared with ICRF 159, toxicity increased even more so that in the final analysis the therapeutic index was lower than for ICRF 159. For 5 180 the Therapeutic Index (LD10)is C:! 20

ED90 for ICRF 159 and~ 1 for ICRF 193.

On L1210 leukaemia the activity of ICRF 193 was about 100 times greater than that of ICRF 159 (See Table 2).

TABLE 1: Effect of ICRF 193 on S 180.

DOSE (mg/kg) ROUTE VEHICLE SURVIVORS T/C

0.5 x 4 i. p. corn oil 5/7 0.0 0.25 x 5 i. p. corn oil 7/7 0.22 0.1 x 5 i. p. corn oil 7/7 0.9 0.5 x 5 i. p. CMC 5/5 1.0

219

220 K. HELLMANN

TABLE 2 : Effect of ICRF 193 on L1210

DOSE (mg/kg) ROUTE VEHICLE SURVIVORS T/C

0.5 x 9 i. p. corn oil 8/8 161 0.25 x 9 i. p. corn oil 8/8 165 0.5 x 7 oral corn oil 8/8 158 0.25 x 6 oral corn oil 8/8 137

It is not yet clear what the optimum dosage regimen should be, but it seems that ICRF 193 like ICRF 159, is active by all routes.

ICRF 193 has also been examined on the Lewis lung carcinoma (Table 3). It inhibits the growth of the primary implant and of the development of pulmonary secondaries, but whether this antimetastatic effect is due to inhibition of growth or whether through some other mechanism as has been the case with ICRF 159, has yet to be determined.

TABLE 3: Effect of ICRF 193 on 3LL.

DOSE (mg/kg) ROUTE VEHICLE SURVIVORS ~

T/C s

0."3 i. p. DMSO 8/8 0.54 0.1 0.4 i. p. DMSO 7/7 0.37 0.1 0.5 i. p. DMSO 7/7 0.71 0.1

REFERENCE 1. Creighton, A.M., Hellmann, K., & Whitecross, Susan.

(1969). Nature, 222, 384-385.

NEW DERIVATIVES OF NITI0300REA WITH A HIGH 'lHERAPEllTIC INDEX FOR

CNCOS'mTISM AND n+1UNQSUPPRFSSICN

1 2 2 _3 J. L. IMBACH , M. HAYAT , E. CHENU , B. SERIDU-,

& G. MATHE2

1Laboratoire de Chimie Bio-organique, 34-M::>ntpellier, ;Institut de Gancerologie et d'Immunogenetique,Villejuif Centre Paul LanBrque, Cliniques St Eloi, 34-M:>ntpellier, France, and (EO~)

Clinically available nitrosoureas : BCNU (1,3-bis-2-chloroethyl) -l-nitrosourea (ICIG 1251), CCNU (chloro-2-ethyl)-1-cyclohexyl-3 ni­trosourea (ICIG 1109) and MeCCNU (chloro-2-ethyl)-1-(methyl-4-cyclo­hexyl)-3-nitrosourea (ICIG 1110) are powerful experimental ani cli­nical oncostatics (7, 10). '!heir action is probably related to nucleic acid alkylation as well as to alkylation that their lipo­philicity is essential in detennining the degree to which they cross cell nanbranes and the so-called "blood-brain barrier". '!he crossing of this barrier explains their evident activity in glioblastomas (9), especially when administered in carbination with another cyt0-static that also crosses this barrier, such as VM 26 (6, 9).

HCMever, their toxicity is high, especially in relation to nor­mal henopoietic cells (7, 10) ; this limits their use at doses below those which seem necessary for once-eradicating action.

Hence the need to synthesize rrore derivatives, Results obtained by Hansch (2) on the structure-activity relationship of a series of nitrosoureas having the general structure indicated in fig. 1 sug­gested that an increase in the hydrophilic content of these can­pounds might make it possible to obtain drugs which are rrore active and less toxic than BCNU or CCNU.

'!he same assunption led us to replace the R groups (cyclohexyl in the case of CCNU) by a sugar IIDlecule belonging to the ribose, xylose, or glucose series.

221

222

Fig. 1.

NO I

o = C .....----N - CH2

~-R

R = Cyclohexyl : CCNU

R = CH2 - CH2 - Cl BCNU

- CH Cl 2

R = Methyl-3 cyclohexyl : Methyl CCNU

J.L. IMBACH ET AL.

The ooject of this paper is to present the preliminary but encouraging results for the four following agents: a) (chloro -2-ethyl) -1- ribofuranosyl-isopropylidene-2'-3' paranitrobenzoate -5 ') -3 nitrosourea or RFCNU (ICIG 1105) ; b) (chloro-2-ethyl-l-dooxy-2' glucopyranosyl tretracetate-14,3' ,4' ,6')-3 nitro-sourea or GCNU (ICIG 1134) ; c) (chloro-2-ethyl) -1 (ribopyranosyl triacetate -2' ,3' ,4')-3 nitrosourea or RPCNU (ICIG 1163) ;

ICIG IR(KBr) cm-I n° ref. R FO

0 JJC=O .JJ amide .uNzO

1105 N02CS H5 CO2 <-->- 133 -139 1770 1515 1480

RFCNU 0 0 ;><.

ACtO

1580

1163 35-37 1785 15S0 1470

RPCNU 1530 OAc OAc

~ 1570

1164 39-40 1780 1550 14S0

XPCNU AcO 1525

OAe

A'£; 1134 OAe )..OAe 130-133 1750 1520 1470

GCNU AcO

Fig. 2. Chemical and physicochemical characteristics ot four nitrosoureas derivatives.

NEW DERIVATIVES OF NITROSOUREA

d) (chloro-2-ethyl)-1 (xylopyranosyl triacetate -2' ,3' ,4')-3 nitrosourea or XPCNU (ICIG 1164) (fig. 2).

223

'!hese agents were sul:mitted to two tests : the Ll210 leukemia curative effect (1) and the herrolytic plaque-fonning cell test (5) •

MA'lERIAL AND ME'IHOOO

1. Cbnpounds

Synthesis of the above four carpounds was carried out accord­ing to Montgarery' s general procedure (3), by reacting the appro­priate arninosugar on chloro-2-ethyl-isocyanate, and afteIWa.rd.s nitrosizing the urea thus obtained. '!he alcohol functions of the various sugars were blocked in order to increase the stability of the intenreiiate and final products.

Of these conpounds, GCNU has already been synthesized (3), and its non-acetylated hoIIDlogue was recently described by M:>nt­gooery (4). The synthesis of RFCNU (ICIG 1105) and RPCNU (ICIG 1163) have also been described (8). '!hat of XPCNU (ICIG 1164) has not yet been reported ; the correspondin:] urea has a melting point of 121-123°C, nitrosation being subsequently effected in fonnic acid.

The group of canpounds displays physico-chemical properties consistent with their structures; in addition to their melting points, the IR facts concernin:J the drugs used in this work are assenbled in fig. 2.

2. Screening

2,1. L1210 leukemia Test for Detecting Oncostatic Action (1)

PI (IEA/2 x C57Bl/6) mice were iroculated with 105 L1210 leukemia cells by the 1. P. route at day O. At days 1, 5 and 9, they were given 5, 10, 20, 30 or 60 rrg/kg of each carpound. The conpounds were injected as oil suspensions.

'!he IIDrtality of the aninlals was studied, and an autopsy in­dicated whether death was due to leukemia or the toxic action of the drug.

Results for the specified doses of each drug were expressed as T/C x 100 (T representing the rredian survival of the group of mice treated, and C, the rredian survival of the control group) • These percentages expressed prolongation of survival (P.S.). We consider a product active when P. S. 125 %. If, in a treated group, IIDre than 50 % of the aninlals are cured, the P.S. value of this group will be t:J'O •

224

2,2.

J.L. IMBACH ET AL.

The Harolytic plaque-fonning Cell Test (PFC) for inmuno­rrodulation detection (1)

Fl (Il3A/2 x C57B1/6) mice ~e given loP. inject:ions of fresh sheep red blood cells (SRBC) obtained frcrn the Pasteur Institute. The mice were born and kept in a pathogen-free aninB.l roan to pro­tect them from the effects of a microbiological envirorment. Eigh­teen animals were used for each drug and divided at randan into 3 groups of six animals. 'l\o.c groups were treated with a single drug injection, the animals were killed and the harolytic plaques counted.

The expression of the results is rendered by the ratio TIC, where T is the rrean nl.ll'!ber of PFC per spleen for the treated group, and C the mean nunber for the controls. '!hese ~ mean values are conpared by the Student-Fisher test. When TIC ratio is less than 1, the product is coosidered as irmrunosuppressive.

RESULTS

1. L1210 Leukemia Test

Fig. 3 shows the results for the specified doses of the 4 pro­ducts. Ordinates are P .S., and abscissa the different doses.

a>

200 CCNU MeCCNU

a_ ,_"_ ' _

~ IIIL. , . 5 30 60 5 30 60.

a>

200

150

125

100 ..ummmJ , , 5 30 60 60 5 30 60 ~ Efficiency interval mmmm Toxicity

Fig. 3. Effect of specific doses of nitrosourea coI1'(Ounds on Ll210 Leukemia.

NEW DERIVATIVES OF NITROSOUREA 225

'!he following remarks may be made: a) all four products are onoostatic on LI210; b) the highest activity for CCNU and methyl CCNU is ooserved at 10 ItIJ'/kg (P.S. = oQ) and toxicity is observed at 15 Il9Ikg; c) the highest activity of RPCNU and XPCNU is obsez­ved at 10 Il9Ikg and 15 Il9Ikg; d) the highest activity of RFCNU is ooserved at 15 ItIJ'/kg and 30 IllJ/kg ; toxicity only appears at 60 ItIJ'/kg.

Hence it can be concltrled that these 3 new nitrosourea deriva­tives are less toxic than CCNU and MeCnru. '!he fourth derivative, GCNU, is less active.

2. The Henolytic PFC Test

Results are indicated in Table I. '!he products were iImunosup­pressive when administered either before or after the antigen, ex­cept for GCNU which was only imnunosuwressive after antigen, and RFCNU which was mt immmosuppressive either before or after the antigen.

SUMMARY

Four new derivatives of nitrosourea : (chloro-2-ethyl)-1 (rilio furanosyl-isopropylidene-2' ,3' ,paranitrobenzoate-S')-3 nitrosou­rea (RF'O-ID), (chloro-2-ethyl)-1 (deaxy-2' gluoopyranosyl tetrace­tate-I' ,3' ,4' ,6')-3 nitrosourea (GCNU), (chloro-2-ethyl)-1 (ribo pyranosyl triacetate-2' ,3' ,4')-3 nitrosourea (RPCNU), (chlaro-2 ethyl)-l (xy10 pyranosy1 triacetate-2' ,3' ,4')-3 nitrosourea (XPCNU) have been suJ:::mitted to our screening for ancostatlln and imnunosup­pression. '!heir effects have been <XI1'pared to those of CCNU and nethyl-CCNU.

The effect on Ll210 leukemia and toxicity have been registe­red for each ooop:>nent for the follCMing doses : 5 ng, 10 ITg, 30 It¥J, and 60 ng per kg. CCNU, Me<XJ, RPCNU and XPCNU cure IOOI'e than 50 % of the animals at the dose of 10 ng/kg, a dose which is 0 % lethal. While the 100 % lethal dose is 15 ng/kg for CCNU and for MeCCNU, it is 30 It¥J/kg for RPCNU and XPCNU.

The 100 % lethal doses for RFCNJ and IG::NU are respectively 30 ng/kg and 60 ItIJ'/kg, which indicates a great increase in tolerance correlated with the structural IOOdificatians, but the doses which cure zrore than 50 % of the animals for L1210 are respectively 15 ng/kg and 30 mg/kg, which indicates a loss in the ancostatic effect associated with a loss in general toxicity. These new ClCllp:>­nents are immmosuwressive in the henolytic plaque fonning cell test.

TA

BL

E

I -

EF

FE

Cl'

OF

CC

NU

AN

D

CY

IHER

NI'I

'IDSO

UR

EA

S 0

0 T

HE

H

EM

)LY

TIC

PL

AQ

UE

ro~G

CE

LL

T

ES

T

BEF

OR

E A

NT

IGE

N

AF

TE

R

AN

TIG

EN

--

----

----

----

----

----

----

----

---

----

----

----

----

----

----

----

--IC

IG

DEN

:r1IN

ATI

CN

STA

TIST

ICA

L ST

ATI

STIC

AL

RATI

O

TIC

RA

TIO

T

IC

SIG

NIF

ICA

NC

E SI

GN

IFIC

AN

CE

1109

CC

NU

0

,13

-+

+ 0

,35

+

1110

M

eCCN

U

0,0

09

-+

+ 0

,39

+

1105

R

FCN

U

0,8

N

S 1

,73

N

S

1134

Q

NU

0

,87

N

S 0

,32

-+

+

1163

R

PCN

U

0,0

14

-+

+ 0

,3

-++

1164

X

PC

NU

0

,07

-+

+ 0

,37

+

-~

'---

-

+

P "

0,05

-+

+ p

-< 0

,01

I i , I I I I I

'" '" 0- !­ :- :s:: co ~ :::c

m

--i

l>

:-

NEW DERIVATIVES OF NITROSOUREA 227

REFERENCES

1. European Organi zation for Research on the Trea1:n'ent of cancer (E.O.R.T.C.). Screening Group. Handl:xJok of materials and rre­thods. Europ. J. Cancer, 1972, .!!' 185.

2. HANSCH C., SMI'lH N., ENGLE R. & WJOD H. Quantitative struc­ture-activity relationships and antineoplastic drugs : nitro­soureas and triazenoirnidazoles. Cancer Chamth. Rep., 1972, 56, part. 1, 443.

3. JOHNS'IDN T. P., McCALEB G. S., OPLIGER P. S., RUSSEL IAS'IER W. & MJN'IG:MERY J. A. Synthesis of };X)tential anticancer agents. 38. N-nitrosoureas. 4. Further synthesis and evaluation of haloethyl derivatives. J. Med. Chern., 1971, 14, 600.

4. JOHNS'IDN T. P., McCALEB G. S. & MJN'IG:MERY J. A. Synthesis of chlorozotocin, the 2-chloroethyl analog of the anticancer anti­biotic streptozocotin. J. Med. Chern., 1975, 18, 104.

5. MATHE G., HALLE-PANNENKO 0., FIDRENTIN 1., BRULEY-IDSSET M., KllME:L M. HIU 1. J. & OOURUT C. '!he second generation of EO~­ICIG experirrental screening for systemic immunity adjuvants. Its significance for cancer llnmunotherapy. A conparison of BCG and its hydro soluble extract. Europ. J. Cancer, 1975, in press.

6. MATHE G., SCHWARZENBERG L., POUILlARI' P., OLDHAM R., WEINER R., JASMIN C., IDSENFELD C., HAYAT M., MISSET J. L., MUSSET M., SCHNEIDER M., AMIEDL J. L. & DE VASSAL F. 'IWo epiJ;X:>dophylloto­xin derivatives VM 26 and VP 16213, in the trea1:n'ent of leuke­mias, henatosarcorras and lyrrpharas. Cancer 1974, 34, 985.

7. MATHE G. & KENIS Y. La chimiotherapie des cancers (leucemies, herratosarCCl!Ies et turoours solides. 3rd ed. I Paris, 1975, Expansion Scientifique.

8. IDNTEID J. L. & IMBACH J. L. Synthese de nouvelles nitrosourees a visee antineoplasiques. C. R. Acad. Sci., Paris, 1974, 279, 809.

9. POUILLARr P., SCHWARZENBERG L., AMIEL J. L., MATHE G., HmUENIN P. M:>RIN P., MIDN A., LAPARRE Ch. & PARIDI' R. Conbinaisons chirniotherapiques de drogues se };X)tentialisant. III. Applica­tion aux turreurs prirni ti ves du sysrerre nerveux central. NJuv . Presse Med., 1975, i, 721.

10. WASSERMl-\N T. H., SIAVIK M. & CARl'ER S. K. Review of CCNU in clinical cancer therapy. Cancer Treat. Rev., 1974, 1:., 131.

228 J.L. IMBACH ET AL.

11. WHEELER G. P., 1n'JOON B. J., GRIMSIEY J. A. & LIDYD H. H. Inter­relationships of roIre chanical, physioochanical, and biological activities of several 1- (2-haloethyl) -l-nitrosoureas. cancer Res. 1974, 34, 194.

EFFECI' ON Ll2l0 IEUKEMIA, ON ANTIBODY FORMING CELIS, AND ON

MACROPHAGE CYTOrOXICITY OF ELLIPTICINE AND 3 DERIVATIVES

.-G. MA'lHE, M. HAYAT, E. CHENU, I. FLDRENTIN, M. BRULEY-

ruSSET, M. JANOT, P. parIER, N. DAT-XUONG, A. CAVE, T.

SEVENET, C. KAN-FAN, J. POISSCN, J. MIET, J. I.E MEN,

F. I.E GOFFIC, A. CDUYETI'E, A. AHOOD, L. DAL'ION, T. CDNNORS

Institut de Cancerologie et d'IImnmogenetique, Villejuif and the ExperinEntal Screening Group of E.O.R.T.C.

In 1970 Mathe et al. (8) published the oncostatic effect of 9-methoxy-ellipticine (lactate) extracted from Ochrosia borbonica, on human acute ~eloid leukaemia. Garcia-Giralt and Macieira­Coelho (4) showed that this agent, active on nrurine turrors (lLl4), inhibits rnA synthesis strongly. But they observed that it' also blocks noticeably RNA and protein synthesis, which probably explains why it is not a cycle dependant agent.

The effect of 9-methoxy-ellipticine ona human tunor, as well as the weakness of this effect and its action on cells in Go, caused us to m:xlify its chemical formula.

The Gif-sur-Yvette group of chemists organized (owing to a C.N.R.S. "Recherche Cooperative sur Programne", jointly conducted with the Institut de Cancerologie et d'IImnmogenetique (I.C.I.G.) Group) :

a) a systemic search for other Ochrosia species, especially in South Pacific Islands, for the preparation of agents by extraction ;

b) and speculated for the preparations of others by synthesis or semi -synthesis on known frequent metabolic routes in phannaco­kinetics (see 13), specially hydroxylation in position 9 of the benzene nuclei oi' ellipticine, and the 0 derrethylation of 9-rnetho­xy ellipticine. These two processes conducted to the sane cart-

229

230 G. MATHE ET AL.

pound : 9-hydroxy-ellipticine. The latter was prepared by derre­thylation of 9-nethoxy-ellipticine with pyridinium hydrochloride.

The other groups also prepared the ellipticine base and several derivatives (Dalton, Connors).

'Ihese carpounds are sul::tnitted not only to the screening tests available at the "Institut de cancerologie et d I Itmunogenetique" for the C.N.R.S. "action cooperative sur programne" and for the "Experi­zrental Screening Group" of E.O.R.T.C. but also to other tests avai­lable in other centers working for this group (3).

Nevertheless, we will only publish in this paper the results obtained on L 1210 leukaemia (because the sensitivity of this expe­riIrental turror is the nearest to that of human leukaemias) , and on the haaoolytic plaque fonning cell (H. P .F • C. ) test, because it is the IlOSt sensitive one to detect the possible imnunosuppressi ve ac­tien of a c:x::npound, which represents a severe side effect in cancer chaootherapy (7), but may make it useful in transplantation.

One of the coopounds, 9-hydroxy-ellipticine, was given by Dat­Xuong, Ie Pecx:1 et a1. who tested it also and found it active on L. 1210 leukaemia (12).

MATERIAIS AND ME'lHODS

'!he nanes of the carpounds, their fonnulae, the dates of their screening laboratOl:Y of the Institut de Cancerologie et d I Jnmmoge­netique are zrentionned on Figure 1.

L 1210 leukaemia test, as we used it, is the one described by E.O.R.T.C. (3), but the carpounds are given intraperitoneally on days 1, 5 and 9, and the results are expressed in prolongation of sw:vival (PS)

'1he H.P.F.C. test is the Jeme test (5), but the results are expressed according to its adaptation by Mathe et ale (9) to the screening of imnuni ty systemic adjuvants.

RESULTS

'!he results are given on Figure 1.

Four CCllP:>l.mds o~ of 20 are able to prolong the survival in mice which received 10 of L 1210 leukaemia cells by thEi intraperi­toneal route, significantly c:x:a:rpa.red to that of the controls. They are, in chronological order according to the dates of their scree­ning :

a) 9-nethoxy-ellipticine (lactate), which gave a PS of 130 % (signj-

EFFECTS OF ELLIPTICINE AND THREE DERIVATIVES 231

ficant (S) at 5 %),

b) e11ipticine base, which gave a PS of 115 % (Dat-Xuong) to 125 % (Dalton) (S at 1 %).

c) 9-hydroxy-e11ipticine, which gave a PS of 164 % (S at 1 %)

d) and 9-amino-e11ipticine, which gave a PS of 120 % (S at 1 %)

These two last compounds were screened simultaneously and 9-hydroxy-ellipticine is found to be significantly more active than 9-amino-ellipticine (S at 2 %).

All these compounds which are oncostatic for L 1210 leukaemia are immunosuppressive in the H.P.F.C. test (see Figure 1). The 9-hydroxy-ellipticine is the most immunosuppressive of these 4 compounds (100 times more than 9-amino-ellipticine).

DIS C U S S ION

Ellipticine (base) and three of its derivatives have a significant oncostatic effect on L 1210 leu­kaemia expressed as prolongation of survival. There is a significant difference, though moderate, between 9-hydroxy-ellipticine and the others, and its effect on L 1210 leukaemia was first observed by Le Pecq et ale (12) •

As we know that 9-methoxy-ellipticine (lactate) is slightly efficient on human myeloid leukaemia, there is a hope that ellipticine itself or other derivatives of it, specially 9-hydroxy-ellipticine, may be more active in human neoplasias, especially on acute myeloid leukaemia.

Because of the immunosuppressive action of these compounds, the patients given them in a phase 1 clini­cal trial (6, 10) should systematically undergo an im­mune investigation for control.

But this immunosuppressive effect of these four compounds detected by the H.P.F.C. test let us hope that they may be of some use in transplantation. Their effect on skin graft and graft versus host reac­tion will be published later.

SUMMARY

In 1970, Mathe et ale published the oncostatic ef­fect of 9-methoxy-ellipticine (lactate) on human acute myeloid leukemia. Garcia-Giralt and Macieira-Coelho

232 G. MATHE ET AL.

Acute

_of HPFC survival ICIG EORTC Source Formula Compound 001. ~~ on Ll210 TIC ... . ...

"" 180 690 Lt Men CM,o()r;:(X) ... CM1.Ct4OK-COOM 0,03

! ' 9- MethollY -ellt phClne (lactic solt I 5-68 200 30S 5% S I ....

, "" "" GIF ~'COOH-ICHOM)I:-COOH

119A 1130 IC.y') Elhpllclne(tortanc salt) 1-71 240 100 NS ! ' , .... 110 1131 GIF oiO ElliptiC,"! bose (synthetic)

0,003 1-71 150 2551%

(Oot Xuong '. ' S IGloo M CM,

111 1132 Gtib'~ Eillphclne hydrochloride (synthetic) 4-71 115 15 S 1% . , H e"l

co, 112 1133 CH10O;W1II

~)., ,. 9-Methoxy- eillptlcine base(synfhetlc) 4-11 150 101 NS

HeMs

" 113 1134 CH'O~'HCl'Hro 9-MefhoKY- eillptlcine (hydrochlol'lde) 4-71 180 101 NS . ' (synthetic)

H CH,

OzOJo N-BenzyI3-(2!.mefhyI3!.lndolyllmethyl 5-71 .1!500 100 NS 116 .. Le Gofflc 4-Plpendone

... 711 Le Gofflc 0:1))'" N-SenzyI3-8-l3'-,rnIoI,llethyi] 5-71 )1500 110 NS .. " 0 ~ 0 I 4-Plpendone

"" 778 .. VW-a N-Senz" 3-E'-13',rnIoI,'lethyi] 5-71 160 100 NS

~ 5." 4-hydroxy 4-ethlnyl Plpendine

119 CH'O~M .. I I

" ' 15'Metho" 3',rnIoI"l.P-p,,,,,t methan~ 5-71 1200 100 NS ,

780 .. o-io I ~I (3-lndolyl )~pyrldyl methanol(synthe1lc) 5-71 1500 100 NS

, 781 ""

Similar to Dolton GctO Eillpticme bose 1218 4-71 · 125 1% 770 (Austraha) ~ ~ '"

~,

782 1279 ~ " ' ~ " 11- Oesmethyl-elhptlcme 4-71 · 100 NS , <0,

183 1280 0:.&' " ' ~ " "'>""

N- Methyl- .llIptlcme 4-71 · 108 NS

Figure lLa The 3 fonus and 19 derivatives or analogs of ellipticine tested and the dates & results of the tests.

EFFECTS OF ELLIPTICINE AND THREE DERIVATIVES

784 1281 Dalton

785 1282

862 Le GoffIC

863 Similar to

782

864

86~

879 1533

899

~~-CH2 V,)oVO

H ~ I

(tfC" I I '" ;'.

i7"o.o,methyt.e,llpttcme

9- Methyl -eillptlcm!

5,11- Desdtmethyl- elhpticme

11- Desmethyl-elltpttClne

N-Benzyl 3-(3'-,ndolyl) methyl4-plperldone

N- BenzyI3-(2'-ethyl 3'-lndolyl) methyl4-plpendone

f3- (3'.lndolyl ) methyl pyridine

4-71 • 104 NS

12-71 1000 89 NS

12-71 350 94 NS

12-71 850 KlO NS

12-71 950 105 NS

3-72 600 100 NS

Similar to It. Dot XuonCj N-Methyl elllptlClne

783

900

927

928

929 1754

1089

N-lsopentyl- eillptlc,"e 4-72 .. 100 NS

3~-melhyl~phenyl)elt!yl<mlOO-~ 6-72 160 100 NS 1,4 dimethyl carbazole

Connors H~~~ V,~

H eH,

9- Hydroxy - elllptICI"!

JlNotevaluited (insuilielen! supply I

** Number not yet attributed T medliln of surVival In trealed group

*** PSI s eva I uited oy Ie" ~ median of survival 11'1 conlrol 9roup • 100

6-72 100 164 S III 0,001 S I%.

11-73 • 120 SillS O,,~

11*** I· mun number of PFC per spleen 01 lesled mlc@ I mean nurpber or PFC per spr~en or tontroh

Figure lib

233

234 G. MATHE ET AL.

described its effect on mA, RNA and protein synthesis, and its action on cells in Go. Since that date, other CC41pJunds of these series have been prepared by extraction or synthesis or semi -synthe­sis in the hope of obtaining cycle-dependent agents. 'lhree folllS and 19 derivatives or analogs of ellipticine have been prepared and screened on L 1210 leukemia test as ~ll as on the hem::>lytic pla­que foIIning cell (HPFC) test, the allogeneic skin graft test and the macrophage cytotoxicity test for the conp::>unds active on the latter. L 1210 leukemia presenting the nearest sensitivity to that of hunan leukemias, we shall only zrention here the four cx:.at{lOunds which are significantly active on it : they are, in chronological order according to the dates of their screening: 9-mathoxy-ellip -ticine (lactate), ellipticine base, 9-hydroxy-ellipticine and 9-amino-ellipticine. These four a:npJunds are inmunos~essive in the HPFC test. '!hey decrease macrophage cytotoxicity at high ooses while they increase it at snall ooses. IbYlever, they do not prolong allogeneic skin graft.

REFERENCES

" 1. BRFARD J., WEINER R. S., OI.LHAM R. K. & mTHE G, Irmune res~n-siveness in acute lynphocytic leukemia patients under chem::>the­rapy and immmotherapy : a preliminary report. In : Cmplications of cancer cherrotherapy, G. Mathe ed., 1 vol., Springer Verlag, Heidelberg, 1974.

2. BURrnENAL J., Murine and hunan leukemias. Pmc. of the VI Int. COngo on carparative leukemia, Nagoya, Ja~n, Sept. 1973 (to be Published) •

3. European Organisation for Research on the Treatmant of Cancer (EORn::), Screening Group. Handbook of materials and methods, Europ. J. cancer, 1972, ~, 185.

4. GAK::IA-GlRALT E. & MACmlRA-<DELHO A. Methoxy-9-Ellipticine II. Analysis in vitro of the mechanism of action. Europ. J. Clin. BioI. Res., 1970, 15, 539.

5. JERNE N. K., NORDIN A. A. & HENRY C.C. '!he agar plaque tech­nique for recognizing antibody producing cells. P. 109, in Cell bound antibodies, vol. 1, Wistar Institute, Philadelph..i.a, 1963.

'" 6. MATHE G. Clinical examination of drugs : a scientific and ethi-cal challenge. Biomedicine, 1973, 18, 169.

;'

7. MA'IHE G. Prevention of the chaIDtherapy oonplications : time, toxicity, phannacokinetic, phanracodynamic and logistic factors, in Cmplications of cancer chenDtherapy, G. Mathe, ed., 1 vol. Springer Verlag, Heidelberg, 1974.

EFFECTS OF ELLIPTICINE AND THREE DERIVATIVES 235

/

8. MATHE G., HAYAT M., de VASSAL F., SOfWARZENBEffi L. I SCHNEIDER M. , SCHUJMB~ J. R., JA£MIN C. & IDSENFEID C. Metmxy-9-ellipti­cine lactate. III. Clinical screening: its action in acute leu­kemia. Europ. J. C1in. BioI. Res., 1970, 15, 541.

9. MATHE G., KAMEL MK, DEZFULIAN M., HALLE-PANNENKQ 0. & I3OURUI' C. An experimental screening for "systemic adjuvants of irrmmity" applicable in cancer inmunotherapy. Cancer Res., 1973, 33, 1987.

10. MA'!'H€ G. & KENIS Y. Logistics of therapeutic trials : the exam­ple of cancerology, Biorredicine, 1973, 18, 181.

11. LE MEN J., HAYAT M., MA'lHE G., GUILIDN J. C., CHENU E., HUMBIDr E., & MASSCN Y. Methoxy-9-ellipticine lactate. I. Experimental study (anCOBtatic and immunosuppressive actions : preclinical pharnaco1ogy). Europ. J. C1in. BioI. Res., 1970, 15, 534.

12. LE PECQ J. B., GOSSE C., [ll\T-XUONG N. & PIOLETTI C. Un nouveau compose antiturroral : l'hydroxy-9-e11ipticine. Action sur la leuc8mie L 1210 de la souris. C.R. Acad. Sci., Paris, 1973, 277, 2289.

13. PACKEVO H. La phanraco1ogie rro1eculaire, 1 Vol., Presses Universitaires de France, Paris, 1973.

14. SVOOODA G. H., POORE G. A. & MCNIFORlI' M. L. Alkaloids of Ochro­sia maculata Jaa:r. (Ochrosia borbonica Qnel) - Isolation of the Alkaloids and study of the anti tunor properties of 9-rnethoxy­E11ipticine, J. Phanra. Sci., 1968, 57, 1720.

ANTITUMOR ACTIVITY OF DAUNORUBICIN DERIVATIVES

G. JOLLES, R. MARAL, M. MESSER and G. PONSINET

Rh8ne-Poulenc,Centre Nicolas Grillet, Vitry s/Seine,

France

Daunorubicin is an anthracycline antibiotic extracted from Streptomyces coeruleorubidus and from S. peucetius. Its activity was proved against experimental mouse tumors and against human tumors. It is widely used for the treatment of acute leukemia in man (BERNARD et al., 1969).

In order to increase the activity and decrease the toxicity of the drug,various derivatives have been prepared by reactions on the aglycone moiety and the amino and keto grou~of daunorubicin.

o

o

OH

OH OR

COCH3

•. OH Daunorubicin

Aglycone

I - DERIVATIVES OF THE AGLYCONE

-R H§J -R = -H NH2

Daunorubicin forms with DNA a complex which seems to be responsible for its cytotoxicity (CALENDI et al., 1965). It was proved that, while the aromatic chromophore is intercalated between the base - pairs of DNA, the amino group forms a salt with the phosphoric acid function of DNA (BARTHELEMY-CLAVEY et al.,197~. Thus modifications of the distance between the amino group and the quinone could modify the interaction with DNA and thus could improve the cytotoxicity.

237

238 G. JOLLES ET AL.

-R i.p. s.c. M. T.D. L12lO CI M.T.D. S-18.Q

-CO-c::r ::>U lUU 7. 50 1.0

-CO-CH2NH2 100 100 % 50 1.0

-CO-( CH2 )2 NH2 50 llO % 50 0.10

-CO-( CH2 )3 NH2 20 100 %

I -CO-(CH2)4 NH2 35 96 % 35 0.60

-CO-CH2(NH2)CH2CH3 100 100 % 100 0.75

-CO~NHCOlC:)rNH' 35 89 % 150 1.0

-co NH2 25 140 % 25 0.10

- COOCH2NH2 6 140 % 25 0.30

-CUNH 2 l2.5 100 % 50 0.20

- COU NH2 l2.5 100 % 50 0.35

-C0lC:)rN( CH3) 2 20 llO % 37.5 0.45

- COO NH2 l2 .5 125 % 25 0.10 OCOCH3

-C0lC:)rNH2 10 180 % 6 25 0.15

-Coa l2.5 156 % 3 25 0.10

-C0"Oo~2 25 150 % 4 50 0.30

-C0lC:)rNHCH2S03H l2 .5 150 % 4 50 0.20

- C0-o-NH2 7.5 155 % 30 0.10 (13-dihydro)

Table I - Derivatives of the aglycone of daunorubicin

M.T.D. Maximal Tolerated Dose; dose (i.p. or s.c.) which induces neither weight loss nor mortality ; mg/kg ; mice

L12l0 Leukemia L12l0 (103 cells grafted i.p.) mean survival time treated/control x 100 ; mice treated at M.T.D.

CI Chemotherapeutic Index (leukemia L12l0) ratio : LD50/ED150

S180 Sarcoma 180 (grafted subcutaneously) mean weight of a tumour treated/control; mice treated Et: M.T.D.

ANTITUMOR ACTIVITY OF DAUNORUBICIN DERIVATIVES 239

a-Aml.no aCl.d used Lp. s.c. M. T.D. L12l0 CI M.T.D. S180

L leucin 1.2 163 7- 10 12.5 0.10 D leucin 10 150 % 4 37.5 0.10 L leucin (13-dihydro) 5 170 % 3 18.5 0.05 L phenylglycin 2.5 140 % 3 6 0.10

Table II - N-a-Aminoacyl derivatives of daunorubicin

Moreover, among the inactive metabolites of daunorubicin in blood serum is the above aglycone (BACHUR, 1971). In order to prepare a more stable conjugate of the aglycone, basic chains were attached to the secondary alcohol function of the aglycone through an ester linkage, more stable than the glycosidic one.

To obtain these compounds N-protected aminoacids were reacted with the aglycone in the presence of benzenesulfonyl chloride in pyridine (RHONE-POULENC, 1968 b). The protective groups, either -COOC(CH3)3 or -COOCHZ-C6H4-0CH3 were chosen for their easy removing by mild acidic treatment.

Table I sums up the structure, toxicity and activity of the new derivatives. One can see from these results that among the most active are the compounds with an amine function included in or attached to a six membered ring and roughly the same geometry as daunorubicin. A chemotherapeutic index close to the value calculamd for daunorubicin could even be obtained. However the specific activities were in each case much lower.

II - DERIVATIVES OF THE AMINO GROUP OF DAUNORUBICIN

When daunorubicin was treated with a amino-acids N-carboxy­anhydrides, amides were obtained directly with a free -NH2. Table II presents the figures obtained for the compounds which have been prepared with leucin and phenylglycin (RHONE-POULENC, 1967 b).

Several N- acylated derivatives of daunorubicin were recently described (YAMAMOTO et al., 1972 and WILSON et al., 1975) : most of them have low or no activity. The high level of activity of the present analogues can be related to the reintroduction of the free primary amino group.

240 G. JOllES ET Al.

Reagent used : H2NNH-K 1. .p • s.c. -R' = M. T.D. Ll2lO CI M.T.D. Sll:SO

-COCH3 7.5 185 % 7 15 0.20 -COC2H5 5 195 % 10 5 0.20

-COCH(CH3)2 2.5 135 % 5 0.25

-CO(CH2h4CH3 1.5 160 % 6 l2 .5 0.10

-COC6H5 1.5 190 % 12 3 0.15

-CO-C5H4N 5 190 % 10 10 0.05 -CO-C6H4( -pOCH3) 5 190 % 5 5 0.05

-CHO 5 185 % 7 l2 .5 0.05 -C(=NH)NH2 15 150 % 5 15 0.60

-CSNH2 15 200 % 10 l2.5 0.10 -CSNHCH3 7.5 170 % 9 15 l2 .5 -CSNH (CH2 h 1 CH3 5 170 % 6 50 0.20

-CSSCH3 3 170 % 5 12.5 0,30 Duborimycin 3 160 % 5-8 7.5 0.25 Daunorubicin 0,75 180 % 5-10 2.5 0.10

Table III - Derivatives of the keto group of daunorubicin

III-DERIVATIVES OF THE KETO GROUP OF DAUNORUBICIN

a) Rubidazone and Analogues

Daunorubicin reacts through its keto group with many reagents leading to hydrazones, oximes, (thio)-semicarbazones (RHONE­POULENC, 1967 a),

Table III shows that most of these compounds retain the activity of daunorubicin while some have a lower toxicity. In this series the best example is given by the benzoy1hydrazone of daunorubicin or rubidazone (MARAL et aI" 1972), the clinical value (BERNARD et al" 1972) and the lesser cardio-toxicity (YOUNG, 1974) of which have been established.

b) Duborimycin

Among the other anthracyclines elaborated py S. coeruleorubidus is 13-dihydrodaunorubicin or duborimycin, 20 798 R.P, (RHONE­POULENC, 1968 a). This compound has also been described under the name of daunorubicinol as one of the metabolites of daunorubicin in the blood (HUFFMAN et aI" 1972) and it can be prepared by microbiological reduction of daunorubicin by Corynebacterium simplex (RHONE-POULENC, 1973), An extensive experimental study showed its activity on a large number of mice tumors (MARAL, 1975) and preliminary clinical trials showed promising results (CHAUVERGNE et al., 1975).

ANTITUMOR ACTIVITY OF DAUNORUBICIN DERIVATIVES 241

CONCLUSION

A survey of these results leads to the conclusion that within certain limits major changes of the daunorubicin formula are compatible with high antitumor activity : the quinone system and the presence of a basic group in an appropiate geometric position seem to have a major importance whereas other functions, especially the keto group on the anthracycline ring or the hydroxy group on the sugar moiety, may be deleted.

The semi-synthetic approach to new derivatives is particularly aimed to find analogues with a better chemotherapeutic index and lower cardiotoxicity. The toxicological behaviour of several of the compounds described above is now actively investigated with these objectives.

REFERENCES

BACHUR, N.R. (1971), J. Exp. Pharm. Exp. Ther., 177,573 BARTHELEMY-CLAVEY, V., MAURIZOT, J.C. and SICARD:-P.J. (1972),

Biochimie, 55, 859 BERNARD, J., JACQUILLAT, Cl., BOIRON, M., WElL, M., GEMON, M.F., IZRAEL, V., SCHAISON, G. and DELOBEL, J. (1972), Nouv. Presse

Med., 1, 2149 BERNARD, J., PAUL, R., BOIRON, M., JACQUILLAT, Cl. and MARAL, R. (1969), Recent Results in Cancer Research, 20 (1969) CALENDI, E., DI MARCO, A., REGGIANI, M., SCARPINATO, B. and VALENTINI, L. (1965), Biochim. biophys. Acta, 103, 25 CHAUVERGNE, J., CARTON, M., BERLIE, J., BRULE, G., CLAVEL, B., GARY-BOBO, J., GUERRIN, J., KLEIN, T. and POMMATEAU, E. (1975), to be published HUFFMAN, D.H. (1972), Cancer Res., 33, 600 MARAL, R. (1975), to be published MARAL, R., PONSINET, G. and JOLLES, G. (1972), C.R. Acad. Sci.,

275 D, 301 RHONE-POULENC (1967 a), Fr. Pat. 1,578,722, Appl. 18 Oct. 1967 RHONE-POULENC (1967 b), Fr. Pat. 1,578,734, Appl. 28 Nov. 1967 RHONE-POULENC (1968 a), Fr. Pat. 1,583,752, Appl. 5 March 1968 RHONE-POULENC (1968 b), Fr. Pat. 1,593,555, Appl. 15 July 1968 RHONE-POULENC (1973), Fr. Pat., Appl. 73, 42191 27 Nov. 1973 WILSON, D.W., GRIER, D., REIMER, R., BAUMAN, J.D., PRESTON, J.F. and GABBAY, E.J. (1975), J. Med. Chern., in press YAMAMOTO, K., ACTON, E.M. and HENRY, D.W. (1972), J. Med. Chern.,

15, 872 YOUNG-,-D. (1974), personal communication.

NEW ANTITUMOUR ANALOGUES OF CYTOSINE ARABINOS]DE A.~ THE EFFECT

AGAINST MOUSE LEUKEMIA L12l0

Michiko Aoshima, Shigeru Tsukagoshi, Yoshio Sakurail ): Jun·-ichi Oh-ishi, ¥inoru Akiyama, Torao Ishida2) Cancer Chemotherany Center, Japane~j Foundation for Cancer Research, Toshima-ku, Tokyo ; Technical Research Lahoratory, Asahi Chemical Industry Co., Ltd., Fuji, Shizuoka2), Japan

Cytosine arabinoside is one of the most reliable drugs for treatment of leukemia and lymphoma, and is also used for the treat­ment of solid tumours as a component of their combin"ation treat­ments. In order to find a better congener of this drug, numerous derivatives have been widely examined for their antitumour activity. This presentation deals with the anti tumour effect of a series of Nll'-acyl derivatives of cytosine arabinoside.

In 1968, Fox and others reported the presentation and anti­tumour activity of N4-acetylcytosine arabinoside, but the deriva~ tives acylated at N4_position with lower fatty acids were not highly effective, while the acyl derivatives with fatty acids of long chain length proved to have a ~reat efficacy against L12l0 mouse leukemia as shown in Table 1. Oleoyl derivative Nas less effective than stearoyl one.

NJ:. ~( .. J N

HOH2V?~

~ + (RCO)20

OH

H20. DIOXANE ,

o •

tSCA

HOH2CV"0~

~ OH

Fig. 1. Synthetic method ofN4_acyl ara-C.

243

244 M, AOSHIMA ET AL.

it Table I, Effect of N'-acyl ara-C Table 2. Effect of N4-, 5'­

di-substituted derivatives of ara-C on the survival time of mice bearing leukemia L12l0.

on the survival time of mice bearing leukemia L12l0.

R (N4-) R' (5'-) TIC % •

CNH2N+1CO 100 200 400

(N+1) (MG/KG)

4 (BUTYRYU H 131 131 139 5 (VALERYL> H 133 171 191

R (N4_)

CNH2N+1CO

(N+ 1)

16 (PALMITOYU 17 (I1ARGAROYU 18 (STEAROYU

R' (5'-)

PHOSPHATE

TIC % •

100 200 400 (MG/KG)

262 267 88 284 323 188 321 362 104

6 (CAPROYL> H 125 133 150 -------------------------------------------------8 (CAPRYLYU H 123 127 163 16 (PALMITOYLl 221 296 129

10 (CAPRYU H 159 188 284 12 (LAUROYU H 177 272 296 14 (MYRISTOYU H 2ll 191 90

17 ( I1ARGAROYLl 250 254 125 18 (STEAROYLl SODIUH- 250 313 200 20 (ARACH IOOYLl PHOSPHATE 213 250 166

15 (PENTAllECANOYU H 246 309 91 22 (BEHEIIOYLl 204 221 204 16 (PAUlITOYL> H 361 242 78 -------------------------------------------------17 (INGAROYL> H 405 152 99 18 (STEAROYL> H 329 123 III 18 (STEAROYL>

19 (IOWlECMOYL> H 298 260 134 20 (ARACHIOOYU H 313 363 329 22 (BEHENOYU H 241 298 342 18 (STEAROYLl

cls-9-C17H33CO (OLEOYL) H 218 214 109 ---------------------------------------------- 16 (PALM 1T0YLl

17 (PIARGAROYU 18 (STEAROYLl

ARA-C CYCLO-C

* inoculation treatment

127 144 159 191 184 163

16 (PALMITOYLl 5 ip 17 (PlARGAROYLl 10 cells/mouse,

18 (STEAROYLl day 2 & 6, ip

*inoculation treatment

Table 3. PreliminarY4estimation of the toxicity of N -acyl ara-C.

DEAD MICE I 3 MICE

(OMPD . 3000 1000 500 300 (MG/KG)

(10 ----- 3 ~ (12----- ) 2 0 0 (14----- 3 3 3 (15----- 3 3 3 0 (16----- 3 3 0 (17----- 3 3 0 0 (18----- 3 3 0 0 (19----- 3 3 0 0 (20 ----- 0 0 0 0 C22 _nn 0 0 0 0

AIIIlNIUH-PHOSPHATE 284 309 125

CHOLlNE-PHOSPHATE 275 304 238

DIETHYL 204 235 167 - 317 366 106

GLYCINATE 275 321 125

DIETHYL- 260 265 167 AHINO- 248 313 199

PROPIONATE 305 345 106

5 10 cells/mouse, ip day 2 & 6, ip

ANTITUMOUR ANALOGUES OF CYTOSINE ARABINOSIDE 245

The samples were synthesized by acylation of cytosine arabino­side by use of about two-fold equivalent amount of the corresponding acid anhydride in the presence of a great excess of water and dioxane, as shown in Fig. 1.

Additional substitution at 5 1 -position of the sugar moiety by anionic or cationic group increased their solubility in water but did not enhance their antitumour effect as indicated in Table 2. Sodium salt of 5 1-phosphate of stearoyl derivative manifested a haemolytic activity, though it showed a high activity on L12l0 tumour.

So far as the saturated fatty acid derivatives are concerned, the optimum length of fatty acid chain was found to be between C16 and.Cl9 , as seen in Fig. 2, but as shown in Table 3, the highest tOX1Clty on normal CDFI mice was detected at C14 . It was worth noting that the most toxic derivative has a shorter acyl chain than the most effective one.

From these findings, 3 derivatives, palmitoyl, margaroyl, and stearoyl derivatives were presently chosen for further investigation. Dose response of these 3 compounds on L12l0 inoculated in CDFI mice, when injected intraperitoneally on day 2 and 6, is demonstrated in Fig. 3. The optimum dose was found between 50 and 100 mg/kg/day. Dose range between minimum effective and optimum dose was fairly wide.

The dose responses with a single injection on day 1 is shown in Fig. 4, in which the maximum T/C values were nearly the same as in the case of 2 injection on day 2 and 6. When they were injected 9 times daily from day 1 to 9, dose response indicated in Fig. 5 was revealed. The maximum T/C values were the same as in above 2 schedules of administration, although the daily doses were as small as 15 to 25 mg/kg.

In short, the maximum effects of these 3 compounds, which were approximately 300% of T/C rate, were similarly attained by the total dose of nearly 200 mg/kg, independent of schedules of administration. The result of experiments is summarized in Table 4. The effect of these compounds by routes of administration other than intraperito­neal injection is shown in Table 5. The palmitoyl derivative showed an antitumour effect by intramuscular or subcutaneous injection as strong as that by intraperitoneal injection with the same dose level and same schedule of administration, but the stearoyl derivative exhibited a fairly good effect even by oral administration on day 2 and 6 with 200 mg/kg/day dose. Correct schedule and dose of these derivatives by oral administration remain to be determined by fur­ther experiments.

246

<T/a) IlOO

200

100

M. AOSHIMA ET AL.

11m 2 I 6. !P. )!DeJY!MY

./ /e ------~ .. "'4e_ .. ea'-----------------------------------------,-------

Fig. 2. Structure-activity relationship of N4-acy1 derivatives of ara-C.

* number of carbon atoms in acyl groups

<TItS)

IlOO

200

100

o

pm 2 & 6. IP

0: t16 o : tIl 6: t18

1.5 3.l 6.3 12.5 25 50 15 100 200 IlOO

(l1li111)

Fig. 3. Dose response curve for the effectiveness of N4_acy1 ara-C against L1210.

ANTITUMOUR ANALOGUES OF CYTOSINE ARABINOSIDE

<TICS) IlOO

300

200

100

!lAy 1 QI1.Y

--.. --. o ~99~-~I"'':''''""~222~--::m=---:500=---=750=-

(""KG)

Fig. 4. Influence o! the schedule of treatment with N -acyl ara-C on the life-span of mice bearing 11210.

<TICS)

IlOO

300

Dty11-9

o : C17 6: C18

~LY><\a: c16

200 .. ____________________________ i~;

o 6.6 9.9 15.0 22.0 31.0 SO.O 75.0

(MlWDAY)

Fig. 5. Influence of the schedule of treatment with N4-acyl ara-C on the life-span of mice bearing 11210.

247

248

Table 4. Optimum dose of N4-acyl ara-C for the treatment of L12l0 bearing mice.

SCHEDllLE g~~~L TOTAL TICS COIIPD. OF TREATllENT DOSEo DOSE ••

DAY 1 1l1li 1l1li 338 C16 DAYS 2 & 6 75 ISO 360

DAYS 1- 9 22 198 m -------------------------------------

DAY 1 222 222 337 C17 DAYS 2 & 6 100 200 3811

DAYS 1- 9 15 135 315 .. - .. __ ... - ... _ .... _---------------_ ... _--------

DAY 1 222 222 372 C18 DAYS 2 & 6 75 ISO 364

DAYS 1- 9 22 198 330

°wr./KG/DAY .OIlG/KG

M. AOSHIMA ET AL.

Table 5. Effect of N4-acyl de­rivatives of ara-C on leukemia L12l0 by sc, im, or po routes.

TIC I 0

CIJIIIDS. ROUTES 100 200 qoo (IIG/KG)

N"-PAIJIITOYL- SC 273 m 295 ARA-C UI 282 253 256

PO 119 12" 132 -----------------------------------------"" -STEAROYL- PO 128 158 151 ARA-C

*inoculation 105cells/mouse, treatment day 2 & 6,

It is well known that cytosine arabinoside promptly deaminated in vivo, and the level of concentration in the blood does not last long, which accounts well for the precise schedule dependency of cytosine arabinoside.

Investigations on the distribution and the fate of these N4_ acyl derivatives in vivo is in progress, but as anticipated from their schedule independency, some of the N4-acyl derivatives of cytosine arabinoside might possibly be developed as a new antitumour agent with a characteristic desirable for practical application.

ip

EXCEPTIONAL RESPONSES TO CHEMOTHERAPY AND/OR HORMONO-

THERAPY OF CASES WITH GENERALIZED CANCER

D. Razis, M. Constantoulakis, M. Dimitriadis, A. Athanassiou, T. Messaropoulos

Diagnostic and Therapeutic Institute of Piraeus, 51, Botassi Street, Pi raeus 30, Greece

SUMMARY

Exceptional responses of several cases of generalised malignant lymphomas and carcinomas are reported. These unusual responses may imply either that we do·not always use the available drugs optimally, or that in generalized cancer there are "patterns" which respond to chemotherapy while others do not. Analysis of such unusual responses may I ead to better desi gn of future thereaeutic regimens or give clues with respect to interractions between chemotherapy or hormonotherapy and host immune reacti ons.

Complete remission, or, sometimes cure, is achieved today in significant percentages in generalized lymphomas. These results, though promising, are still far from satisfactory. The figures of complete remission and cures in generalized carcinomas, at least in adults, i.e. in the most common tumours, are not just unsatisfactory but depressingly low.

In order to make therapeutic decisions and estimate prognosis it is essential to know the rate of expected responses with chemotherapy, in lymphomas and solid tumours. As exceptional response we refer to (a) an occasional complete remission or even cure, in cases where such a response is not usually expected, (b) an unusually high general response rate, or (c) in the sense that an unusually good response and or an excessive toxicity, follows a small dose of chemotherapeutic agent.

It is our purpose to present exceptional responses to chemotherapy and/or

249

250 D. RAZIS ET AL.

Figure 1:

Patient A.L., before chemotherapy

Figure 2:

Patient A.L., 20 months later in complete remlSSlon with chemotherapy

EXCEPTIONAL RESPONSES OF CASES WITH GENERALIZED CANCER 251

hormonotherapy in patients with generalized carcinomas and lymphomas treated during the first 7 years of operation of the Cancer Institute of Piraeus. Analysis of such unusual responses may lead to better design of future thera­peutic regimens or give clues with respect to interractions between chemo­therapy or hormonotherapy and host immune reacti ons.

Breast Cancer

24 cases of Iymphangitic spread to the lung were seen in 250 cases of generalized carcinoma of the breast. This serious complication is notoriously known for not responding to conventional treatment. 60% of our cases, however, responded to combi nati on chemotherapy, the response lasti ng for more than a year in all cases (Razis et al ., 1973). In fi gures 1 and 2 we see the case of a 65 year old lady (A. L.) with soft tissue and pulmonary metastases and 20 months later (figure 2) the patient is in complete remission. E. K. is the case of a 61 year old lady who developed Iymphangitic spread in the lungs and responded partially to "5 day - 5 drug" combination chemo­therapy. After recurrence the chemotherapy was changed to once weekly with complete remission. We consider this patient cured - she is fully active without any evidence of disease (and without any treatment) four years after the development of pul monary Iymphangiti c metastases.

The most common visceral metastasis in our material is pulmonary and next is hepatic, lout of 4 of the patients develop liver metastases. We treat massive liver metastases with a combination of hormonal and non-hormonal agents wi th more than 50% response lasti ng for at I east a year (Razis et al . , 1972; Kaufman and Ochoa, 1974). Some responses were exceptional and a few patients we believe cured. In figures 3 and 4 we see the liver scans of a catholic nun treated with this combination of hormonal and non-hormonal agents and, on recurrence,with a modified Cooper's regimen. She finally developed osseous metastases treated with adrenalectomy. During this procedure only scar tissue was found where metastatic disease was previously demonstrated by liver scanning (Lissaios and Razis, 1972).

It is interesting that in some cases the combination of phosphates and hormonotherapy gave quite satisfactory results in both hypercalcemia and cancer, though hypercalcemia was hormone-i nduced in some of these pati ents.

Excepti ona I responses we have a Iso observed in soft tissue and osseous disease and in two remarkable cases of recurrence after adrenalectomy treated wi th androgens and now in compl ete remissi on for more than 5 years. We consider six cases of generalized carcinoma of the breast, living and well without evidence of disease for more than 5 years, as "probably cured" as in

252 D. RAZIS ET AL.

J. G • 30·9.68

•

• •

Figure 3:

Patient E.K., before chemotherapy

11.1.71

J.G

Figure 4:

Patient E.K., recurrence after remission with "5 day - 5 drug" chemotherapy

EXCEPTIONAL RESPONSES OF CASES WITH GENERALIZED CANCER 253

a few cases after years of complete remission recurrence finally developed.

Ovarian Cancer

We treat ovarian cancer stage III and IV (F.I.G.O.) with combination chemotherapy - either a modified "5 day- 5 drug" Cooper's regimen, or the "4 drug weekly" regimen, recently modified by substituting Methotrexate with Ac.tinomycin-D. 64% of 44 patients responded objectively to chemo­therapy while 20% demonstrated complete remission without any evidence of disease, the average duration of response lasting 7 months. 3 patients however are considered as "probably cured". One (H. B.) with widespread abdominal disease and ascites proven on exploratory laparatomy, was treated with cycles of combination chemotherapy and six years later now, she is I i vi ng free of the disease. Another pati ent ( I • K.) was a Iso found to have widespread intra-abdominal disease on exploratory laparotomy and treated with weekly combination chemotherapy. 13 months later on a second look operation, no disease was found.

Non-Hodgkin's Lymphoma

We treat Non-Hodgkin's Lymphoma, III and IV B, usually with COP; Cycling agents, Methotrexate, Thioguanine, ARA-C, and Bleomycine being recently added. Cases are calssified as LSA and RCS, the Rapaport's class­ification applied in our Hospital only recently. We observed objective response in 80% of 70 patients with 36% 2-year survival and 21%( 15 patients) probable cures. These supposedly cured patients are free of disease for more than two years (11 more than 5 years). Cured cases are both maintained and unmaintained. One patient with very extensive intra-abdominal lymphoma is cured with only 5 cycles of COP. In another very remarkable case the disease disappeared completely after only one cycle of chemotherapy. This patient was recently re-checked in the Hospital and there was no evidence of disease three years after the one cycle of chemotherapy. The disease was very extensive in several cured cases.

Soft Tissue Sarcomas and Other Tumours

We treat soft tissue sarcomas with several combinations of Adriamycin, DTlC, Vincristine, Actinomycin D, Cyclophosphamide and Methotrexate. We consider as probably cured two pati ents with embryonal rhabdomyosarcoma; a patient with very extensive, intra-abdominal and pulmonary Wi/m's tumour J a remarkable case of very extensive retroperitoneal sarcoma with pulmonary

254 D. RAZIS ET AL.

disease in a middle aged woman/and a boy with metastatic malignant giant cell tumour of the bone. Impressive is also the case of a choriocarcinoma of the testes after treatment with 8 day continuous i .v. infusion of Bleomycin (Krakoff, 1974) in combination with Vincristine and Methotrexate.

In another case a patient with massive liver metastases from colonic carci­noma lived two years free of disease wi th chemotherapy and on recurrence cirrhotic changes were found in the liver probably related to the continuous chemotherapy. Several other exceptional responses were observed in multiple myeloma and in tumours not usually responding to chemotherapy such as carcinoma of the cervix and clear cell carcinoma of the kidney. In a few remarkable cases, minute doses of chemotherapy resulted in excellent response but in some of these cases in severe toxicity too.

COMMENTS

Exceptional responses of various generalized malignant neoplasms to chemotherapy and hormonotherapy are presented. There is no apparent explanati on for these exceptional responses. It is possibl e that we do not always use optimally the available drugs, and or that there are patterns of generalized cancer who respond exceptionally to therapy for immunological or kinetic reasons. We have often seen MIF positive patients become, as expected, negative after chemotherapy but we have also seen exactly the opposite, i.e. MIF negative patients become positive after therapy. It is also interesting that exceptional responses were seen after aggressive chemo­therapy but also after minute doses of chemotherapy, in cases where aggressive chemotherapy has previously failed. There are finally the cases of very satisfactory partial remissions, with the patients practically asymptomatic, treated with small doses of chemotherapy.

Analysis of the exceptional responses might lead to more effective thera­peutic schemes, more knowledge of the natural course of this disease, more understanding of the host-immune reactions.

REFERENCES

1. Kaufman, R.J. and Ochoa, M. (1974). Abstracts, Xlth International Cancer Congress, 3-599.

2. Krakoff, I. H. (1974). Abstracts Xlth International Cancer Congress, 1-367.

3. Lissaios, B. and Rizis, D. (1972). Proceedings of the Vllth Inter-nati onal Congress of Chemotherapy, 463.

EXCEPTIONAL RESPONSES OF CASES WITH GENERALIZED CANCER 255

4. Razis, D., Tsats-Jronis, A., Triantafyllou, D., Christodoulopoulos, J. and Constantoulakis, M. (1972). Proceedings of the Vllth International Congress of Chemotherapy, 459.

5. Razis, D., Constantoulakis, M., Dimitriadis, M., Georgakou, A., Tsatsaronis, A. and Cortessis, A. (1973). Proceedings of the 1st Hellenic Radiology Congress, 513.

CLINICAL CONSIDJmATIONS IN MYELOMATOSIS

J.B. HEALY

ST. LUKE'S HOSPITAL, DUBLIN 6

The common pattern of response of ~eloma protein during treatment is one with an initial fal~then a levelling off at a figure still above the normal and lastly a rise. Dr. Sidney Salmon has suggested that as the total number of plasma cells is reduced) the cells begin to divide more rapidly so that jon a constant dosage of drug a plateau is reached when destruction and reproduction are/ balanced. Have we evidence of this clinically? Could the levelling off be equally well explained by a rising clone of resistant cells, which by addition to the falling sensitive clone would form a valley? The valley could be wide and shallow so as to resemble a plateau. I looked through records of 40 fairly recent cases. I defined a plateau as a levelling off of the M globulin which could not be accounted for by addition from a subsequent logarithmic rise. (The drug dosage should be constant over a long period). If addition of two curves, one falling, one rising could account for the findings, I called the result a valley. Of the 40 cases only 12 gave usable data (others excluded because of inconsistent dosage, insensitivity, descent of curve to normal level or being Bence Jones only). There were 5 good valley patterns and 3 fairly good. Fig. 1 Shows a valley case. There were two doubtful plateaux and only two clear ones (fig. 2 and 3). Fig. 3 shows another expected plateau property; if the cells are dividing more rapidly, then increasing the dosage should lower the plateau. A resistent clone is not likely to respond in this way. Of the 8 "valleys" 3 showed clear failure on more intensive chemotherapy, 3 had inadequate data but two improved. This problem should yield to simple clinical testing.

257

258

.. " c.

o

10

... c:

" CJ .. " c.

o

10

5

f--- Mel. + Prado

\ I

I"

/

/

Upper Normal IgA

i. v. Mel.

1.0~-~-+--_-~+--+-~-~--­Jan. April July Oct. Dec.

Time (months)

Figure 1

/

V'

O.l~---------_O--_-___ ""-Time (months)

Figure 2

J.B. HEALY

CLINICAL CONSIDERATIONS IN MYELOMATOSIS

> .. 'C ~ .. Co

; " 0 ., " u

" " III

E .. G

5.0

1.0

~ 0.5

E C!I

Pred. + Mel. every 6 weeks----..P. + M. alternating

.Witn End:x.

Upper Normal Level of IgA Globulin

0.1~---~'----+-"""'--"'----_""_ 1973 1974 1972 Time (units of 2 montns)

Figure 3

10 Melp./Pred. A.C. T.H.

8 'lU J U ,.

'Endox./Pred. Melp./Prad. 7 , ,""'" .....

,/1 6 '/' , I

I 5 1/1 /: 4

I 3 /1 2

I Endoxana tjt .. '''''6thHU'''' H

i~ :~ ~ /

0~~ __ ~~~~~-4~~~-+-+~~~~~ __ ~~+-+-+-___

~ 1971 1972 1973 1974 1976

Time (units of 2 montns)

Figure 4

259

260

.. " " " :. Q.

cj

3

2

0.5

Pred., M./P. M./P. .... I 6/52 6/52

.. .s " o E Ol

M./P. (End. + M./P .

..... I .... II ... I.IJ.~~IeIeI.U 3/52 I I

I I

Time (units of 2 months)

Figure 5

J.B. HEALY

We examined the initial M globulin fall. Of 40 cases, 6 were Bence Jones only, 9 had inadequate t life data and in two there was no globulin fall. In the other 22 cases t life was checked against survival. Result:

Half Life Short (2/12 or less) Long (> 2/12 )

Alive at 1 year 7 8

Deceased at 1yr. 7 o

There was no relation between survival and the initial level of M globulin or between survival and a rough M globulin formation figure (Total M Globulin 7 t time of fall).

There is normally a rise in M globulin over some months towards the end of the disease course but we noted ~ cases with a clear fall in the month before death. The falls varied between 20% and 55%. In 40 cases, 12 are still alive, 2 had low near normal levels, 6 had no data in their last month, and 20 were assessed.

CLINICAL CONSIDERATIONS IN MYELOMATOSIS

" iii (,)

(/)

g .J

+

scale in months.

scale in 2 months.

+

__ -+-~_",--+----+---+_~_ scale in 2 months .

scale in 2 months.

__ -...-_---+--+-<--~ ......... -+-I scale in Y:t months.

Figure 6

T· ;ItHINAI. PALL YF1> "yes"

90 1

IN M GLOBULIN. NO "no"

2 2

261

"yes" - 5 were in first phase of treatment, l died of a coronary,one of acute monocyte leukaemia. "no" - 1 in first phase. I died drug induced leucopoenia.

262 J.B. HEALY

220

200

180

160

140

120

100

80

60

40

20

01L---------~----_t--------------~ 1 month 1 Y, months 5 months

Figure 7

Bence Jones/protein fall was examined. 21 patients had no Bence Jones. 6 had a trace. 13 had a gram or more in 24 hour urine. (1died within 3 days). 12 assessed.

HALF TIM!!! OF PALL IN' BENCE JONES

2 days

6

2 weeks

3x x One had ! time in two weeks, in the other

Several

3xx

two the was not clear.

months.

actual t time

xx 2 of these were Bence Jones only and in the third IgA never tell in 30 months.

CLINICAL CONSIDERATIONS IN MYELOMATOSIS 263

treated with Prednisolone alone, 4 responded. Of the 12, 5 were 6 It " It Melphalan + Prednisolone, 6 responded 1 was II It Melphalan alone and responded.

The responses to Prednisolone alone were quite clear cut, i.e. the Bence Jones secretion fell to less than 25% of the initial. I then looked at the other 28 cases. There were various initial treatments. I shall only deal with those who had Prednisolone and Melphala n initially.

INITIAL TRF.ATMENT M GLOBULIN RESPONSE

Prednisolone alone Melphalan + Prednisolone

4 sensitive 15 It

1 fail 5 It

Prednisolone is suitable in patients with initial leucopoenia. Dose was usually 4Omg. daily for a few days then reduced. Melphalan was given 12mg. daily for 4 days every six weeks with Prednisolone. In cases resistant to Melphalan and Prednisolone various drugs were tried.

MI~LPlIALAN/pnEDNISOI..oNE RESIStANT CASJ~

Endoxana alone 1 sensitive 3 fail Endoxana + Mel/Pred 2 It 1 " C.C.N.U. alone It 5 " C.C.N.U. + End + Mel/Pred " 5

Fig. 4 shows a clear example of the first type. Fig. 5 of the second Overall a third of the resistaut cases responded to some other alkylating agent. Our data wi~h antimetabolites and yeast extracts are inadequate.

lie tested Asparaginase in 6 cases. Fig. 6 shows the overall pattern of M globulin response. Clinically 4 of the patients deteriorated rapidly, none improved. The drug seems to cause a sudden rise in M globulin. l:e measured hydro~proline in 24 hour urine in these patients (on a 2 day diet). Fig. 7 shows a remark­able reduction in 5 cases (the other case had her first test done during the treatment, not beforehand). Hydroxyproline tests seem to be of prognostic importance. I list here our last hydro~proline result in each of 17 cases.

LAST HYDROXYPROLINE TEST (mgm/24hrs) Dead. 234,173,152,82,81,60,46, 33x (The last died of a heart attck when myeloma doing well)

Alive. 60x , 'i~, 55~ 521', 33, 27, 27, 24, 10. P.T.O

264 J.B. HEALY

x Test done at an early s~ag~ in trea~nen~. ~ In these two the score is falling from a higher level. ~ In this case score has started to rise, M globulin also rising.

I think hydroxyproline tests will prove a useful guide; above 6Omg/daily on a suitable diet is an ominous sign. Comment: We should investigate the plateau/valley pattern in myel­oma more th~roughly as it should give us information on growth rate and resistance. We have shown absence of cross resistance to two nitrogen mustard derivatives; the two drugs can be combined together. Asparaginase would appear to be harmful in myeloma.

CHRONIC GASTRITIS, ATYPICAL EPITHELIA IN BIOPSIES AND

THERAPEUTIC CONSEQUENCES

J. Zangger and M. Taufer

Dept. Experimental Cell Research and Oncology, Institute of Pathology, University Graz, A 8036 Graz, Austria

SUMMARY

Examination of gastroscopic biopsies reveals a large percentage of chronic atrophic gastritis. In these cases there may often be increased proliferation of mucous glands and also the appearance of atypical epithelia. We are classifying these patients in a separate category as "patients with an increased risk". They are controlled over short periods by gastroscopy and selective gastrobiopsy. In this way early diagnosis and treatment of gastric carcinoma is possible, and facilities for prophylactic chemotherapy are thereby given.

In accordance with a critical study published by Lambert (1972) the clinical term "chronic gastritis" is commonly used when gastric symptoms occur which are not associated with focal lesions in the stomach. On the contrary gastroscopic diagnostic criteria of chronic gastritis frequently are divergent. Myren and co-workers (Myren and Serck-Hanssen, 1974) for example, reported that endoscopically reduced folding of the mucosa is a valuable criterion for the diagnosis of chronic atrophic gastritis. Other investigators describe the main diagnostic criteria as mucosal reddening or increased vulnerability of the mucosa and some other variations. There is no conformity butwebelieve that the clinical diagnosis of atrophic gastritis is important with regard to early diagnosis of gastric cancer.

Since Konjetzny (1921) chronic atrophic gastritis has been totally integrated into the precarc inomatous state but the discussion about it is worldwide. Our experience like that of Thaler (1972) forces us to the following compromise:

265

266 J. ZANGGER AND M. TAUFER

Fig.

(Explanation see text)

CHRONIC GASTRITIS AND ATYPICAL EPITHELIA IN BIOPSIES 267

the exclusivity of Konjetzny's theory surely is wrong, but in principle it is likely to be correct.

In our own practice we have surveyed the histology of more than 3000 biopsies of the stomach. In 29% we found chronic atrophic gastritis. It is difficult to make a uniform classification of histological types of gastritis. A number of classifications have been suggested often with overlapping between types. In chronic atrophic gastritis the lesions are distinct, but it is not possible to introduce a quantitative element in every case. The character­istic histological picture shows atrophy of the mucosa associated with round­cell infiltration in the lamina propria (see figure 1a). In addition degener­ative changes of the surface epithelium, particularly intestinal metaplasia with goblet cells. The degree of atrophy is estimated as moderate, subtotal or total, accordi ng to the number of pari etal cells. In our own study, we observed in numerous gastric biopsies an intensified proliferation of glands (see figure 1b and 1c). The normal cells are substituted by undifferentiated and atypical cells. The cell nucleus is polychrome and sometimes polymorph. The cytoplasm shows basophi I ia. An important characteristi c is that the basement membranes of the glands are not broken. No invasion takes place as long as the membranes are intact. It is a very difficult decision and involves great responsibility on the part of the microscopist to diagnose these alterations as described. When all the facts have been assessed, the correct histological diagnosis is: "chronic progressive and atrophic gastritis with intensified proliferating and atypical epithelia ll .

Following this diagnosis we classify and register these patients in a separate category. This category is "Patients with increased risk ll . This means that in view of the risk of malignant degeneration the clinician has to make a gastroscopic examination with multicentric biopsies at frequent intervals. In this way (with excellent co-operation between gastroscopists and pathologists) by examination of patients with an increased risk we found last year 16 carcinomas in the earliest stages, i. e. "carcinoma in situ". This means that we found 28% of all gastric cancers during this period.

The so called lIearly cancer", introduced by the Gastroendoscopical Society of Japan, is never an "early cancerll. This tumour is growing destructi ve, i nvasi ve and frequentl y metastasi n9 . We must focus our attention on every pre-carcinomatous symptom, clinical and morphological.

Our findings have implications for prophylactic chemotherapy of patients with excessive atrophic gastritis and atypical proliferation of glands. Nearly six months ago we started a trial with a group of patients who have shown histological alterations of the stomach as described above. The standard agent for treating carcinomas of the gastrointestinal tract is 5-Fluorouracil.

268 J. ZANGGER AND M. TAUFER

Our patients receive a weekly regimen of 15 mWkg body weight intravenously and we repeat this prophylaxis at intervals of 3 months. Toxic complications have not been observed up to naw, but neither is it possible to say as yet whether this chemotherapeutic trial is effective in reducing the incidence of stomach cancer.

Incidence and mortality rates for gastric cancer worldwide have not declined during the past 20 years. In Germany and in Austria 50% of patients coming into hospital with cancer of the stomach are inoperable and incurable. Only ten to twenty percent of surgically treated patients with gastric cancer survive more thon 5 years. In view of these depressing statistics it is necessary to get an early clinical and bioptic diagnosis. We hope that this will be successful.

REFERENCES

Konjetzny, G. E. (1921). In Anschutz-Konjetzny. Stuttgart 1921. Lampert, R. (1972). Digestion, 7, 83. Myren, J. and Serck-Hanssen, A.(1974). Scand.J.Gastroent., 9, 457. Thaler, H. (1972). Deutsche Med. Wschr. 97, 51. -

CLINICAL STUDIES ON CHANGES IN SERUM GLYCOPROTEINS IN CANCER

CHEMOTHERAPY

Kunihiro Funahashi

R. Esaki, J. Hayashi, T. Itoh, K. Shibata The First Department of Surgery, Nagoya City University Medical School, Mizuho, Nagoya, Japan

Serum glycoproteins of patients with cancer(). - Acidglyco­protein, ~I -Antitrypsin, a~-Haptoglobin, ~~-Ceruloplasmin ) were determined with the Immunoassay Partigen method and the values were observed to be remarkably higher than those patients with benign diseases. Further, it was noted that these values tended to increase with the progress of tumor. Accordingly, it was consid­ered that the progress of cancer could be comprehended objectively through the changes of the values of serum glycoproteins and that these changes might be helpful for the evalation of the effect of anticancer agents administered. Besides, these serum glyco­proteins might be a material coating lymphocytes, closely asso­ciated with immunoinsufficiency found in patients with cancer. The number of cancer patients examined was 108 in total, which included 33 cases of stomach cancer, 33 cases of colon and rectal cancer, 22 cases of liver, biliary and pancreatic cancer, 8 cases of breast cancer, 5 cases of lung cancer, 6 cases of malignant lymphoma. On the other hand, the benign diseases included 14 cases of gastric, duodenal and cholelithiasis. Classification of the changes of values of alpha,AG by the disease revealed that in benign diseases they ranged between 40 to 70 mgjdl, remaining almost normal, while in the case of stomach cancer, colon and rectal cancer, they showed a stairlike increase in accordance with the groups aggravating from radical operation group ( A ), palliative operations group ( B ) and inoperable cases ( C ). In particular, in the cases of malignant lymphoma, the values recorded were characteristically high in palliative operatiotls group. However, in the cases of breast cancer, hardly any correlation was observed between the progress of the neoplasm

269

270 K. FUNAHASHI

and the values of ~I-Acidglycoprotein. Observation of the changes of~,-Antitrypsin, on the other hand revealed a stair-like increase almost in all cases except for lung cancer, indicating that the increase was closely correlated wi th the aggravation of cancer. Obs'ervation of the change of values of~~-Haptoglobin showed an increase simultaneously with the progress of tumor, but in inoperable cases, the one showing remarkably high and the other remarkably low, indicating perhaps the collapse of homeostasis at the terminal stage of cancer. Furthermore, in the cases of malignant lymphoma and lung cancer, the values were characteristically high, while in the cases of breast cancer, hardly any changes were observed. Changes of values of ~~ -Ceruloplasmin were also examined,and their stair-like increase in accordance with the growth of tumor was noted in all cases except for breast cancer. In particular, in liver, biliary and pancreatic cancer the values recorded were characteristically high. Based on presumption that serum glycoproteins are a blocking fac­tor coating their host lymphocytes and inactivating them, the in­crease in the values of serum glycoproteins may decrease the immu­nity of the host, acting very unfavorably toward the host. Further examination was made how serum glycoproteins changed by the excision of tumor. Post-operative values were determined in principle 2 weeks after operation, a period during which the influ­ences of operation are disappeared. As a result, 0., -AG, o..-AT, 0...1. -HP, 0.1 -CP altogether showed an im­provement toward a normal range after excion.

POSTOPERATIVE CHANGES OF GLYCOPROTEIN IN CANCER PATIENTS ( TUMOR RESECTION )

"" - Acidqlycoprotein 0(,- Antitrypsin

mq/dl ""J/dl

200 1000

100 500

NORMAL

Figure I

I .iORJotAL

CHANGES IN SERUM GL YCOPROTEINS

lIIg'/dl

500

250

POSTOPERATIVE CHANGES OF GLYCOPROTEIN IN CANCER PATIENTS ( TUMOR RESECTION )

HaptO<]robin lIIg'/dl Ceruloplasmin

50

40

30

2G

Figure 2

l~

If complicated by infection, the values tended to increase after operation. This phenomenon is considered to indicate that the effect of the excision of tumor on the host is valuable. Besides, the follow up of the changes of values of serum glyco­proteins after excision of tumor may greatly serve the purpose of the recognition of recurrence. Next examination was made on the effect of administration of an­ticancer agents to the inoperable cancer patients through the the changes of values of serum glycoproteins, and it was found that in effective cases where tumor definitely shrank, the im­provement in 0.1 -AG, d.1 -AT, d.i -HP, 0.,2. -CP, was observed, while in aggravating cases, a remarkable increase in the values of serum glycoproteins was noted. Therefore, clinical effect

271

due to administration of anticancer agents and the values of serum glycoproteins were found to be changing in good correlation. However, in the cases where~I-AG increased remarkably high be­fore chemotherapy, therapeutic effects due to administration of anticancer agents were hardly obtainable and therefore, it is considered that for such cases further efforts were necessary example to improve the immunity of the host. In summary, we observed 1) the values of serum glycoproteins

showed an increase simultaneously with the progress of tumor, and this phenomenon was closely correlated with the decrease of the immunity of the host in the cases of progressive cancer 2) the values of serum glycoproteins once increased were reduced to a normal range by the excision of tumor, 3) the clinical effects produced by the administration of anticancer agents and the changes of values of serum glycoproteins in inoperable cancer showed a good correlation, and 4) in the cases where ~I-AG was

272 K. FUNAHASHI

POSTCHEI!OTHERAPEUTIC CHANGES OF GLYCOPROTEIN IN II«lPERABLE CANCER PATIENTS

119/41

200

100

O.-Acidglycoprotein

IMPROVED CASES

aoq/d1

I

Figure 3

0,- Anti trYPlin

I NORMAL

POSTCHEI!OTHERAPEUTIC CHANGE~ OF GLYCOPROTEIN IN INOPERABLE CANCER PATIEtITS

( IMPROVED CASES

Haptoglobin Ceru!opl ••• 1n

1119/41 aoq/41

500 60

400 50

300 40

~I~ 200 30

100 ~I-~ 10

Figure 4

CHANGES IN SERUM GL YCOPROTEINS

POSTCHEI1OTHERAPEUTIC CHANGES OF GLYCOPROTEIN III 1I10PERABLE CANCER PATlEIHS

( ADVERSE CASES

ct. -Ac ldqhc:oprotein ~I- Ant l trypain mg/dl

200 1000

100 500

..

Figure 5

POSTCHOOTHERAPEUTIC CHANGES OF GLYCOPROTEIN IN lHOPERABLE CANCER PATIENTS

ADVERSE CASES

Hap t o9 1ol>ln Ceru lopI ••• 1n mg/dl mq/dl

500 GO

400 50

300 40

200 30

100 IHO~ 20

10

Figure 6

273

274 K.FUNAHASHI

particularly high, chemotherapy alone did not produce desired effects, and further efforts in terms of the improvement of the immunity seemed to be necessary.

REFERENCES

1) Esaki, R. et al. Activity on Cancer Cells and Distribution in Tissue of Anticancer Drugs. 11,334,1970. Tokyo.

2) Esaki, R. et al.Sensitivity of Anticancer Drugs and Their Concentration in Tissues. Advances in Antimicrobial and Antineoplastic Chemotherapy.11.383,1972.Praha.

3) Surgery and Chemotherapy in Children with Malignant Neoplasmas: With Special Reference to Drug Sensitivity and Drug Concentration in Tumor Tissues. Progress in Chemotherapy.111.637,1974.Athens.

DOUBLE-BLIND TRIAL WITH LEVAMISOLE IN RESECTABLE

LUNG CANCER

* W. Amery

Janssen Pharmaceutica, Research Laboratoria

B-2340 Beerse, Belgium

ABSTRACT

This second interim analysis reveals that intermittent treat­ment with levamisole 50 mg t. i. d. orally for three consecutive days every fortnight, started three days before surgery, only seems effective in patients weighing 70 kg or less. In these pa­tients the post- surgical recurrences are markedly diminished. Other possibly relevant factors are discussed.

INTRODUCTION

Levamisole is an antianergic chemotherapeutia agent which restores host defence mechanisms when these are inefficient.

The present paper reports the results from a second interim analysis.

* on behalf of the Study Group for Bronchogenic Carcinoma whose composition is as follows: Prof. J. Cosemans, Dr. H. C. Goos­zen, Prof. E. Lopes Cardozo, Dr. A. Louwagie, Dr. J. Stam, Prof. J. Swierenga, Dr. R. G. Vanderschueren, Dr. R. W. Veldhuizen (investigators); Eng. G. De Ceuster, Dr. L. De­splenter J. Dony, Prof. A. Drochmans, Dr. P.A.J. Janssen, Dr. W. Tanghe (consultants); E. Denissen (co-ordinator); Dr. W. Amery (chairman)

275

TA

BL

E I

. R

esu

lts

as

rela

ted

to

th

e p

ati

en

ts'

weig

ht

Weig

ht

PO

SS

IBL

E R

EC

UR

RE

NC

ES

P

RO

VE

N R

EL

AP

SE

S

CA

RC

INO

MA

TO

US

DE

AT

HS

Lev

am

iso

le

Pla

ceb

o

Lev

am

iso

le

Pla

ceb

o

Lev

am

iso

le

Pla

ceb

o

~

60

kg

1/6

(1

6.7

%)

5/1

0 (

50.0

'10)

0

/6

(0.0

%)

5/1

0 (

50

.0'1

0)

0/6

(0

. 0

%)

4/1

0 (

40

. 0

%)

61

-70

kg

1/1

9(5

.3%

) 1

2/2

5 (

48.0

'10)

1

/19

(5

.3%

) 1

0/2

5 (

40

. 0

%)

1/1

9 (

5.3'

10)

6/2

5 (

24

.0%

)

71

-80

kg

8/2

5 (

32.0

'10)

4

/21

(1

9.1

%)

8/2

5 (

32.0

'10)

1

/21

(4

.8%

) 6

/25

(24

.0'1

0)

1/2

1(4

.8%

)

> 8

0 k

g 6

/15

(40

.0'1

0)

6/1

6 (

37.5

'10)

5/1

5 (

33.3

'10)

5

/16

(3

1.

3'10

) 3

/15

(2

O.

0%

) 3

/16

(l8

. 8

%)

TA

BL

E I

I.

Su

peri

ori

ty o

f le

vam

iso

le i

n r

ela

tio

n t

o t

he p

ati

en

ts'

weig

ht

Bo

dy

P

RE

VIO

US

IN

TE

RIM

AN

AL

YS

IS*

P

RE

SE

NT

IN

TE

RIM

AN

AL

YS

IS*

P

ara

mete

r P

-P

-w

eig

ht

Lev

am

iso

le

Pla

ceb

o

valu

e

Lev

am

iso

le

Pla

ceb

o

valu

e

susp

ecte

d r

ecu

rren

ces 1

/15

(6

.7%

) 1

3/2

7 (

48

.1%

) <

0.0

1

2/2

5 (

8.0

%)

17

/35

(4

8.6

%)

<0

.00

1

:::; 7

0 k

g p

rov

en

rela

pse

s 1

/15

(6

.7%

) 1

2/2

7 (

44

.4%

) <

0.0

5

1/2

5 (

4.

0%

) 1

5/3

5 (

42

.9%

) <

0.0

01

dis

ease m

ort

ali

ties

1/1

5 (

6.7'

10)

7/2

7 (

25

.9%

) n

. s.

1

/25

(4

.0%

) 1

0/3

5 (

28

.6%

) <

0.

05

susp

ecte

d r

ecu

rren

ces 8

/33

(2

4.2

%)

8/2

8 (

28

.6%

) n

. s.

1

4/4

0 (

35

.0%

) 1

0/3

7 (

27

.0%

) n

. s.

>7

0 k

g p

rov

en

rela

pse

s 8

/33

(2

4.2

%)

6/2

8 (

21

.4%

) n

. s.

1

3/4

0 (

32

.5%

) 6

/37

(1

6.2

%)

n.

s.

dis

ease m

ort

ali

ties

6/3

3 (

18

.2%

) 3

/28

(1

0.7

%)

n.

s.

9/4

0 (

22

. 5

%)

4/3

7 (

10

.8%

) n

. s.

* th

e t

ota

l n

um

ber

of

pati

en

ts i

s n

ot III (p

rev

iou

s an

aly

sis)

or

14

7 (

pre

sen

t an

aly

sis)

sin

ce t

he w

eig

ht

was

no

t re

co

rded

in

a

few

pati

en

ts.

Fo

r m

ore

deta

ils

reg

ard

ing

th

e p

rev

iou

s an

aly

sis.

se

e r

efe

­re

nce (

1)

~

'-I

0. ?E

» s::

m

::c

-<

LEVAMISOLE IN RESECTABLE LUNG CANCER

TABLE III. Differences between levamisole and placebo in pa­tients weighing::; 70 kg

P-value: levamisole ~ placebo· Parameter

Initial PPD <lOmm ~10mm

Initial DNCB 50 }lg no induration induration

Initial DNCB 100 }lg no induration induration

Epidermoi'd carcinoma Adenocarcinoma Others + mixed

Largest tumor diameter ::s; 3 cm 4-6 cm ~ 7 cm

G · 1 ** rouplng cat. a Other categories

Grade I Grade II

suspected recurrences

< 0.10 < 0.05

< 0.10 n. s.

n. s. < 0.10

< 0.10 n. s.

< 0.05

n. s. < 0.05

= 0.05

n. s. < 0.01

n. s. < O. 01

proven relapses

< 0.10 < O. 05

n. s. n. s.

n. s. < 0.10

< 0.05 n. s.

< 0.10

n. s. < 0.05 = 0.10

n. s. < 0.01

n. s. < O. 01

carcinomatous deaths

n. s. n. s.

n. s. n. s.

n. s. n. s.

n. s. n. s. n. s.

n. s. n. s. n. s.

n. s. n. s.

n. s. n. s.

:. all trends are in favor of levamisole this is the only category of patients for which there is no evi­dence that the tumor might have spread, or has already spread beyond its primary site. For definitions see reference (1)

277

278 W.AMERY

FA TIENTS AND ME THODS

The criteria for the selection of the patients and the study de­sign have been described at length elsewhere (1). This report concerns the data from the first 147 patients (69 on levamisole and 78 on placebo) who had been operated at least one year befo­re the present analysis was made.

One tablet, containing 50 mg of levamisole or placebo, is gi­ven three times daily on the last three days before surgery and a similar three day-course of treatment is given every second week thereafter. The medication is strictly double- blind and se­parately randomized for each of the three co-operating centers into levamisole or placebo.

The end-points of the study are the first sound suspicion of recurrence, the first proof of relapse, and death from the di­sease. The major purpose of the trial is to find out whether le­varni sole in the dosage used reduces the incidence of these end­points. If a beneficial effect of levamisole is observed, the trial should also provide some information on the factors (especially delayed type hypersensitivity skin tests and several tumor cha­racteristics) which help to predict this effect. For the latter purpose, patients are skin-tested with several doses of DNCB and with 101. U. of PPD. The tumors are classified per histo­logical diagnosis by means of the WHO criteria, their regional extent is assessed by a category grouping system which is a slight modification (1) of Slack's system (2). Eventually the tumor diameter and the regional extent category are combined into a two-grade system for describing the pre-operative tumor burden (l).

The statistical analysis is perfor:med by :means of the Fisher exact probability test, two-tailed, using a Wang 2200 computer.

RESULTS

For the three end-points, there was a statistically non- sig­nificant trend in favor of levamisole, and this trend was present in the three co-operating centers.

The previous interim analysis revealed (1) that the beneficial effect of levamisole was not related to the skin test reactivity data, but that the histological diagnosis appeared to be relevant to this effect (significant differences were only found in patients

LEVAMISOLE IN RESECTABLE LUNG CANCER 279

with epidermoid carcinoma whilst no trend in favor of levamisole was found in adenocarcinoma) and that levamisole superiority to placebo was limited to patients with a higher tumor load (size> 3 cm; patients for whom there is evidence that the tumor might have spread or has already spread outside its primary site; and patients with a grade II tumor burden). At first sight, these factors were not substantiated by the present analysis.

Further analysis, however, revealed that the weight of the patients is of extreme importance as regards the differences be­tween levamisole and placebo, as shown in Table 1. So, it be­came evident that levamisole, in the dosage used, was onlyac­tive in patients with a weight of 70 kg or less (Note that the me­dian weight of the patients included in the present analysis is 72 kg). Table II shows that levamisole is significantly superior to placebo for the three end-points studied in patients of the lower weight (i. e. ~ 70 kg) category but not in the other patients. Moreover, going back to the data used in the previous analysis (1) we found out that the same difference was already present at that time (Table II). Note that the seeming trend to the dis­advantage of levamisole in the higher weight category is due to the unexpected low recurrence and mortality rate with placebo in patients weighing from 71 to 80 kg (compare figures with those of other placebo patients in Table I) and this trend is, therefore, very likely due to coincidence.

A separate analysis of the data of the lower weight category reveals (Table III) that in these patients the conclusions from the previous interim analysis are largely confirmed. Moreover, the only proven relapse amongst the levamisole patients was a case of intrathoracic recurrence. Amongst the 15 placebo pa­tients with proven relapses, the relapse was an intrathoracic recurrence in four and a distant metastasis in eleven patients. Therefore, distant metastases (none in 25 levamisole patients, eleven in 35 placebo patients) were strikingly reduced (P < 0.01) in the levamisole group. This again confirms the data from the previous analysis (1) which suggest a beneficial effect (p = 0.06) of levamisole as regards distant metastasis but not as regards intrathoracic recurrences.

CONCL USIONS

The dosage of levamisole used is seemingly only beneficial in patients weighing 70 kg or less. This finding strongly suggests that the dosage used was too low for the other patients and that

280 W.AMERY

in future trials, therefore, the dosage should be better adapted to the patients' weight (e. g. a dosage of 2.5 mg/kg per day would appear to be reasonable).

Other predictive factors as regards the beneficial effect of levamisole are especially to be looked for amongst the tumor characteristics (histological diagnosis and tumor extent). Also, the site of recurrence might be of primary importance in evalu­ating the effect of levamisole.

REFERENCES

1. Study Group for Bronchogenic Carcinoma: Immunopotentiation with levamisole in resectable bronchogenic carcinoma. Bri­tish Medical Journal (in press, 1975).

2. Slack, N. H. : Bronchogenic carcinoma: nitrogen mustard as a surgical adjuvant and factors influencing survival. Cancer ~, 987 (1970).

CARCINO-EMBRYONIC ANTIGEN DETERMINATIONS

AND CHEMOTHERAPY IN CANCER PATIENTS

J. HUYS and P.M. VAN VAERENBERGH

Kliniek voor Radiotherapie en Kerngeneeskunde

Akademisch Ziekenhuis, Gent, Belgium

The aim of this report is to present an attempt to determine the role of serial plasma CEA determina­tions and the therapeutic monitoring of cancer patients, especially bronchus-, breast- and gastro-intestinal tumours. In other words to examine if a correlation between CEA levels and the clinical course after a che­motherapeutic treatment can be found.

PATIENTS AND METHODS

All the patients in our study were untreated or with clinical evidence of evolving recurrent disease when the first CEA was done. One hundred and nine pa­tients were actually studied, all adults with a histo­logical proven carcinoma.

Table 1 gives the number of patients for each diagnosis subdivided according to the presence or ab­sence of metastases.

The patients with a lungcancer received a combined radio-chemotherapeutic treatment consisting of an irradiation of the primary tumorarea and a five drug combination medical treatment. The breast and gi tract tumors only had a palliative chemotherapy with the exception of the breast (3) and oesophagus (6) cancers without metastases. These patients were also irradiated. Patients with a suspected survival of less than two months were excluded from this trial.

281

282 J. HUYS AND P.M. VAN VAERENBERGH

DIAGNOSIS MO M1 TOTAL

BREAST CANCER 3 2B 31

BRONCHUS CANCER 13 2B 51

G. I. TRACT CANCER 6 21 27

TOTAL 22 B7 109

Table 1

The CEA assay was performed using the Hansen Z-gel method (CEA-Roche) while each test was done in duplicate. With this method values above 2.5 nanograms/ml are con­sidered as abnormal. The CEA plasma level was deter­mined before any treatment, weekly during the period of the induction treatment (6 toB weeks) and afterwards at each monthly follow-up consultation. Each time the disease status was assessed by the same investigator without knowing the CEA results.

RESULTS

The CEA values before any treatment correspond very well with the figures which have already been published by various authors. Indeed 64%, 39% and B5% of the metastasized cases with respectively breast-, lung- and GI tract cancer have initial values above 5 nanograms/mI.

Four patterns of serial CEA determinations can be found during any cancer treatment; CEA values which are progressively increasing or decreasing and values which remain consistently normal or elevated.

The aim of serial CEA plasma assays is in the first place to study if there is a correlation between the antigen value and the clinical assessment of the patient, and secondly to see if the changes in the titer appear more rapidly than the usual methods for evaluating the tumor growth (physical eXEmination, biochemistry, radio­graphy, nuclear medicine). In other words to investi­gate if the CEA variations can reliably predict the further evolution of the disease.

CARCINO-EMBRYONIC ANTIGEN DETERMINATIONS 283

The correlation is classified as total, partial or non-existing according to the following criteria. A high CEA value increasing with a deterioration of the patient or decreasing with a clinical improvement is of course considered as a complete correlation. The same is also true for a low CEA increasing with an evolving disease or a low CEA which remain~stable with a clinical amelioration. All the other combinations are classified as cases of no correlation except for the patient with a high CEA value remaining stable with a deterioration. We consider this a partial correlation.

Table 2 gives us the results of this classification where we can see that there was no correlation in 43%, 19% and 22% for lung, breast and GI tract cancers. Most of these patients showing no correlation usually had low initial CEA values which failed to become abnormal in spite of an increasing tumor.

We also looked for a correlation between the CEA value and the survival of the patient. Table 3 shows this survival at 6 months for all the cases with a metastasized tumor, according to an initial CEA value of lower or higher than 5 nanograms. Although the difference is not statistically significant due to the small number, the chances to have a longer survival seems better for the patients with a low pretreatment value.

LUNG BREAST G. I.

TOTAL 24 22 17

PARTIAL 5 3 4

NO 22 = 43% 6 = 19% 6 = 22%

Table 2 Correlation CEA/Clinical Status

284 J. HUYS AND P.M. VAN VAERENBERGH

CEA (5 )5

BREAST 4/8 6/16

GI TRACT 1/4 2/17

LUNG

TOTAL

Table 3

9/21 3/12

14/33 11/45

42% ALIVE 24%

Correlation Survival at 6 Months/CEA Metastasized Patients

DISCUSSION

Serial plasma CEA determinations have been used with success by different authors in the follow-up and the detection of recurrence after surgical treatment of cancer patients. On the contrary very little information is known about the evolution of the CEA titer after a radiotherapeutic and/or chemotherapeutic treatment. Besides the conclusions of the scarce material which has been published are not always the same : some authors deny the role of CEA monitoring in cancer chemo~ therapy while others conclude that these determinations may have some value.

We belong to the latter group : the correlation between the CEA monitoring and the clinical result of the non surgical treatment is in our series acceptable especially for those cases with an increased initial CEA value. The mean duration for the normalisation of CEA is 2 to 3 months although we frequently see a de­crease after 2 or 3 weeks.

CARCINO-EMBRYONIC ANTIGEN DETERMINATIONS 285

On account of our experience we now th1nk that after an induction chemotherapeutic regimen of two months with no change at all in the CEA titer, that it is advisable to change the treatment. Maybe even quicker when the CEA continues to rise during the obser­vation period. On the other hand when we find a raised CEA on two consecutive samples (without an obvious non­neoplastic reason) in a patient in clinical remission we actually do change the chemotherapeutic treatment without waiting for the recurrence to be detected by other means.

CONCLUSION

As a conclusion for this study we may say that serial CEA determinations have some value in the moni­toring of chemotherapeutic treated cancer patients, that the evolution of the titer can predict more or less reliably the ultimate result of the therapeutic regimen and at last that it is possible to detect a metastasis or a recurrence before any symptoms have appeared.

We must however emphasize that not every cancer can be followed by serial CEA determinations. Indeed even for those patients where stat~stically the highest percentages of elevated titers can be found almost 1/3 never have an abnormal CEA during a progressive disease. The annoying problem is that we actually have no means to differentiate and exclude these cases in advance from serial CEA assays.

Aspects of Chemo-Immunotherapy in a Controlled Clinical Study

for the Treatment of Bronchogen i.c Cancer

Ch. Cerni. O. Kokron. M. Micksche. R. Titscher. H. Wrba

Institut fUr Krebsforschung der UniversiHit Wien Ludwig-Boltzmann-Institut fUr klinische Onkologie 1090 Wien. Borschkegasse 8a

It is a well known fact that patients suffering from advanced cancer usually have a decreased immunocompetence.

As far as the therapy of advanced bronchogenic carcinoma is concerned, chemotherapeutic drugs and immunotherapy are pro­mising treatments, but no miracle should be expected at present. ASide from the question whether the decreased immunocompetence in patients with far advanced cancers is caused by the progressive growth or whether it causes the progressive growth, most chemo­therapeutic drugs act as immunosuppressives, too. Since the question which is the best therapy in far advanced cancers of the lung is still open, we investigated in a randomized study 5 differ­ent types of therapy on patients with inoperable bronchogenic carcinoma.

A so-called immunoprofile was determined in patients suf­fering from lung cancer before the start of therapy, and we in­vestigated the effects of various therapies on the immunestatus and on the survivaltime.

I should like to describe first the five types of therapy and the criteria for classification of the patients. then I shall briefly mention the immunological in vivo and in vitro tests which were performed. and finally discuss the results.

Table 1 shows the five different therapy types: 1) Biological therapy (control group) 2) Cyclophosphamide + Vitamin A 3) Irradiation + Vitamin A 4) Irradiation 5) Immunotherapy

The first group is the so-called biological therapy which acts as the control group. These patients receive no special anticancer

287

288 Ch. CERNI ET AL.

treatment but anabolica, drugs supporting the heart, etc., and influenza vaccination for nonspecific stimulation.

We Justify this kind of therapy knowing from many previous studies that beyond a certain tumor size, the absence of any therapy does not significantly influence the survival time of lung cancer patients.

The second group was treated with a combination of cyclo­phosphamide and Vitamin A in a concentration of 1,5 mill units/day.

The third group was irradiated and received additional Vitamin A as scheduled in group 2.

The fourth group received irradiation only. The fifth group was treated with immunotherapy. These

patients received subcutaneously a tumor extract which was pre­pared in the following way: pooled bronchogenic carcinoma of different histological types and blood groups were minced in saline solution. The resulting supernatant was centrifuged at 22 OOOg and the proteinconcentration was adJusted to 1,5 mg/ml. The patients received 1 ml of this extract subcutaneously twice weekly.

To be included in our study the patients had to fulfill the following criteria:

prerequisites for inclusion in this study 1) clinical inoperability of lung cancer 2) male patients only 3) histological confirmation of carcinoma 4) diameter of the tumor not larger than 8 cm 5) no detectable metastasis 6) fair cHni cal condi Hon 7) no previous treatment of the neoplasm

These are the immunological in vivo and in vitro tests which were applied: in vivo methods: (skintests)

Tuberkulin Streptokinase-Streptodornase Mumps Bronchasma DNCB (Dinitrochlorobenzol) Phytha emaggl u tinin

in vitro methods: Lymphocyte transformation with PHA Lymphocyte migrationinhibition Tuberkulin

The schedule of the tests and the therapy: day 1 Admission to the hospital day 2 a) Skintests with tuberkulin and DNCB

b) Immuneprofile I

day 14 day 18 day 32

Examination for evaluation of patients to be included in the study Challenge with DNCB Start of therapy Immuneprofile II

Tumorextract

CHEMO-IMMUNOTHERAPY IN BRONCHOGENIC CANCER

da;y 63 Immuneprofile III day 64 Release from the hospital Every 6 - 8 weeks later: re-admission to the hospital for re­examination. Immuneprofile IV.

The prerequisites for being included into the study are relatively strict. In a period of 3 years 77 male patients have been accepted and divided into five therapy groups at random.

SURVIVAL· TIME CURVE OF PATIENTS WITH

INOPERABLE BRONCHOGENIC CARCINOMAS

289

- I Biological Therapy In.131

OIl

"§ I

'" > .~

::I

'"

-- ][ Cyclophoaphamide + VitA In.21) ---- I Irradiation + Vii. A In.17I - I!rlrradiallon (n.ll I •••• ]llmmunotherapy (n.15)

O~----~----<A----~~----~----~----~=---~~--2 40 60 80 weeki

Figure 1

As you can see there are no differences among the survival times of the control group, the Vitamin A and irradiation group, the irradiation only I and the immunotherapy group. Only the straight line which represents the survival time of the group of patients treated with Vitamin A and cyclophosphamide shows a more unfavourable picture, but the difference between the sur­vival time table of the group treated with Vitamin A plus cyclo­phosphamide and the biological group is not significant.

This implies that the various therapies have little effect on the survival time of our patients with far advanced lung cancer. We now evaluated the influence of therapy on the immunocom­petence of the patients. As far as the evaluation of immunological

290 Ch. CERN! ET AL.

reactivity of these patients is concerned, the number of patients with a complete immuneprofile, that means the immunestatus was determined at least 4 times, is decreased for technical reasons, by indolence or death.

Each immunestatus consists of a battery of sklntests, lymphocyte transformation with PHA and lymphocyte migration­inhibition with tuberkulin and with soluble membrane extracts of a cell-line of a squamous cell carcinoma of the lung, called E-14. The presence of these membrane antigens has been de­monstrated by indirect immunofluorescence.

The next table demonstrates the immunological results of the biological therapy whereby patients are treated with non­specific anticancer drugs only.

T.I.

160

140

100

80

60

40

20

I BIOLOGICAL THERAPY

;.;: % migration Inhibition by

~~~: E 14 - tumour antigen

....

t o 2 6 8 10 12 14 16 \8 20 22 24 26 28 30 32 34 36 38 weeks

Figure 2

The lines represent the lymphocyte transformation rates after incubation with PHA for 72 hours, pulsed with radioactive thymidine. These columns represent the results of lymphocyte migration expressed as a migration-index, that is Area of migration antigen 100 Area of migration without AG x An inhibition of more than 15% is considered to be specific.

CHEMO·IMMUNOTHERAPY IN BRONCHOGENIC CANCER

As you can see the values of lymphocyte transformation do not change very dramatically with this therapy. Only in cases where patients died shortly after the start of the therapy the transformation-indices decreased. As far as the specific cell mediated immunity is concerned (that means specIfic inhibition of the lymphocyte migration after addition of E-14-antigen) patients with squamous cell carcinoma, who do react, will con­tinue theIr reactivity during therapy. Patients with other than squamous cell CA histology of their lung cancers showed of course no reactivity towards the E-14-antigen.

Quite different is the immunological reactivity In patients treated with Vitamin A and Cyclophosphamide.

n CVCI..DPH08FIWI + VIT. A

atart of therapy I ... migration I nhibUIOn by . E 14 - tumour antigen

11 ... ' : .:: I

160 .~ + 140

120

100

60

40

20

291

o 4 6 8 10 12 14 16 18 20 22 ~ 28 26 30 32 34 311 36 weeh

Figure 3

All these patients who were followed up by immunological tests showed a decrease of the transformation-indices after therapy, with a minimum of stimulation after about 2 weeks following the start of therapy, however, the lymphocytes do recover after about 5 - 10 weeks. If they do not recover, the prognosis is rather poor, a fact which has been described by HarrIs & Stewart and others. However, the specific cell mediated immunity does not increase again. Lymphocytes of patients with speCific

292 Cn. CERNI ET AL.

sensitization to tumor antigen before therapy lose that ability during therapy. We could not detect specific mIgration inhibition after this therapy in any of the investigated patients.

The next table demonstrates the results of the immuno­therapy group.

T.\.

,60

,40

'20

'00

o

lZ IMMUNOTHERAPY

.(::: % migration inhibitIOn by i:~ pooled tumour antigens

6 a 10 ' 2 14 16 18 20 22 24 26 28 40

Figure 4

42 44 weeks

As you see, the transformation indices do not change very much. Patients with low transformation indices remain low. But the specific immunoreactivity increases. The columns represent migration-inhibition toward the injected antigenpool, which in­creases later in three patients, in two patients the specific cell mediated immunity increased initially and dropped again afterwards.

The conclusion we can draw from these results are the following: First: in patients with far advanced, inoperable lung cancer none of the five different types of therapy in our study had any appreci­able effect on the survival time of the patients. Only the group treated with Vitamin A and Cyclophosphamide showed a shortened survival time but the difference is also not significant.

CHEMO-IMMUNOTHERAPY IN BRONCHOGENIC CANCER 293

Second: Immunological changes of some consequence could be observed in the chemotherapy group. After starting the therapy the ability of lymphocytes to become nonspecifically transformed decreases, but recovers while the specific cell mediated immunity remains lower than in the first immuneprofiles.

Third: Patients receiving tumorextracts develop an increased immunologic reactivity toward the injected antigens, but this reactivity is not- constant. Although there is some evidence that lymphocytes of these patients could be sensitized toward the anti­genpool in vitro, the evidence that this reactivity is beneficial in vivo is missing. This demonstrates once more the complexity of tumor immunology and the need for additional knowledge, not only of the single components, but also of the interplay between these various factors.

PULSE-CYTOPHOTOMETRIC MONITORING OF THE INTENSIVE CHEMOTHERAPY

OF ACUTE LEUKAEMIA

S. Pawelski and S. Majx

Institute of Haematology

00-957 Warsaw, Poland

SUMMARY

Pulse-cytophotometric investigations of bone marrow cells were performed during synchronization therapy of acute myeloblastic leukaemia in adult patients. Hydroxyurea or vincristine were used as synchronizing agents and cytosine - arabinoside or rubidomycine as a second cytostatic drug. It was shown that pulse-cyto­photometry was a very valuable method for establishing the effects of drugs on DNA histograms of bone marrow cells during cytost&tic therapy of acute leukaemia in adults.

Chemotherapy of acute leukaemia in adults has not been as successful as in children. The results of treatment remain un­satisfactory, which explains why so many different programs of drug combinations are being used at the present time. Some of them are based on cell kinetic studies (1).

There is a general belief that synchronization therapy is a more successful way in the treatment of cancer (5,7,8,9,10). Chemotherapeutic agents have their maximal effects during a defined phase of the cell cycle, usually in the DNA synthesis ~hase (10, 11). A number of useful methods for obtaining synchronized pop­ulations of mammalian cells in vitro and in vivo have been develop­ed. The principles of most of the methods in current use involve blocking of the cells at some point in the cell cycle usually in DNA synthesis or mitosis (5,9,10). Appropriate criteria of syn­chrony are needed for judging the success of a given method.

Recent biological investigations utilizing the principle of pulse-cytophotometry have demonstrated its usefulness in analysis

295

296 S. PAWELSKI AND S. MAJ

of cell cycle distribution of proliferating cell population (2,6, 10, 12).

We have started to apply the method of synchronization therapy to the treatment of acute leukaemia in adults. The purpose of this report is to present our results of treatment and determine the usefulness of pulse-cytophotometry in the evaluation of cell synchrony.

MATERIAL AND METHODS

Twenty adult patients with acute myeloblastic leukaemia (AML) between ages of 17 and 81 were admitted to the present study during the 10-month period from October 1973 through July 1974. Ten patients had been previously treated with other combinations of drugs.

The induction drugs were cycled in 4 or 7 day courses (Table 1) •

Fifteen patients were given 2 mg of vincristine (VCR) for 2 days and 5 patients received 13 - 24 g of hydroxyurea (HU) p.o. in 4 divided doses every 6 h, followed by cytosine - arabinoside (ARA-C) 100 mg daily for 5 days (in 16 patients) or rubidomycine (RBD) 40 - 60 mg daily for 2 days (in 4 patients). The total number of courses of the treatment varied from 2 to 6 (average 4) in each patient. Courses were repeated after interval of 6-14 days depending particularly on the degree of bone marrow toxicity.

In all cases prednisonel .2-2.0 mg/kg/day was given by mouth with the purpose of preventing or treatment of thrombocytopenic purpura. Transfusions of packed erytrocytes and platelet con­centrates were given if indicated, and infections were treated with appropriate antibiotics.

Table I: Treatment during study

I Synchronizing agent Vincristine 2 mg/day for 2 days Hydroxyurea 13.0-24.0 g for 24 h

II Cytostatic drug Cytosine arabinoside 100 mg/day

for 5 days Rubidomycine 40-60 mg/day for

2 days

PULSE-CYTOPHOTOMETRIC MONITORING OF CHEMOTHERAPY 297

Once remission has been induced, the maintenance therapy was initiated with 6-mercaptopurine 1.5-2. mg/kg daily p.o. and methrotrexate 15 mg twice weekly p.o.

Bone marrow samples were taken three times during each course of the therapy: before and after treatment with synchronizing agent and 1-3 days after the last dose of therapeutic drug. Part of the aliquot was smeared on glass slides for May-Grunwald­Giemsa staining, the remainder of the aspirate (0.5-1.0 ml) was taken for pulse-cytophotometric examinations. Erythrocytes were sedimented in ACD-Macrodex solution. Bone marrow cells were washed, fixed in 70% ethanol and stained with ethidium bromide (10 mg in 1000 ml of Tris-buffer, pH 7.5). The pulse-cytophotome­try was performed with PHYWE ICP 11 Impulscytophotometer.

Results and discussion

Results of the synchronization therapy are presented in Table 2.

a/ Synchrony regimen with HU.

Synchronizing effects of HU was seen in 4 out of 5 patients. DNA histograms showed an elimination of late Sand G2-M phase cells and the narrowing of Gl peak (Fig. 1).

Table 2: Results of the synchronization therapy.

Drug Number of Synchrony CR PR NR patients + -

VCR 15 5 10 3 4 8

HU 5 4 1 1 1 3

ARA-C 16 - - 3 4 9

RBD 4 - - 1 1 2

Total 20 9 11 4 5 11

298 S. PAWELSKI AND S. MAJ

Counts/Channel

0.8

0.6

0.4

0.2

GF CML·BC 22 XII 1973 BEFORE TREATMENT

40 60

29 XII 1973 AFTER HU

40 60 20

411974 AFTER ARA·C

40

Channel

60

Fig. 1: DNA histograms of bone marrow cells in a patient G.F. /CML-BC/ treated with HU/13,0 g for 24 h/ and ARA-C /100 mg! day for 5 days. In the left: before treatment.

~:y~:f~~=d~~~r:~~~ination of late Sand G2-M cells 2

With this technique synchronization of cells occurs in early S phase. An S phase specific drug ARA-C was administered at the time, that partially synchronized cells entered S phase (15-Z0 h). During the treatment with ARA-C there was a complete disappearance of Sand GZ-M phase cells. The main effect of ARA-C in the doses employed seems to be an arrest of the cells in DNA synthesis phase, because Z-3 days after stopping the therapy there was a reappear­ance of cells in Sand GZ-M phase (see Fig. 1). This may rep­resent also some svnchronization and may be explained either by moving synchronously through the cell cycle the cells accumulated at the Gl-S boundary or by recruitment of cells from GO to S phase (4) •

bl Synchrony regimen with VCR

The accumulation of cells in GZ-M phase was seen only in 5 out of 15 patients (Fig. Z).

VCR is a well known to be a potent mitosis blocking agent. After synchronizing with VCR, the leukaemic blasts blocked at

G2-M phase should progress around the cell cycle in a partially synchronized manner, reaching S phase Z-3 days later. It is supposed that the mean cell cycle time of blast cells is around 50-100 h and the duration of S phase usually l5-Z0 h, GZ and mitosis about Z h.

PULSE-CYTOPHOTOMETRIC MONITORING OF CHEMOTHERAPY

Counts/Channel

0.8

0.6

JJ AI 22 XII 1973 BEFORE TREATMENT

21 1974 AFTER ARA-C

299

-L ________ -.l:C=hannel

40 60 80 20 40 60 80 100 20 40 60 80

Fig. 2: An accumulation of cells in G -M phase/in the middle/ after VCR and partial elimination of these cells after ARA-C treatment/on the right/ in a patient J.J. with AML.

Seven patients of this group were treated with ARA-C and 4 patients with RBD.

Treatment with RBD had no influence on DNA histogram in 2 patients (Fig.3) and in 2 other patients an accumulation of cells in S-G2 phases was observed (Fig.4).

RBD is a cycle phase non specific drug (3). It is a potent inhibitor of RNA and DNA synthesis. From this studies it appears that RBD may blcok cells in G2•

Influence of ARA-C on DNA histograms in this group was similar as in the synchrony regimen with HU (Fig.5)

After the first course of the treatment, the percentage of leukaemic cells in bone marrow fell only slightly. Sequential courses of treatment were given until bone marrow remission or/ and hypoplasia were achieved.

The clinical results of synchrony therapy were the following: 4(20%) patients achieved complete remission (CR), 5(25%) achieved partial remission (PR) and 11(55%) patients failed to show any response (NR) and died during study from progress of the disease. Nearlyall deceased patients died of either overwhelming infect­ion or haemorrhage in association with pancytopenia in the presence

300

Counts/Channel

0.8

0.6

0.4

0.2

20

SM AI 1911974 BEFORE TREATMENT

40 20

21 I 1974 AFTER VCR

40 60

S. PAWELSKI AND S. MAJ

2311974 AFTER RBD

40

Channel

60

Fig. 3: Nearly stable DNA histogram of bone marrow cells during the treatment period with VCR and RBD in a patient S.M. with AML did not respond to the therapy.

of extensive leukaemic infiltration. The remissions lasted from 2 to 10 months. All patients were alive at the conclusion of the study. Younger and previously untreated patients experienced remissions more frequently. Although the dosages used were kept deliberately low to minimize bone marrow toxicity, the major side-effects of the treatment was bone marrow depression with related haemorrhagic and infectious complications.

There was no correlation between the final results of the therapy, particularly of the remission rate and the synchronizing effect.

CONCLUSIONS

Forty-five per cent of adult patients with acute myeloblastic leukaemia achieved remission with synchronization therapy. In this rather small group of patients the remission rate was not better than after other programs of combination therapy recently reported. Most of the remissions occured in younger and previously

PULSE-CYTOPHOTOMETRIC MONITORING OF CHEMOTHERAPY

Counts/Channel

0.8

0.6

0.4

0.2

BAAL 6 III 1974 BEFORE TREATMENT

81111974 AFTER VCR

301

L..........L--2~0---4-0--~60""' ... 2-0---4~0--~60---' 2~0 __ ---:,c-______ -,-=Channel

Fig. 4: An accumulation of cells in Sand G -M phase after RBD treatment in a patient B.A. sufferi~g from AML.

Counts/Channel

0.8

0.6

0.4

0.2

20

KBAI 8 I 1974 BEFORE TREATMENT

40 60 20

1011974 AFTER VCR

40 60 20

1611974 AFTER ARA-C

Channel

40

Fig. 5: Disappearance of cells in Sand G2-M phase after ARA-C treatment in a patient K.B. with AML.

302 S. PAWELSKI AND S. MAJ

untreated patients. Main cause of the death in non responders were infection and haemorrhage.

Pulse-cytophotometry offers a rapid, simplified and very valuable method for establishing the effect of drugs on DNA hist­ogram of bone marrow cells. But its usefulness could be increased if we take serial bone marrow samples more frequently, at hour intervals. This way we could really regulate the dose and duration of the therapy up to the first effect - pronounced synchronization. Then we could introduce at the right time the phase - specific cytostatics and obtain perhaps optimal therapeutic response.

Unfortunately such procedure is verY troublesome for the patients and they refuse so frequent bone marrow punctures. Therefore further intensive studies are needed for the more accurate evaluation of synchronization therapy in the treatment of acute leukaemia in adults.

SUMMARY

Pulse-cytophotometric investigations of bone marrow cells were performed during synchronization therapy of acute myeloblastic leukaemia in 20 adult patients. Hydroxyurea (HU) or vincristine (VCR) were used as synchronizing agents followed by cytosine ara­binoside (ARA-C) or rubidomycine (RBD) as a therapeutic drug.

During the treatment with HU or ARA-C the DNA histograms showed an elimination of late Sand G2-M phase cells and the narrowing of Gl peak. Synchronizing effect of VCR (an accumulation of G2-M cells) was seen in 5 out of 15 patients. Treatment with RBD had no in­fluence on DNA histogram in some patients, but in others an accumu­lation of cells in S-G2 phase was observed.

The overall results of the treatment (the remission rates, toxicity) were not better than after other kinds of combination therapy recently reported.

Pulse-cytophotometry is a very valuable method for establish­ing the effects of drugs on DNA histograms of bone marrow cells during the synchronization therapy of acute leukaemia in adults, but serial bone marrow samples should be taken during the treat­ment period at a short time interval.

REFERENCES

1. Beard M.E.J., Fairley G.H. Seminars Haematol., 1974, 11,5. 2. Buechner T.: Blut, 1974, 28,1. 3. Ernst P.: Scand. J. Haemat., 1973, 11,13. 4. Ernst P., Faille A., Killmann S.A.: Scand. J. Haemat., 1973, 10,

209.

PULSE-CYTOPHOTOMETRIC MONITORING OF CHEMOTHERAPY 303

5. Klein H.O., Lennartz K.J., Gross R., Eder M., Fischer R.: Dtsch. Med. Wschr., 1972, 97, 1273.

6. Maj S, Lutz D, Stacher A.: in M.Andreeff Impulscytophotometrie­Symposium Heidelberg, 1973, Springer-Verlag Berlin - Heidel­berg - New York, 1975.

7. Mauer A.M., Lampkin B.C., McWilliams N.B.: Transplantation Proc., 1973, 5, 1181.

8. Nitze H.R., Vosteen K.H., Ganzer D.: Acta Otolaryng. 1971, 71, 227_

9. Pouillart P., Mathe G., Schwarzenberg L.: Bull. Cancer 1973, 60, 187.

10.Rajewsky M.F.: Bull. Cancer 1973, 60, 143. 11.Skipper H.E. et al.: Cancer Chemoth. Rep., 1970, 54, 431. 12.Steinkamp J.A., Romero A, Horan P.K., Crissman H.A.: Exp.

Cell Res., 1974, 84, 15.

TREATMENT OF ADENOCARCINOMA OF THE OVARY WITH

COMBINED IMMUNOTHERAPY AND CHEMOTHERAPY

Kalpaktsoglou, P.K., Kondyli, A.P., Ioannidou, G.B., Soulpi­Margariti, K.E., Comninos, A.C. and Andritsakis, G.P. From the Immunobiology Research Center and the B' Clinic of Obst. & Gynaec. at the "MARIKA ELIADI" Maternity Hospital and the Cancer Institue "AGHIOS SABBAS" 2 Helena Venizelos Square, Athens 601, Greece

Carcinoma of the ovary continues to be considered as one of the most lethal neoplasms. Apparently this is due to the lack of methods available for an early diagnosis of ovarian carcinoma.

In our previous studies we have shown that a combined treatment of immuno-chemotherapy results in a low overall mortality in mice with transplantable (1) or spontaneous tumor (2, 3). Preliminary results of the combined immunotherapy and chemotherapy for a re­stricted period of time in carcinoma of the reproductive system of the female showed that interruption of treatment is against the wellbeing of patients, therefore we changed the initial scheme of treatment (4).

The purpose of the present work is to study the influence of prolonged immunotherapy and chemotherapy in cases of adenocarcinoma of the ovary stage III and IV (F.I.G.O.).

Our aim was (a) to trigger T cells, macrophages and B cells by stimulating the humoral and cellular immunity in the hope to reduce the possibility of blocking antibody formation and (b) to protect the lymphohaemopoietic tissues from the side effects of cytotoxic agents.

305

306 P.K. KALPAKTSOGLOU ET AL.

MATERIAL AND METHODS

The scheme of treatment applied consists of two parts: First the stimulation of the humoral immunity by a battery of antigens in four consecutive courses of treatment and second the stimulation of the cellular immunity by S.C.G. administered per os. Figure 1 shows the whole scheme of treatment applied. The active non-specific humoral immunity is divided into four consecutive sessions, as follows:

re e, Di-re ., Influenza.ll- Mumps' , , cyc/o __ _ 60mgYkg (200mgs/d8Y I.V.)

BeG. 10mgs per os (total 60mgs)

cYc/o 25mgs X 2 per os /da y

1st session •

2nd .. •

3rd It • *

4th • ...

••••••

15 12 - 18 10

Day 5 of treatment Figure 1

TREATMENT OF ADENOCARCINOMA OF THE OVARY 307

1st session: Tetanus antigen (Te 1 ml S.C.); 2nd session: Diphthe­ria - Te (Di-Te 1 ml S.C.); 3rd session: Di-Te, 3 days later influ­

enza antigen; 4th session: Di-Te, 3 days later influenza and another 3 days later mumps antigen.

Chemotherapy starts three days after each session of immuno­therapy with the administration of cyclophosphamide in a total dose of 60 mg/Kg I.V. per course of treatment, divided into daily doses each one not exceeding 200 mgs.

A period of 10 days free from any medication is always left between each session of immuno-chemotherapy.

The stimulation of cellular immunity starts 10 days after the 4th session of active humoral immunization. We administer 10 mgs of B.C.G. per os every second day up to the total dose of 60 mgs. After three days free from medication the patient receives 50 mgsp.o. of cyclophosphamide per day for 12 to 15 days. This last session of treatment is repeated up to the end of the first year of immuno­chemotherapy. An interval of 10 to 15 days between each session of treatment is left free from medication. The same annual scheme of treatment starts again with the difference that cyclophos-phamide is administered per os and in a daily dose of 50 mgs for a period of 10 to 15 days.

During the first four sessions of treatment the patients under­go a check-up every 5 to 7 days and thereafter every 10 to 12 days. This includes the WBC, Ht, platelets, serum levels of IgG, IgA and IgM, and NBT positive cells. The latter are tested by a modified nitroblue-tetrazolium test (NBT) method (5). Administration of cy­clophosphamide is discontinued whenever a drop of WBC below 4500 to 4000/cmm is noticed. Antibiotics are given whenever the NBT po­sitive cells exceed the absolute number of 1000/cmm or 18 percent. All patients are under continuous vitamins A, E, B complex and C treatment.

The above scheme of treatment has been applied in seven cases of adenocarcinoma of the ovary stage III or IV (F.I.G.O.).

RESULTS

Table 1 shows the response of our seven cases to the above scheme of immuno-chemotherapy. The first case was treated only with active humoral non-specific immunotherapy and chemotherapy (four sessions). Although the patient did not have hysterectomy, she was under complete remission for six months. Following that, she started having partial bowel obstruction and died 12 months after the initial treatment.

308 P.K. KALPAKTSOGLOU ET AL.

Our fourth case who belonged tnr'stage IV, died wi thin two and a half months of acute anuria.

The f i f t h sase, who had an exploratory laparatomy without hysterectomy, showed Pap-smears positive, class 5, 15 months after the beginning of treatment. As she did not accept operation she had only one session of local radiotherapy which resulted in a signif­icant drop of the lymphocyte count and in a very low number of spontaneous rosettes forming cells. Recently, 21 months after initial treatment,she had hysterectomy. Macroscopically we observed scattered small masses of malignant cells in the abdominal cavity. Postopera­tively she is under immunotherapy and intense chemotherapy (cyclo­phosphamide 200 mgs/day I.V.).

The remaining cases are doing well, are free from clinical or laboratory findings and have a normal and active life.

Patients' Reg.f\t). Age

78937/73 58

79511/73 28

25811/73 ED

80017/73 53

80413/73 63

341EB/74 47

83173/74 45

TABLE 1 Seven cases of adenocarcinCJna of the ovaries wi th e~ansion

to the pelvic and extra-pelvic abdominal organs and ascites

Time elapsed before treat- Hyster-

Inwnuno­

chemo­

therapy ment(months) ectomy

one March 1973

one + ,April 1973

one + June 1973

one + Aug. 1973

one Sept. 1973

+ March 74

2 Y2 + June 1974

6

length of remission (months)

27 complete

23

2 partial

22

16 complete

13

Survival (months)

12

alive

alive

alive

alive

alive

Figure 2 presents serial determination of the absolute number of lymphocytes. The lower levels of lymphocytes were observed in the two cases without hysterectomy. Also the fourth case showed low le­vels of lymphocytes after local radiotherapy.

TREATMENT OF ADENOCARCINOMA OF THE OVARY 309

All our cases show a normal absolute number of granulocytes with the exception of the first stage of treatment, where a relative increase of the absolute number of granulocytes is noticed.

Figure 3 demonstrates the levels of serum IgG. We very rarely obtain levels of serum IgG below 100 I.U/ml. In normal controls the serum lev8Lof IgG ranges between 100 and 150 I.U./ml.

E 5 E 0

~ 0

X I/)

tI .... >. 0 0

.&. c.. E >.

I 0

J

Iii I ' 3 6

period

J i , ! i period

, T iii iii 9 12 15

after treatment (in months)

Figure 2

i , I 18

start

i T iii iii i i r i ~ 12 15 1l:S

after treatment start ( in months)

Figure 3

i I 21

310 P.K. KALPAKTSOGLOU ET AL.

Figure 4 shows the serum levels of IgM and IgA. The high levels of serum IgM observed in two cases during the first months of treat­ment correspond to cases which developed hepatitis after surgery. The very low levels of IgM correspond to the 4th~ase who belonged to stage IV, and had a survival of only 2 Y2 months. In normal con­trols the serum levels of IgM range between 150 and 200 I.U./ml.

Regardinn the serum levels of IgA it is interesting to point out that the 4th case who belonged to stage IV shows the highest levels of serum IgA. In the rest of our cases the serum IgA ranges within the normal levels (130 - 160 I.U./ml).

150

E 1'100

l: 50 01

r 0

I I 3

period

I i '\2 i iii i \ i I 2~ 15 18 21

after treatment start ( in months)

Figure 4

TREATMENT OF ADENOCARCINOMA OF THE OVARY 311

CONCLUSIONS

The applied scheme of immuno-chemotherapy in cases of adeno­carcinoma of the ovary,stage III (F.I.G.O.),resulted in the rapid amelioration of the patients' general condition, the reduction of the abdominal tumor masses, the reestablishment of the normal function of the bowel and a prolonged remission free of any sub­jective complaints and objective findings.

The favorable results of the applied treatment depend on the reduction of the tumor masses by surgery, preferably performed prior to immuno-chemotherapy and on the continuous treatment of immuno-chemotherapy under clinical and laboratory control, even during complete remission of the desease.

REFERENCES

1. KALPAKTSOGLOU, P.K. and GOOD, R.A., 1971. Effect of pertussis antigen and cyclophosphamide on myeloma tumor. Cancer Res., 30, 2841 - 2846

2. KALPAKTSOGLOU, P.K. The effect of ~hemotherapy an9 comb~ned immunotherapy and chemotherapy In mammary carClnoma In C3H mice. Progress in Chemotherapy. Proceedings of the 8th Int. Congress of Chemotherapy, Athens 1973 G.K. Oaikos, Hellenic Soc. of Chemotherapy 1974,p.477-481

3. KALPAKTSOGLOU, P.K. Immunotherapy or combined immunotherapy and chemotherapy applied on mammary carcinoma of C3H mice. 1975, 2nd National Hellenic Congress of Ongology (abstract) p. 128, 1975 Athens

4. COMNINOS A.C., LEKOU, S.I., APALAKIS, M. and KALPAKTSOGLOU,P.K. Immunotherapy and chemotherapy in gynecological canc~r Proceedings of the 8th Int. Congress of Chemotherapy, Athens 1973, G.K. OAIKOS Hellenic Soc. of Chemotherapy p. 400 - 404 1974

5. KALPAKTSOGLOU, P.K., PAOIATELLIS, C.P., SOFATZIS, J.A. and METAXA C.B. 1974. Evaluation of nitroblue-tetrazolium-test in low-birth-weight infants. J. Pediat., 84, 441 - 443

CLINICAL AND EXPERIMENTAL STUDIES ON IMMUNOCHEMOTHERAPY

USING OK-432, A NE~ STREPTOCOCCAL PREPARATION

T. Hattori, M. Niimoto, S. Yamagata, T. Tohge

Dept Surgery, Research Institute for Nuclear

Medicine and Biology, Hiroshima City, Japan

It is well known that many of the existing anticancer agents can improve dramatically the clinical picture of patients with advanced cancer. But the frequency of improvements is small and the duration short. OK-432, a new type of anticancer agent prepared from Streptococcus hemolyticus by Okamoto et al (1966, 1967) in Japan, seems to have both direct cytocidal and indirect host­mediated anticancer activity. This is a preliminary report of a clinical study of the combined use of Mitomycin-C, 5-FU, Cytosine arabinoside with or without OK-432. Results of experiments with large dose intra­tumoral OK-432 administration are also given.

1. CLINICAL

Materials and Methods

Fifty-seven patients with malignant disease refractory to conventional therapy, who were admitted to Surgical Division of Research Institute for Nuclear Medicine and Biology of Hiroshima University, were given Mitomycin-C, 5-FU, Cytosine arabinoside with or without OK-432. The patients consisted of 39 with gastric cancer and 18 with other malignancies (Table 1). Two administration schedules of OK-432 were explored. In schedule I small doses of OK-432 were given intramuscularly, starting from 0.2 KE (Klinische Einheit, 1 KE contains 0.1 mg of lyophilized Streptococcal constituent), increasing up to 2.0 KE within 8 days and then a maintenance dose of 2.0 KE was given daily as long as possible. Schedule II

313

314 T. HATTORI ET AL.

involved large-dose intratumoral administration, in which 100 KE of OK-432 was given directly into tumor tissue with multiple injections under general anesthesia during the laparotomy.

In the cases of continuous intramuscular injection of OK-432 further chemotherapy was combined. 0.08 mg/kg Mitomycin-C, 10 mg/kg 5-FU and 0.8 mg/kg Cytosine arabino­side were mixed and given intravenously once a week for 10 weeks in most cases. This group was designated as MFC-O group. Most of the cases of large-dose intratumoral injection of OK-432 were followed in the postoperative course by the combination therapy of MFC or MFC-O (designated as "large-dose + MFC" or "large-dose + MFC-O" group respectively). In gastric cancer patients were randomized into either MFC or MFC-O.

Results

Toxicity No deaths were caused directly by toxicity. In 14 patients there were no toxic effects; in 43 def­inite single or multiple side effects occurred. Anorexia and general weakness were the most common and seen in about one third of the cases. There were mild, trans­ient and did not necessitate discontinuance of the loading course in the most cases.

Tumour Grout;!

Large-dose Large-dose Total MFC MFC-O + MFC + MFC-O

Gastric 10 10 10 9 39

Esophageal 1 1 2 Color ectal 5 5

Pancreatic 1 4 5 Bile duct 1 1 Mammary 1 1 Pulmonary 1 1

Retroperitoneal 2 2 Malignant lymphoma 1 1

TOTAL 11 15 10 21 57

Table 1 Diagnosis of 57 patients treated by MFC or MFC-O

OK-432, A NEW STREPTOCOCCAL PREPARATION 315

Karnofsk~ criteria O-C

and Group Entered Evaluable 0-0 O-A 0-£ Q-Cl-A Category

1 MFC 11 10 5 3 2 0 0 0

MFC-O 15 14 8 3 0 2 1 3 ( 2'1.4%) Large-dose 10 10 3 1 1 3 2 5 (50.0%) + MFC Large-dose

21 19 3 7 1 5 3 8 ( 42.1%) + MFC-O Total 57 53 19 14 4 10 6 16 (30.1%)

Table 2 Response rate of 57 cases to MFC or MFC-O therapy

Therapeutic effects. Of 53 evaluable patients 16 had both subjective and objective responses and were judged O-C or Category 1 according Karnofsky criteria. Of these 53 cases, 43 were combined with either intra­muscular or large-dose intratumoral OK-432 or both. 16 of them wer,e judged O-C and Category 1 for a response rate of 37.2 %. It is to be emphasized that all cases of O-C and 1 belong to the group combined with OK-432. Of 53 cases, 31 were given the initial large-dose intra­tumoral injection. 13 of them were judged O-C or I for a response rate of 44.8 % as compared with 12.5 % in the group in which the initial large-dose was not done (Table 2).

II. EXPERIMENTAL

Materials and Methods

Experimental design. 2.5 X 106 Ehrlich carcinoma cells was firstly inoculated subcutaneously and on the day 15, when the solid tumor was palpable, large-dose of OK-432 was injected intratumorally and at the same time, the same number of tumor cells was inoculated intraperitoneally. The combined treatment of small-dose OK-432 (4 consecutive day injection of 50 KE/kg intra­peritoneally) and/or Mitomycin-C was performed from the day 17. The survival time was observed.

Tre

atm

en

t

Larg

e-d

ose

Sm

all

-do

se

Su

rviv

al

day

s

T/

d)

Su

rviv

ors

OK

-4}

2a

) OK

-4~2

b)

MO

t 0

CC

) (m

ean

::!:

S

D)

C

e)

1 o

my

cln

-at

day

5

0

KE

/kg

K

E/k

g

mg

/kg

1

,00

0

50

X

4

No

ne

35

.6

::!:

11

.6

19

6

3 /

10

1

,00

0

No

ne

1.0

2

9.6

+

1

4.6

1

63

3 /

10

1

,00

0

25

X

4

0.5

2

4.8

±

13.6

1

37

2

/ 1

0

1,0

00

5

0

X

4 1

.0

37

.4

::!:

12

.5

20

6

4 /

10

No

ne

50

X

4

No

ne

27

.2

+

12

.3

15

0

2 /

10

N

on

e

No

ne

1.0

2

6.0

+

10

.0

14

3 1

/ 1

0

No

ne

25

X

4

0.5

3

0.4

!

13

.4

16

7

2 /

10

N

on

e

50

X

4

1.0

3

3.0

::!:

1

2.2

1

82

3

/ 1

0

No

ne

No

ne

No

ne

18

.1

+

4.8

-

o /

10

a)

Larg

e-d

ose

OK

-43

2

was

giv

en

in

tratu

mo

rall

y

on

d

ay

1

5.

b)

Sm

all

-do

se

OK

-43

2

was

giv

en

in

trap

erit

on

eall

y fo

r

4 co

nsecu

tiv

e

day

s

fro

m

day

1

7.

c)

Mit

om

ycin

-C

was

giv

en

in

trap

erit

on

eall

y

on

d

ay

1

7.

d)

T/C

=m

ean

su

rviv

al

tim

e

of

treate

d

gro

up

/

mean

su

rv

ival

tim

e

of

co

ntr

ol.

e)

Mic

e

su

rviv

ing

o

ver

50

d

ay

s

were

calc

ula

ted

as

50

d

ay

su

rv

ivo

r.

Tab

le

3 C

om

bin

ed

tr

eatm

en

t o

f in

tra-tu

mo

ral

larg

e-d

ose

OK

-43

2 in

jecti

on

an

d M

ito

my

cin

-C

Co)

0- :-l

:J: ~ o ::tJ m

-l » r

0 '" T

reatm

en

t S

urv

ival

day

s T

/ d

) S

urv

ivo

rs

~

Larg

e-d

ose

Sm

all

-do

se

Co)

M"

t "C

c)

day

5

0e

) .r.

:!

OK

-4]2

a)

OK

-4,2

2b

) (m

ean

~

SD

) C

at

» 1

om

ycln

-Z

K

E/k

g

KE

/kg

m

g/k

g

m

:E

1,0

00

5

0

X

4 N

on

e 2

5.6

+

1

2.6

1

40

2

/ 1

0

en

-i

50

0

50

X

4

No

ne

27

.5

+ 1

0.3

1

51

*

1 /

10

::0

10

0

50

X

4

No

ne

29

.4 +

12

.9

16

1*

2

/ 1

0

m

""0 -i

No

ne

50

X

4

No

ne

28

.4 +

12

.1

15

6*

2

/ 1

0

0 (")

0 1

,00

0

No

ne

1.0

2

6.7

±

7.5

1

46

*

o /

10

("

) ("

)

50

0

No

ne

1.0

3

2.1

+

1

2.1

1

76

**

3

/ 1

0

» r 1

00

N

on

e 1

.0

26

.0 +

4

.8

14

2*

*

o /

10

""

0 ::0

N

on

e N

on

e 1

.0

24

.4 +

10

.0

13

4 1

/ 1

0

m

""0 »

50

X

4

16

7*

*

::0

1,0

00

1

.0

30

.5 ±

7.1

1

/ 1

0

» -i

50

0

50

X

4

1.0

3

9.3

+

1

3.2

2

15

**

6

/ 1

0

(5

10

0

50

X

4

1.0

3

0.4

+

13

.8

16

7*

2

/ 1

0

z N

on

e 5

0

X

4 1

.0

28

.8 +

12

.1

15

8*

2

/ 1

0

No

ne

No

ne

No

ne

18

.2

+

5.5

-

0 /

10

*p

< 0

.05

*

*p

( 0

.01

a)

Larg

e-d

ose

OK

-43

2

was

giv

en

in

tratu

mo

rall

y

on

d

ay

1

5.

b)

Sm

all

-do

se

OK

-43

2

was

giv

en

in

trap

erit

on

eall

y fo

r

4 co

nsecu

tiv

e

day

s b

eg

inn

ing

fr

om

d

ay

1

7.

c)

Mit

om

ycin

-C

was

giv

en

in

trap

erit

on

eall

y

on

d

ay

1

7.

d)

T/C

=

mean

su

rviv

al

tim

e

of

treate

d

gro

up

/

mean

su

rviv

al

tim

e

of

co

ntr

ol.

e)

Mic

e

su

rviv

ing

o

ver

50

d

ay

s w

ere

calc

ula

ted

as

50

d

ay

su

rviv

or.

Tab

le

4 E

ffect

of

larg

e-d

ose

intr

atu

mo

ral

OK

-43

2 in

jecti

on

as rela

ted

to

its

do

se

Co)

- "

318 T. HATTORI ET AL.

Results

Combined treatment of large-dose OK-432 and Mito­mycin-C. As shown in Table 3, the best results were seen in the group, in which full dose of consecutive small­dose OK-432 and Mitomycin-C were combined after initial large-dose intratumoral OK-432 injection. The difference was, however, not significant(Table J).

Effect of large-dose intra tumoral OK-432 as related to its dose. In order to determine the suitable dose of OK-432 as large-dose injection, three dose regimen, 100 KE/kg, 500 KE/kg and 1,000 KE/kg were examined using the same design. From the results shown in Table 4, 500 KE/ kg was suggested to be the most effective and suitable dose as large-dose intratumoral OK-432 injection(Table 4).

Discussion

Therapeutic effects of OK-432 are not always better than those produced by the other existing anticancer drugs when used as a single agent, but having no bone marrow depression suggests to use OK-432 in combination with the other conventional carcinostatic agents. As above stated, large-dose intratumoral injection of OK-432 resulted in the increased therapeutic effects when combined with MFC or MFC-O therapy. From the experimen­tal results it was also confirmed that initial large­dose intra tumoral OK-432 was benificial to the follow­ing combined treatment of Mitomycin-C and 4 day conse­cutive small-dose OK-432. The most effective and suit­able large-dose was indicated as 500 KE/kg in mice. 500 KE/kg is just 50 times as much as conventional dose of 10 KE/kg in mice. On the other hand, conventional dose of OK-432 in man may be 1.0 - 2.0 KE per day. From these it may be that 50 N 100 KE should be the corresponding large-dose in man.

Summary

Streptococcal preparation (oK-432) was given to the patients with advanced cancer combined with Mitomycin-C, 5-FU and Cytosine arabinoside. Of 53 evaluable cases, 31 were given initial large-dose intratumoral injection. 13 of them were judged O-C and 1 for a response rate of 44.8 % as compared with 12.5 % in the group without the initial large-dose intratumoral injection. Some experi­ments were successfully done in mice to verify effects of large-dose intratumoral OK-432 injection.

PHASE I AND PHASE II STUDIES IN THE TREATMENT OF CANCER PATIENTS BY RADIOTHERAPY, CHEMOTHERAPY AND METHANOL EXTRACTION RESIDUE OF AN ANTI-TUBERCULOSIS VACCINE (MER)

E. Robinson, R. Haasz, A. Bartal, Y. Cohen Department of Oncology Rambam University Hospital and The Aba Khoushy

School of Medicine The Technion, Haifa, Israel

This work was supported in part by a donation in memory of the late Luise GOterman, administered by the Office of the Administrator General and the Medical Research Fund.

The concept that tumor growth may be susceptible to control by host defense mechanism dates back to the beg-inning of this century. But interest in immunotherapy was reopened in the past two decades with the conclusive demonstration of tumor specific antigens. Specific and non-specific immunotherapy has been given widely to cancer patients. As it is not always possible to obtain material for specific immunotherapy non-specific immunotherapy is used more often. B.C.G. is known to be a potent immunological adjunct to increase host immune response, (1,2,3). Its prophylactic value in preventing neoplasm, has been documented in animal tumor systems. MER is a methanol extraction residue of B. C. G •.. lJe have previously reported that in mice with tumors, radio­therapy or chemotherapy combined with MER was more effective in reducing tumor volume and prolongin~ the survival of the animals than radiotherapy alone (4). The results of Phase I and Phase II trial whe~e MER was given to cancer patients are reported here.

MATERIALS AND METHODS

Only patients with histological proven malignant neoplasia were included in this study. There were 35 patients in Phase I.

319

320

Diagnosis

Melanoma Lung Bladder Larynx Colon Tongue Pancreas Retroper i t. Ovary Oesophagus

E. ROBINSON ET AL.

No. of Patients

14 7 3 2 3 2 1 1 1 1

35

TABLE 1 : Diagnosis of Patients.

Table 1 shows the diagnosis of the patients. 21 patients had inoperable epithelial cancer and 14 patients had malignant melanoma. Patients were examined at least every two weeks and received conventional treatment according to protocols used in our department. In Phase II there were 21 patients with lung cancer. They were randomized into two groups. One group of ten patients was treated by radiotherapy and/or chemotherapy and the other group of 11 patients received in addition to the conventional treatment immunotherapy by MER.

Test antigens. The patients were skin tested by intra­dermal injection 0.1 ml of 3 recall antigens. The anti­gens were Purified Protein Derivative (Ministry of Health, Israel, 2 TU), streptokinase (Lederle, U.S.A. 40~/10~) and Candida (Institute of Biology, Nes-Ziona, 0.1%). The skin tests were performed simultaneously on the forearm of the patients and repeated monthly. The average diameter of induration at 48 hours was recorded in millimeters.

No. of patients

Improvement 10 9 competent

No change 11 2 not competent

Deterioration 5

26

TABLE II: Change in the Skin Reactivity after MER treatment.

COMBINED THERAPY USING MER 321

MER: MER (Merck, U.S.A.) was given intradermally on the back by 10 injections each containing 0.1 mg for a total of 1.0 mg per patient. Two schedules of treatment were used. 20 patients were given MER once a month (schedule A) and 11 patients bi-weekly (schedule B). 4 patients received only one course of MER. A total of 125 treat­ments of MER were given. 25 patients received 3-10 courses of MER, while the remaining 10 patients received 1-2 courses only. Skin reactivity of MER was recorded one week after each course of injections. On each exam­ination previous sites of MER injection were evaluated. The intensity of the skin reaction to MER was graded according to average diameter of induration obtained by two right angle measurements: no response, weak response, -induration up to 4mm, medium response, -induration of 4-1Dmm, medium response-induration of 4-1Dmm and strong response IDmm of more of induration with caseation and ulceration.

In Phase II, MER was given once a month.

Lymphocyte Culture. The lymphocytes were cultured accor­ding to our previously described technique (Mekori et al, 1974). The following agents were used for stimulation:-

1. Purified Protein Derivative (PPD)(Tuberculin Section, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Weybridge~ Surrey, England, 5D~g in 0.1 diluent per culture tubes).

2. Streptokinase-Streptodornase (SK-SD) or Varidase (Lederle Laboratories, Division, Pearl River, N.V., U.S.A., 40 U-SK/IDU-SD in 0.1 ml diluent per culture tube).

3. Candidine (Cand.) obtained from Dept. of Epidermiology Nes Ziona Institute of Biology, was used for stimulation of lymphocytes at a concentration of 1% saline solution (0.1 ml volume).

4. Phytohemagglutinin (PHA) ("Wellcome" Research Labora­tories, Beckenham, England, 0.05 ml per culture tube).

All patients in Phase II were immunologically mon­itored before treatment, after radiotherapy and/or chemo­therapy and monthly thereafter (14 days after each course of MER or saline). The following immunological parameters were followed: cutaneous delayed hypersensitivity to 3 recall antigens (PPD, SK-SD and Cand.), and in vitro lymphocyte transformation to PHA and to the 3 above mentioned antigens. Previously described techniques were used (T. Mekori, 1974).

322 E. ROBINSON ET AL.

statistical Evaluation of Significance. The results were assessed by means of the Student's test (one-tailed).

RESULTS

The skin reactivity after at least 3 MER treatments is seen in Table II. In 10 patients an improvement was seen, in 11 patients there was no change and in 5 patients a deterioration occurred. Patients receiving bi-weekly immunotherapy with MER have shown more frequent im~rove­ment in their skin reactivity (5 out of 6 ~atients) than patients treated once a month (9 out of 18). The strength of the reaction at the MER injection site was evaluated in the 27 patients. After the first administration of MER 11 patients had a weak, 13 a medium and only 3 a strong reaction. Of the 20 patients treated with at least three courses of MER, the reaction was found strong in 10 ~atientE (change from weak to medium or to strong from medium).

MER induced in most patients a local inflammatory reaction whibh sometimes progressed to necrosis, ulceration and squama formation. The injection sites usually became slightly painful, secreting variable amounts of caseous materials. After repeated injections of MER, flare up phenomenon of the previous sites of injection was observed. Most lesions resolved in a period of 3-6 weeks.

In 3 patients with repeated injections out of the 35, regional lymph nodes became palpable and tender and re­gressed spontaneousoY after 6 weeks. 4 patients suffered from fever up to 39 C with malaise that lasted 24-72 hours after injection.

PHASES COMPARED

8efore conventional treatment after second MER injection

After conventional treatment after second MER injection

LEVEL OF SIGNI­FICANCE OF SKIN

* TEST VALUES

0.05 (PPD)

LEVEL OF SIG­NIFICANCE OF LYMPHOCYTE STIMULATION INDEX VALUES*

0.02 (PPD)

0.05(PPD) 0.01 (Candi-

dine)

TABLE III: Statistical Significance of Difference in Immunological reactivity of MER treated patients.

* Antigens for which significant results are obtained are shown in brackets.

COMBINED THERAPY USING MER 323

Table III shows the result of the skin tests before and after radio - and/or chemotherapy and after various administrations of MER or saline.

Skin reactions to PPD and after MER treatme~were significantly greater than before treatment (at 0.05 level). No significant changes were found in the skin reactivity to PPD, SK-SD, and Candidine in the control patients (Table III).

Table III shows the lymphocyte stimulating index in the control and MER treated patients. In the group receiving MER, the stimulation index rose significantly when PPD (0.02 level) and Candidine (0.01 level) were the stimulants. When PHA was the stimulant the increase found in the index was not significant due to great standard deviation. The~e was no significant change in the control group.

The immunological reactivity after MER treatment was stronger than that of the control patients (saline treated) The skin test and the lymphocyte stimulation index to PPD rose significantly in the MER treated patients above the values obtained in the control patients (0.02 and respect­ively 0.05 level) (Table IV).

PHASES COMPARED

After second MER injection, after saline injection

LEVEL OF SIGNI­FI CAN CE OF SK IN TEST VALUES*

0.02 (PPD)

LEVEL OF SIGNI­FICANCE OF LYMPHOCYTE STI­MULATION

INDEX VALUES*

0.05 (PPD)

TABLE IV: Statistical significance of difference in immunological reactivity between MER and control patients.

* Antigens for which significant results are obtained are shown in brackets.

DISCUSSION

Immunotherapy has been given to cancer patients by BCG. Some investigators reported improvement but also side effects to this treatment have been found (1).

MER is a methanol extration residue of the

324 E. ROBINSON ET AL.

attenuated antituberculosis vaccine BeG. It has the advantage over BCG of being a non-viable material. In mice it has been proven to be of value and with no side-effects. In this work we have shown that MER can be given bi-weekly or once a month with no serious complications. Patients who were anergic to a battery of skin tests had a low or no reaction to the MER injection.

In 10 out of 26 patients the skin reactivity became stronger during treatment. 11 patients had no change in the cutaneous reactivity and in 9 patients immunocompete­nce was retained. This occurred more often in bi-weekly treated patients (five out of six) than in patients treat­ed with MER once a month (5 patients out of 20 improved in skin reactivity and 9 patients retained their normal reactivity). Due to local reaction, MER immunotherapy cannot be given more frequently.

The danger that overstimulation of the immune response would enhance tumor growth should also be taken into consideration when the number and schedules of treatment are decided. In Phase II studies in lung cancer patients we found that MER increased the skin reactivity to PPD and stimulation index to PPD and Candidine became higher.

It can be concluded that MER has no deleterious side effects. It is well tolerated by the patients.

MER was shown to be an immune stimulator in patients with advanced tumor. Further trials in patients with no evidence of disease but known to have poor prognosis, should be undertaken.

SUMMARY

Immunotherapy by a methanol extraction residue (MER) of the tubercle bacilli was given to patients with mal­ignant neoplasia.

Two schedules of treatment were used - bi-weekly or once a month administration of MER. Repeated injections were given to some patients. The skin reactivity to a battery of recall antigens, as well as to injected MER was used to monitor the immune response. Improvement of the skin reactivity occurred in 10 out of 26 patients tested with recall antigens. In 9 other patients skin reactivity remained intact. 5 out of 6 patients treated bi-weekly, improved their immune response, while out of 20 patients on monthly schedule, 5 improved and 9

COMBINED THERAPY USING MER 325

retained normal cutaneous reactivity. Thus, repeated and bi-weekly injections were more effective in stimulating the immune response than few injections of MER. The few side side effects were tolerable.

In Phase II studies in lung cancer MER induced aD improvement in the skin reaction and in the lymphocyte stimulation index. No prolongation of survival was found in the treated group as compared to the control.

REFERENCES

1. Nathanson, L. Use of BCG in the treatment of human neoplasma. A. Review. Seminars in Oncology 1, 337-350, 1974.

2. Bast, R.C., Fbar, B., Borosos, T., Rapp, H.J •• BCG and Cancer. New England J. Medicine 290, 1413-1420 and 1458-1469, 1974.

3. Crispen, R.G. BCG vaccine in perspective. Seminars in Oncology, 1, 311-317, 1974.

4. Vron, 1., IJeiss, D.IJ., Robinson, E., Cohen, D., Adelberg, M.G., Mekori, T. & Haber, ,. Immunothera­peutic experiments in mice with the MER fraction of BCG. Studies with solid tumors. Nat. Cancer Inst. Monog. ~, 33-54, 1973.

5. Mekori, T., Sher, S., & Robinson, E. Suppression of the mitogenic response to PHA in malignant neo­plasia. Correlation with clinical stage and therapy. J. Nat. Cancer Inst. g, 9-12, 1974.

ADDITIONAL THERAPY WITH TRENIMON IN TREATMENT OF CARCINOMA

OF THE UTERINE CERVIX

M. Kaether and G. Franz

2000 Hamburg 76, Finkenau 35 - Frauenklinik Finkenau

West Germany

The object of our studies was to investigate the additional use of cyto­stati c agents in primary therapy of pati ents with carci noma of the uteri ne cervix. During the period of 1961 to 1966 a total of 146 women were treated with such agents, mostly Trenimon, in addition to the conventional basic regimen. The influence of this procedure upon the 5-year-cure-rate could be investigated.

Since 1946 primary treatment of collum-carcinoma at the Gynaecological Hospital Finkenau was performed as follows: Stage I - patients were treated by surgery using Wertheim's hysterectomy. In patients stage II to IV, combined radiation therapy was used. X-ray therapy was within ortho-volt­range with an average dose of 15000 Roentgen surface dose, distributed to 5 to 7 areas. Treatment with radium was applied intrauterine and intra­vaginally mostly with four carriers - the total dose was about 7000 milligram el ement-hours .

The 5 -year-cure-rate increased from 48.9% between 1946 and 1950 to 63.6% in 1961. Considering the last 5 years, the cure-rate was 59.6% on average. There is a marked correlation with the decrease in stage I as expressed in percentages.

In our opinion the reduction of the 5-year-cure-rate is primarily associated wi th the decrease of Stage I and stage II pati ents from 66.7% to 60.7%, observed during the period 1951 to 1966.

Cytostatic agents as concomitant medication of the basic therapeutic

327

328 M. KAETHER AND G. FRANZ

regimen of collum-carcinoma were in use mostly between 1961 and 1966. As in other hospitals at that time, we expected an improvement of our results from this procedure, however, we could not ascertain this by evaluation of our cure-rates.

In a total of 146 female patients we used either Trenimon, or its pre­decessor Bayer 3231, or the Podophyllin-derivative Proresid, or - in a few cases - the antimetabolic agent, DG 428, mostly as mono-therapy and not in combination. The average dose of Trenimon was 2.0 mg, mostly given intravenously.

Evaluation of matched pairs of women receiving cytostatic agents versus those not having this additional medication was done in the largest group under our observation, namely the 131 patients having received Trenimon. Out of this group of 131 women, 108 had been subjected to typical surgery or radi ati on as basi c therapeuti c measure.

These are designated as "Group A". Using the "matched-pair-method" this group A is compared with "Group B", not having received Trenimon. Excluded from comparison were 23 patients having received Trenimon, because in these cases either basic treatment was not performed in typical ways (for example little or no radiation at all), or because they had multiple carcinomata, etcetera. For these reasons we did not think them to be suitable cases for comparison .

Group A, consisting of 108 patients having received Trenimon was compared as matched pairs, at a relation of one to two, with Group B, consisting of 216 women without additional medication 'of Trenimon. For comparison we used patients of same stage, same histological findings, same age as far as possible, and generally speaking the same overall conditions. For exampl e one pati ent of Group A wi th uteri ne stump cancer, undergoi ng Wertheim's operation, subsequent radiation and treated with Trenimon was compared to two pati ents of Group B wi th cervi cal stump cancer, havi ng the same treatment, however, without additional Trenimon.

The influence of additional use of Trenimon on the 5-year-rate may be seen from the following results. In Group A, which was treated with Trenimon this rate was 66.7%. Group B without Trenimon treatment was 71.3%. As for stage I patients with a frequency of surgical intervention of 91%, the 5-year-cure-rates were almost identical in both groups. In stage II and especially in stage III this rate was found to be unfavorable for those patients having additionally received Trenimon. The Chi-square-test did not show significant differences however. Finally it should be pointed out, that in Group A at least a part of the patients received additional cytostatic agents

ADDITIONAL THERAPY WITH TRENIMON 329

because of an unfavorable prognosis based on other criteria than staging of the disease alone.

In conclusion it should be said, that additional Trenimon medication to the basic therapy of collum-carcinoma results in no improvement of the 5-year-cure-rate but merely a decrease, as compared to controls without cyto­static agents. The difference of 4 .6% was arranged in the "four-fold-table" and showed no significance.

INTERFERENCES OF RADIOTHERAPY AND CHEMOTHERAPY ON THE

BINDING OF3H-17~-OESTRADIOL WITH ITS SPECIFIC RECEPTORS

E. GENAZZANI, G.L. SANNAZZARI, G. CONTI, F. DI CARLO

INST. OF PHARMACOLOGY AND II CHAIR OF RADIOLOGY UNIVERSITY OF TURIN, ITALY

A clearly lower number of hormone-dependent tumours in the metastatic breast cancers submitted to radiothera­py was observed in our laboratory (see table 1).

We have wondered whether radiotherapy or chemothera­py may eventually influence the hormone-dependence of breast tumours o In order to answer this question we have studied the effects of irradiation or chemotherapy on an­other estrogen target organ, the rat uterus.

METHODS

[6,7-3HJOestradiol-17~ (40 Ci/mmole) was obtained from the Radiochemical Centre, Amersham.

The buffer solution contained 1 mM-EDTA and 10 mM Tris-HCl pH 7,4. The dextran-charcoal suspension contai-

Irradiated Unirradiated

N. of cancers

10 6

N. of hormone-dependent cancers

1 6

(10'70) (100%)

TABLE 1e Hormone-dependence in metastatic breast tu­mours obtained from patients previously submitted or not to radiotherapy.

331

Z

H

W

~~

sP-<

gc5 ti

l

c5~

H;>

< '1

20

BO

~~

Eo<

S ti

l _

_

Wtl

l IW

X

,..J

C

"")

0 ::<: ~

40

o

, /

--"

/ --

"

------

-.---

_. .-

...,..

""",,-'-

--.

-. ~

........

........

........

.. "

....

r

.A

75

r

"" ""

.,

-.e

'12

5

r

---

I --

I 2

B

'10

'1

4

DA

VS

FIG

. 1

-E

ffects

o

f ir

rad

iati

on

o

n estr

ad

iol

rat

ute

rin

e re

cep

tors

. T

he

an

i­m

als

w

ere

ir

rad

iate

d w

ith

2

5r,

7

5r

or

12

5r

an

d sacri

ficed

afte

r

2,6

,10

o

r 1

4 d

ay

s.

w

w .....

m

Cl

m

Z » N

N » z m

-I

» r

INTERFERENCES BY RADIOTHERAPY AND CHEMOTHERAPY 333

ned 0.0025% dextran and 0.25% activated charcoal in buf­fer solution.

Female rats (200-220 g) previously submitted to ab­dominal irradiation or treated with adriamycin, methotre xate, vincristine or cyclophosphamide were sacrificedby cervical dislocation. The uteri were removed immediately and placed on ice. Groups of two uteri were homogenized together in 5 m1 of buffer. The homogenates were centri­fuged at 105,000 g for 60 minutes to obtain the superna­tant cytosol fraction. 100 ~1 of cytosol were incubated with increasing quantities of 3H-17~-oestradio1overnight at 4°Co Then 0.5 m1 of the dextran-charcoal suspension were added and after 30 minutes of incubationat 4°C the mixture was centrifuged for 10 minutes at 3000 g.The su­pernatant containing the bound 3H-17~-oestradio1was ad­ded to 10 m1 of scintillation fluid (Instage1 Packard).

The radioactivity in each sample was determined in a liquid scintillation counter WALLAC LKB.

The results determined3by Scatchard analysis were expressed as femtomo1es of H-17~-oestradio1 bound/mg of cytosol protein. Protein content was determined accord­ing to Lowry et a1.

RESULTS

The number of 17~-oestradio1 uterine receptors is reduced by a single irradiation of 75 or 125 r.

This reduction of uterine receptors content is re­markable on the 6th day and lasts until the 14th day aft­er irradiation (fig o 1)0

Adriamycin, vincristine, methotrexate and cyclophos­phamide employed at therapeutic doses clearly reduce the number of oestradiol uterine receptors in rats treated i.mo or i.v. 1,3,6,24,48 hours before (fig o 2,3,4,5).

These results show that radiotherapy or antineoplas­tic chemotherapy influence the binding of oestradiol with specific uterine receptors.

334 E. GENAZZANI ET AL.

• • 0.5 mg/kg Lv.

ADRIAMVCIN _ - .. 1 mg/kg Lv.

120

o '1 ------------'1--------,----------------,-------, 1 3 S 24 4B

hour.

Fig. 2 - Effects of adriamycin on oestradiol rat uterine receptors. The animals were treated with 0.5 or 1 mg/kg i.v. and sacrificed after 1,3,6,24 or 48 hours.

INTERFERENCES BY RADIOTHERAPY AND CHEMOTHERAPY 335

120

so

40

o

•• - ...... 4 mg/kg Lm.

CYCLOPHOSPHAMIDE _ ..... 40 mg/kg Lm.

.... ___ -4"

1

I 3

--I B

-----~-- .....

114

I 4B

hours

Fig. 3 - Effects of cyclophosphamide on oes­tradiol rat uterine receptors. The animals were trea-ted with 4 or 40 mg/kg i.m. and sacrificed after 1, 3,6,24 or 48 hours.

336 E. GENAZZANI ET AL.

1110

¢:i [1:1 E-<

olil ~p..

al"" 5l BD

""~ 8>-~u E-<~ (I) --[1:1(1)

4D 1[1:1

::C"" '" ~ Iz..

o

METHDTREXATE

1 3 &

0,25 mg/kg Lrn.

114 I

48

hours

Fig. 4 - Effects of methotrexate on oestradiol rat uterine receptors. The animals were treated with 0.25 mg/kg i.m. and sacrificed after 1,3,6,24 or 48 hours.

INTERFERENCES BY RADIOTHERAPY AND CHEMOTHERAPY

z ..... '<1 E-< o

Cl ex:

120

§ ~ BO 0""': C'l0

Cf)

"";0 OE-< ..... >-

~':o E-< E t3 -;;, 40 IW

:r: ....; M 0

L:

(,..

o

VINCRISTINE

------ .......... -

• .0.05 mg/kg Lm.

_ - _0.15 mg/kg Lm.

/

I I

-...----- I -I

1

I 3

I B

I 24

337

I 4B

Fig. 5 - Effects of vincristine on oestradiol rat uterine receptors. The animals were treated with 0.05 or 0.15 mg/kg i.m. and sacrificed after 1,3,6, 24 or 48 hours.

Therefore we mayreasonably assume that the above mentioned therapies can induce similar effects on oes­tradiol specific receptors of other target organs and presumably also of breast tumours. This could be the reason why in our statistics most irradiated breast tu­mours have been found to be non hormone-dependent o

MECHANISM OF ANTITUMOR ACTION OF HEMOLYTIC STREPTOCOCCAL PREPARATION OK-432 (NSC-Bl16209) FOR MALIGNANT PLEURAL OR PERITONEAL EFFUSION BY INTRATHORACAL AND INTRAPERITONEAL INJECTION

Kazuo Ota, Atsushi Oyama

Aichi Cancer Center

Nagoya, Japan

In 1868, Busch(l) first reported that a human ma­lignant tumor regressed when the patient was infected with erysipelas. Since then, many investigators(2)-(9) have tried to use hemolytic streptococci to find a new approach to the treatment of human cancer. Okamoto et al(lO), investigating on an anticancer activity of hemo­lytic streptococci, developed streptococcal anticancer preparation, OK-432, in which a low virulent strain, Su, of streptococcus hemolyticus (group A) were treated with penicillin G and heated at 4 SoC and lyophilized. The preparation lost the ability to grow in vivo or in blood-agar medium, and failed to produce streptolysin S.

This preparation has direct anticancer effect in vitro, showing cytocidal effect(lO) and inhibition of RNA and DNA synthesis(ll) as well. When animals are pretreated with this preparation before inoculation of tumors, a significant prolongation of life span is ob­served, indicating its host mediated anticancer effect (12) .

From the above results, it appears that this pre­paration has both chemotherapeutic and immunotherapeutic activity in cancer treatment. Clinical trial has shown tumor shrinkage by direct injection into the tumor, es­pecially in malignancy of the head and neck(13), and ben ficial effects have been observed in the G.l. cancer by intravenous injection(14). The mechanism of action of the preparation however, is still obscure at present in the clinical application. In this paper, cytological changes in malignant effusion of cancer patients treat­ed with intrathoracal or intraperitoneal injection of

339

Tab

le

1 E

ffect

of

OK

-432

A

gain

st M

ali

gn

an

t P

leu

ro-p

eri

ton

eal

Eff

usi

on

~Age

jse,

D

ise

ase

C

an

ce

r

1.1

52

M

lu

ng

2'160

M

L

ung

3.

37

M

lun

g

4. I

50

I M

I lu

ng

5. I

35

I F I

lu

ng

6. I

64

I F I

Sta

moc

h

7. I

51

I F I

Sto

mac

h

a 1

63

1M

llu

ng

9. I

69

I F

I l

un

g

10.

I 50

I F

I O

vary

7 lu

ng

2

Sta

mac

h 1

Ova

ry

Pre

vo

Rx.

MFC

MFC

MFC

MFC

FA

NT

5 N

one

Site

R.pI

lop(

l.p

l

l.p(

l.p

l

lop(

R.p

l

R.pI

Lp(

l.P

(

Per.

9 P

I. 1

Per,

OK

-43

2

Dos

isId

I No

.oIl Pe

riod I C

ombi

ned

(KE

) In

j.

(m)

Rx.

1-1

0

5-1

Q2-

Q5

20

-5

1-2

2 025-

25

1-5

2 02-2

0 (1

-5)

24

I 5

6 I

2

7 11

.5

3 I

1

4 1

4d

.

11

I 1

22

12

5

4 I

1

9 I

2

18

I 3

28

I 7

MFC

IV

O

K43

2 1M

AD

IT

(l)

MFC

IT

MFC

IT

MET

VFC

IV

MFC

IP

3-28

11od

-7m

I6 N

one

1 M

FC I

V

OK

1M

2

MFC

IT

1 M

FC I

P

Eff

usi

on

E

ffe

ct

Fev

er

WB

CI

(C)

VoI

l.m I

Can

cer I Ne

utro

·llym

ph·1

Dec

.oIl

Su

bj.

I Du

ra-

' C

ell

Tum

or

1fr4:l

rCM

? t,

on(m

)

II

!l -- T ! ! 11 T 1 31

1 2

l 3

-2

f

! 1

11 ! Jl 1 !1

U 1 11

11 1 611

4 l

II

11 IT tt T

tT tl fl

Tl TT

9fT

1 1

fl rr Tl TT

T

-+

3fT

1 f

6-

++

? ? + +

1#

2

+

5-

2?

++

++

+

+ 2++

2+

6

-

6 5 8-

8-

2-8-

1 1 13

8'

T

39

'

--+

I

39D

~ '3

9'

T .-

1 I

38

'

T .-

fl'­

r .-

2ft

15+

4t

5-

3-

1 !

Co

)

~ A o -t » » z o » o -< » s:: »

ANTITUMOR ACTION OF PREPARATION OK-432 341

OK-432 are presented.

Case No.1, 52 years old, male, with adenocarcino­ma of the lung with right pleural involvement(Table 1).

The patient was first treated with combination che­motherapy MFC by intravenous and intrathoracal injec­tions and then with combination MET by intrathoracal injection for 4 months. Despite of these treatments. his pleural effusion increased and this had to be re­moved by frequent taps. Dyspnea and chest pain became worse and his general condition deteriorated. Conven­tional anticancer drugs could not be used any more, be­cause of leucopenia due to previous chemotherapy. The treatment with OK-432 was then instituted byintrathoracal injection. Pleural fluid decreased quickly, and then chest pain, dyspnea and edema of the back disappeared, and cancer cells in pleural fluid disappeared. One month after the treatment with OK-432, pleural effusion ceased to accumulate and his general condition improved greatly. Two months after the start of therapy, left supraclavicular metastatic tumor, 2 cm in diameter, dis­appeared, and he was able to ambulate after 6 months of bedridden condition. Four months after the start of therapy he could be discharged from the hospital. White blood cell count increased to the maximum of 17,600 5 months after the start of therapy.

Case No.2, 60 years old, with adenocarcinoma of the lung with left pleural involvement.

The patient was trea~ed first with carbazilquinone, then MFC, bleomycin. The tumor however became resistant to conventional anticancer drugs. At that point, injec­tion of 5 KE of OK-432 into pleural cavity was started once weekly. After 5 doses of intrathoracal injection of OK-432 pleural fluid ceased to accumulate. A large number of adenocarcinoma cells were observed in the pleural fluid before the injection of OK-432. Many neu­trophils and macrophages appeared one week after the in­jection of 5 KE of OK-432. Degenerated cancer cells were attached by lymphocytes and neutrophils 2 weeks after the start of therapy, that is after 2 injections of 5 and 3 KE of OK-432. Neutrophils and cancer cells have disappeared and many lymphocytes are now infiltrat­ed 5 weeks after the start of therapy. These changes appear very similar to the destruction of cancer cells by inflamatory reaction observed in the case of injec­tion of BCG into a tumor mass. His general condition became very well and he could be discharged from the hospital after 3 months of OK-432 treatment.

Case No.6, 64 years old female, with stomach can­cer with metastases to the lung and pleural cavity.

342 K. OTA AND A. OYAMA

She was operated on stomach cancer seven years be­fore the entry. She complained of cough and dyspnea due to 1ymphangitic lung metastasis and pleural effusion in both sides. Many adenocarcinoma cells were found in the bloody fluid in both pleural cavities. 2 KE of OK-432 was injected first into the left pleural cavity. One week after the start of therapy, that is, after 4 injec­tions of 2 KE each of OK-432, the bloody fluid of the left side changed to yellow in color. No cancer cell was present and a large number of lymphocytes were infiltrat­ed. Nowever, the fluid of right side stayed bloody with many cancer cell. At that time 300 m1 of gp1eura1 fluid was removed from the left side and 5 x 10 cells of lym­phocytes were prepared. This was injected into the right pleural cavity. If the lymphocytes had been sen­sitized to cancer cells by immunotherapeutic action of OK-432, they could have had an ability to destruct can­cer cells in the fluid of the right side. However, the trial did not succeed. Then injectioq of OK-432 was started into the right pleural cavity. Bloody fluid became clear rapidly and cancer cells disappeared. Cytological change was dramatic. A large number of neu­trophi1s and some macrophages appeared in the fluid of right side obtained one day after the injection of 2 KE of OK-432. It was observed that the cells had 65% of lymphocytes 7 days after the start of therapy. In this case improvement of pleural effusion was observed, but lung metastases stayed as the same and beneficial effect was not obtained.

Case No.10, 50 years old female, with carcinoma of the ovary with peritoneal involvement.

She presented with a mass in the abdomen and the diagnosis of carcinoma of the ovary with peritoneal in­volvement with bloody ascites was made. For the first three months, she was treated intravenous injection of combination chemotherapy METVFC and intraperitoneal in­jection of 2 KE of OK-432 once weekly. For the next 4 months she was given combination MFC and OK-432. One month after the institution of therapy, ascites ceased to accumulate, and the abdominal mass regressed in size gradually. Her general condition became very well and she was discharged from the hospital 7 months after the start of therapy.

The results of 10 cases treated with OK-432 were as follows. 0.2 to 20 KE of OK-432 was given into pleural and/or peritoneal cavity for the period of 4 days to 7 months. In some case 1 mg of OK-432 was injected every day for 4 days. In 3 of 10 cases, no pleural fluid was recovered after the therapy. In all cases cancer cells

ANTITUMOR ACTION OF PREPARATION OK-432 343

in the fluid became small in number and in 6 cases no cancer cells could be found. In all cases, marked infil­tration of neutraphi1s was observed. This nook place very quickly and in 3 cases a large number of lymphocytes appeared. Significant regression of tumor in the dis­tant focus was seen in only one case. Benificia1 effect was observed in 4 cases, in which 2 cases were treated with OK-432 alone and 2 cases with combination of OK-432 and other anticancer drugs. Duration of the response of the former 2 cases was 6 and 5 months respectively. In 2 of 10 cases, marked leukocytosis in the periphiera1 blood was observed. In 5 of 10 cases, high fever was not observed, which was encountered in all cases treated with intravenous injection of OK-432. No significant changes in total protein, S-GOT and S-GPT was observed. In 2 cases, T-cell and B-ce11 subpopu1ation of lympho­cytes in the pleural fluid after the treatment was exa­mined, which showed increased T-cell subpopu1ation.

In summary, mechanism of action of OK-432 was dis­cussed from the point of cytological changes in the pleural and peritoneal fluid when given into pleural and peritoneal cavity. In addition to its direct antitumor effect, the sequential changes, namely, quick infiltra­tion of neutrophi1s and macrophages, degeneration and de­crease in number of cancer cells, and then infiltration of lymphocytes, suggest local host immune reaction as well.

Intrathoraca1 or intraperitoneal injection of OK-432 to pleural or peritoneal involvement of malignance seems promising was to obtain a quick improvement of the effu­sion without severe toxicity either with this preparation alone or combination with other anticancer drugs.

REFERENCES 1. BUSCH, W. Niederrheinische Gese11schaft fur Natur­

und Hei1kunde in Bonn. Aus der Sitzung der Medi­cinischen Section vom 13, November 1867. Berlin K1in Wschr 5:137-139, 1868.

2. VON FEHLEISEN Ueber die Zuchtung der Erysipe1kokken auf kunsti1chem Nahrboden und ihre Uebertragbarkeit auf dem Menschen. Deutsch Med Wschr 8:553-554, 1882.

3. COLEY, W.B. Contribution to the knowledge of sarcoma. Ann Surg 14:199-220, 1891.

4. NAUTS, H.G., FOWLER, G.A., and BOGATKO, F.H. A re­view of the influence of bacterial infection and of bacterial products (Coley's toxins) on malignant tumors in man. Acta Med Scand 145 (supp1):1-103, 1953.

344 K. OTA AND A. OYAMA

5. JORDAN, R.T., RASMUSSEN, A.F., and BIERMAN, H.R. Effect of group A streptococci on transplantable leukemia of mice. Cancer Res 18:943-946, 1958.

6. CHRISTENSEN, E.A. Infection and malignant tumours. 1. Growth of Brown-Pearce carcinoma in rabbits treat­ed with living or killed haemolytic streptococci. Acta Path Microbiol Scand 46:285-295, 1959.

7. GINSBURG, l., and GROSSOWICZ, N. Effect of strepto­coccal haemolysins on Ehrlich ascites tumour cells. J. Path Bact 80:111-119, 1960

8. HAVAS, H.F., DONNELLY, A.J., and PORRECA, A.V. The cytotoxic effects of hemolytic streptococci on as­cites tumor cells. Cancer Res 23:700-706, 1963.

9. FERTMAN, M.H., FERTMAN, B., and FURST, A. The ex­perimental use of streptococcus as an anticancer agent. Proc West Pharmacol Soc 6:27-28, 1963.

10. OKAMOTO, H., SHOIN, S., KOSHIMURA, S., ET AL. Stud­ies on the anticancer and streptolysin S forming ablities of hemolytic streptococci (a review). Japan J Microbiol 11:323-336, 1967.

11. ONO, T., KURATA, S., WAKABAYASHI, K., ET AL. In­hibitory effect of a streptococcal preparation (OK-432) on the nucleic acid synthesis in tumor cell in vitro. GANN 64:59-69, 1973.

12. SAKURAI, Y., TSUKAGOSHI, S., SATOH, H., ET AL. Tumor-inhibitory effect of a streptococcal prepara­tion (NSC-Bl16209). Cancer Chemother Rep 56:9-17, 1972.

13. TOYOTA, B., MAESAKA, A., KEYAKI, Y., ET AL. Effect of streptococcal preparation "PC-B-45" on malignant head and neck tumors. J Otolaryngol Jap 72:1332-1338, 1969.

14. KUROKAWA, T., HATTORI, T., and FURUE, H. Clinical experiences with the streptococcal anticancer pre­paration, OK-432 (NSC-B116209) Cancer Chemother Rep 56:211-220, 1972.

THE SIGNIFICANCE OF REDUCTION SURGERY IN THE TREATMENT OF

ADVANCED CANCER PATIENTS

Ryusetsu Esaki, Kiyohito Shibata, Kunihiro Funahashi

First Department of Surgery, Nagoya City University Medical School, Mizuhoku, Nagoya, 467 Japan

It is generally recognized that pati ents with advanced or recurrent cancer usually come to their terminal stage without any effective and active treatment. This is because these types of pati ents show I i ttl e response to any chemotherapy and radiotherapy on account of their deteriorated general condition and insufficient immunological tolerance.

However, in surgery, we sometimes have the experience that patients with advanced cancer unexpectedly survive for prolonged periods after resection of the tumour tissue (with additional appropriate treatment) even if the surgical treatment was a non-curative resection. When patients with advanced cancer were operated to diminish the volume of their tumour, a similar trend was observed as with remission induction therapy for acute leukaemia and the prognosis of the patients then mainly depends upon the chemotherapy taken for the distant metastases and lymph node metastases both of which are amenable to surgical resection. I designate "Reduction Surgery" non-curative resection adapted for incurably advanced cancer (including primary main tumour) which is not responsive to chemotherapy.

There are several problems to be studied in Reduction Surgery for advanced cancer, especially in response of the remnant tumour tissue or the distant metastases to the chemotherapy taken. One of the probl ems is the relati on­ship between growth of the tumour and the penetration of the anticancer agent into the tumour tissue. For example, incorporation of H3-5-fluorouracil transferred into the primary metastatic lymph nodes of rats with subcutaneously transplanted Yoshida Sarcoma was gradually reduced as the lymph node metastases grew. The same was found in 44 T -nephroblastoma transplanted into T6 mice.

345

346 R. ESAKI, K. SHIBATA,AND K. FUNAHASHI

Figure 1

DRUG (5FU) CONCENTRATION IN GASTRIC CANCER TISSUE AT DIFFERENT STAGE

STAGE 1 STAGE 2 STAGE 3 STAGE 4

Figure 2

REDUCTION SURGERY IN ADVANCED CANCER PATIENTS

15

~

" ~ ~

10 :z: 0 ... ~ Eo< :z: r.:I u :z: 5 0 u 0 r.. 11'1

DRUG (5FU) CONCENTRATION IN INVOLVED LYMPH NODE OF GASTRIC CANCER AT DIFFERENT STAGE

STAGE 2 STAGE 3 STAGE 4

Figure 3

347

In patients with gastric carcinoma given 1000 mg of 5-fluorouracil one hour before the operati on, drug penetrati on into the tumour was decreased as the stage of the gastric carcinoma proceeded. A similar tendency was observed not only in the primary lesion, but also in the involved lymph nodes.

We have previously reported that fibrin debris and micro-thrombosis prevent anticancer agents from penetrating the tumour tissue as the stage of carcinoma proceeds, and that fibrinolytic enzyme is consequently indispensable to make the drugs penetrate into the tumour tissue at that time. This treatment is, however, too poor at such an advanced stage. Therefore we tri ed to perform surgery for reduction in volume of the tissue.

It was observed that the incorporation of H3-5-fluorouracil per unit weight transferred into tumour tissue decreased as Yoshida Sarcoma inoculated sub­cutaneously into the back of rats grew. When the tumour weight reached 2.2 g (on average 15 days after the inoculation) resection of about 90 per cent of the tumour caused the amount of the drug transferred into the tumour tissue and retained to recover rapidly.

A similar phenomenon was observed in the case of the mice transplanted with 44T-nephroblastoma and given H3-Actinomycin D.

The survival rate of mice transplanted with 44T -nephroblastoma was greater in the group of mice given Actinomycin 0 after resection of the tumour tissue than two other groups of mice that had resection only or were treated by chemotherapy alone.

_ 1

0

" "- '" "" ~u 0 .... :< ;: .. iI! 0 "" '" 0 u i!l " ~ I

~ :I:

INFL

UE

NC

E

OF

TUM

OR

RE

SEC

TIO

N

ON

THE

AM

OU

NT

OF

HJ-

5F

U

IN

TUM

OR

TIS

SU

E

OF

YO

SHID

A

SAR

CO

MA

TUM

OR

WEI

GH

T

10

15

20

DA

YS

AFT

ER

IN

OC

UL

AT

ION

(D

AY

)

" "-2

00

0

E "" '"'u

20

0 .... z

~ 0 ;:: iI!

.. :0: "

10

00

i

0 0. '" 8 e:;

10

'" 0 E " ..

Q I Z i i!l t '" I ::

Fig

ure

4

INFL

UE

NC

E

OF

TUM

OR

RE

SEC

TIO

N

ON

TH

E A

MO

UN

T O

F H

l-A

CT

INO

MY

CIN

-D

IN

TUM

OR

TIS

SU

E

OF

44T

-NE

PH

RO

­B

LAST

OM

A

TUM

OR

WEI

GH

T

10

2

0

30

DA

YS

AFT

ER

IN

OC

UL

AT

ION

(D

AY

)

Co

)

~

~

!::

C i

;:c

0::

m

~ C

I)

" »

.. " ~ C

I)

::z::

al » -I !> » 2 0 ~

"T1

C

2 » ::z:: G;

::z::

REDUCTION SURGERY IN ADVANCED CANCER PATIENTS

..

%

SURVIVAL RATE OF 44T-NEPHROBLASTOMA BEARING

MICE(T6) AFTER REDUCTION SURGERY

RS:90% REDUCTION SURGERY CT:ACTINOMYCIN-D

100 I---~-..... :--""f"'~-_

RS+CHEMOTHERAPY+OK432

RS+CHEMOTHERAPY

30 40 50 60

DAYS AFTER INOCULATION (DAY)

Figure 5

349

In order to judge the curability by surgery, we estimated the amount of a2-haptoglobin, that is, the subfraction of a-glycoprotein which shows a significantly close relation with the degree of progression of the cancer. These proteins in patients with carcinoma rapidly decreased to the normal level following resection of the tumour tissue. A remarkable finding concerning a-glycoprotein indicates that this protein is a blocking factor against cell mediated immunity.

When a carcinoma has been advancing, the change in a-glycoprotein and lymphocyte blastoid transformation seems to show negative correlation. The mean value of blastoid transformation was higher in the group of patients with curative operability than in the group without any curability.

There are many cases who recovered normal values of blastoid transformatior after the resection of tumour tissue, and also, several cases with rapid decrease of the val ue were found. These cases were considered as the result of con­comitant immunity and not as an indication for Reduction Surgery.

Finally, we studi ed the survival rate over one year in 24 cases of stage IV gastri c carci noma treated by Reducti on Surgery and 11 cases who had only

350 R. ESAKI, K. SHIBATA, AND K. FUNAHASHI

ALPHA-GLYCOPROTEINS

1)

2)

Al-ACID GLYCOPROTEIN Al-ANTITRYPSIN A2-CERULOPLASMIN A2-HAPTOGLOBIN

INDICATOR OF CURABILITY OF TUMOR RELATING PROTEIN -- SURGERY , CHEMOTHERAPY

MARKER OF TUMOR GROWTH AND/OR TUMOR VOLUME REDUCTION

BLOCKING FACTOR TO CELL RESTORATION OF IMMUNOLOGICAL MEDIATED IMMUNITY -- DEFENSE MECHANISM AFTER REDUCTION

SURGERY

Figure 6

exploratory laporatomy. A remarkably higher survival rate was found in the group administered (for more than three courses) 5-fluorouracil after the resecti on of tumour compared wi th the other group.

It was also observed that, among the cases of col oni c and rectal carci noma with liver metastases, even if both tumour resected group and exploratory laparotomy group were given 5-fluorouraciI for the same number of courses, the group treoted with chemotherapy after the resection of primary lesion showed a remarkably higher survival rate than the other group.

CONCLUSION

1) It is quite possible that "Reduction Surgery" makes chemotherapy more effecti ve to the remnant tumour tissue i ncl udi ng the metastases.

2) It is possible by "Reduction Surgery" to predict the drug sensitivity of

20

0

;:; C "- " ?;. :'; '"

10

0

!; " 0. 0 U ,. ..l § ~ I ;;:

CFF

:CC

T

OF

TUM

OR

RESECTIO~

ON

A

LP

HA

-GL

YC

OP

RO

TE

IN

OF

C

AN

CE

R

PA

TIE

NT

10

00

GL

YC

O­

PR

OT

EIl

J

~ "- ~ ~ '"

~-Ij

l

Al-

AN

TIT

RY

PS

IN

50

0 .d

c:~< >

2'5i

aI

50

0

;:; c "- " ?;. ~ 2

50

'" 0 ..

l " 0 .. Ii

~ ,.

'" Fig

ure

7

E,'

FE

CT

O

F

TU

MO

R

RE

SE

CT

ION

O

N

AL

PH

A-G

LY

CO

PII

OT

EH

I O

F

CA

NC

ER

P

AT

IEN

T

Ii

50

;:; C ~ Z Z 2

5

~ S

&l '" Y

:;c

II

::0

m

C

C

(')

-I

0 Z

en

C

::0

G)

m

::0

-< Z » c < » z ('

) m

c (') » z (') m

::0 " » -I

m

Z

-I

en

w

VI

12

"'0

10

M

x '" ... ~ ~ o M ~

0.

U

EFF

EC

T

OF

TUM

OR

RE

SEC

TIO

N

ON

LYM

PHO

CY

TE

BL

AST

OID

T

RA

NSF

OR

MA

TIO

N

:j:

PRE

OPE

RA

TIV

E

21

DA

YS

AFT

ER

O

PER

AT

IO>!

(D

AY

)

10

0 M X '" ... ,. ~ 0:

:.: ,.

~..,

0 M "- :.:

0.

U

Fig

ure

8

CO

RR

EL

AT

ION

B

ETW

EEN

SE

RU

M

AL

PHA

-GL

YC

OPR

OT

EIN

L

EV

EL

&

LY

MPH

OC

YTE

B

LA

STO

ID

TR

AN

SFO

RM

AT

ION

IN

C

AN

CER

PA

TIE

NT

•

•

SO

10

0

AI-

AC

ID

GL

YC

OP

RO

TE

IN(M

G/D

L)

W

01

."

,

::0

m

en » A

A

en

::J:

tIl » -I

J> » z o A " C Z » ::J: ~

::J:

REDUCTION SURGERY IN ADVANCED CANCER PATIENTS 353

PRIMARY TUMOR RESECTED NO TUMOR RESECTION

(REDUCTION SURGERY)

* NO. OF COURSE MEAN SURVIVAL RATIO OF SURVIVED NO. OF COURSE MEAN SURVIVAL RATIO OF SURVIVED (5FU) (MO.) FOR OVER 1 YEAR (5FU) (MO.) FOR OVER 1 YEAR

(/) (\) V) (\)

0 4.7 0/3 0 0 1.8 0/3 0

1 5.3 0/4 0 1 2.0 0/3 0

2 9.3 2/4 50.0 2 2.7 0/3 0

3 16.8 6/6 100.0 3 4.0 0/1 0

4 19.8 4/5 80.0 4 5.0 0/3 0

5 16.5 2/2 100.0

* ONE COURSE = 500mg/Day x 5 of 5FU

Figure 9. Relation between Reduction Surgery & Survival Period of Advanced Cancer of the Stomach with Liver Metastases

PRIMARY TUMOR RESECTED NO TUMOR RESECTION

(REDUCTION SURGERY)

* NO.OF COURSE MEAN SURVIVAL RATIO OF SURVIVED NO. OF COURSE MEAN SURVIVAL RATIO OF SURVIVED

(SFU) (MO.) FOR OVER 1 YEAR (5FU) (MO.)

FOR OVER 1 YEAR V) (\) V) (\)

0 4.5 0/2 0

1 10.0 1/2 50.0 1 3.5 0/2 0

2 8.7 2/5 40.0 2 8.0 1/~ 33.3

3 14.1 4/7 57.1 3 10.0 2/4 50.0

4 16.3 7/8 87.5 4 7.5 0/2 0

5 16.2 4/5 80.0 5 16.0 2/3 66.7

• ONE COURSE = 500mg/Day x 5 Of 5FU

Figure 10. Relation between Reduction Surgery & Survival Period of Advanced Cancer of the Colon & Rectum with Liver Metases

354 R. ESAKI, K. SHIBATA, AND K. FUNAHASHI

REDUCTION THERAPY MAINTENANCE THERAPY

REDUCTION SURGERY

REDUCTION SURGERY makes:

1) CHEMOTHERAPY more effective to the remnant tumor

tissue and metastases

2) predictive DRUG SENSITIVITY to the tumor tissue

3) estimable DRUG TruL~SFERABILITY into the tumor tissue

4) effective to restore IMMUNOLOGICAL DEFENSE MECHANISM

Figure 11

tumour and the transferabi I i ty of the drug into the tumour tissue, both of which can be the indeces of effectiveness of chemotherapy taken after the resection.

3) Reduction in volume of the tumour tissue in a host is effective to reapir the defence mechanism immunologically.

REFERENCES

1. Esaki, R. et al. (1970). Activity on Cancer Cells and Distribution in Tissue of Anticancer Drugs. Progress in Antimicrobial and Ant i cancer Chemothera py, 11, 334 , Tokyo.

2. Esaki, R. et al. (1972). Sensitivity of Anticancer Drugs and Their Concentration in Tissues. Advances in Antimicrobial and Anti­neoplastic Chemotherapy, 11, 383.

3. Surgery and Chemotherapy in Children with Malignant Neoplasms: With special reference to drug sensitivity and drug concentrations in tumour tissues. Progress in Chemotherapy, III, 637, 1974 Athens.

INTERMITTENT LONGTERM POLYCHEMOTHERAPY AS AN

ADJUVANT TO SURGERY OF BRONCHOGENIC CARCINOMA

Karrer, K. and Pridun, N.

Cancer Research Institute of the Univ. of Vienna I. Surg. Dep., Hospital of the City of Vienna - Lainz A-1095 Vienna, Borschkegasse 8a

The Viennese group has been studying the "adjuvant chemotherapy in bronchogenic carcinoma" since 1954.

Theoretically, the application of a combination of cytostatic drugs after radical surgery seems to be the best treatment at present. Since 1969 as a result of preliminary studies we have used regu­larly a new form of treatment of long term intermittent poly­chemotherapy. This treatment is expected to produce a better balance between the tumor inhibitory effect and undesirable immunosuppression. The highest possible drug dosage must be administered in order to attain in the body maximum destruction of tumor cells in small groups. The cytostatic drugs presently available are not capable of destroying all tumor cells completely in the organism and even by administration of maximum dosages only a certain number of tumor cells can be eliminated per treat­ment. Therefore , the therapy should be continued over an ex­tended period of time. In addition we try to define exactly the different groups of bronchogenic carcinomata and the reaction of patients to uniform therapy. This was accomplished by a definite description and classification of the tumor at the time of resection. We have used the TNM-system (stage I = T1NOMO) and the Fein­stein categories (this is an attempt to correlate anamnestic findings with the growth rate of the tumor). In order to gain unbiased results we divide our patients into 2 groups, one to be treated, the other untreated acting as control group. Evaluation takes place every 6th month to examine the patient for possible early noxious effects. The study was started in autumn of 1969 and we are now presenting the evaluation up to September 1974.

355

356 K. KARRER AND N. PRIDUN

METHODS

We selected two groups of patients at random - one group was treated with chemotherapeutic agents - the second group received no specific therapy after radical operation. Both groups of patients are controlled at equal intervals and treated in the same non­specific symptomatic fashion. The indication for surgical operation and the methods of operation are the same for both groups. The histological reports are supplied by the same pathologist. Chemo­therapy was carried out as follows: 1 - 2 weeks after surgery the first intravenous infusion of 500 ml of 5% Laevulose including 12 mg/kg of cyclophosphamide, 12 mg/kg of 5-FU, 0.5 mg/kg methotrexate and 0.1 mg/kg of vinblastine was administered. The infusions were repeated a second and third time in intervals of 6 days each. The procedure requires the administration of 13 series of infusions within 3 years after surgical operation. One series consists of 3 infusions each. At least the first series of infusions is given while the patient is hospitalized. After dis­missal from the hospital, further infusions are given to the patient in the out-patient department. The hematogram must be done before treatment with cytostatic drugs is started. The minimum hematological status should be: 4 mio erythrocytes, 4000 leucocytes/mm3 and 100.000 platelets/mm3• During therapy, leucocyte-control is required twice a week. To carry out this program, we try to cooperate as much as possible with the re­ferring family physician.

126 patients were treated surgically in 1974 in our department in accordance with the methods established by the Vienna school of surgery. 123 patients were treated surgically and with chemo­therapeutic agents. On table 1 you can see the division in TNM stages, main-cell types and Feinstein categories. In accordance with the Viennese school of surgery (DENK), the oat cell carcinoma is also included in surgical treatment in our department. A rather large number of patients are classified as TNM-stages I and II; this might possibly find its significant explanation in the pre­selection of patients done in private practice and essentially beyond our control. Most of these cases have been diagnosed as squamous cell and adenocarcinomata. The ratio of patients treated chemotherapeutically is actually identical with those who were operated on only. The distribution ratio according to age and sex shows a significant peak at the ages of 60 to 65. There are only a few patients older than 70 years. The male-female ratio is 1/5.

RESULTS

Generally, the treatment administered is tolerated quite well. Side effects observed are vertigo and vomiting, loss of hair occurred relatively rarely. Leukopenia was noticed in this form of treatment comparatively rarely, as tabulated in table 2. In

INTERMITTENT LONG-TERM POL YCHEMOTHERAPY 357

TN M - Stages

Z I II III IV ~uAdOatDiv SquAd Oat Dh Squ Ad Oat Div Squ Ad Oat Div

1 23 4 2 2 2 6 3 1 1 2

~2 ~

20 6 3 4 3 1 2 1

~3 52 ~7 3 1 4 5 5 1 1 5 1 4 2 3 ~

2 ~4 24 6 1 2 3 1 4 1 1 2 1 ~

5 4 2 1 1

33 10 2 8 16 12 1 2 15 1 4 1 8 5 4 1 t 123 53 31 21 18

Tab. 1: Bronchial-Carcinoma radically operated with Chemo­therapy

TN M - Stages

I II III IV Z r $~ ~ ....: . ~ ...... . ...; 'E . . ...... ....: I:: ...... I:: ...... ...... +' I:: rn »0 cd '.-1 I:: cd • .-1 I:: cd '.-1 I:: cd • .-1 I:: rn I:: C)r-i 0. . '.-1 0. . '.-1 0. . • .-1 0. . '.-1

'El 0 00) ...... I:: ...... ...... I:: ...... ...... .S ...... ...... I:: ...... ..-1 C),.Cl 0 • .-1 0 0 '.-1 0 0 0 0 ..-1 0 0) rn

S S S S • .-1 .8 ::l+, . . . . . . . +' 0)0 0 ] 0 0 '0 0 0 '0 0 0 '0 0 cd .S ~I:: Z Z Z cd Z Z cd Z Z cd Z 0.

1- 1- 1- 1-2000 f45 42 541 28 30 333 ~1 33 208 13 34 128 107 1210

2- 14 15-1500 3 13 17 3 33 62 - - - 3 26 36 9 115

3-1000 4 32 49 - - - - - - 1 28 28 5 77

250 1 11 11 - - - - - - 1 6 6 2 17

L: 1- 1- 1- 1-53 42 618 31 33 395 21 33 208 18 34 198 123 1419

Tab. 2: Bronchial-carcinoma of radically operated patients with or without leucopenia after polychemotherapy

~

~ '" 1

0

~ 5

0

... 4

0

'1l 25

30

>

. ~

:.- ; 2

0

'1l

10

-1-5

3

ch

em

oth

erap

y

_._.

b3

ran

do

miz

ed

co

ntr

ols

~

'.

'. , ...

. -

31

-2.

6-._

.

g e

s \ .,

2,(

-25

" _._

.

., ...

... II

I

..... -. \ '. .. .

,

0'

I ~'--~--

__

--~~

.. 1'

2 1'8

2'

4 6

1'2

1'8

2'4'

(;

i2

1'8

i4

'.

..t8

-12

.. _

._.

6

,

1'-

' . '._._

._.-

mo

nth

s

aft

e r

op

era!

on

1

2

18

2

4

LI

Sp

l. 7~

Fig

. 1

: C

om

pari

son

of

life

-tab

les

fro

m p

ati

en

ts w

ith

or !:.~h2~

ad

juv

an

t

ch

em

oth

era

py

aft

er

rad

ical

surg

ery

of

bro

nch

ial

carc

ino

ma

Co

) U

1

00

?' '" » ::t

J ::t

J m

::tJ » z o Z

-0

::tJ o C z

10

0

90

80

~ 7

0

~ ~ 6

0

0-

.;:

:i 0

:r.

3 --i

0

'E :~

o

~ 2

0

Fl

, , , ",

'''., " '

--\ '.

.

" ", '. " ,

F2

, \

, " ' ...

........

...... ,

---_

.,\.\ .....

..

F3

, , , , , , , ",

\

'" , , " . " .....

.........

12

:3--

Clw

mO

llw

!'ap

y

20

--

1 O~

, :1

2--

:i;;

---r

an

do

llli

zp

d (

'oll

tl'o

ls

2--

i---

:38 ---

.---

, 6

1 2

18

24

6

1 2

1 8

24

6

1 2

1 8

24

m

on

ths

aflP

f'

op

t'l'

ali

on

24

--

:!:3 ---

, , '"

"\ \ \ .....

.... "

.

6 1

2

18

2-

-i

L:S

P1

,7--

i

Fig

. 2

: C

om

pari

son

of

life

-tab

les

fro

m p

ati

en

ts w

ith

or

wit

ho

ut

ad

juv

an

t ch

em

oth

era

py

aft

er

rad

ical

surg

ery

of

bro

nch

ial

carc

ino

ma

TN

M-st

ag

es

I -

IV s

ub

div

ided

in

to F

EIN

ST

EIN

' s-

cla

ssif

icati

on

Z

-!

m

::D

3: ~ m

Z

-!

r o Z

G'l .!oj m

::D

3:

-0

o r -<

(')

J:

m

3:

o -!

J:

m

::D » ~

Co)

IJ

I -0

360 K. KARRER AND N. PRIDUN

most cases leukopenia can be brought under control by extending the intervals between infusions.

In the hospital the dose of chemotherapeutic agents given always followed exactely the established routine. Many of our patients live outside the city of Vienna, therefore, some patients received only one or just a few infusions. All these patients are also in­cluded in the study, this should be considered in the evaluation of the results.

The survival rates are calculated according to the life table method. Figure 1 shows the survival rates of treated and untreated patients in TNM stage I - IV. The steepness of the curves explains the correspondence of the prognosis with the TNM stages. Evaluating these findings carefully, we can state that the patient seems to benefit from this approach, especially in TNM stage II and IV. Figure 2 shows the same curves for the Feinstein categories 1 - 4. In the first group (Feinstein 1) - accidentally diagnosed tumors - we see a slighly positive trend in patients treated by the identical therapeutic method. In the second group (Feinstein 2) -slow growing tumors - no effect of therapy can be seen. The third group (Feinstein 3) - the fast growing tumors - show the most pronounced positive effect in the treated group. The fourth group (Feinstein 4) - patients with pulmonary and general symptoms -similar to the third group also show a positive trend in the treated group. It means that the Feinstein categories are helpful in establi­shing the prognosis and in evaluating the therapeutic effect of any given therapy.

DISCUSSION

This adjuvant chemotherapy is well tolerated. In some cases the therapy had to be discontinued for extraneous reasons; mainly because patients in Austria are not informed of their diagnosis. Evaluating the results of the described method of treatment, we feel encouraged to continue the study. Considering the variety of prognostic factors and the type of tumor involved including differen­ces in sensitivity to a given drug and/or dosage, it might become necessary to select various chemotherapeutic agents as specific as possible for a specific kind of tumor. The results shown in figure 2 seem to support the hypothesis that the dose-schedule used was not effective in slow growing tumors.

Therefore, it seems indicated to change the dose- schedule for this group of tumors or possibly use a different kind of adjuvant treat­ment. This does not necessarily mean that the dosage used is optimal effective in faster growing tumors. We believe that with certain modifications in procedure the results could be improved. Obviously, such decisions can only be made when the results of larger groups of patients will be available for comparison.

POLY CHEMOTHERAPY FOR ADVANCED LUNG CARCINOMA: CLINICAL

RESULTS AND FURTHER CONSEQUENCES

J. Kuehboeck, P. Aiginger and P. Poetzi

II. Medical Clinic, University of Vienna

Garnisongasse 13, A-1090 Vienna. Austria

Summary. 66 patients with advanced lung carcinoma of different histologic types have been treated with a polychemotherapy with 6 cytotoxic substances. In 58 % objective remission and in 21 % subjective improvement was obtained, further 21 % remained uninfluenced. Com­paring the patients with clinical remission with the patients without response the mean survival time of the first group is significantly prolonged.

The high incidence of bronchogenic carcinoma and the low rate of patients curable by surgical or radiation therapy have made cytotoxic treatment of lung carcinoma to one of the most important topics of cancer therapy.

Materials and Methods

Therefore we have initiated a combination chemo­therapy as a modification of a method formerly described by Israel et al. (1968). This polychemotherapy consists of 6 cytotoxic drugs, namely procarbazine and cyclophos­phamide, 6-mercaptopurine, 5-fluorouracil and methotrex­ate as well as vinblastine. They were administered par­enterally according to the following schedule (Fig. 1) in cycles of 1 week duration each, up to 4 cycles with a mean of 2,4 cycles. In 15 patients a second course was administered because of neoplastic recurrence. During the treatment period exact clinical control and haemato­logic observation was performed, including biochemical and radiologic parameters.

361

362 J. KUEHBOECK, P. AIGINGER, AND P. POETZI

DRUG SINGLE DOSE DAV OF ADMINISTRATION

rng f. 2. l. 4. S. 6. 7.

6-MERCAPTOPURINE 50 • • • • • • • PROCARBAZINE 125 - 250 • • • • • • • c.YClOP~OSPWAMIDE 300 - LtOO • • • 5-I=LUOROURACll 250 • • • MET~OTREXATE 5 • • ._--

VI NBlASTI NSUlFATE 5 • Fig. 1. Schedule of polychemotherapy (1 week-cycle)

(Kuehboeck et ale 1968)

The side effects of our polychemotherapy consisted of inappetence and nausea, emesis, and rarely mental alteration. Bone marrow depression could be observed in nearly all cases, depending on the number of cycles and on the kind and dosage of pretreatment agents or radio­therapy respectively and finally on the individual sensi­tivity.

All of our 66 patients (59 males and 7 females, aged 39 - 84 years with a mean of 62,9 years) had ad­vanced lung carcinoma with radiologic evidence of exten­sive stage. Table 1 shows the histologic types of the 66 cases with exception of 11 patients, in whom biopsy was not possible. In 44 patients the existence of 1-2 meta­static tumor localisations could be proved in the follo­wing organs (patients): regional lymph nodes and medi­astinum (8), lungs (7), pleural effusion (7), cerebral metastases (7), liver (13), bones (18).

The duration of disease before treatment in the whole material was 7,4 months in the mean. The pretreat­ment period is in connexion with the histologic tumor cell type, being shortest in alveolar cell type (2,0 months) and small (oat-) cell type (5,6 months) ( - which may be also the greater part of the cases with unknown histology - 5,5 months), longer in adenocarcinoma (6,) months) and large cell carcinoma (8,4 months), up to a maximum in squamous cell carcinoma (11,4 months).

POLYCHEMOTHERAPY FOR ADVANCED LUNG CARCINOMA 363

Table 1. Histologic type, therapeutic response and duration of remission and survival in 66 patients with

lung carcinoma treated by polychemotherapy

No. Response Remission Survival

Histologic Type Duration (Months) Pts. CR PR SI Ne P (Months)

Small cell carcinOia 14 3 10 - - 1 3.2 7.2 Large cell carcinOia 25 - 13 5 4 3 1.7 3.4 Squamous cell carcinoma 9 - 5 3 1 - 3.5 7.3 Adenocarcinoma 6 - 2 1 2 1 1.0 4.2 Alveolar cell carcinoma 1 - - - - 1 - 2.0 Unknown 11 1 4 5 1 - 1.6 4.1

Total 66 4 34 14 8 6 2.4 4.9

Results

The results of our treatment consisted of complete remission (CR' or partial remission (PR) and subjective improvement (SI) on the one side, a stable state = no change (NC) and progression (P) on the other side.

According to the histologic classification objective improvement (CR + PR) could be observed in 93 % of small cell type carcinoma, in 52 % of large cell type carcino­ma, in 56 % of squamous cell type carcinoma, in 33 % of patients with adenocarcinoma and in 46 % of patients with unknown histologic tumor cell type.

The duration of remission proved different relating to the histologic type of tumor (Table 1): Shortest re­mission in adenocarcinoma as well as in large cell carci­noma and carcinoma without classification is in contrast to a remarkably longer duration of remission in small cell carcinoma and squamous cell carcinoma.

Calculating the duration of survival after success­ful treatment according to the quality of response we have observed the longest survival (6,7 months) at CR and a shorter one (5,5 months) at PR, decreasing to a lower level in SI (4,4 months) and NC (3,7 months), while in cases of P the duration of survival lasts only 1,0 month in the mean. So the group of responders (CR + PR,

364

10

10

" .... \\

... , \ , , , , ,

~'" '\

'\ .~-- ....

--'"' "-

J. KUEHBOECK, P. AIGINGER, AND P. POETZI

.............. ,

~EjPOND&S (N -~9) NONRESPOND6f5(N ,. 21)

.... _-----., , '.

'-------------~--~------. 6 ~ ~ 9 M ~ ~ ~ ~ ~ M ~ m ~ ~ SUIN/VAL r/ME (MONlff5)

Fig. 2. Survival curves of patients with lung cancer, separated on the basis of therapeutic response

mean 5,8 months) separates itself from the group of SI (mean 4,4 months) and the group with NC + P (mean 2,4 months) or the collective group of nonresponders (SI + NC + p) respectively with a mean survival of ),5 months. Comparing the group of responders with the group of non­responders, the difference in the mean survival time bet­ween these two groups is statistically significant (p< 0,01). In a similar manner the total duration of dis­ease varies considerable between the responders (mean 1),7 months) and the subgroups of nonresponders (SI 10,4 months and NC + P 9,2 months).

The influence of some pretreatment on the results of the later polychemotherapy is demonstrated by the alternative between foregoing radiation and/or chemo­therapy, the latter show a decrease of survival time of 1,1 month. This difference may be explained by the better clinical condition in cases suitable for radiation the­rapy.

Following polychemotherapy in 10 patients a radio­therapy was performed, while in 15 patients a further cytostatic chemotherapy was administered. This resulted

POLYCHEMOTHERAPY FOR ADVANCED LUNG CARCINOMA 365

Fig. 3. Patient S.W. 65 y. Diagnosis: Small cell carci­noma. Chest roentgenogram. Left: Bilateral enlargement of the upper mediastinum. Right: Complete regression of the mediastinal tumor (after 3 cycles po]ychemotherapy)

in a longer survival time (7,4 months) after radiothera­py than after repeated cytotoxic chemotherapy (5,2 months), probably because of the first group being com­posed of more favorable cases compared with more poor risk cases in the chemotherapeutic group.

Discussion

Our results confirm the effectiveness of polychemo­therapy in patients with lung cancer. Depending on the histological type we have observed a maximal response rate of 93 % in the group of small cell carcinoma and the minimal of 33 % in adenocarcinoma.

A further prognostic factor especially in cases with epidermoid carcinoma shall be the patient's age with a diminished survival rate in the group above 65 years (Alberto 1974). We can not confirm this view (Fig. 3).

The importance of early chemotherapy to prevent metastasis was discussed. But patients with advanced bronchial carcinoma especially of the small cell type have a relatively short survival time. In such patients

366 J. KUEHBOECK, P. AIGINGER, AND P. POETZI

we observed a definite increase of survival time if the polychemotherapy was initiated within the first 6 months. As Newman and Hansen (1974) stated in average within 6 months the onset of brain metastases occurred.

Our relatively high rate of partial or complete response and the observed increased survival time par­ticularly in patients with small cell carcinoma justify the continuation of our kind of polychemotherapy.

References

Alberto, P. (1974), Schweiz. med. Wschr. 104, 268. Eagan, R.T., Maurer, L.H., Forcier, R.J., and Tulloh, M. (1973), Cancer 32, 371. Israel, L., Delobel, J., Sors, Ch., and Bernard, E. (1967), 5. Internat. Congress of Chemotherapy, Proceed. 11/2. Kuehboeck, J., Pokorny, D., Steinbach, K., und Eggerth, G. (1968), Wien. Ztschr. inn. Med. 49, 449. Newman, St.J., and Hansen, H.H. (1974), Cancer 33, 492.

CHEMOTHERAPY IN CONSERVATIVE TREATMENT OF lUNG CANCER

PATIENTS

I.V. Kasiananko, A.I. Pozmogov, E.l. Jerusalimsky

Institute for Oncology Problems

Kiev, U.S.S.R.

There are many reasons for chemotherapy to be consid­ered the third method to treat the patients with malignant tumour. It is used both independently and in combination with radiotherapy and surgery. We employed chemothera~y in 690 inoperable lung cancer patients (stages 3 and 4). 340 patients received antitumour drugs only and 350 patients were alloted to chemotadiotreatment; 152 patients were assigned to repeated courses of conservative treat­ment. In all the cases the diagnosis was based on clin­ical, roentgenological, bronchoscopical and morphological findings. The results of treatment were evaluated on clinical, roentgenological and on survival rate. Object­ive effects were classified according to a three scale system.

The general characteristics of the patients are presented in Table 1. Patients with disseminated disease or with contra-indications for radiotherapy were given 2 or 3 antitumour drugs with different mechanisms of action (alkylating, antimetabolite, vinca alkaloids) and 20mg of prednisolone daily.

Patients of the first group were allotted to two polychemotherapy schemes. One sub-group of patients rec­eived 0.4-0.6 g cyclophosphamide i.v. every other day with a preliminary dose of 5 mg of methotrexate p.o. The other sub-group was injected with 0.6 g of cyclophosphamide i.v. every fourth day with 0.5 g i.v. injections of 5-fluorou­racil in the interval. In all the other patients of this group the injection of 0.4-0.6 g of cyclophosphamide inter-

367

TREA

TMEN

T N

O.

OF

SEX

LO

CA

TIO

N

OF

STA

GE

HIS

TO

LO

GIC

AL

ST

RU

CTU

RE

PAT

IEN

TS

M

F TH

E TU

MO

UR

III

IV

OF

THE

TUM

OUR

cen

-p

eri

ph

-E

pid

er-

Ade

no

Sm

all

An

apl-

tral

era

l m

oid

cell

asti

c

Che

mot

h er

apy

3

40

26

7 73

2

60

8

0

19

7

14

3

14

5

77

93

25

Che

mo-

rad

ioth

era

py

3

50

2

81

69

2

52

9

8

23

4

11

6

14

7

79

10

4

20

To

tal

69

0

54

8

14

2

51

2

17

8

43

1

25

9

29

2

15

6

19

7

45

TAB

LE

I G

ener

al

ch

ara

cte

risti

cs

of

lun

g

can

cer

pati

en

ts

ass

ign

ed

to

co

nse

rvati

ve

treatm

en

t.

Co>

0-

0>

:< A » en

);

z » z A

.0

!>

:- "'tI

o N

s:: o G) o .< » z o m

r- c.... m

::0

C en » r s:: en

A -<

CONSERVATIVE TREATMENT OF LUNG CANCER 369

changed with the injection of 0.5 g of 5-fluorouracil.

The second group of patients was treated with three drugs according to the following schemes. The first half of the course: 1st day - 10-15 mg of vinblastine, 2nd day - nil, 3rd-7th day - 400-600 mg of cyclophosphamide with 5 mg of methotrexate daily, 8th day - nil, 9th day - 10 mg of vinblastine. After an interval of two days the second half of the course was given.

The second scheme specified the injection of 400-600 mg of cyclophosphamide with 5 mg of methotrexate every 4th day and in the intervals we gave 500 mg of 5-fluor-ouracil. Polychemotherapy in the second group was used in combination with 20 mg of prednisolone daily. The control group of patients was treated with cyclophosphamide only. The result and the summary of the dosage scheme are presented in Table 2. The results show that three drugs are slightly more effective than two drugs in all patients. Patients in stage III gave better immediate results than those in stage IV. The chemotherapy frequently gave toxic effects on the alimentary canal, myelogenous haemopoesis and the cardiovascular system. The combination of cyclo­phosphamide with two antimetabolites produced considerable gastro-intestinal toxicity (vomiting, stomatitis, diarr­hoea) while with the combination of cyclophosphamide, methotrexate and vinblastine, leukopenia and thrombo­cytopenia occurred more often.

In evaluating treatment on the survival rate, it should be noted that survival during the first 6 months after the treatment was better with three drugs. Longer survival and median survival rate did not depend vitally on the amount of the administered drugs. Nevertheless 42% of patients (out of 270) survived for more than 6 months, 14.9% of patients for more than a year and 3.3% of patients for more than 18 months (Table 3). Survival and median survival rate were somewhat higher in patients with well differentiated cancer.

Analysis of the data obtained from patients receiv­ing polychemotherapy courses repeatedly shows (Table 4) that the number of patients with objective effect becomes smaller the greater the number of courses received. Nevertheless, subjective improvement and stabilization of the malignant process in some patients was achieved as a result of the repeated courses. Survival of these patients increased in comparison with patients administ ered only one course of polychemotherapy.

TR

EA

TM

EN

T

TOT

Al

DO

SE

NO

. O

F O

BJE

CT

IVE

E

FF

EC

T

SU

BJE

CT

IVE

E

FF

EC

T

(g)

PA

TIE

NT

S

3 2

1

I Cy

clo

ph

osp

ham

ide

4.0

-6.0

Meth

otr

ex

ate

5

0-7

0

mg

11

8

2 7

17

2

9

( 5

-flu

oro

ura

cil

) 3

.0-4

.0

26

II

Cy

clo

ph

osp

ham

ide

4.0

-6.0

Meth

otr

ex

ate

5

0-7

0

mg

(5-f

luo

rou

racil

) 3

.0-4

.0

15

2

5 1

5

33

4

6

Vin

bla

sti

ne

30

-40

m

g 5

3

Pre

dn

iso

lon

e

III

!cy

clo

ph

osp

ham

ide

4.0

-8.0

70

0

4 8

15

1

2

IV OT

Al

34

0

91

90

TA

BL

E

2:

Ch

em

oth

era

py

o

f lu

ng

C

an

cer

Sta

ges

III

an

d

IV

i ,

w ~ :< A » en

l>

z » z A

,0

!>

"'tJ o N ~

o G) o < » z o m :- <

­ m

:tI

C

en » r ~

en

A -<

CONSERVATIVE TREATMENT OF LUNG CANCER 371

TREATMENT NO. OF SURVIVAL IN MONTHS PATIENTS 7-12 13-18 18-24

Cyclophosphamide Methotrexate 90 38 12 3 (5- fluorouracil)

Cyclophosphamide Methotrexate Vonblastine 115 55 20 4 (5-fluorouraci 1) Prednisolone

Cyclophosphamide 65 22 8 2

TOTAL 270 115 40 9

TABLE 3: Survival of Lung Cancer Patients, Stages III and IV assigned to Chemotherapy.

Combination of drugs with irradiation also §ave good results. The total doses of radiation and drugs are given in Table 5. The best objective effect was observed in patients in stage III with combined treatment of telegammatherapy and antimetabolites. The worst effect was obtained where irradiation was used alone. Clinical improvement in patients with squamous or adenocarcinoma was better as a result of the combined treatment with telegammatherapy and antimetabolites, but in the ana­plastic form it was better with cyclophosphamide. The effectiveness of chemoradiotherapy depended directly on the total irradiation dose and the stage of the disease. Chemoradiotherapy prolongs to a small degree (up to 18 months) the life of stage III lung cancer patients but the extent depends on total irradiation dose, drugs used and morphological type of the tumor. 204 out of 319 patients under observation survived for more than 6 months 109 more than a year, 31 more than 2 years (Table 6).

Two to 14 months after the end of themoradiotherapy 70 patients underwent 1-3 poly chemotherapy courses (Table 7). The number of patients having objective benefit decreased with every additional course, but there was subjective improvement stabilization of the malignant process in a percentage of patients. The median survival rate in patients treated with courses of maintenance polychemotherapy increased, but these data need further verification.

372 LV. KASIANANKO. A. I. POZMOGOV. AND E.L. JERUSALIMSKY

NO. OF COURSES

ALL OBJECTIVE EFFECT STABILIZED SUBJECTIVE

I

in 2-10 months

II

in 2-5 months

III

82

82

22

(in numbers) PROCESS EFFECT 321

4 12 20 13 29 36

o 3 13 24 26 16

o o 3 3 10 3

TABLE 4: Results of Repeated Polychemotherapy courses in Lung Canc er.

GROUP TREATMENT DOSES NO. Or. PATS.

EFFECT Objective Subj 321

il.

2

3

5

Telegamma 3000-8000R Cyclophosphamide 3.0-8.0

Telegamma 5-fluorouracil

Telegamma Methotrexate

Telegamma

TOTAL

3000-8000R 3.0-8.0

3000-8000R 80-140 mg

3000-8000R

75 8 18 19 45

77 13 26 17 56

115 15 38 33 86

83 5 11 21 37

350 41 94 82 217

TABLE 5: Results of Chemoradiotherapy in Lung Cancer Stages III and IV.

20

13

27

26

86

The rate of complications during the repeated cour­ses of polychemotherapy and maintenance courses of poly­chemotherapy after radiotherapy did not increase by comparison with the patients given only one course. We found no serious toxicities and treatment was only dis­continued in isolated cases.

CONSERVATIVE TREATMENT OF LUNG CANCER 373

~REATMENT NO. OF SURVIVAL IN MONTHS PATS.

7-12 13-18 19-24 25-30 31-36 37-42 43-4E

~elegamma Cyclophos- 65 38 18 7 4 2

phamide

Telegamma 69 54 34 18 12 5 4 1 5-FU Telegamma Metho- 100 76 37 20 10 4 2 trexate

~elegamma 75 36 20 9 5 3 1

~OTAL 319 204 109 54 31 14 7 1

TABLE 6 Survival of Lung Cancer Patients, Stages II I and IV assigned to Chemoradiotherapy.

NO. OF ALL OBJECTIVE EFFECT STABILIZED SUBJECTIVE COURSES (io oumbaI:s) PROCESS EFFECT

3 2 1

I Chemoradio 70 5 25 20 5 8 4-12 months 50

1 Chemotherapy 70 0 0 11 14 32 4-15 months 11

11 Chemotherapy 22 0 0 0 5 12

TABLE 7: Repeated courses of Treatment (Chemoradio I and Polychemotherapy II, III)

EFFECTIVE CHEMOTHERAPY FOR BRONCHIAL CARCINOMA

E.W. Street

Bromley Group of Hospitals Department of Medicine, Farnborough Hospital Farnborough Common, Orpington, Kent, BR6 8ND

If the progress of cancer chemotherapy has to depend entirely on the results of prospective, randomized trials it will be exceedingly slow. I am sure my audience is familiar with the articles of Freireich2 and Gehan3 which were published last year on the subject of non-randomized, historical controls. I make no apology for using a consecutive series of historical controls in this report on the first 33 cases treated by combination chemotherapy using intravenous emetine and cyclophosphamide. As an individual clinician, working in a district hospital, a randomized trial on patients suffering from a lethal disease is almost unethical, and all the more so when the physician is already convinced that one of the alternative treatments is better than the other.

This trial was not preplanned. It started by chance when a young woman patient, with rapidly advancing malignant disease ln both lungs, was given emetine hydrochloride as supplementary treat­ment to intravenous cyclophosphamide. Open lung biopsy had been reported as squamous carcinoma, and an unexpected, prolonged remission followed. The next few cases (Street 1972)4 responded surprisingly well to this treatment also, and it has since evolved as my standard therapy for nearly all cases of inoperable bronchial carcinoma. Well over one hundred cases have now been given courses of combination chemotherapy using once weekly intravenous emetine throughout and intravenous cyclophosphamide for most or all of the time. I have aimed at an unbroken course of 12 weeks as my standard regimen, but this has been varied as expediency demanded and, from the early days, Methotrexate was used additionally in some cases. This year I have started using Vincristine and Fluorouracil also, but always in combination with emetine and all drugs have been

375

376 E.W. STREET

given by direct intravenous injection, neVer by mouth or by drip infusion. The dosages used are intended to avoid myelosuppression of sufficient degree to require interruption of the course. The same principle applies to emetine which must be stopped if there is evidence of myocardial toxicity, the cardinal sign of which is tachycardia.

A healthy respect for the potential cardiotoxicity of emetine hydrochloride, with WhlCh I started, led to a switch to dehydroemetine in December 1972. In addition to the cytostatic combination therapy I give supplementary supportive therapy during the first three weeks of the course which is designed to minimize the side effects and ensure symptomatic improvement from the start of treatment.

Long term corticosteroids were given to the earlier cases, but this adds to the immunosuppression and increases the risk of opportunist infections. There were four deaths from this cause during the first year, but none during the second year. The following tables show how the detailed protocol of treatment was changed during this two year period.

TABLE 1

EMETINE/CYCLOPHOSPHAMIDE SCHEDULE - JUNE 1972

EMETINE HYDROCHLORIDE

1.5mg.per kg. given once weekly by slow I.V. injection.

CYCLOPHOSPHAMIDE

8mg. per kg. I.V., age 50 - 70

Each dose increased by 50mg. under age of 50 Each dose decreased by 50mg. over age of 70

1st dose given on DAY 2 of EMETINE, then daily for 1 week.

2nd - 5th weeks - Twice weekly 6th - 10th weeks- Once weekly, but dose stepped up by 100mg.

PREDNISONE

20mg. daily for 5 weeks, reducing thereafter.

EFFECTIVE CHEMOTHERAPY FOR BRONCHIAL CARCINOMA

TABLE 2

DEHYDROEMETINE/CHCLOPHOSPHAMIDE SCHEDULE - JANUARY 1974

CYCLOPHOSPHAMIDE

8mg. per kg. I.V., age - 50 - 70

Each dose increased by 50mg. under age of 50 Each dose decreased by 50mg. over age of 70

DAILY for first 3 days and on days 5 - 7 2nd - 5th weeks TWICE WEEKLY 6th -12th weeks ONCE WEEKLY but dose stepped up by 100mg.

DEHYDROEMETINE (starting on DAY 4)

2.5mg. per kg. given once weekly by slow I.V. injection throughout 12 week course.

PREDNISONE

20mg. daily for 1st week 15mg. daily for 2nd week 10mg. daily for 3rd week and finish.

OXYMETHOLONE

100mg. daily for 1st week 50mg. daily for 2nd week and finish.

Emetine as a single agent is certainly a weak cytostatic

377

drug. The use of such a drug in combination therapy has recently been aired in the correspondence column of Cancer Chemotherapy Reports, (Bunn 1974) land, in defence of my use of emetine I compared it to PAS in antituberculous chemotherapy. (Street 1974) 5 It is well known that this drug is of very limited efficacy when used alone, but it held its place as one of the three first line drugs ln combination therapy for all of 20 years.

My claim that combined chemotherapy with emetine is effect­ive in the treatment of advanced bronchial carcinoma must be support­ed by statistical evidence of increased survival as this is something which can be measured. It has taken nearly three years for the evidence on survival to build up, but I was convinced very early in the first year that this was the treatment of choice for nearly all cases of unresectable bronchial carcinoma. The only explanation I can offer is that my experience of this common disease. amounting to one new case each week on average. taught me to appreciate an improvement in the quality of these patients' all too short lives. and thus to persist with this new method of treatment.

378 E.W. STREET

The only treatment for unresectable bronchial carcinoma which has been proved to prolong life is radiotherapy. This is definite in the oat cell variety, but doubtful in the other cell types. I would have liked to present you with straightforward com­parative survivals between my combined chemotherapy and radiotherapy, but the latter is suitable for less than half the cases of inoper­able lung cancer, and chemotherapy has its greatest application in cases of disseminated disease. This is not to say that combined chemotherapy is any less effective than radiotherapy when the disease has spread no further than the mediastinal glands. It will relieve superior venacaval obstruction just as certainly and more quickly than radiotherapy. The pain of bony metastases, which are nearly always multiple and not amenable to radiotherapy, can usually be relieved in a dramatic manner by this combination the~apy.

Eighteen of the control series completed a full course of radical radiotherapy and, for a fair comparison, I would need an equal number of cases which were capable of being treated by this method who have completed my standard 12 week course of combined chemotherapy. I am not able to do this at present, so both series are made up of a mixture of this type of case and those with more widespread disease. The emetine series has only 13 cases which were judged capable of being treated by radical radiotherapy, the remainder having more advanced and disseminated disease. This table shows the basic statistics of the two series compared and gives the mean survival figures in days from the date of histological diagnosis. The difference is about 60% in favour of the emetine series with four still surviving. None of the other comparative statistics - such as proportions of different cell types - is weighted in favour of the emetine series.

Males Females Mean age at diagnosis

CELL TYPES

Squamous (Epidermoid) Oat (Small cell) Large undifferentiated Adenocarcinoma Mean survival (days from diagnosis)

TABLE 3

Emetine Series

25 8

61. 5

15 13

3 2

293·5

Control Series

31 2

62

13 12

3 5

186

EFFECTIVE CHEMOTHERAPY FOR BRONCHIAL CARCINOMA

TABLE 4

COMBINED CHEMOTHERAPY TRIAL

Reasons for exclusion from survival statistics

1. Histology not established before start of treatment.

2. Primary tumour not certainly of bronchial origin.

3. Antecedent primary treatment of a different nature, or subsequent D.X.T.

4. Minimum course of 30 days not completed.

379

I am putting forward this new combination therapy as the definitive primary treatment for most cases of unresectable bronchial carcinoma. I must therefore exclude not only cases which are unproved histologically but also those with malignant disease which is not certainly of bronchial origin. Patients who have had additional treatment, such as radiotherapy, before or after the chemotherapy, have also been excluded. The final category of exclusions has been added because a number of frankly terminal cases were given a short trial of combined chemotherapy as palliative or placebo treatment. Some unexpected responses were obtained in apparently hopeless cases and chemotherapy was then continued for more than four weeks. The mean survival of those excluded under all four categories put together works out exactly the same as the control figure - 186 days. They total 26 cases in all and, if they were included with the 33 strictly controlled cases, the mean survival of the combined chemotherapy series would be lowered but still remain well above that of the control series.

The graph shows the actuarial survival of the two series compared. Two features stand out. One is the closely parallel course of the two groups over much of the graph, and the other is the increasing divergence of the two curves towards the bottom. This feature will be even more marked when the four remaining survivors in the emetine series have been followed until death. Note that the divergence begins at number 25 of the 33 cases. May I conclude from a study of these early figures that the 60% overall increase in mean survival is due to about 20% of the cases treated with emetine combination, some of whom are achieving remissions of quite surprising duration.

380

'-' z

> '" " '"

30

1'":iJ 300 4'":iJ 600 7'":iJ 900 DAYS FROM DIAGNOSIS TO DEATH

(0- SURVIVORS OF EMETINE SERiES ON 1.7.75)

Graph of Comparative Acturial Survival

E.W. STREET

1O'":iJ

REFERENCES

1. Bunn P.A.,Jr. (1974) Cancer Chemotherapy Reports, 2§., 127-128.

2. Freireich E.J., and Gehan, E.A. (1974) Cancer Chemotherapy Reports.Pt.l, ~,623-626

3. Gehan E.A. and Freireich E.J. (1974) New England Journal of Medicine, 198-203

4. Street E.W. (1972) Lancet, 2, 381-382

5. Street E.W. (1974) Cancer Chemotherapy Reports, Pt. 1, ~, 621-622

MULTIDISCIPLINARY CURATIVE ASSAULT ON DISSEMINATED CARCINOMA

OF THE BREAST

P. Mannes, R. Derriks, R. Moens, C. Laurent and J. Dalcq

Department of Clinical Oncology Clinique Saint-Jean Brussels, Belgium

SUMMARY

84 cases of disseminated breast carcinoma were treated with the association right from the beginning of hormonal and combination chemo­therapy. Hormonal treatment included castration for women still menstruating, estrogens for women menopaused for more than 5 years and androgens or progesterone for women menopaused for less than 5 years. Combination chemotherapy consisted of monthly courses of 5 days: 5FU, 500 mg day, days 1-5; Cytoxan, 300 mg day, days 1 and 4 i Vi ncri sti ne, 1 mg day, days 2 and 5 i Methotrexate, 100 mg day 1, foil owed by Leucovori n, 12 mg day, days 2 and 3. Overall response rate is 83.3%: 43 complete remissions (51%) and 27 partial remissions. Median survival time lasts 17 months, 6 months for the 14 failures, 12 months for the 27 P.R. and 27 months for the 43 C.R. 15 patients are still alive in C.R., one after 16 months, one after 17, two after 19, one after 23, 24, 25, 31, 35, 37, 38, 43, 48, 68 and 70 months.

Disseminated breast cancer used to be treated by hormonal therapy. Primary endocrine therapy consists of castration in premenopausal women and treatment with androgens, estrogens and progesterone in postmenopausal women. Secondary endocrine therapy consists of adrenalectomy, hypophysectomy, or massive doses of cortico-steroids.

Chemotherapy was started only after failure of hormonal therapy.

Since 1968 we applied to metastatic carcinoma of the breast a multi-

381

382 P. MANNES ET AL.

di sci pi i nary treatment. The pati ents were treated wi th the associ ati on, right from the beginning, of hormonal and non-hormonal combination chemo­therapy.

MATERIALS AND METHODS

Eighty-four cases of disseminated carcinoma of the breast were studied. We made no selection and no case of the series has been rejected, however important was the severity of the disease. There were: 64 cases of thoracic metastases; 13 cases of bone metastases i 4 cases of centra I nervous system metastases i 3 cases of hepati c metastases.

Hormonal Treatment

Hormonal treatment included: castration for women still menstruating; estrogen therapy, diethylstilbestrol, 15 mg day or ethinylestradiol, 1.5 mg day for women menopaused for more than 5 years; androgens, delta-l-testo-I olactone, cal usterone, or progestati onal agent, medoxyprogesterone acetate, 100 to 300 mg day, orally, for women menopaused for less than 5 years.

Combination Therapy

We utilized a four drug combination including 5-FU, Cytoxan, Metho­trexate and Vincristine by monthly courses of 5 days, according to the foil owi ng schedul e: 5-FU. 500 mg i.v. day, days 1 t05; Cytoxan, 300 mg i . v. day, days 1 and 4; Vincristine, 1 mg i .y. day, days 2 and 5; Methqtrexate, 100 mg i • v. day I, followed by Ci trovorum factor, 12 mg day

days 2 and 3.

After one year, for the patients in complete remission, the courses were progressively spaced up to every 6 weeks, every 2 months, and, after two years of treatment to every 3 months.

RESULTS

For the entire group of 84 patients (Table 1), the median survival time is 17 months. The overall response rate is 83.3% (70/84). There are 43 complete remissions (C.R.) with a median survival time of 27 months. 15

MULTIDISCIPLINARY CURE OF BREAST CARCINOMA 383

patients in C.R. are still alive after 16 to 70 months.

There are 27 partial remissions (P.R.) with a median survival time of 12 months. 4 patients in P.R. are still alive after 16 to 31 months. 14 patients did not respond and their median survival time is 6 months.

The median duration of the complete remission is 20 months, of the partial remissi on 7 months.

Thoraci c metastases (64 cases)

Among the 64 patients, 12 premenopaused were submitted to castration.

The median survival time is 16.5 months (Table 2). The overall response rate is 84.3% (54/64) .

There are 34 C.R. (53.1%) with a median survival time of 27 months. 11 patients, in C.R., are still alive one after 16 months, one after 17, two after 19, one after 23, 31, 35, 38, 43, 68 and 70 months.

There are 20 P.R. (31.2%) with a median survival time of 10.5 months. One patient in P.R. is still alive after 16 months.

10 patients did not respond: median survival time is 6 months.

The median duration of complete remission is 18 months and of partial remission 6 months.

Bone Metastases: 13 cases

7 C.R., median survival time 22 months. 3 patients are still alive, two after 24 months and one after 48 months.

Central Nervous System Metastases: 4 cases

Two C.R., one died after 46 months and one is still alive in C.R. after 37 months. Two failures, survival time 4 and 7 months.

Li ver Metastases: 3 cases

One P.R., survival time 18 months. Two failures, survival time 3 months.

384 P. MANNES ET AL.

Table 1. Summary of the results obtained for the 84 patients.

Th ora ci c metastases (I ung, pi eura, peri cordi um, lymph nodes, chest wall, ski n)

Bones Central Nervous System Liver Overall response rate Median survival Number C.R. 43, median survival

15 patients are still living, one after 16, one after 17, two after 19, one after 23, 24, 25, 31, 35, 37, 38, 43, 48, 68 and 70 months

Number P.R. 27 median survival 4 patients still living after 16-31 months

Number failures 14 median survival Median duration of complete response Median duration of partial response

64 cases

13 cases 4 cases 3 cases

83.3% 17 months 27 months

12 months

6 months 20 months

7 months

Table 2. Summary of the results obtained for the 64 patients with thoracic metastases.

Overall response rate Median survival Number C.R. 34 median survival

11 patients are still living: one after 16, one after 17, two after 19, one after 23, 31, 35, 38, 43, 68, 70 months

Number P. R. 20 median survival one patient is still living after 16 months

Number of fai lures 10 median survi val Median duration of complete response Median durati on of partial response

84 .3% 16.5 months 27 months

10.5 months

6 months 18 months 6 months

MULTIDISCIPLINARY CURE OF BREAST CARCINOMA 385

DISCUSSION

To reach the highest rate of response we studied a regimen associating, right from the beginning of the treatment, hormonotherapy and cytostatic poly­chemotherapy.

Hormonotherapy alone failed to achieve more than a 35% response rate (1,2,3,4,5) .

Combined chemotherapy alone achi eved about a 55% response rate (6,7) •

We obtained by associating the two methods a higher rate of response and a noti ceably longer durati on of the remissi on.

These results are bringing the breast cancer among the chemiosensitive tumours, after leukaemias and lymphomas. It will be necessary to carry on studies relative to: the choice of optimal drug combinations; the regimen of drug administration, either continuous or intermittent; the possibility to resort to a different drug association after exhausting the effectiveness of the first drug combination.

We have applied the most used drug combination: 5-FU, Cytoxan, Metho­trexate and Vincristine. Some authors add systematically Prednisone, others cancel Vincristine. Anyhow it appears that Cytoxan, 5-FU and Methotrexate are the three most important drugs in this combination.

Since Adriamycin (8) was introduced, various trials have been made combining Adriamycin, 5-FU and Cytoxan or Adriamycin and Vincristine or Adriamycin and CCNU.

We think, like Bonadonna (9), that it is necessary to study the use of two associations: firstly MTX, Cytoxan and 5-FUi secondly Adriamycin, Vin­cristine, or Adriamycin, Vincristine and CCNU.

We can begin with 6 to 8 courses of Adriamycin and Vincristine, or Adria­mycin, Vincristine and CCNU and thereafter switch to the combination 5-FU, MTX and Cytoxan. We can also alternate the two drug combinations. The greatest difficulty we have met is the psychic reaction caused by alopecia which appears, with Adraimycin in almost all cases.

Finally I it is difficult to choose between a continuous or an intermittent drug administration. We believe that an intermittent regimen has a superior activity, a lesser toxicity and is less immunosuppressive.

386 P. MANNES ET AL.

REFERENCES

1. Frachia, A.A., Farrow, J.H., Depalo, A.J., Connolly,D.P. and Huvos, A.G. (1969). Surg. Gyn. and Obst., 128, 1226-1234.

2. Frachia, A.A., Randal, H.T., Farrow, J.H. (1967). Surg. Gyn. arid Obst., 125, 747-749.

3. Volk, H., Deupree, R.H., Goldenberg, 1.5., Wilde, R.C., Carabasi, R.A. and Escher, G.c. (1974). Cancer, 33, 9-13.

4. Kennedy, B.J, Theologides, A., Fortuny, I., Fole~ J. and Brown, J. ( 1964)'. Cancer Chemotherapy Reports, 41, 11-14

5. Huys, J., Moens, R., de Thibault de Boesinghe, T., Mannes, P., Van Vaerenbergh, P.M., (1975). Acta Therapeutica, 1,5-18.

6. Carter, S.K. (1972). Cancer, 30,1543-1555. -7. Broder, L.E. and Tormey, D.C. (1974). Cancer Treatment Reviews,

1, 183-203. 8. Gotlieb~ J.A., Blumenshein, G.B., Gutterman, J.U., Freireich, E.J.

and Cardenas, J. (1975). Adriamycine Review, European Press Medikon, Ghent Belgium, pp 249.

9. Delana, M., Brambi Ila, C., Morabi to, A. and Bonadonna, G. (1975) • Cancer, 35,1108-1115.

POI'ENI'IATION OF DRUGS USING SEQUENI'IAL CHEM)THERAPY AGhlNST DISSEMINATED BRFAT, B:RCNCHIAL AND CENTRAL NERVOUS SYSTEM SOLID TlJM)RS

.. +P. POOlIJARI', L. SCHWARZENBERG, J.L. AMIEL, G. MATHE ++P. HUGUENIN, Ph. IDRIN, A. BAIDN, Ch. IAPARRE, R. PARRaI'

+Unit~ FrErl-Siguier de OOveloppanent Therapeutique HOpital Paul Brousse & Service d'Hematologie de l' Institut Gustave Roussy, 94800 VILLEJUIF, France

++Centre M€dico-Chirurgical de Bligny, 9164D-Briis-sous-Forges, France

'!he role of chE!lOtherapy in the treatment of solid turrors re­IIains irrprecise, che1rotherapy is only usErl when other treatments have been triErl and have failed, such as surgery and/or radio­therapy. This limitation of charotherapeutic indications to the only palliation of advanced tunors might explain the inconstancy of its results. An :irrp:>rtant toxicity is often the price to pay for a greater efficiency, sought by awlying several drugs, the choice, the association and the administration metIDds of which are often errpirical (2, 3, 24). We have been trying to detennine a cherrotherapeutic protocol for patients suffering fran solid tu­mrs, in accordanoe with what we consider as the association chE!lO­therapy principles: a) the need to avoid cumulative toxicity; b) the use of drugs which previous trials have shown to be s0me­what efficient for the treated turrors (1, 5, 6, 13, 14) ; c) the increase of the pel:Centage of turror cells destroyed at each thera­peutic cycle by the use of drugs which p:Jtentialize for cell kine­tic reasons {l7, 18, 19, 22, 23) and for pharmacodynamic reasons (21) •

Administering vincristine or VM 26, aside their therapeutic activity results in a rrodification of the proliferation kinetics of a eolid or liquid experimental tunor (17, 18) through 'b.o me­chanisms : a) it blocks partially and terrp:>rarily the tunor cells in phase M ; b) it increases the proportion of tunor cells in

387

388 P. POUILLART ET AL.

phase S, 18 murs after the administration. The sane mechanism was observed in nan, on leukemic cell populations which were re­sistant to the administration of vincristine (22, 23).

This new cell distribution in the cell cycle awears to be the consequence of a double phenanenon of partial synchronization and direct recruitment of the cells in phase S. It partly ex­plains the greater efficiency of a phase-dependent drug adminis­tered after vincristine. It is independent of the activity of vincristine or VM 26.

The phannacodynamic potentialization phencmenon was studied in vitro by Goldman & Fyfe (9). Our recent stulies in arrimals have enlarged the concept of pharrracological potentialization. Apart fl:al\ any IOOdification of the kinetics of tunor proliferation, v:in::ristine potentializes the effect of various drugs such as cyclophosphamide, methotrexate, 5-flroro-uracil, mitaT\Ycin C,fl:al\ the 8th to the 72nd hour after administration (21). Urder the sane experimental conditions, 5-flroro-uracil potentializes the action of alkylants (?) and nitrosoureas (21). VM 26 as well as vincristine potentializes the action of 5-FU, alkylants (?) and nitrosoureas (20).

'lbese ch.em:>therapies are based on the same principles, i.e. intermittent cherrotherapies which sequentially associate the drugs; first vincristine or \1M 26, in order to benefit fran cell recruit.­nent and partial synchronization, and secondly a drug or a drug association phase or cycle dependent, the action of which can be­nefit fl:al\ this recruitment and fnn this partial synchronization. The drugs given second are also chosen far their phanracological actions which are potentialized by the action of vincristine or VM 26 given first, and in the case of an association, for their action of reciprocal potentialization.

This pattern can be adapted to each kind of tUIlDr in order to take into account all specific conditions of the tunor, particu­larly the access of the drugs to the tunor tissue, am the specific sensitivity of each turror to each drug (this sensitivity is known through prel:imi.nary trials which use each drug separately~.

We will describe three trials of this therapeutic pattem, adap­ted to disseminated breast cancers which are ho:rnonotherapy resis­tant, to bronchial tunors beyond the possibilities of surgery and radiotherapy, to central nervous system tunors in which surgery and radiotherapy cannot be utilized either.

concerning breast tunours, we chose vincristine, follCMed by a CCNU. The liposolubility of VM 26 enables it to clear the harane­ningeal obstacle, and this product is efficient in gliana evolution. AIoong the drugs which can kill the cells which are recruited and

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 389

TABIE 1 - Breast cancer. Previous treatment, existing lesions at the start of the sequential charotherapy trial on 27 patients, am the results.

nO Initial TnlatllEnt PreVious IJ:x:al1satiDn of les:la1s Results

chom>thel:IIpy and/or

Imm:x1D\:hempy Local Skin Lymph Pleural LIm9 Li_ Bona Per1- at> 50 '(50' 'l!' relapse M nodes M M M M~

M

1 !Iurqety CMIDtherapy + + + + +

2 SUrgmy + radio ~+ + -tI1eroW iI>l:In:lrother + + + + +

3 !Iurqety + radio ~+ + + + + 1:het'IIVi iI>l:In:lrother

4 1lod1ot:I1azapy iI>l:In:lrother + + + 0>IIIDthenpy + + + +

5 !Iurqety + Radio iI>l:In:lrother + + -tI1eroW

+ + + + +

6 !Iurqety + + +

7 su"-y + RId10 -tI1eroW

+ + +

IIurgcy + RIId10 + + + -tI1eroW + + + +

9 1lod1ot:I1azapy + + +

10 !Iurqety + + + +

11 SuEgaty + RIId10 ~ + ~ + +

12 1lod1ot:I1azapy iIDI:IIaI:>t:be + + a..othenpy + +

13 !Iurqety a..othenpy + + +

14 1lod1ot:I1azapy + + +

15 IIurgcy + RIId10 + + tIwroIw + +

16 1lod1ot:I1azapy + + + + + + +

17 1lod1ot:I1azapy + + + +

18 """->' Chso:>therapy +

19 !Iurqety + Radio - + + + + +

20 !Iurqety + RIId10 -tharg + + + + +

21 !Iurqety + RIId10 -theI:aw + +

22 !Iurqety + RIId10 -tIwroIw + +

23 su"-y + Radio -tI1eroW + +

24 SUrgety 0I0m:>therapy& + + + + + +

25 Surgery + RIId10 -thermw + + + +

26 su"-y + Rad1oth.Chaa>therapy + + + +

27 IIurgcy + Rlldiotb. + + + + + + +

M • _. , 'l!' • 1bt:al failure , CR - Cl:IIp1ete nmia.ion , :m = total regression

390 P. POUILLART ET AL.

synchronized by VM 26, and whose action is also p::>tentialized by that of VM 26, nitrosoureas alone fulfill the corrl1tion of free access to the tUl10urs of the central nervous systan.

PATIEN'IS & ME'HfODS

A. '!he Patients

Sixty patients entered this trial, Le. 27 breast cancers, 16 bronchial tUl1Ours, 17 primitive tUl10urs of the central nervous system. In all these patients, surgery, radiotherapy and honrono­therapy were out of the question, either because of previous failu­re, or because of the volume, the location or the catpOsition of the tUl1Our.

'lWenty seven entered this trial in March 1972 ; their ages ranged fran 46 to 82 (naiian age: 63 yr). At the tine of the che­notherapeutic trial, all these patients had at least tvK> different xretastatic localisations (Thble I), they were all castrated or Ire­

nopaused. They had been initially treated by surgery alone in six cases, irradiation alone in six cases, surgery and irradiation in 15 cases; twelve of them had been initially treated by surgery alone in six cases, irradiation alone in six cases, surgery and irradiation in 15 cases; twelve of than had already received che­rrotherapy (6 cases) or an association of charotherapy and drug hor­rrotherapy (5 cases), or ho:rnonotherapy alone (1 case). All these treatments had remained or had becane inefficient before the patients entered the trial.

Arrong the xretastatic localizations, there were 13 local relap­ses or controlateral relapses, 14 skin localizations, 11 ganglionary localizations, 20 pleural localizations, 13 pulmonary localizations, 5 bone localizations (this nUll'ber is probably underestimated in the absence of bone scintigraphy), 2 peritoneal localizations.

16 patients entered this trial which started in June 1973 ; 15 had an epidennic cancer and one had an anaplasic cancer. The age of the patients ranged fran 41 to 71 years old : naiian age : 66 yr ('!able 2). When they entered the trial, tw::> patients were relapsing after a pnetmectany, three patients were relapsing after a radiotre­rapy, seven patients had already received an inefficient charothera­py. The initial lesion was associated, in seven cases with contra­lateral Img xretastasis, in ten cases with liver xretastasis, in tw::> cases, with bone metastasis and in two cases, with brain xretastasis ('!able 2).

."

0 -i m

.'!!!

!!...

l -P

rev

iou

s T

reat

men

t, e

x1st

in:J

lesia

ls a

t th

e s

tart

of

the

seq

uen

tial

ch

eml:

her

apy

tr

ial

Z

-i

on

16

Pati

en

ts,

and

the

msu

lts

5> -i

u:x

:al1

sati

an o

f th

e l

esi

on

s P

rese

nt

0 nO

1

q!

Init

ial

Pre

vio

us

Z

'l're

atI

IeJt

<llSIDther~

IDea

l U

lng

P

leu

ral

SIb

-Cl

Liv

a-

Bo

ne

B

rain

C

R

'lR

'lR

iF

Reg

ress

ion

0

rela

pse

M

M

M

M

M

M

50

%

50

' u

nd

er

" <

llen

Dth

era

py

0 ::0

l.

62 y

r R

!Id1

othe

mpy

0

+

+

+

120

day

s C

G

')

2.

68

yr

80 d

ay

s C

J)

+

+

+

+

+

+

Z

3.

71 y

r +

+

+

+

+

30

day

s C

J)

m

4.

67 y

r +

+

+

+

+

70

day

S

0 C

m

5.

59 y

r +

+

+

+

21

0 d

ay

s Z

-i

6.

69 y

r +

+

+

90

day

s 5> r

7.

65 y

r S

urg

ery

+

+

+

+

21

0 d

ays

-i ::c

m

8.

41 yr

+

+

+

+

220

day

s ::0

» 9

. 69

yr

+

+

+

95 d

ays

."

-< 1

0.

58

yr

Rad

ioth

erap

y

+

+

+

120

day

s

11.

69 y

r +

+

+

+

+

+

12

0 d

ays

12

. 53

yr

Su

zg

ery

+

+

+

8

0 d

ay

s

13

. 65

yr

+

+

+

210

day

s

14

. 7

lyr

+

+

+

+

+

150

day

s

15

. 53

yr

Ra

dio

the

rap

y +

+

+

+

+

+

50

day

s

16

. 56

yr

+

+

+

+

+

150

days

M =

Me

tasta

sis

S

Ib-C

l =

Slb

-clA

v1

cula

r ly

u¢

ro

de

C

R =

CcII

ple

te r

emis

sio

n ;

TR

= 'n

>ta

l. re

gre

ssio

n

iF ,

. 'J

l)ta

l fa

ilu

re.

w ~

TA

BlE

J -

PA

TII

!Nl'S

Wl'l

H

PR

IMIT

IVE

~

OF

'lH

E C

Dn

'RA

L N

ERV

OU

S SY

STE

M T

RFA

TE

D w

rm C

lIEH

Y.m

ER

AP

Y

w

'<I

'->

N°

AGE

Dia

grD

sis

Pre

vio

us

ca

tple

te

Part

ial

Neu

rolo

gic

al

'lb

tal

'lb

tal

Aft

er

the

Tre

atm

ent

Rem

issi

on

R

eg

ress

ion

S

tab

ilis

ati

on

Fail

ure

Su

rviv

al

Rel

apse

1 45

yr

Gli

ob

last

ata

?

SU

t:ge

zy

+

600

day

s 24

0 d

ays

2 50

yr

Gli

cb

last

an

a

Su

rger

y

+

900

day

s 1

80

day

s

3 44

yr

Gli

ob

last

ata

S

urg

ery

+

8

40

day

s 12

0 d

ay

s

4 54

yr

Gli

ob

last

an

a

Su

rgery

+

+

540

day

s 24

0 d

ays

Rid

ioth

era

py

5 6

44

yr

Gli

ob

last

an

a

+

120

day

s 1

20

day

s

6 66

yr

Gli

ob

last

ata

+

90

day

s 9

0 d

ays

7 61

y

r G

licb

last

an

a

+

70 d

ay

s 70

day

s

8 56

yr

Gli

cb

last

ata

S

urg

ery

+

63

0 d

ay

s 24

0 d

ays

9 63

yr

Gl.

iob

last

ana

Su

rg&

y +

+

51

0 d

ays

510

day

s Ridiothera~

10

45

yr

Astr

ocyta

na

?

Su

rger

y

+

90

0 d

ays

180

day

s

11

. 36

yr

Astr

ocyta

'IB

S

urg

ery

+

1

320

day

s 27

0 d

ay

s

12

6

yr

Astr

ocyta

'IB

S

urg

ery

+

+

1 32

0 d

ay

s 63

0 d

ays

Ra

dio

the

rap

y

13

30

yr

0xm

Xm

a ?

S

urg

ery

+

8

70

day

s 21

0 d

ay

s ~

14

56 y

r 0

l0rC

bta

S

urg

ery

+

1

080

day

s 10

0 d

ay

s " 0 C

15

1

6 y

r C

1Dti

Iooa

S

urg

ery

+

+

1 3

20

day

s 57

0 d

ay

s r

Ra

dio

the

rap

y

r » 1

6

30 y

r M

edu

llab

last

ana

? S

urg

ery

+

+

1 17

0 d

ay

s 36

0 d

ay

s :0

Ra

dio

the

rap

y

-t

m

17

72

yr

Fib

l:o

saro

ana

? S

urg

ery

+

+

630

day

s 26

0 d

ay

s -t

~

» !

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY

Seventeen patients entered this trial which started in May 1972, their ages ranged fran 6 to 72 years old (Table 3).

Eight of these patients were relapsing after an association

393

of surgery and radiotherapy, three of these patients had received no treatrrent but the turrour was considered as ineradicable because of its size, its location and its nature. When they were sul::mitted to this chenotherapeutic protocol, the patients had only received syrrptanatic treatment and all surgery and/or irradiation had been refused.

B. Charotherapies

1. ~~§asL£~~

'fue charotherapy 2 involves the intralltenous administration of vincristine at 1 ng/m on the first and second day of the cycle2 and the muscular administration of cyclophosphamide at 300 ng/m on the third, fourth, fifth and sixth day of the cycle, and a one and a half hour venous perfusion of 500 ng/m2 £lucra-uracil on the third, fourth, fifth and sixth day of the treatment cycle.

'!hese treatrrent cycles are renewed only when the blood count is back to the pre-charotherapy figures. '!he period between two cy­cles can go from 22 to 34 days, with an average of 25 days. The treatment is stopped when the failure is canplete (lesion progress) or in the case of intolerance. Otherwise, it is indefinitely nain­tained.

The chenotherapy involves the intravenous administration of vin­cristine at 1 ng/m2 on the first and second day, the bone adminis­tration of CCNU at 60 ngjm2 on the third and fourth day, the venous perfusion of 5-£lwro-uracil (500 ng/m2) on the third, fourth and fifth day of the treatment cycle.

The treatrrent cycles are renewed only after the leucocyte and blood platelets have returned to the pre-chenotherapy figures.

3. ~~t!Y~U:~!¥..2_QLEh§_£~tfal_!!~~_2Y~~

The chenotherapy is intennittent, cyclic and sequential. Each four day cycle involves the perfusion administration of VM 26 (60 ng/ m2) on the first and second day, and the absorption of CCNU at 60 ng/ m2 on the third and fourth day. The period between two cycles can go from 15 to 30 days with an average of 20 days. '!he treatrrent is stowed in the case of total failure or rrajor intolerance to VM 26 administration. Otherwise, it is indefinitely rraintained, under

394 P. POUILLART ET AL.

TABLE 4. Results obtained in 27 patients with dissemi­nated mammary tumours treated by sequential chemotherapy.

Number of patients

27

Complete remission

9

(33.3 %)

Partial regression Total failure

50 % 50 %

11 6 1

(40.8 %) (21 %) (4 %)

All the patients presented in this table were treated for more than 14 months.

TABLE 5. Sensitivity of breast cancer metastasis to sequential chemotherapy depending on the localisation

Metastasis Number of Total Partial Regression Total cases regression failure

50 % 50 %

Skin 14 5 7 1 1

Lymph nodes 11 1 8 2 0

Pleural 20 6 8 5 1

Lung 13 5 4 4 0

Liver 9 4 3 2 0

Bone 5 0 3 2 0

Perinoneal 2 1 1 0 0

Study done on 27 patients treated for more than 14 months.

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 395

regular haematological oontrol.

During each treatIrent cycle and eight days after the end of the chenotherapeutic cycle, the patients also receive a hypertonic glucosis sermt perfusion as well as 40 ng of nethyl prednisone and 0.5 g of acetazolamide. '!he patients also receive a daily intrcnrus­cular injection of ACl'H. This therapy is maintained between the cycles for three nonths at 1 ng every two days.

RESULTS

A. Breast cancers

'!he patients received fran 5 to 15 cycles, the median nurrber being 7 cycles. The two first cycles were administered to hospita­lized patients, the other cycles in outpatients.

'!he haernatological tolerance is generally good, each cycle being follCM'ed by a leucopenia phase between 500 to 1500 leu:::ocyt.es, the nean duration of which is 6 days, restoration is spontanoous. In two cases the administration of the first treatment cycle was follCM'ed by a leucopina, carplicated by infections which had to be treated by antibiotherapy.

'!hese leucopenias were spontanEDusly restored and the cycles oould be renewed after a reduction of the doses. We did not note any death due to haanatological carplications of the treatIrent.

'!he period between cycles ranged fran 22 to 34 days, but this varied according to the patient, tb:>ugh cycles were nore or less similar for the sane patient.

During the first treatment cycle, we noted 18 cases of diges­tive troubles : anorexia, nausea, vomiting; these troubles generally decreased during the follCMing cycles.

Neurological carplications, essentially oonsisting in distal paresthesia, were noted in three patients; they were tb:>ught to re­sult fran the use of vincristine and this product was replaced by VM 26. '!hey disawea,red several weeks later,

Alopecia is an alIIDst pennanent carplication and has been nore or less noted in 26 patients out of the 27 patients in the trial.

'Iherapeutic results are presented in '!able 1, 4, 5. OUt of 27 patients we obtained 9 catplete remissions, 11 regressions of

396 P. POUILLART ET AL.

the lesion volume of rrore than 50 %. We integrated in this group of patients, in whom the regression was superior to 50 %, all the cases consisting in the maintenance of an isolated radiological or biological perturbation, without any other detectable lesion. We obtained six less than 50 % regressions and a complete failure. On the whole we obtained 20 very good results out of 27 patients (74 % of the cases). All types of lesions were notably affected by the treatment as shCMn. on '!able 3. We noted the irrprovenent of the Itmg and pleural lesions, of the bone lesion, and of the liver and skin lesions. '!he carplete or very good results (rrore than 50 % regressions) are skin lesions (86 %), lynph node lesions (82 %), pleural lesions (70 %), Itmg lesions (70 %), liver lesions (77 %) and bone lesions (60 %). '!he localisation of the lesions thus does not seem to affect their sensibility. The maxi­rm:an therapeutic effect is generally obtained after the third treat­ment cycle. The remissions and regressions obtained can be maintain­ed under treatment for eight to 17 rronths. We have not yet noted any relapse tmder treatment.

B. Bronchial cancers

All the patients who entered this trial have received, up to now, f:rom four to seven treatment cycles. All these cycles were applied to oospitalized patients. Haanatological tolerance is good during the four first cycles and the mean period between ~ cycles is 28 days (extremes f:rom 23 to 36). After the fourth rronth of treat -ment there are rrore and rrore thronix>penias due to a delayed toxi­city of CCNU and the perioos between tw:::> therapeutic cycles have to be lengthened. The patients treated according to this pattern did not suffer f:rom any inp:)rtant haarorrhagic accident. All patients suffered from digestive troubles, anoraxia, nausea during the first cycle but never to the point where the treatment had to be interrup­ted.

No neurological trouble was mted in this series of patients.

Alopecia is seen in all the patients woo entered this trial.

Therapeutic results are presented in Table 2 and 6. Out of 16 patients in the trial, two are in camplete remission and seven have a turrour regression superior to 50 %. In four cases, the tUlIOur re­gression was slow and inferior to 50 %. We had three carplete fai­lures. On the whole we obtained nine good obj.ecti ve results out of 16 patients (56 %) and in 13 cases out of 16 (81 %) we noted an im­portant subjective irrproverrent. Liver or pleura lesions observed upon first examination were mtably affected by the treatment.

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 397

In this limited trial we did mt note any significant. diffe­rence in the sensitivity of the lesions acoording to their locali­sation' except for the patients which had brain netastasis.

Out of the sixteen patients who entered the trial, 12 are pre­sently alive. The average set back for all the patients of the trial is superior to 125 days. The therapeutic effect is rapidly ob­tained, generally in less than 50 days, i.e. as soon as the seoond chelotherapeutic cycle.

c. The primitive tUlIOurs of the central nervous systan

The patients have received fIan 3 to 15 cycles. All the charo­therapeutic cycles were administered to hospitalizErl patients. Haena -tological tolerance is good during the four first cycles. Because of the risk of a delayed toxicity due to CCNU, with thrombopenia, the periods between the cycles must then be lengthened to an average of 30 days. No i.rcp::>rtant haatOrrhagic accident related to this toxi­city occurred in this series.

Digestive troubles with nausea, amrexia, vaniting are mt very i.rcp::>rtant, they are always transitory and occur when the chatOthera­peutic cycles are being appliErl.

Alopecia is frequent. Finally, we did mt note any VM 26 into­lerance in this series.

2. :fh~a~~!~!£_~2~~

The therapeutic results are presented in Table 3 and 7. 'Ib eva­luate the results which are presented in these tables, one must take into accotmt the chatOtherapeutic evolution of the objective neutolo­gical troubles of the patients when they entered the trial, the nodi­fication of the synptans in relation to an intracranian hypertension, and the nodification of the garma encephalography linages they can be regularly obtained.

Out of nine patients with glioblastans, we obtained three COItr'

plete remissions and two partial regressions, three neurological stabilizations which lasted more than three months, and a complete failure.

Out of eight patients with degenerate astrocytanes (three), choroomes (three), rredulloblastomes (one), meingeal fibrosarcara. (one), we obtained two conplete remissions, four partial tUlIOur regressions, one neurological stabilimation which lasted more than three months and one complete failure.

398 P. POUILLART ET AL.

On the whole, five c:x:rnplete remissions out of 17 patients treat­erl were obtained at the neurological level (Le. 29 %), six par­tial regressions (Le. 35 %) were obtained, as well as four neuro­logical stabilizations (Le. 24 %) which have lasted for nore than three nonths, and tTNO total failures (12 %).

We called catplete remissions the total regression obtainerl in 5 patients ooncerning neurological synptans which had acoarpmied the tunour relapse. In three cases the disawearance of the gamra encephalographic images oonfi1:mEd the clinical result. In b.o other cases the neurological stabiliEation is rraintained in spite of the treatment internption nine nonths ago.

In all these cases the therapeutic effect came after at least three nonths of treatment. The regression of the objective neurolo­gical synptat\'3 is always slow although the subjective one is quite rapid. Presently 12 out of the 17 patients wID entered the trial are still alive with follCM-ups that go from 2 to 21 nonths after the beginning of the chem:>therapy treated relapse.

The mean survival t:ime of the patients with glioblastana, fol­lowing their entry into the chem:>therapeutic protocol, is superior to 200 days, and seven patients out of nine are still alive. The mean survival period of the patients wID have another type of brain tunour is presently superior to 277 days arx1 five patients out of eight are still alive (Table 3).

DISCUSSIOO'

'!he results of this therapeutic trial seem very encouraging be­cause of the mmber of c;:pod results obtained on maI'IIIBl:Y tmours, and of the existence of good results in the bronchial tmours and the cen­tral nervous systan tunours which are generally resistant to chE!1D­therapy.

Concerning breast tunours, the positive elanents are, besides the nunber of very good results (75 %), the action of the treatment upon all types of localizations, the persistance of the remissions and regressions obtained.

These results are quite superior to those obtained with the chem>therapies which use only one drug on a pennanent basis (see 19). OUr results can be oonpared with those of Cooper (6, 7) arx1 Greenspan (10) wID used vincristine, cyclophosphamide, 5-flwto­uracil, rcethotrexate and prednisone, according to a pattern which differs from ours. OUr protocol seems to be toleratErl for a longer time and the remissions obtained seem longer.

In the case of bronchial tunours positive results are frequent (56 %) and quickly obtained.

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 399

In all positive cases, an inp:>rtant cbjective reaction was ob­tained after "t:ID trea1::Iralt cycles and the prolongation of charothe­rapy did not enable us to obtain the regression of a resistant tu­nour. It is thus {X)ssible to increase the intervals between the cy­cles and to delay and even suppress the haematological mmifestations of a delayed toxicity of CXNJ. In these conditions of adndnistra­tion we have been able to extend this charotherapy without any ma­jor complication.

Using only one cytostatic product, even when chenotherapy can carplemant the action of radiotherapy, is not very rewarding (11, 12, 15). 'Ihe results obtained in this trial are superior in level and duration of regressions to those obtained by administrating each of the drugs of this combination (6,8,12,13,14,25,28).

The extent of the turrours treated and the presence of metasta­sis obviously constituted a counter-indication to any surgery or ra­diotherapy attenpt. In comparable groups of patients, the mean sur­vival tiIre of a {X)pulation of 17 patients (Hanser and Selawry, 11) was 123 days. Our results are better as after 130 days of treatment 13 patients out of 16 are still alive and two are in conplete remis­sion. One must nevertheless note that the brain rretastasis, in spite of CCNU, have mt regressed.

The results obtained in the treatIralt of prllnitive brain turrours must be considered in relation with the very bad prognosiS of such tunours.

In the Baltinore series presented by walker, out of 150 patients wID entered the trial protocol (surgery, radiotherapy and a BCNU treat­rrent), mne of the patients lived longer than the 14th nonth (26). Our results seem superior to those obtained with BCNU and CXNJ alone (4, 27).

Out of 17 patients submitted to this charotherapy with mother therapeutic {X)ssibility, we only had two total failures and five pa­tients are presently in conplete remission with regression that go fran 3 to 17 nonths.

The diffusibility of nitrosoureas, due to .their great liposo­lubility, explains why drugs are active in the treatment of brain tu­nours. VN 26 is also li{X)soluble. We were able to shcM, in a patient suffering from glioblastoma, that the intravenous administration of VM 26 quickly gave a drug concentration at the level of the turrour tissue which was higher than the drug concentration in venous or ar­terial blood at the same rranent.

The therapeutic results obtained with these therapeutic protocoI.s do mt prove that the starting hyp::>theses were correct. '!hey none the less encourage us to continue alonq these lines and suggest carparable

400 P. POUILLART ET AL.

TABLE 6. Bronchial cancer. Therapeutic results obtained after 4 chemotherapeutic cycles.

Number of patients

16

Complete remission

2

(12 %)

Tumour regression

50 % 50 %

7

(44 %)

4

(25 %)

Total failure

3

(19 %)

TABLE 7. Results obtained in the treatment of malignant brain tumours by sequential cyclic intermittent chemotherapy

VM 26-CCNU

Complete Partial neurological total remission regression stabilization failure

Glioblastoma 3 2 3 1

Other primitive brain tumours 2 4 1 1

Total 5 6 4 2

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 401

patterns for other types of solid tunours.

'!hese results also lead us to reoonsider the problem of the place of ch.enDtherapy in the trea:tIrent of rolid tunours. Because of the efficiency of such protocols, their quick results, the low toxic oost, the duration of the remissions am regressions, we are encou­raged to examine new cherrotherapeutic protoools in tunours which have been treated by surgay and/or radiotherapy in an apparently radical rnarmer, but which have a bad prognosis, such as breast can­cers with a quick doubling tbre.

Thus trials in continwus charotherapies using sinple drugs have not been very positive up to row, but the rrore efficient and less imnunodepressing cherrotherapeutic protocols arployed at present give us hope for rrore favorable long tenn results.

SrnMARY

We have been using intennittent chenotherapies CX>IlPrising cycles separated by intervals. '!he cycles oonsist of a first agent which pennits phase recrui1:m:mt and partial synchronization of tunor cells, then of a second agent or a carbination of agents known for their reciprocal phamaoological potentiation and which are also kmwn to be potentiated by the first product. In disseminated breast tUTOrs, the cycle formula consists in a sequence of vincristine and the can­bination of 5-FU and cyclophosphamide. Regressions (carplete or in­conplete but rrore than 50 % of the tunor volume) were obtained in 24 % of the cases. The treatnent can be naintained for several years. Regressions last rrore than one year. The cycles acbpted in the case of bronchial cancers which cannot be treated by surgay am radio­therapy consist in a sequence of vincristine and the carbination of 5-FU and CCNU ; regressiOns were obtained in 56 % of the cases. In the case of central nervous system tunors which cannot be treated by surgery and radiotherapy, the cycles consist of a sequence of VM 26 and CCNU. Regressions were obtained in 65 % of the cases (30 % appa­rently conplete) •

REFERENCES

1. Ansfield, F.J.: Current status or 5-fluoro-uracil. Therapy of breast cancer. In Proceedings of the Che­motherapy Conference on the-Chemotherapy of Solid Tumors An appraisal of 5-fluoro-uracil and BCNU (S.K.Carter, ed) Washington, January, 1970, p; 48-57.

2. Ansfield, F.J., Corbitz, B.C.and Davis, H.L.Jr.: Five drug therapy for advanced breast cancer. A phase II study. Cancer Chemoth. Rep., 55: 183-194, 1971.

402 P. POUILLART ET AL.

3. Carter, S.K.: Clinical trials and combination chemothe­rapy. Cancer Chemoth. Rep., ~:81-97, 1971. (part 3).

4. Carter, S.K. and Newman, J.W.: 1,3-bis (2-chloroethyl) -l-nitrosoures (NSC-409962,BCNU) and 1-(2-chloroethyl) -3-cyclohexyl-l-nitrosoures (NSC-79037,CCNU). Clinical brochure. Cancer Chemoth. Rep. l: 115-151, 1968.

5. Cole, M.P., Todd, I.D.H. and Wilkinson, P.M.: Cyclophos­phamide and mandrolone deacanoate in the treatment of advanced carcinoma of the breast. Results of a compara­tive controlled trial of the agents used singly and in combination. Brit. J. Cancer, ~: 396-399, 1973.

6. Cooper, R.G.: Combination chemotherapy in hormone resis­tant breast cancer. Proc. Amer. Ass. Cancer Res., 10: 15, 1969. (abstract)

7. Cooper, R.G.: Five drug combination approach in breast cancer. In-Proceedings of the chemotherapy conference on the chemotherapy of solid tumors. An appraisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed) Washington, Ja­nuary, 1970. p. 149-156.

8. De Vita, V.: Studies at the National Cancer Institute with the nitrosoureas. In Proceedings of the chemotherapy con­ference on the chemotherapy of solid tumors. An appraisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed) Washington, January 1970, p. 181-188.

9. Goldman, 1.0. and Fyfe, M.J.: Ftee intracellular methotrex­ate (MTX) is required for maximum inhibition of DNA synthe­sis augmentation by vincristine (VCR) Proc. Amer. Ass. Can­cer Res., .!.!: 50,1973 (abstract 199)

10. Greenspan, E.: Combination cytotoxic chemotherapy in advanced disseminated breast carcinoma. J. Mount Si­nai Hosp. N.Y. ll: 1-27, 1966.

11. Hansen, H.H., Muggia, F.M., Andrews, R. and Selawry, O.S.: Intensive combined chemotherapy and radiothe­rapy in patients with non resectable bronchogenic carcinoma. Cancer lQ: 315-324,1972.

12. Hansen, H.H., Selawry, O.S., Muggia, F.M. and Walker, M.D.: Clinical studies with 1-(2-chloroethyl)-3-cyclo­hexyl-l-nitrosoures (NSC 79087). Cancer Res., 31: 223-227, 1971.

POTENTIATION OF DRUGS IN SEQUENTIAL THERAPY 403

13. Holland, J.F., Scharlau, C., Gailani, S., Krant, M.J., Olson, K.B., Horton, J., Shnider, B.I., Lynch,J.J. Owens, A., Carbone, P.P., Colsky, J., Grob, D., Miller, S.P. and Hall, T.C. Vincristine. Treatment of advanced cancer. A cooperative study of 392 cases. Cancer Res., 11, 1258-1264, 1973.

14. Horton, J.: Comparison of three weekly dose schedules of 5-fluoro-uracil without a loading dose in the treat­ment of solid tumors. In Proceedings of the chemotherapy conference on the chemotherapy of solid tumors. An ap­praisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed) Washington, January, 1970 p. 112-119.

15. Hyde, L., Yee, J.,Wilson, R. and Patno, M.E.: Cell cy­cle and the natural history of lung cancer. J. Amer. Med. Ass. 193: 52-60, 1965.

16. Jacobs, E.J.: Weekly 5-fluoro-uracil without a loading dose. In Proceedings of the chemotherapy conference on the chemotherapy of solid tumors. An appraisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed) Washington, January 1970, p. 102-111.

17. Klein, 0.: Synchronization of tumor cell proliferation and the timing of cytostactic drugs. Europ. J. Clin. Biol. Res. 1I: 835-838, 1972.

18. Klein, H.O., Lennartz, K.J., Teichmuller, W. and Gross, R.: Cytostatic treatment of partially synchronized tumor cells in animals and men. Vllth Inter. Congr. or Chemo­therapy, Prague, 1971, 1 vol., Munich, Urban und Schwarzenberg, 1972.

19. Mathe, G. and Kenis, Y.: La chimiotherapie des cancers a l'usage du praticien. 3rd. ed., Paris, Expansion Scientifique Fran~aise, 1975.

20. Pouillar, P., Hoang Thy Huong, T. and Lheritier, J.: La leucemie L1210, modele experimental pour la poten­tialisation des drogues applicables aux leucemies aigUes. Bull. Cancer, ~: 509-526, 1974.

21. Pouillart, P., Lheritier, J. and Hoang Thy Huong, T.: Etude experimentale de la potentialisation pharmaco­dynamique de drogues anticancereuses administrees se­quentiellement. Biomedicine, 1975, in press.

404 P. POUILLART ET AL.

22. Pouillart, P., Mathe, G. and Schwarzenberg, L.: Essai de recrutement cellulaire par synchronisation par­tielle pour la combinaison chimiotherapique. Bull. Cancer, ~: 187-196, 1973.

23. Pouillart, P., Schwarzenberg, L., Mathe, G., Schneider, M., Jasmin, C., Hayat, M., Weiner, R., De Vassal, F., Amiel, J.L., Beyer, H.P. and Fajbisowicz, S.: Essai cli­nique de combinaisons chimiotherapiques basees sur la notion de tentative de synchronisation cellulaire. Nouv. Presse Med., l, 1757-1762, 1972.

24. Spigel, St. C., Coltman, C.A. and Costanzi, J.J.: Disse­minated breast carcinoma : treatment with combination chemotherapy. Arch. Intern. Med. 132, 575-577, 1973.

25. Vermund, H.: Radiation and 5-fluoro-uracil in bronchoge­nic and head cancer. In Proceedings of the chemotherapy conference on the chemotherapy of solid tumors. An ap­praisal of 5-fluoro-uracil and BCNU (S.K. Carter, Ed), Washington, January 1970, p. 128-135.

26. Walker, M.D.: BCNU therapy of brain tumors. In Proceedings of the chemotherapy conference on the chemotherapy of so­lid tumors. An appraisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed), Washington, January, 1970,p. 227-235.

27. Walker, M.D. and Hurwitz, B.S.: BCNU (1,3(2-chloroethyl-1-nitrosoures NSC 409962) in the treatment of malignant brain tumor. A preliminary report. Cancer Chemoth. Rep. 54: 263-271, 1970. --

28. Wolf, J.: BCNU therapy of Lung cancer. In Proceedings of the chemotherapy conference on the chemotherapy of solid tumors. An appraisal of 5-fluoro-uracil and BCNU (S.K. Carter, ed), Washington, January 1970, p. 203-211.

ANIMAL AND HUMAN STUDIES WITH ORAL MITOMYCIN c(6): A PRELIMINARY

REPORT

Boasberg, P.D. (1), Hall, T.C. (1,2,3,4), Odujinrin, O. O. (1), Benjamin, R.S. (1,5), Lowitz, B.B. (1), Nevinny, H.B. (3,7) and Maddock, Charlotte L. (3,8)

1. Los Angeles County-University of Southern California Cancer Center

2. University of Rochester, School of Medicine 3. Children's Cancer Research Foundation 4. Recipient of ACS Research Professorship No.PRP 47 5. Present Address: University of Texas System Cancer

Center, M.D.Anderson Hospital and Tumor Institute, Houston, Texas 77025.

6. Supported in part by NIH Grants Ca 16014 and Ca 6506 7. Present Address: Weiss Memorial Hospital, Chicago,

Illinois 8. Deceased

INTRODUCTION

Mitomycin C, a product of Streptomyces caespetosus, is an anti­neoplastic antibiotic marketed for intravenous usage (1). Extra­vasation outside the vein at the site of the injection results in painful necrosis of the surrounding tissue, and daily intravenous injections are a difficult method to use when continuous low dose therapy is desired. Consideration was given to the development of an oral form of Mitomycin C. This paper reports results from our initial animal and human trials. We later learned that oral Mito­mycin C has been used clinically in Japan.

MATERIALS AND METHODS

Animals

DBA/2 mice with Pl534 ascites lymphatic leukaemia were given Mitomycin C by gastric instillation or intraperitoneal injection

405

406 P.O. BOASBERG ET AL.

to groups of 10 animals each at the dose levels shown on Table 1.

At 17 days, mice from each group were selected for white blood counts, since at this time, the leukaemic cell count in the peri­pheral blood of tumour-bearing animals was uniformly high.

Humans

Mitomycin C was supplied in capsules of 2.5 mg and 0.5 mg by Bristol Laboratories and administered to 15 patients in a Phase I trial. One patient had carcinoma of the cervix and 14 patients had adenocarcinomas (17 gastric; 4 colorectal; 2 pancreatic; 1 unknown primary). Ten patients had previous therapy with 5-fluorouracil. The Mitomycin C dose levels are shown on Table III. Patients receiving the 0.125 mg/kg five-day course were also given 50 ~gm/kg of NaHC03 in tablet form five minutes before taking Mito­mycln C.

RESULTS

Animals

Table I indicates that Mitomycin C at 8 mg/kg/day by gastric instillation (group 5) produced tumour size inhibition similar to that induced by the intraperitoneal route, but with only comparable mean survival times. Table II shows that oral drug at 8 mgm was as effective in reducing leukaemic white cell count at day 17 as was intraperitoneal drug given at one-eighth the daily dose. At lower doses by both routes, less effect was seen. It is of interest that the higher intraperitoneal dose appeared to be more toxic than the highest oral dose, since none of the former animals survived.

Humans

Results of the human studies are shown in Table III. Seven patients received 0.125 mg/kg/day for five days every four weeks. One patient developed thrombocytopenia (50,000). Two patients developed moderate nausea, vomiting and anorexia; one of these patients developed thrombocytopenia below 50,000 after one course which was elevated to 0.20 mg/kg/day for 5 days after a month at the initial dose with no toxicity. Two other patients had thrombo­cytopenia and severe gastrointestinal toxicity consisting of bloody stools, associated with nausea and vomiting in one patient. Two patients did not develop toxicity after one course at the initial level.

STUDIES WITH ORAL MITOMYCIN C(6) 407

Table I. P1534 Treatment with Mitomycin C.

Group Tumour sizes % cm2 (Day 14) inhibition

1 0 19.6 2.0

2 Img/kg x 6 lop. 18.2 0.81 +51

3** 2mg/kg x 6 lop. 14.9 0.58 +11

4 4mg/kg x 8 gastric 19.5 1.23 +39 instillation

5 8mg/kg x I gastric 16. 1 0·12 +64 instillation

*Mean Survival Time

** No animal survived 14 days

Table II Antileukaemic Activity of Mitomycin C: Effect on Mouse WEC (WEC/mm3) P1534 - Day 11

Group 1 Group 2 Group 3 Group 4 Group 5

Mitomycin C ° Img/kg i.p. * 4mg/kg 8mg/kg gastric gastric

Mouse 1 61 ,000 6,400 * 21,100 5,000

Mouse 2 43,000 11,800 * 10,000 6,400

Mouse 3 30,000 3,500 * 9,000 4,100

* No animal survived 11 days

Tab

le III.

Pt.

C

an

cer

Do

sag

e T

yp

e

1 S

tom

ach

O

.lO

mg

/kg

d

ail

y

2 C

olo

n

"

3 C

olo

n

"

4 S

tom

ach

0

.05

mg

/kg

d

ail

y

5 S

tom

ach

"

6 C

olo

n

" 7

Co

lon

"

Pri

mary

8

" u

nk

no

wn

To

xic

ity

o

f P

ati

en

ts

on

O

ral

Mlt

om

yci

n

C

Pre

vio

us

To

xic

ity

T

ota

l d

ose

R

X

to to

xic

ity

H

aem

ato

log

ic

leu

ko

pen

ia

(3,3

00

) th

rom

bo

-

5FU

cy

top

en

ia

(15

,00

0)

, 99

mg

dia

rrh

oea,a

no

rex

ia,n

au

sea,

vo

mit

ing

, d

eath

nau

sea,v

om

itin

g,d

iarr

ho

ea,

5FU

an

ore

xia

5FU

N

o T

ox

icit

y

leu

ko

pen

ia

(1,3

00

),th

rom

bo

-

Non

e cy

top

en

ia

(26

,00

0)

10

9m

g

dia

rrh

oea,a

no

rex

ia,n

au

sea

& v

om

itin

g,

death

.

leu

ko

pen

ia

(3,3

00

),th

rom

bo

-5F

U

cy

top

en

ia

(64

,00

0),

93

mg

nau

sea

& v

om

itin

g,a

no

rex

ia

5FU

N

o T

ox

icit

y

5FU

N

o T

ox

icit

y

5FU

N

o T

ox

icit

y

Fir

st

day

T

ox

icit

y

1:8

thro

ug

ho

ut

36

27

co

nt ..

.

..,..

o 00

:"

0 txl

0 » en

txl

m

::c

G>

m

-t » r

Tab

le II

I co

nti

nu

ed

.

Pt

9

10

11

12

13

14

15

Can

cer

Typ

e

Sto

mac

h

Pan

cre

as

Sto

mac

h

Sto

mac

h

Pan

cre

as

Sto

mac

h

Cer

vix

Dos

age

0.1

25

mg

/kg

x

5 d

ays

'1

4 w

ks

It

It

It

It

It

It

Pre

vio

us

RX

Non

e

Non

e

5FU

5FU

5FU

Non

e

Non

e

To

tal

do

se

To

xic

ity

to

to

xic

ity

h

aem

ato

log

ic

Th

rom

bo

cyto

pen

ia

(76

,00

0)

30m

g

nau

sea,v

om

itin

g,a

no

rex

ia

nau

sea,v

om

itin

g,a

no

rex

ia

thro

mb

ocy

top

enia

(2

8,0

00

) b

loo

d sto

ols

, n

au

sea,

67m

g v

om

itin

g,

an

ore

xia

thro

mb

ocy

top

enia

(1

00

,00

0)

25m

g b

loo

d st

oo

ls

No

To

xic

ity

No

To

xic

ity

Fir

st

day

T

ox

icit

y

24

du

rin

g

5 d

ay p

eri

od

It

59

26

~

c o m

en

:E

=i

::I: o :D

» r s::: ~ s:::

-< (') z (') § ~

410 P.O. BOASBERG ET AL.

On the 0.05 mg/kg/day schedule there was one drug related death secondary to diarrhoea, leukopenia, and thrombocytopenia. One patient developed nausea, vomiting and prolonged leukopenia and thrombocytopenia. Three patients did not develop tocixity while on continuous therapy for 4 weeks. Their doses were then escalated to the 0.10 mg/kg level. Thereafter one patient died of disease without drug toxicity one week later, one patient had no toxicity after 3 weeks, and one patient developed gastrointestinal bleeding after 0.05 for 4 weeks, 0.10 for 4 weeks and 0.15 mg/kg after 4 weeks, but without haematologic toxicity.

On the 0.10 mgm/kg daily schedule there was one drug-related death from diarrhoea, leukopenia and thrombocytopenia. One patient developed severe nausea, vomiting and diarrhoea requiring the cessation of therapy. One patient showed no toxicity. There was no apparent alopecia or other host drug toxicity such as renal impairment in any of the treatment groups.

None of the fifteen patients treated demonstrated partial remission, but this is not remarkable since this was a phase I dose seeking trial, and measurable disease was not required for entry.

DISCUSSION

The results obtained in previous animal toxicology studies had suggested that oral drug was absorbed and active (2). Our animal data suggest that antitumour effects can also be seen. In P1534 these were reflected both by decrease in tumour size and decrease in number of circulating leukaemic cells. At high, toxic doses, both the oral and parenteral routes of administration in animals caused dose-related haemorrhagic intestinal lesions producing watery and bloody diarrhoea (2). Such changes were probably res­ponsible for the diarrhoea and gastrointestinal bleeding seen in our patients. We also noted leukopenia and thrombocytopenia sim­ilar to previous reports for intravenous therapy (1,3). Platelets appear to be particularly sensitive to oral as well as parenteral Mitomycin C, and at daily doses of over 0.10 mgm only the bone marrow toxicity was prolonged, and life threatening, having contributed to the death of two patients on continuous dose regimens. The group of patients on high, intermittent courses appeared to have more gastrointestinal and less serious marrow toxicity.

Some patients demonstrated no toxicity at dose levels res­ponsible for marked toxicity in other patients - implying variable drug absorption or residual marrow impairment from previous therapy. Mitomycin C is unstable in an acid media and its absorption from the gastrointestinal tract may be enhanced by antacids. However, in our patients on antacids, variable toxicity was seen; this was the group on high intermittent doses with sodium bicarbonate.

STUDIES WITH ORAL MITOMYCIN C(6) 411

Further work needs to be done to evaluate the role of antacids.

Due to the variable toxicity, it appears that chronic daily therapy should be initiated at a low dose and escalated, treating to mild toxicity with dosage adjustments made during careful moni­toring. Intermittent high dose therapy seems to have less bone marrow toxicity, and the gastrointestinal toxicity might be decreased by addition of NaCH03. We next plan to institute higher dose inter­mittent trials with and without NaHC03' to add NaHC03 to a group of patients receiving low dose 0.05 mgm/kg oral daily drug, and to monitor blood and urine levels with the N.S.P. test. Mitomycin C is active orally and further study of the oral route is indicated.

REFERENCES

1. Carter, S.K. (1968). Mitomycin C (NSC-26980)- Clinical Brochure Cancer Chemotherapy Reports, Part 3, Vol. 1., 99-104.

2. Philips, F.S., Schwartz, H.S. and Sternberg, S.S. (1960). Pharmacology of Mitomycin C. I. Toxicity and Patho­logic Effects. Cancer Research, 20, 1354-1361.

3. Whittington, R.M. and Close, H.P. (1970). --Clinical Experience with Mitomycin C. (NSC-26980) - Cancer Chemotherapy Reports, Part 1, Vol. 54., 195-198, No.3.

CHEMOHORMONAL THERAPY OF BREAST CANCER - A PILOT PHASE I-II STUDY

Odujinrin, 0.0., Benjamin, R.J., Hardy, R.E., Boasberg, P.D. and Hall, T.C. Los Angeles County/University of Southern California Cancer Center, Los Angeles, California 90033, U.S.A.

INTRODUCTION

There are few studies reporting the combined use of hormones and non-hormonal agents. In addition, in vitro methods are now available to predict the effectiveness of oestrogens, fluorouracil, methotrexate and adriamycin. We wished to undertake a trial of methotrexate, adriamycin, fluorouracil, and oestrogen in patients with breast cancer following determination of their cellular pre­dictive indices. However, it was first necessary to determine the tolerability of the drug regimen. This paper reports the results of our preliminary phase I trial.

CLINICAL MATERIAL AND METHODS

Ten patients with advanced breast cancer were treated (Table 1). There were nine female patients and one male patient. six of the' nine female patients were postmenopausal, two were perimenopausal and one was premenopausal. All the patients had dissemination to local soft tissues, viscera or bone. Eight patients had failed conventional therapy including combination chemotherapy (Table 1). Two patients treated received MAFE as first chemotherapy.

The initial treatment regimen, regimen 1 (Table 2), consisted of Adriamycin 50 mg/m2, 5-Fluorouracil 500 mg/m2 , and Methotrexate 25 mg/m2 given i.v. Methotrexate and 5 Fluorouracil doses were repeated on day 8. Diethystilbesterol 5 mg was given orally three times daily continuously. Each cycle of therapy was given every twenty-eight days depending on marrow recovery. Six patients received this regimen. Toxicity considerations led to change in

413

414 0.0. ODUJINRIN ET AL.

Table 1. Patient Characteristics

Patient Age years Metastatic sites

C.D.** 58 Axillary Nodes,Bones

M.D. 69

Vi.B. 32

G.S. 55

E.G. 53

C.F.

E.M. 52

E.F. 48

C.R. 64

N.D. 52

Local skin, Pleural effusion

Liver, Bone, Other breast, axillary and supraclavicular nodes

Parenchymal lung, bones

Parenchymal lung, Pleural effusion

Liver, Bones

Bones- other breast, Pleura

Brain, Bones, Parenchymal lung, Orbit

Axillary nodes, Other breast

Brain, skin - local and distant

** male patient

RT: CYT: VCR: 5FU:

Radiation Therapy Cyclophosphamide Vincristine 5 Fluorouracil

MTX: DES: POSTOP:

PreviouE Therapy

Mastectomy + Post-op RT Orchiectomy,RT to verte­brae

Mastectomy + post-op RT DES, Cyt.+ VCR + 5FU

RT to breast primary

Mastectomy + post-op RT Cyt.+ VCR + 5FU + Prednisone, RT to brain

RT to breast primary, axilla and chest wall, DES, Cyt.+ VCR + 5FU + Prednisone

RT to breast primary, axilla and chest wall, Estradiol valerate, 5FU, Cyt.

Mastectomy, RT to spine

Mastectomy, Oophorectomy 5FU, Cyt.+ VCR + MTX + 5FU RT to breast primary, RT to brain

Mastectomy + post-op RT, RT to brain, ventriculo­jugular shunt.

Methotrexate Diethylstilbesterol Post operative

CHEMOHORMONAL THERAPY OF BREAST CANCER 415

Table 2. Treatment Regimen I

Day 1 8 15 22 28 29

Adriamycin 50 mg/M2 None ~ 1.V. (l)

No Cytotoxic 'd

5-FU 500 mg/M2 500 mg/M2 (l) l.V. l.V. Therapy '"' c+

MTX 25 mg/M2 1.V. 25 mg/M2 1.V. 0 '<: 0

DES 5 mg TID, p.o. r-' (l)

Table 3. Treatment Regimen II

Day 1 8 15 22

Adriamycin 40 mg/M2 1.V.

5-FU 500 mg/M2 1.V. No Cytotoxic Therapy Repeat Cycle

MTX 20 mg/M2 1.V.

DES 5 mg TID,p.o.

Table 4. Pattern of Toxicity ln Treated Patients

Treatment Regimen

I II

Toxicity Types No. of patients No. of patients

Alopecia 6/6 IIi Stomatitis 4/6 311 Nausea and Vomiting 6/6 3/, Water Retention 3/6 2/,* Change in Cardiac Function 0/6 111* Median Nadir WBC count 2.5 4.0

cells/mm3 x 103 Range - cells/mm3 x 103 0.4-5.9 0.9-6.0

Median Nadir Platelet count 90.0 200.0 cells/mm3 x 103 range - cells/mm3 x 103 6.0-340.0 21.0-985.0

*Toxicity seen only ln patients treated with Regimens I and II consecutively

416 0.0. ODUJINRIN ET AL.

treatment schedule, treatment regimen II (Table 3). This consisted of Adriamycin 40 mg/m2 , 5-Fluorouracil 500 mg/m2 , Methotrexate 20 mg/m2 given once i.v. and repeated every 22 days depending on white blood cell count, diethylstilbesterol 5 mg orally three times a day continuously. Four patients received treatment regimen II only, while three of the six patients who tolerated several courses of treatment regimen I and who demonstrated no evidence of progressive disease were switched to treatment regimen II. The dose of Adria­mycin was reduced by half in patients with abnormal liver function tests. A minimum of 2 courses of therapy was given to each patient. Thereafter, evidence of progression of disease prompted change of therapy.

Estrogen binding studies were obtained on the tumour tissue of 2 patients using both the saturation analysis (dextran coated char­coal) and sucrose gradient analysis techniques. Serial "carcino­embryonic antigen" studies were done on three patients. Complete blood counts were obtained at weekly intervals. Other laboratory studies were repeated during the course of therapy as clinically indicated. Cardiac hepatic and renal function remained normal in all treated patients. Measurements were taken before therapy and at intervals during therapy. Responses were classified according to objective changes in measurable disease (1).

RESULTS

A. Toxicity (Table 4)

Non-haematological toxicity consisted of alopecia in all ten patients, stomatitis in five, nausea and vomiting in eight. Stoma­titis occurred in four of six patients while on treatment regimen I, in one patient on treatment regimen II and in two patients on con­secutive therapy with both regimens, while nausea and vomiting occurred in six, two and one patient(s), respectively, in these treatment groups. Three patients experienced water retention mani­fested as oedema of the lower extremities; their serum proteins were normal and there was no evidence of heart failure. Serial testing of cardiac function revealed a change in one patient's left ventri­cular systolic ejection time and pre-ejection period suggestive of early cardiac failure, after a total Adriamycin dose of 370 mg/m2. This necessitated withdrawal of Adriamycin from the treatment regimen in this patient. There were no drug related deaths.

Haematologic changes consisted of median nadir white blood cell count of 3000/mm3 (range 400-6000), median platelet count of 90,000/ mm3 (range 6,000-985,000). The nadir for both was maximal on day 12 and rose to pretreatment levels by day 22 in most patients. There was no decrease of more than 5 vols % in haematocrit on either

CHEMOHORMONAL THERAPY OF BREAST CANCER 417

treatment regimen. The six patients on regimen I manifested the greatest haematologic toxicity. The median nadir white blood cell count in this group was 2,500/mm3 (range 400-5,900) while median nadir platelet count was 90,000/mm3 (range 6,000-340,000). One patient (E.G.) with extensive bony and visceral disease, includ­ing brain metastasis experienced the most severe toxicity. Patients in treatment group II tolerated this regimen rather well without much suppression of haematologic parameters. Only one patient (N.D.), also with extensive disease, manifested major toxicity on this regimen. Two of the three patients (C.D. and M.D.) who initially manifested myelosuppression on treatment reg" regimen I maintained white blood cell and platelet counts within the normal range while on treatment regimen II.

Serum calcium in eight patients. initiating therapy on therapy (Figure

Table 5.

Treatment

B. Effect on Serum Calcium

remained within the normal range (9-11 mg/100ml) Two patients with hypercalcemia prior to

(C.D. and E.M.) developed normocalcemic values 1) .

Treatment Regimen and Tumour Response

Tumour Response Patient

& Regimen Total No. courses (in months)

C.D. I and II 16 Partial Response (13+)

M.D. I and II 10 Partial Response (6)

W.B. II 3 Partial Response (2+)

G.S. I and II 11 Stable Disease (5)

E.G. I 9 Stable Disease (5)

C.F. II 9 Stable Disease (10+)

E.M. II 5 Stable Disease (5+)

E.F. I 4 Progressive Disease

C.R. I 4 Progressive Disease

N.D. II 2 Progressive Disease

14

...J ~

11

0 rl

.......

(.!)

~

c(

10

w ~

::3

IlC:

U.I

9 U

)

8

SERU

M C

ALCI

UM V

ALUE

S IN

2

HYPE

RCAL

CEM

IC

PATI

ENTS

TR

EATE

D

WIT

H MA

FE

L

PRET

REAT

MEN

T

Pa

tie

nt

LM

.

t 5

10

15

STAR

T OF

TR

EATM

ENT

20

WE

E K

S

Fig

ure

1

25

30

35

40

o N

T

HE

RA

P Y

45

48

t;

co o 9 o o c c..

Z

::0

Z m

-I » r

CHEMOHORMONAL THERAPY OF BREAST CANCER 419

C. Tumour Markers

"Carcinoembryonic antigen" was obtained serially on three patients. One patient had a fall in titre while on therapy. Another patient had values that did not change significantly, while a third had a marked rise in titre consistent with disease progresslon while on therapy.

Tumour tissues from two patients (W.B. and E.M.) were studied for the presence of oestrogen binding receptor protein (ER). Both patients were ER negative.

D. Tumour Response

Table 5 outlines the pattern of response seen ln the treated patients. Three patients had progressive disease while on adequate courses of therapy and did not appear to have derived benefit from therapy. Four patients had stabilization of previously progressive disease lasting 5 to 10+ months. Three patients had measurable objective evidence of tumour regression lasting 2+ to 13+ months.

The two patients with negative ER, one of whom is premenopausal, responded to therapy.

DISCUSSION

The use of additive chemotherapeutic and hormonal agents ln breast cancer is not a new concept (1,2,3,4,5).

We have treated one menstruating premenopausal woman (W.B.) and she had no deleterious effect attributable to the oestrogen, and has had a 2+ months regression of metastatic tumour.

The two patients whose tumour tissues we studied both had negative ER (6). Traditionally these patients would have been subjected to ablative hormonal therapy (in W.B.) or additive hormonal therapy (in E.M.) for a period of 3 months before lack of effectiveness of such therapy is confirmable. Use of chemotherapy in such patients in addition to hormonal therapy may obviate the delay in instituting such therapy.

The present study is a preliminary one aimed at developing treatment information. Even though the number of patients studied is small, certain conclusions can be drawn from the study. There was no evidence of accelerated growth of disease. Also hormone induced hypercalcemia did not constitute a problem in this study, as a matter of fact the two patients with initial hypercalcemia became normocalcemic on therapy. This would appear to confirm

420 0.0. ODUJINRIN ET AL.

the lack of other hormone-induced ontoward side effects. Treatment regimen II appears to be a safe and tolerable regimen in terms of drug dosage and treatment schedule. Tumour regression also appears to be obtainable on this regimen. We would however still recommend a reduction in initial drug dosage in patients with extensive disease involving the bones and in patients with extensive prior radiation treatment.

REFERENCES

1. Carter, S.K. (1972). Study design principles for the clinical evaluation of new drugs developed by the chemotherapy programme of the National Cancer Institute. In: The Design of Clinical Trials in Cancer Therapy. (Editor, Staquet, M.) Edition Scientifique Europeennes, Brussels, p. 265.

2. Kiang, D.T., Kennedy, B.J. (1971). Combination of cyclo­phosphamide and oestrogen therapy in DMBA-induced rat mammary cancer. Cancer, 28, 1201-1210.

3. Kennedy, B.J., Kiang, D.T. (In:press). The effect of short­term cyclophosphamide in oestrogen therapy in metastatic breast canc er. Medic'al and Pediatric Oncology.

4. Taylor, S.G., Pocock, S.J., Schnider, B.I., Colsky, J.C., Hall, T.C. (In Press). Clinical studies of 5-Fluorouracil and Premarin in the treatment of breast cancer. Medical and Pediatric Oncology.

5. Stoll, B.A. (1972). Castration and Oestrogen therapy. In: Endocrine Therapy in Malignant Disease (Stoll B.A., Editor) Saunders, 139-163.

6. McGuire, W.L., Carbone, P.P., Sears, M.E., Escher, G.C. (1975). Estrogen Receptors in Human Breast Cancer. An Overview. Raven Press.

THE RESULTS OF BLEOMYCIN TREATMENT IN 90 PATIENTS WITH

MALIGNANT DISEASE

J. Christov and T. Donchev

Centre of Oncology

Sofia, Bulgaria

In 1962 in Japan, Hamao Umezawa et al. isolated the antibiotic Bleomycin from Streptomyces verticilus. Clinical trials started in 1965.

We studied the response and the side effects of bleomycin on squamous carcinoma of the lung, the cervix, the oesophagus and on a limited number of patients with other kinds of malignant disease.

METHODS AND MATERIALS

We have used bleomycin on 90 patients: 56 with squamous carcinoma of the lung; 12 with epidermoid carcinoma of the cervix; 9 with carcinoma of the oesophagus; 4 with astrocytomas (2nd and 3rd stage) and 9 with other malignant diseases. 85 per cent of the patients were more than 71 years of age. All patients in stages 3 and 4 were unsuitable for operative treatment as metastases were found in many of them. Bleomycin was administered intravenously 30 mg twice a week and in 71 patients (80 per cent), a total dose between 250-300 mg (or more) was administered.

RESULTS AND DISCUSSION

The results are given in Table 1. 27 per cent of the patients with a primary tumour had begun to improve (mostly 25-50 per cent of the initial size). The best effect was obtained on carcinoma of the oesophagus ($'7 responded). Carcinoma of the lung ( 12/56 responded), carcinoma of the

421

422 I. CHRISTOV AND T. DONCHEV

cervix responded only poorly. 68 patients (75.5%) had a subjective response and 47 (52.2%) an objective response.

Table 1. Results of Treatment

Local i zati on No. of Primary Tumor Metastases of carci noma pati ents No. pati ents Improv. No. patients Improv.

Lung 56 56 12 16 9 Cervix 12 5 1 7 3 Oesophagus 9 7 5 2 1 Astrocytoma

4 0 - 0 -(stage 2-3) Others 9 6 2 6 3

90 74 20/27% 31 16/51.6%

In Table 2 the dependence between the dosage and effect of bleomycin can be seen. The coefficient effect (CE) is the ratio between the improved and the toto I number of the treated patients calculated in percentage. The CE at a course dose of 135-200 mg is small (9'1'0). From Table 2 it can be seen that the CE is greatest (30%) at a dose of 300 mg or more.

Table 2. Dose and effect against primary tumour and metastases.

Course dosage No. of No. of patients Response

Coeffi c i ent (mg) patients evaluated Effect (CE)

135-200 11 8 1 9% 201-250 16 15 4 25% 251-300 43 40 10 23.2% 301-435 20 18 6 30%

SIDE EFFECTS

The most frequent side effects seen were on the ski n (hyperpi gmentati on and skin sclerosis) and increase of temperature. Gastro-intestinal effects were frequent (nausea, vomiting and anorexia). Leukopenia and lung fibrosis occurred in 2.2% of patients. A side effect, not previously mentioned, was hypotension down to 90/60 mm in 5.5% of the patients. There were no side effects in 7.7% of the patients.

RESULTS OF BLEOMYCIN TREATMENT 423

SUMMARY

The effect of bleomycin on 90 patients mostly with carcinoma of the lung, cervix, oesophagus, astrocytoma, stage 2-3 has been observed. In 27% of the primary tumours and in 51.6% of patients with metastases there was reduc­tion of between 25-50% of the size. The best effects were observed in carcinoma of the oesophagus and the lung and a moderate response was seen in carcinoma of the cervix. The best results needed a course dosage of 251-300 mg and more. The most frequent side effects were on the skin, with increased temperature and gastro-intestinal disturbances. Leukopenia was 3.3% and lung fibrosis 2.2%. In 5.5% moderate hypotension was found.

CIS-PLATINUM DIAMMINODICHLORIDE IN THE TREATMENT

SQUAMOUS CELL CARCINOMA AND OTHER MALIGNANT DISEASES

Loeb,Ellen,M.D.;Hill,J.M.,M.D.;MacLellan,Ayten,M.D.;Hil1, N.O.,M.D.;Khan,M.D.;King,J.J.,M.D.;Speer,R.,Ph.D;Ridway,H. Granville C.Morton Cancer and Research Hospital of the Wadley Institutes of Molecular Medicine, 9000 Harry Hines Blvd.,Dallas,Texas 75235

Two Hundred Nineteen (219) patients with various malignant diseases have been treated with cis-platinum diamminodichloride (PDD) during the last 4 years at the Wadley Research Institute. Early animal trials were done by Rosenberg (1)(5) et al. and Speer and Ridgway (ll).Clinical usefulness of PDD has been shown by Hill (10) et al.,Talley (6),Welsch (7),(9) and Higby (13). Commonlyem­ployed dosage of PDD was 1/2 mg. to 2.5 mg/kg. Schedules have varied from 1/2 mg/kg. b.i.d. to the maximum single dose of 2.5 mg/kg. Total course doses varied ordinarily from 2 to 4 mg/kg. with an in­terval between courses of at least one month. Treatment was pur­sued until limiting toxicity or complete remission was observed. The toxic reaction causing the most concern is dose related renal tubular damage. BUN, creatinine, uric acid and creatinine clear­ance were closely followed. Urine creatinine clearance less than 50 cc/min.disqua1ified the patient from PDD treatment. Side effects related to hypersensitivity were only seen in one case recently re­ported by Dr.Khan (15) et al. PDD was used alone and in combina­tion with other drugs such as amethopterin (AME) cyclophosphamide, thioguanine (Protocol designated as PACT) or followed by 5 FU,cyclo­phosphamide,vincristine, AME, given over 5 days or bleomycin, vin­cristine or vinblastine.

RESULTS

As previously reported, PDD was removed rapidly from the cir­culation and widely distributed in the tissues with less than 10% of the platinum remaining in the plasma one hour after administra­tion. The half-life in the blood stream, after this time, varied from 2 to 5 days. Elimination of platinum was chiefly by the kidney with the peak of excretion in the first 48 hours.

425

426 E. LOEB ET AL.

Tissue distribution following PDD administration was as illustrated in

TABLE I. These tissues were removed at autopsy and showed the distribution pattern at 2 and 25 days after treatment. Both of these patients received a single dose of 2 mg/kg. Note the wide­spread distribution with a tendency for the greatest concentration to be in the kidney shortly after administration, and later in the liver.

TABLE II. Side effects. The following tables are results of 86 non-leukemic patients treated with PDD alone. The most fre­quent side effect was nausea and vomiting, and rarely observed was tinnitus and deafness.

Toxic effects as seen in other chemotherapeutic drugs such as alopecia, stomatitis, cardiac or pulmonary changes or neuropathies have not been observed. The following results taken directly from computer printout will show the laboratory changes during and follow­ing PDD treatment.

TABLE III A AND III B. The effect of PDD on leukocyte counts revealed a nadir of depression occurring on about the 28th day and recovery on about the 33rd to 35th day. Similar analysis demon­strated platelet counts with the nadir of depression at the 17th or 18th day and recovery at the 23rd to 30th day, but not severe enough to require platelet transfusion except in one case with a course dose of 5.5 mg/kg. a platelet count of none seen was found for a 3 day period.

TABLE IV. The BUN changes showed only 4.6% of these patients with BUN above 60 mg.per cent; 6.9%, 41-60 mg. per cent; 36% showed 21-40 mg. per cent BUN; 52% showed no change at all. Recovery usually was seen in 2 to 3 months. The patients received multiple courses of treatment, maximum of 10.

TABLE V. In squamous cell carcinoma treated with PDD alone, 32 patients were treated, there were 5 or 15% in good improvement, 6 or 19% in slight improvement, and 21 or 66% showed no response.

TABLE VI: Results of 18 patients with squamous cell carcinoma treated with PDD and combination chemotherapy, 6 or 33% had good improvement, 3 or 16% had slight improvement, and 9 or 50% had no response.

TABLE VII. PDD with combination chemotherapy treatment of 18 patients, 1 with carcinoma of the esophagus was in the good im­provement group, 10 patients with head and neck carcinoma showed 4 or 40% good improvement, 3 or 30% slight improvement, 3 or 30% had no response. Seven (7) patients with lung carcinoma, 1 or 14%

TREATMENT WITH Cis-PLATINUM DIAMINODICHLORIDE

TABLE I PDD-TISSUE ANALYSES

MICROGRAM PT./GM. DRY TISSUE DIAGNOSIS DOSAGE MG/KG DAYS AFTER RX.

HODGKINS LYMPHOSARCOMA

ORGAN LIVER COLON STOMACH SMALL INTESTINE KIDNEY TESTES PANCREAS LYMPH NODE SPLEEN BRAIN

2 2 2 25

9 4 7 4

15 9 4 9 6 2

TABLE II SIDE EFFECTS

24 3 5 4 6 9 6

< 3.6

< 6 2

86 Patients Receiving PDD Alone

SIDE EFFECTS No.of Patients %

Nausea 35 40 Nausea, severe 3 3 Vomiting 26 30 Vomiting, severe 5 5 Diarrhea 3 3 Tinnitus 3 3 Deafness 2 2

427

428

I '5C~ ,.."'500 C"'-fhOI)O

",'500 1'101)0 "Soo 'r-_ t15DO

~ ltOOO ..... ,,... ..... I ....

co ,,'!oo

"'" .....

'- "'00 <> '70'" <> 1.'500 .. -..CI .",15'50., 11-

~E - 1."00 l&Oo,J

E 1,'501) ,,00.,

"- N~= -11'500

0 ,,000

0 '0'500 ... " 0 "'!,Oll ... ...... >< 0'" ., .. .-u , ... II) ,'\0()

<D .... ~ .. .., .-....

'000

"" .., ... , -.. " 1'500 ,00. ", 0

o 0 ...... LDII'~ . ."..

11 '0000 .--. ... -l1li0000I;I

11- (IOU 221 - <2010 411 · (4100 111 - )4010

,.,. t ... "" '¥ "' ., '1'''''

TABLE III A & III B

PDD • effect on WBC AVERAGES - 78 PATIENTS

EXCLUDING LEUKEMIA

, . " . -u II - 11 • II - • . II

I . • 1' 1

E. LOEB ET AL.

I I I I I I I I I I I I I

I II 'W I I I 'W I'W II 'W I 'W I I

: : ; : : : T : t " ~.-,;:-~ ____ , T ., " ... ., ., T .,. .. L L '" I. L ,. 'I' " ., ., " " "I' l L L II. L. l l. L T , T .... , ., , , , .. ",,.,,,TY'!'lLllllLl"'!''''''''''T\,. 1" •• 1:11'

...... ---~~..u~~1 " ......... ' '' ... 'LLLLLlf..Llfl''-I.'''yTLt't'' ..... '"'·'''''' I.L lll..\.''I..I.'t'LL.''f l TI. I'TY)I; llIlI:''' ., ., ., ., ., " ,. ., I. L .. L I,. .. L I.. .. T t ., '" I. • T r 1 T L r t , or If I )C .. 'II I ......... ., ., ,. ., .. l l l L L .. I. L '" ., r T I. I.. , T r 1 T I. • , ., 'III ,.. • 11 ]( " I I ' '''''''Y'''l.llI.LlLI.I.tTTLI., T r'TI.''''IC:f.;W:II'"lf ""T"""'"LlLLllLI.'f''-I. • .,r'Tl"T1iXlI'JI][vll "T"'T"' LLI. I.I.I."L.lTr'I.I."'Trl.T"''IIW~JlW''11 ",. ... '.,.,"'" t." """"L.lTI''' ... ' ' •• fl.r''JlJllJ1X'Y11 ., ., ... , " ., , , l II. " " ... " I. ... l ., , , " .... , , 1 , I. 1 , , 11 11 lilt ,. I 1 " ...... ",."l\." ... l" ...... "",\.I.'",' ... ',' ••••• ,.11 ,.,,.,,,,.,,, ......... \. ... l\. ...... "'\.L.""''''''' .. I:II.',.11

T 1 v if 1 v I I I I v 'I 1 'W I I

v , I v 't T ,. I I It I 11" '11" I I '11 1 1 1" '" 'W I I 'I 1 v I I 1 ,. v 1 v I I v I v I I I J 'W ,. T ,. J 1 v I v 1 I I I 'W V I 'W 1 I v 1 v 1 I 1 I v V' I v I I II" I "I 1 I I V'V I II" 1 I It t V' 1 1 I I v V' , 'W I I

~~~;~g6~~; ~ :~~!;1~lj : ~~;lIi!~~~ , ::~~,:~,: o~ ~;:;:;:;.,t':I:~:;O o 10 20 3D 40 50 80

days after first POO Rx

PDD· effect on PLATlETS 78 PATIENTS

EXCLUDI NG L EUKEM IA .... . IIZOOoo lC 'W

C.,OI)oO lC 'W I I "'CJ C.OOI)OQ 11'" I I : . iiE ' I: I : ~ : I

",00, 171 - <21100 :: :

: ~: 'l'.,"'''''''''' 221 - <I.OGI : ~ : E ~gg:" ., ., r 'W , , ,. .z," - <IOalGO 't 1 I

E ~J.,IO,',!~ !;:::::: t ~ us - )'O.IGO , : ! : = "........ :; : _ "'V """"""I.l "JI:.I:I: , ,. "Ivl

"- C")::gg:g :;;::;:: t t " ; : ~ ~ : : : :--. 'I V' : : : :

C) ~!~= ::::::::ttt t::~~:::: :-!--- ... r-t:: c t'~ ""'''''''''''''''' I." " ,,1"111' 1(1'( VI ,., V'WIVI

o ~;~;:g ::::::::ttt"ttttL ,tt:!~:~t!~~:: :::: :::: ~::~l >< c~:= :; ::::: : t t t t t t t t ~ : : ! t t ~ : ; : : t : ~ ~ : : : : : : ~::: I :::: ~

Q toooco ,,..,, T ,. , , ... , l " L " " II. l , ., , .. l , , , , , l , ., , , 1 I • I: " V' I v I 1 1 Y"! Y I

(I) N~:gggg ~::;::;; t t t ~ t t t t ~ ~ : ~ t ~ ~ ~ : ~ ~ t ~ ~ ~ ! : : : : : : i ::: l :::::: I-- 170000" , , ¥ " , " '¥ L ..... L l ... L ... I. I , , l II. , , , , , I. , , , , It lC • 11 \' ,. I ., I I I 'W V' 1 V I I 1.&.1 1.~:)QO,. , , .., 'W , , , I. l ... " .. " I. ... I. , , , .. I. , , , , , l , , , , 11 • l( 11 '¥ ¥ I V' 1 It ... 'I I 'W I I --' 150000 '¥ '¥ , l' l' , , , " \. " " \. \. I. l II.. I , , II.. .. 1 • , T 1 .. T , , , II lC II II ... ¥ I v I It ... V' t ... , I

~ ::: : : : : : : ! t t t ~ t t t t t ! ~ ; t t ~ ~ : ~ ; t ~ : : ~ : : : : : ::: : :: :::: ~ : II( ItO'ilOCl" , 't ., , ,. v ., l L L l L l " L L. , , , L I. ., , , • , " , , , , 11 I( II 1; It ,. I "I I 1 It" I V' I I ....I c:t IU'JOO ,~,.'''..,." .. L l ... L \.. l l 1. , "" L.' , , f'" T" I lC Il 11 X It Y I "I I I It" I It 1 I

A. ~15: ::::::;: t t t t ~ ~ t ~ t : i ~ t t ! ~ ~ ! ~ t ~ ~ ~ ~ : : : : : ::: ! ! i ! : : i : i ! '(tOO:l ,,.,.·,. ... "l " .. ll.""Ll'. ' .. '"" 11 '''''''1I'1I1I;1I'' \'1 YII,I""""II 4000Q • ."· ...... ''' I." ... ''L''''''"'''''"''''' .. '",1I •• ,,. 'IT '1,111""'''11 ,.,OOa "",·,.",,,,, ...... ,,I.\..,,\..\."'\.l""'",,,,1I1I.1I''' "1 'I I III"V''''II .,,001) ,,,,,,.,.., l' L l L " .. I. L l I. , T , L. I. , , , , , II. , , 1 , 11 11 II' • '" , V' I It I I I 1 ,. 'I I ,. I , '0000 ,., .. .,"'"lL"I.I."I.I."'I."'I"'L"".:':llIYII VI ¥IIII,.'II"II l~ ""'''''·'''Lll.l.I.lLl.r..'''I.''''''IL"I".II' 'lIvll 111 "IIII"~I"II

C) loOOo ~;~:~:~:~:~:~;~:~'~'~:~~~:.:~:~:~:~:~·~I~:~:~:~~~.~T~:~;~;~:~~~;~;~:~:~:~:~:~:~:~:~:~,~.~.~.~:~.~:.:~:~:~:~:~:~:~:~~~:~.~.~.~.~.~ Il:J·'!"~·01 I:1 · ,.:r""0Il),!o,:r .. tO":lIlS,:r"'OIZ').!o'J.90IrJ.56'''''0

o 10 20 30 40 50 80 d IYS after first POD Rx

TREATMENT WITH Cis-PLATINUM DIAMINODICHLORIDE

%

100

90

80

70

til 60 H Z

~ 50 ~ Po; 40

30

20

10

o

52%

o

TABLE IV

86 Non Leukemic Patients Treated with PDD Alone

20

36%

40 B.U.N. Levels

6.9%

429

I 4.~'Y- I

60 80

430 E. LOEB ET AL.

TABLE V PDD ALONE Rx RESULTS IN

32 SQUAMOUS CELL CARCINOMA PATIENTS

NO. %

More than 50% Improvement 5 15%

25 - 50% Improvement 6 19%

Less than 25% Improvement 21 66%

Average dose total 5.90 mg/kg. PDD

TABLE VI PDD + COMBINATION Rx RESULTS

IN 18 SQUAMOUS CELL CARCINOMA PATIENTS

NO. % More than 50% Improvement 6 33%

25 - 50% Improvement 3 16%

Less than 25% Improvement 9 50%

Average dose total 5.75 mg/kg. PDD

TREATMENT WITH Cis-PLATINUM DIAMINODICHLORIDE 431

showed good improvement, and 1 or 14% showed slight improvement, 72% no response.

TABLE VIII. Results with PDD alone in squamous cell carcinoma in 32 patients. Squamous cell carcinoma of head neck,16 patients treated, 3 or 19% had good improvement, 2 or 13% slight improvement and 11 or 68% showed no response. Nine (9) patients with primary lung involvement only 1 showed slight improvement. Bladder and vag­inal, 2 or 50% good improvement, 4 patients with esophageal carcin­oma, 50% slight improvement and 50% good improvement.

They received 5-17,5 mg/kg.PDD and lived 4-24 months.

DISCUSSIONS

During 4 years of investigation of the antitumor activity of PDD it has been shown to be most promising in the treatment of squamous cell carcinoma, lymphosarcoma, adenocarcinoma of the pan­creas,testicular carcinoma and in endometrial carcinoma. The smal­lest dose, causing tumor regression has been 2.5 mg/kg. and the largest dose, given over a period of two years has been 18 mg/kg. This patient with squamous cell carcinoma of head and neck obtained complete tumor regression with remission of disease without any further treatment during the last 2 1/2 years. After 2-2.5 mg/kg at monthly intervals, very little bone marrow depression was observed and certainly did not require discontinuance of further treatment. The only limiting factor with this dosage is kidney tubular damage, which may be aggravated by prior kidney disease or concomitant use of nephrotoxic drugs such as antibiotics. Nausea and vomiting can be controlled by antiemetis.

PDD shows no apparent cross resistance with other chemotherapy. A patient with squamous cell carcinoma of the lung was previously treated with multiple chemotherapy yet responded well to PDD in com­bination with bleomycin. It is interesting to note that 1 patient with carcinoma of the pancreas showed no evidence of tumor during a second look operation after 6 courses of treatment, but he did not return for follow-up treatment during 16 months; after that time multiple organ metastasis caused a rapid deterioration and death 29 months after diagnosis.

SUMMARY

PDD has been evaluated in a variety of malignant tumors. PDD alone as well as in combination with other drugs has shown definite tumor regression. Further exploration for analogues is in progress. Phase I studies with cis-dichlorobiscyclopentylamine platinum II suspended in oil and aqueous suspension as well as with platinum blue have been initiated in our Institute,but reports are yet in­conclusive.

Recently 2 patients received platinum II, 1,2 diaminocyclo-

432

RESULTS

ESOPHAGUS

BLADDER/VAGINAL

LUNG

HEAD & NECK

RESULTS

ESOPHAGUS * BLADDER/ VAGINAL

LUNG

HEAD & NECK *

TABLE VII

PDD IN COMBINATION

NO. <: 25%

1

7 (5) 72'f.

10 (3) 30%

TABLE VIII

PDD ALONE

NO. < 25%

4

4 (2) 50%

9 (8) 89%

16 (11) 68%

25 - 50%

(1) 14%

(3) 30%

25 - 50%

(2) 50%

(1) 11%

(2) 13%

* one patient had two different carcinomas

E. LOEB ET AL.

> 50%

(1) 100%

(1) 14%

(4) 40%

> 50%

(2) 50%

(2) 50%

(3) 19%

TREATMENT WITH Cis-PLATINUM DIAMINODICHLORIDE 433

hexane malonate (P.H.M.) 1.5-10 mg/kg. one terminal patient with bronchogenic carcinoma and brain metastasis, another paitnet with testicular embryonal carcinoma who developed a hypersensitivity reaction to PDD now seem to have responded to P.H.M.

BIBLIOGRAPHY

1. Rosenberg,B.,Van Camp,L. and Krigas,T.,Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature, 205,p. 698, 1965

2. Rosenberg, B.,VanCamp, L.,Grimley,E.B. and Thomson,A.J. The inhibition of growth of cell division in Escherichia coli by different ionic species of platinum complexes.J.of Biol.Chem. 242, No.6, p.1347,1967

3. Rosenberg,B.,Renshaw,E.,VanCamp, L.,Hartwick,J.and Drobuik,J. Platinum induced filamentous growth in Escherichia coli.J. of Bacteriology 2l, p. 716,1967.

4. Rosenberg,B.,VanCamp,L.,Trosko, J.E. and Mansour, V.H.Platinum Compounds:A new class of potent antitumor agents.Nature 222, p.385,1969

5. Rosenberg,B.and VanCamp,L.The successful regreSSion of large solid sarcoma 180 tumors by platinum compounds. Cancer Research, 30, p. 1799,1970

6. Talley, R.W.Chemotherapy of a mouse reticulum cell sarcoma with platinum salts.Abs.Proc.of the A.A.C.R.,11,6lst annual meeting1970

7. Welsch, C.W.Effects of cis-platinum diamminodichloride II on growth of 7, 12 dimethylbenzanthracene (MBA) induced tumors in female rats. Abs.proc. A.A.C.R., 12, 1971

8. Welsch,C.W. Growth inhibition of rat mammary carcinoma induced by cis-platinum diamminodichloride.J.Nat.Cancer Inst.47,p.l07l, 1971

9. Welsch,C.W. cis-Platinum diamminodichloride (II) induced re­gression of carcinogen induced rat mammory tumors. Advances in Antimicrobial and Antineoplastic Chemotherapy, Vol. II, Univ. Park Press,Balt., p. 255, 1972.

10. Hill,J.M.,Speer, R.J.,Loeb,E.,MacLellan,A.,Hill,N.O. and Khan, A. Clinical experience with cis-platinous diamminodichloride (PDD).Advances in Antimicrobial and Antineoplastic Chemotherapy, Vol. II, Univ.Park Press, BaIt., p. 255, 1972.

434 E. LOEB ET AL.

11. Speer,R.J.,Lapis,S. ,Ridgway,H., Meyers,T.D. and Hill,J.M. cis­Platinous diarnmine dichloride (PDD) in combination therapy of leukemia L12l0.Advances in Antimicrobial and Antineoplastic Chemotherapy, Vol. II, Univ. Park Press,Balt., p.254,1972

12. Hill,J.M.;Loeb,E.; MacLellan, A.; Hill, N.O.; Khan ,A. and Kogler, J. Further clinical experience with cis-platinous diaminodichloride."Platinum Coordination Complexes in Cancer Chemotherapy" (Ed. by T.A. Connors and J.J.Roberts)Springer­Verlag, Heidelberg, in press,1974.

13. Higby, D.J.; Wallace, H.J.; Albert, D. and Holland, J.F. Diarnminodichloroplatinum in the chemotherapy of testicular tumors. J.of Urology, 112, p. 100,1974.

14. Talley, R.W.; O'Bryan, R.M.; Gutterman, J.; Brownlee,R.W.; McCredie, K.B. Phase I clinical evaluation of toxic effects of platinum diarnminodichlorocis. Cancer Chemotherapy Reports, Part I, 57, No.4, 1973.Also in "Platinum Coordination Com­plexes in Cancer Chemotherapy" (Ed. by T.A.Connors and J.J. Roberts)Springer-Verlag,Heidelberg, 1974 in press.

15. Khan, A., Grater, W., Hill, J.M., Loeb, E., MacLellan, A.S.; Hill, N.O. Atopic Hypersensitivity to cis-Dichlorodiarnmine Platinum II and Other Platinum Complexes. Cancer Research, in press, 1975.

ANHYDRO-ARABINOSYL-FLUOROCYTOSINE HYDROCHLORIDE A PHASE I STUDY

P. ALBERTO and R. MEDENICA

Division of Onco-haematology, Dept. of Medicine

Hopital Cantonal, GENEVA, Switzerland

2.2' -anhydro-l-(B-D-arabinosyl)-5-fluorocytosine hydrochlo­ride (fluorocyclocytidine, AAFC). is a pyrimidine analog with anti­tumor activity in experimental animal tumors. AAFC is hydrolysed to arabinosyl-fluorocytosine wicht as arabinosyl-cytosine. inhibits DNA-polymerase activity. AAFC is also deaminated to arabinosyl-fluorouracil. wich possibly also inhibits thymidilate synthetase.

In a phase I study. AAFC was administered in one weekly in­travenous injection for six weeks to 24 patients. and in one weekly two-hour perfusion for six weeks to 14 patients. Starting from 10 mg/kg/week. dose escalation was determined according to a modified Fibonacci schedule to a maximum of 50 mg/kg/week. The most important toxic manifestations were anemia. leukopenia. throm­bocytopenia. nausea and vomiting. The proposed dose for further clinical trials is 33 mg/kd/week x 6. The two-hour perfusion route appeared slightly more toxic than the direct injection route. A possible anti-tumor activity was observed in a few cases of epidermoid head and neck / carcinomas and in one digestive adenocarcinoma.

A phase II trial is presently in progress for patients with epidermoid head and neck and lung tumors. adenocarcinoma of the gastrointestinal tract. breast carcinoma and small cell carcinoma of lung.

435

IFOSFAMIDE IN THE TREATMENT OF LUNG CANCER AND METASTASES

OF SOLID MALIGNANT TUMOURS

Wrbo, H., Kokron, O. and Titscher, R.

Institute for Cancer Research of the University of Vienna, Borschkegasse 8a,A-1090 Vienna Ludwig Boltzmann Institute for Clinical Oncology, Wolkersbergenstra~e 1, A-1130, Vienna

During the years 1969 and 1970 new N-Iost phosphamide-esters have been tried clinically in West Germany within the framework of a large study. This study carried out by 24 authors and more than a dozen hospitals resulted in the abandonment of Ifosfamide (substance Z4942) for clinical use because it was found to be inferior compared with Endoxan. Ifosfamide (If.) does not have the characteristic structural properti es of the N-Iost compounds. Neither of the ethylchloride functions are situated at the same N-atom, but are separated. In fact, it is a secondary amide in which the alkalinity of both N-atoms is largely neutral ized through phosphorylati on. The ethyl­chloride groups are situated at the cyclic and extra-cyclic N-atom (Fig. 1).

Theoretically, it would be desirable to find substances, in which the toxic effects are reversed more rapidly, in which, however, the therapeutic effects are cumulative. In the If. this goal has been accomplished to a much larger degree than in most other alkylating compounds. The accumulation of toxic components, however, is reduced.

The above mentioned cooperative study concluded, that the dosage potential of this substance apparently has not been fully utilized as yet, due to certain side effects. Therefore, we have undertaken in cooperation with the Boltz­mann Institute for Clinical Oncology in Lainz (Austria) and the Radiological Clinic Hoefer-Janker in Bonn (West Germany), a clinical investigation of If.

437

438

CI--CH --CH 2 2 ...........

-­CI--CH·-- CH 2 2

N

H. WRBA, O. KOKRON, AND R. TITSCHER

N--CH /" 2, P = 0 CH2

"'-O-CH/ 2

Cyclophosphamide

CH-H-- CH2-- CI I 2

CI--CH--CH N--CH 2 2" / 2,

N-P= 0 CH2

"'-O-CH/ 2

Isophosphami de

Figure 1.

Ifosfamide

It was found that high doses of If. in quantities in excess of 80 mg/kg were effective on tumours which up to now were considered chemoresistant. Therefore, If. was used at first in cooperation with the Radiation Clinic Janker in Bonn - which had already amply tried the substance on a number of patients with lung metastases of malignant tumours (Table 1) .

The joint publication of results at the Clinic in Bonn as well as at our Institute in Vienna show, that in primary bronchial carcinoma and lung' metastasis originating in other localizations, of a total of 119 treated patients, 32 showed total remissions and 57 showed partial remissions, more than 60 percent response.

At the radiation Clinic in Bonn, the If. application was combined with a series of radiation treatments, in Vienna the If. was administered without additional radiation therapy. In Bonn 5 x 60 mg/kg If., as a rule, was applied once during 5 consecutive days, in Vienna 3 x 35 mg/kg If. were injected at 12 hour intervals. This intensive therapy was repeated after 1 week. Altogether 4 such series of treatments within intervals of 6 weeks were undertaken.

After administration of this dosage, leukopenias appeared at 300 mg/kg

IFOSFAMIDE IN TREATMENT OF LUNG CANCER 439

Table 1. Results of treatment with Ifosfamide

Total Partial Not remission remission Failure evaluable

l. Bronchogenic Cancer

a) Squamous cell 1 2 2 b) Oat-cell 10 19 7 3 c) Adenocarci noma 2 d) Alveolar cell 1

2 1 1

l. Lung Metastases in:

Mammary carcinoma 5 11 5 2 Cervi cal carci noma 1 Adenocarcinoma uteri 1 Ovarian carcinoma 1 Chori onepithel i oma 1 1 Seminoma 4 Malignant teratoma 3 Terato carcinoma 2 1 Embryonic-cell-carcinoma 1 Chori onepi thel i oma 1 Renal adenocarcinoma 1 1 Hypernephroma 6 3 Carci noma col i 1 2 Oesophageal carcinoma 1 Sarcomas 5 4 2 Melanoma 1

32 57 24 6

440 H. WRBA, O. KOKRON, AND R. TITSCHER

Table 2. Study of oat-cell bronchial carcinoma (inoperable, metastasizing)

Criteria:

Male sex Histological and or cytological verification Oat-cell carcinoma with distant metastases No previous tumour therapy (patient seen and diagnosed for

the first time) No other tumour therapy No relevant secondary disease Extensive assimilation of symptomatic therapy

RANDOMISATION

1. Biological Group 2. Ifosfamide group

Intens-ive care by internist regular control

identical care by internist identical controls + Ifosfamide

17 patients 17 patients

Table 3. Study of oat-cell metastasizing bronchial carcinoma

Results

Complete remission

Partial remissi on

Slight regression

No changes

Progressi on

Biological

o

6

8

Ifosfamide

1

4

5

7

o

IFOSFAMIDE IN TREATMENT OF LUNG CANCER 441

If., which approximately corresponds to a leukotoxic effect of 60 mg/kg Endoxan. The time factor in the appearance of leukopenia is identical with Endoxan.

The most dangerous complication is a haemorrhage from the urinary tract. in 31 percent of all cases we found microhaematurias. This occasionally forces a reducti on in dosage. The foil owi ng measures were necessary in order to combat the urinary tract haemorrhage: 1, exclusion of urinary obstacles; 2, cure of chronic infections; 3, alkalization of the urine starting the day before the first injection, continued until 24 hours after the last If. dosage. The alkalization of the urine should reach a pH value of 7 or higher, since complications in the urinary tract were observed primarily in urine with high acid content; 4, immediately prior to injection a permanent catheter was inserted; 5, If. was given in the above-mentioned dosage in the form of a 5-10 percent solution intravenously, in addition Ringer's solution was given by infusion; 6, the minimum diuresis within 24 hours should amount t04 litres. When these precautions are carefully observed, no serious complications are expected.

Nausea and vomiting occur occasionally, however, less frequently than with Endoxan therapy. Loss of hair has been observed regularly.

During the preliminary investigations of lung metastasis of various origins as well as primary tumours of the lung, we found that the introduction of If. represents progress in chemotherapy especially when directed against previously largely therapy-resistant tumours. Consequently, we have initiated a prospective study for the treatment of metastasizing oat-cell bronchial carcinoma (Table 2) .

Patients with metastasizing inoperable oat-cell bronchial carcinoma were admitted to the study. All diagnoses were made by the same pathologist. The pati ents were between 47 and 76 years old. The average age was 61 years. Further pre-requisites for admission to the study were: 1, only male patients were admitted; 2, primary tumours diagnosed for the first time without having received any previous tumour-specific therapy were required; 3, no other decompensated ill nesses . The pati ents were sel ected at random in two groups. By the application of If. to one group or by a "biological therapy" (i .e. a control group) treated with roborant medication only in the other group. In both groups anabolic hormones and antibiotics in the same dosage and frequency were applied in order to avoid any variation in treatment. Clinical controls and the treatment-free intervals were identical in both groups. All therapeutic measures were always carried out by the same team of physicians in an identical manner.

A course of treatment consisted of three fractionated dosages given at

40

3

5 3

5

~~~ J

35

35

35

~~~

[

I --

:=1

AN

TIB

IOT

IKA

35

35

35

mg

/kg

i.

v.

~~~ L

IQU

ID I

NT

AK

E

C:=

====

=:JI

AL

KA

LIZ

AT

ION

DA

YS

'--_

_ --'I

PE

RM

AN

EN

T C

ATH

ETE

R

....... .,

Figu

re 1

. IF

OSFA

MID

THE

RAPY

~ ::I: :e :0

~ 9 S " :0 o z » z c :0

-I ~ ::I

: m

:0

IFOSFAMIDE IN TREATMENT OF LUNG CANCER 443

weekly intervals (Figure 2). These three dosages were repeated affer 6 weeks, a I together 5 ti mes. There were 34 pati ents in the study after 2 years ( 17 in each therapy group). One patient in the biological group had to be excluded from the study later one, because a squamous cell carcinoma was definitely diagnosed during a renewed evaluation of the histological findings. One total remission (i .e. a complete regression of all reoentgenological findings) was found in one patient treated with If. Partial remissions (i .e. a regression of more than 50 percent of the tumour size) were found in four cases treated with If. (Table 3). The duration of the remission, however, is relatively short, lasting between 1 week and 2 months. In two patients we were able to accomplish the induction of remissions 3 times renewing the application of If. In the group treated with If. a decidedly prolonged survival time was noticed. The median survival time for the treated group is 113 days, compared with 73 days in the biological control group.

From these results, it can, therefore be concluded that in the treatment of metastasizing oat-cell bronchial carcinoma a concentrated If. therapy is of real value, to attain remission. The purely symptomatic treatment did not show similar results. The side effects are tolerable.

It is noteworthy, that ina number of pati ents the treatment with If. apparently caused a decrease of their survival time. The explanation of this phenomenon seems to be the oncolyti c effect of the cytostati c drug whi ch apparently leads to a tumour-disintegration-symdrome in patients with large tumour masses. The evaluation of these cases reacting unfavourably does not show a frequent occurrence of large primary tumours in patients who survived from 1-30 days following If. therapy. They did, however, show generalized metastasizing with the presence of a large, accessible tumour-mass. The findings in all these cases have been confirmed by autopsy. We shall, there­fore, in the future exclude all patients with generalized metastases from treatment with If.

Further clear indications in favour of the use of IF. are patients with cancer of the ovaries and testicular tumours of various origins. These cases also show very clearly a high rate of total remissions, as mentioned in the first summary.

All four treated seminoma cases showed complete remissions in malignant teratomas, 3 partial remissions were accomplished in 3 cases treated, in terato carci noma, 2 partial remissi ons were seen three cases treated. Chori on epitheliomas respond particularly well to the If. therapy.

The complication of a massive haematuria can be practically excluded if Acetylcystein is used at the same time and in equal dosages with If (according to an oral communication from the Janker Clinic in Bonn). We have so far

444 H. WRBA. O. KOKRON. AND R. TITSCHER

no experience with this method in combatting the side effects.

In conclusion it can be stated that the treatment with If. accomplished a great number of complete remissions in patients with oat-cell carcinoma, testicular- and ovarian tumours and apparently also in lung metatasis of various origins, and that it also has a beneficial effect on a number of other cases. In evaluating these results, one gains the impression, that If. is particularly well suited to induce remissi ons and that the drug can be and should be combined beneficially with substances which may stabilize and maintain the accompl ished remissi ons.

It is one of the purposes of this report to prevent this substance from being lost, especially since it must be helpful in suitable combinations.

PRECLINICAL AND PHASE-I STUDIES OF IFOSFAMIDE FOR ITS MASSIVE

DOSE CUMULATION SCHEDULE

KANJI KUBO, M. D.

Department of Surgery, National Magoya Hospital

Koku-ritsu Byo-in, Maka-ku, Nagoya 460, Japan

The rate of accumulation of repeatedly administered drugs depends primarily on the persistence of the drug after a single administration. Agents with a long lasting effect would show a steep rate of accumulation while short acting agents would only have a slow rate of accumulation even when both types are given at equal intervals (Fig. 1).

This allows the possibility of dissociating the anti-tumor effect of Ifosfamide from its toxic effect. In the favorable situation where the curative effect lasts longer than the toxic effects, one can expect a steep accumulation of anti-tumor effect and slow accumulation of toxicity when the repeated doses are given during the particular time when the host is recovering from toxicity and the growth of the tumor is still inhibited. This particular administration schedule can be designated the 'cumulation schedule' (Fig. 2).

Shorl Last ing

Fig. 1. Effect-cumulation with equal interval.

445

446 K.KUBO

=:(p;1~=== =I.,~;rhreshold

Too Long Appropriate Too Short

Fig. 2. The cumulation of curative and toxic effects are closely related to the administration intervals of Ifosfamide treatment.

Some types of breast cancer respond in an impressive way to a 'single push' of massive doses of alkylating agents, quite close to the lethal dose. The repeated administration of a proportion of this large dose could bring about a similar response by effect cumulation. The purpose of this study has been to explore the maximum dosage and the appropriate interval in the cumulation schedule of Ifosfamide with special reference to mammary carcinoma.

PRECLINICAL STUDY

Animal experiments were performed using DMBA-induced mammary tumor bearing young female rats (SD-JCL) unless otherwise mentioned.

When Ifosfamide was given intraperitoneally at 40Omg/kg to rats with the tumors ranging from 10 to 20mm in diameter, marked tumor shrinkages continued longer than 32 days whereas the body­weight losses recovered at 30 days after the treatment. This result indicates that the curative effect of Ifosfamide remains after recovery from the toxic damage (Fig. 3).

When Ifosfamide at 5Omg/kg was given intraperitoneally for eight times, at four-day intervals, up to total dose of 400mg/kg, the tumor regressions were not so remarkable compared with the preceding single-push experiment. However, the tumor-growths were significantly suppressed as compared with control group. With this repeated-doses schedule, body-weight losses were not so conspicuous. This result led to the clinical application of a similar dose schedule (Fig. 4).

With repeated doses of 5Omg/kg at intervals less than four days, the rats showed marked emaciation, suggesting detrimental

PRECLINICAL AND PHASE-I STUDIES OF IFOSFAMIDE

01 400 mg/kg x 1 01 50 mg Ikg x 8 q_4d_

• J i .. H ... •

I Tumor Suppression N=4

20 10 -~J--- 20 10 -,t-----~~

I I o 0 ......... ----...- 0 0 .... '--------:.:.::..:... 1 5 10 15 20 30 d. 10 20 30d.

Fig. 3 Fig. 4

447

cumulation of toxicity. With repeated doses of higher than 5Orng/kg at four day intervals, similarly conspicuous emaciations were observed.

To find out the relationship between effect-cumulation and the cumulation of the drug per se, tissue distribution curve of Ifosfamide was studied, employing the nitrobenzyl-pyridine method. The result obtained with lOOrng/kg intraperitoneal administration showed fairly rapid disappearance from the tissues (Fig. 5). This result would indicate that the long continuation of both curative and toxic effects are due to the morphological and biochemical changes caused by the contact with the drug.

25011----- 1----- ~--- 1---- I-irir-----

Blood Tumor Intestine 200t----- t----- 1----- 1-----,::--- 1---lI~~I--

1501----- 1----- 1------- I-'~~r- r;~~~I--

100HI~~- I--(l----\\--- h'f---J~- I-fl------'\\---- H/-:j~~~-

o L.:;._--....;~ I-__ ~ 1--_--10-. '-~~~. '--__ _ JI. )') 1 4 8h~ y, Y2 1 4 8h~ y, y, 1 4 8hr. y, Y2 1 4 8hr y, Y2 1 4 81T

Fig. 5. Tissue distributions of Ifosfamide, t, !, 1, 2, 4, 8hrs after i.p. administration of lOOrng/kg.

448 K.KUBO

Incidentally, extremely high distributions in the liver and the kidney in this study were found. Accordingly it was decided to exclude patients with any hepatic and renal disorders from the clinical trial of Ifosfamide.

CLINICAL STUDY

Women of all ages who were admitted to our hospital with primary advanced inoperable breast cancer, recurrent lesions after mastectomy or distant metastases were the subjects of this study. Ifosfamide was given intravenously, diluted with 500 ml of 5% dextrose in water on each administration.

Since we estimated the maximum dosage around lOO-120mg/kg, in the preliminary pilot study of sing-push schedule, we started once a week schedule with repeated doses of 5Omg/kg upwards. At this administration interval, the maximum dosage was estimated at about 7Omg/kg. Following this, a twice-weekly schedule was studied in the same manner. At this interval, patients well tolerated more than ten repeated doses of Ifosfamide of 5Omg/kg. The major reasons for discontinuation of the treatment were leucopenia, nausea, vomiting, general weakness and hemorrhagic cystitis (Table 1).

l/wk 2/wk Dosage Pt. Toxic Respalse Tol~a~t'd !!!II kg Hem. Gol Uro. Ad.mn Istr

Dosage lOxicRespcllse Tolerated Pt. No of ~ HanG.I. Uro.Ad.mnlstr

50 KA - + + 7 0 30 A.F. + 10 0 57yo,41 kg ./o3yo, 43k9 -60 s2~,q2k9 + + 10 0 40 IN.

52yo,64kg + + - 11 0

70 65:'~k9 + 9 0 K.T + + + 10 0 57YO,55kg

80 yy - ** 2 • N.I + + 0 38yo,49kg 32yo,5' k9 10

90 46;'~;kj* + 7 • M.M + + 10 0 59yo,50kJ

36~O',~8kg * ** 2 • 50 KS + + 10 0 44yo,52k9

5O;,T"", * '"' 2 • MW. + 10 0 100 49yo,51kg

'l!i~'.~5kg + * 2 • K.T. .. + 11 0 53yo,46

TT 3 0 53~O~;k9 + 41yo,51k] + - 12 0

Further Administrat'n Tolerable: 0 Intolerable: •

Table 1. Tolerances in l/wk and 2/wk schedules according to dosage levels. Toxic responses: hematological (Hem) WBC + = less than 4,OOO/cmm, ++ = less than 3,OOO/cmm; gastrointestinal (G. I. ) nausea and vomiting; urological (Uro) hemorrhagic cystitis.

PRECLINICAL AND PHASE·I STUDIES OF IFOSFAMIDE

l/wk

20-40 ~ /G.I. Upset 11'/, mg/kg 67... '

'\ Cystitis S'/, (N: 12) Lassitude 0'/,

30-40 mglkg

(N :19)

2/wk

/ G.L Upset 16'1,

- Cystitis 5'1,

\lassitude 16'/,

50-70 mglkg

/GL Upset '1J.'/, 50 mg/kg

'\. Cystitis WI,

lassitude 20'1,

/ G.I. Upset 27'1,

\. Cystitis 19'1.

lassi tude 16'/,

/ G.L Upset 33'/,

'\. Cystit is 29'/.

lassitude 3'0'1,

Alopecia: 9/24 pt.(3S·/.) 1 I wi< : 4 114 (28 ·I.)

21wk : 5/10 (50·1.)

449

Table 2. Subjective reverse effects on each drug administration, including the cases of preliminary study.

As for the incidence of subjective reverse effects, SOmg/kg x 2/wk schedule showed almost identical rate as compared with SO-7Omg/kg/wk schedule. This would imply the advantage of shortening the interval from a week to half a week at this dosage level.

Fig. 6 illustrates the results of hematological studies at SOmg/kg x 2/wk schedule. Roughly speaking, myelosuppressions were cumulated but they were reversible. Leucopenia was gradually enhanced with repeated doses; the majority of the subjects reached to 3000 levels at 10th dose. This possibly suggests the permissible repetition is 10 doses in this schedule.

w • I ' j..{U J J J // :~ II J JJ JJ J

C ~." fA' NM~ '001_ - _ ." _ _ "000. _

1000_ :c"'-- - - , )0"- _ _

.000- _ :~ . , _ ~ -:- "ot_ .... ~ =- _ --:: ... " ... ,,-JOOO . - --- 1000 __ -- t '~ _ _ tooo .

P L JJ JJJJJ ,

4 S twlu 0 4 S twlu 0 • S twill

Fig. 6. Granulocyte (GRN) and lymphocyte (LYM) decrease their counts almost identically. Platelet count (PLT) were sparing.

450

No. Pt. Trealm .... 1 Foci Size ~ DJrationMaint~-Ons~ flCUU'

~ -----~ -===:::;;; ~ ~ ~

1 K.l,S7y.Cl 2.8gx10 r:" ;" T: SxS PR 4wk 11mo S~ SSkg. tSrrr q. 3--4d. 'is.:t N: 2.2 •• . . ~;.~

2 N.1.,32y.Cl 2.7gx10 ~ T: 8x6 CR ~;;- 5~ _ 54kg.1.5m' q.3--4d. t-~ N(::: _._.~. mo. ~:.~ 3 M.M,S9y.a 2.5gx10 r:.-~ T:7x6 No

_ SOkg. 1.5m' q.Nd. ~..t ~ Crang~

4 K.S.,44yCl 2.Sgx10 (!Jf-:-t mullipl~ PR. 6wk. 6 mo. ~~ _ 52kg,1.Sm' q.:r4d. _, __ 1) _________ ~

5 .MW,49y.o. 2.5gx11 (:':~ T:l0x9 PR. 3wk. 2mo. 51kg. 1.4m' q. 3~4d . ~ '. S~· 2.5gxll r:' .~ R5x4 - - - -

6 rrf ~ r;, N:nhtJ PR. 6wk. 1 mo. _ 4 19,1.4 q.:r4d. _\",_ , ___ " ___ _

7 MA.,S3y.o, lSgx 12 t:-.',>\ T:20x1S No _ -> 1 N's" 67kg.1.6rrr q.:r4d. )", ' h' Chang~

K. KUBO

Table 3. Anti-tumor effects of the patients treated with 5Omg/kg x 2/wk schedule . CR: complete remission, PR: partial remission.

All through this trial there were no abnormal values of biochemical tests which included GOT, GPT, LDH, AI-P, serum total protein level, bilirubin value and so on.

Anti-tumor effects among the seven patients treated with 5Omg/kg x 2/wk schedule are listed in Table 3. One complete remission and four partial remissions were experienced.

CONCLUSION

'Ihe repeated administration of Ifosfamide with "5Omg/kg x lOq. 3-4 days" schedule can be considered worthy of further clinical investigations from the stand point of effect-cumulation.

REFERENCES

1. Friedman,M.A. and Carter,K.S.: Ifosfamide (NSC-I09724) Brochure, National Cancer Institute, 1970.

2. Allen,L.M. et al.: Pharmacokinetics of Ifosfamide, Clin. Pharm. & Therap. 17(4): 492-498, 1975.

3. Creaven,P.J. et al.: Clinical pharmacology of Ifosfamide, Clin.Pharm. & 'Iherap. 16(1): 77-85, 1974.

4. Ahman,D.L. et al.: Phase II clinical trial of Ifosfamide in patients with advanced breast cancer, Cancer Chemotherapy Reports. 58(6): 861-865, 1974.

CLINICAL PHARMACOLOGICAL STUDIES WITH FORMYL-LEUROSIN IN MALIGNANT

DISEASES

S. Eckhardt, I. Hindy, and E. Farkas

National Institute of Oncology

Budapest, Hungary

New cytostatic agents may be new mole­cules or analogues of existing antitumour drugs. In a recent survey the ratio between these two types of compounds was estimated 1 : 8, which means that far more analogues are under clinical investigation, than original substances. It is one of the most difficult tasks to assess the clinical value of an analogue in comparison to the parent compound. Nevertheless, it is possible to evaluate its advantages if the new ana­logue is more active and/or less toxic.

In the group of Vinca alkaloids research was initi­ated some years ago by Jovanovics and Szasz (Richter G. Limited) in order to find more active and/or less toxic compounds. Vincristine and Leurosine were chosen as pa­rent molecules. After a careful analysis of a certain number of derivatives the research team of the Onco­pathological Institute in Budapest (Somfai, Nemeth, Ga1,Bencze,Ke11ner and Sugar) could demonstrate, that Formy1-Leurosine is an active compound possessing anti­tumour activity similar to that of Vincristine with fa­vourable toxicological properties.

Formy1-Leurosine can be distinguished from the na­turally occuring Leurosine by the absence of a methyl group at the Vindo1ine ring which is substituted by a formyl group, as it is in Vincristine (Figure 1).

451

452 S. ECKHARDT, I. HINDY, AND E. FARKAS

VINCRISTINE F-LEUROSINE

Figure 1

Table 1. lists comparative toxicological data con­cerning Vincristine, Vinblastine and F-Leurosine. The new analogue is approximately 7 times less toxic than Vincristine and 4 times less toxic than Vinblastine in acute toxicity experiments carried out on rodents. It is remarkable to note, that even sublethal doses did not show any signs of neurotoxicity an any of the expe­rimental animals.

Formyl-Leurosine exerts striking tumour inhibitory effect upon a series of ascitic tumours as it is demon­strated in table 2. Moreover, chemotherapy resistant solid tumours, such as Harding-Passey melanomas as well

Table 1

ACUTE TOXICITY IN SWISS MICE

/Data of Somfai, Nemeth, Gal, Bencze, Kellner, Sugar/

Agent LDSO mg/kg 1 x

Lp. i.v.

Vincristine 4,0 3,0 Vinblastine 7,6 15,0 F-Leurosine 28,8 32,5

CLINICAL PHARMACOLOGICAL STUDI ES WITH FORMYL·LEUROSIN

Table 2

EFFECT OF F-LEUROSINE ON ASCITIC TUMOURS /Data of Somfai, Nemeth, Gal, Bencze, Kellner, Sugar/

Tumour Do8is Schedule E f f • c t TUIIOurfr •• mg/kg i.p. Survival Tumour , inhibition

• NK/Ly 8 x a,s daily 250 - 2/10

Ehrlich 24 x 0,15 daily ll8 - 3/10 cc. 4 x 0,6 each other day 288 - 7/10

7 x 3,0 daily 244 - 3/10

s 37 4 x 2,5 daily 100 4/6 8 x 1,0 daily 268 1/6

Yoshida 9 x a,s daily 143 0/6 sare. 9 x 1,0 daily 173 2/6

9 x 2,0 daily 243 4/6

Novikoff 6 x 2,0 daily 166 0/6 hepatoma 4 x 3,0 each other day 196 1/6

Table 3

EFFECT OF F-LEUROSINE ON SOLID TUMOURS /Data of Somfai, Nemeth, Gal, Bencze, Kellner, Sugar/

Tumour Dosis Tumour growth Spleen Evaluation mg/kg Lp. inhibition weight day

% %

S 180 9 x 2 53 + 39 14 sarc. 6 x 4 69 + 52 14

Harding- 14 x 5 47 - 20 19 Passey 20 x 7 41 + 26 30 melanoma 11 x 10 37 + 46 24

Rhabdo- 11 x 2 ~ -22 15 myosarc. 8 x 3 25 tox 18

Guerin 8 x 1 78 - 26 11 CC. 8 x 2 81 - 27 11

8 x 3 81 - 2 14

453

454 S. ECKHARDT, I. HINDY, AND E. FARKAS

Table 4 EFFECT OF F-LEUROSINE ON L 1210 I.P. LEUKEMIA ON BOF1~

/Oata of Somfai, Nemeth, Gal, Bencze, Kellner, Sugar/

Oosis Mean survival Survival mg/kg Lp.

Schedule in days %

S x 1,25 daily 27,7 131 S x 2,5 daily 25,0 127 S x 5,0 daily 23,0 109 S x 7,5 daily 19,5 56 tox

S x 2,5 4Sh interval 22,4 137 S x 5,0 4Sh interval 24,0 126 S x 10,0 4Sh interval 21,7 80 tox

8 x 2,5 72~ interval 12,0 48 8 x 5,0 72h interval 22,1 123 8 x 7,5 72h interval 20,2 117 8 x 10,0 72 interval 22,3 145

8 x 5,0 96~ interval 10,8 35 8 x 7,5 96h interval 19,2 95 8 x 10,0 96 interval 23,6 l36

as Guerin tumours responded also favourably to the treatment.

L-1210 Leukemia in BDFI mice was also sensitive to the treatment. It became evident from the experiment that small doses administered daily or high doses given twice weekly are equally potent.

The haematological parameters showed an interes­ting pattern. The number of granulocytes was lowered. by 50% if 0,6 mg/kg F-Leurosine was given, while that of the lymphocytes decreased only after administration of 15,0 mg/kg. No significant changes were observed in the platelet count at similar dose level.

On the basis of these and several other findings a Phase I trial was initiated.

In the escalation period 16 patients, in the eva­luation period 14 patients were treated. The patient selection met following criteria: a/ all types of leukemia, b/ all patients previously treated without success, c/ no X-ray or chemotherapy was administered 4 weeks prior to therapy, d/ at least 8 weeks follow­-up. In the escalation period 0,01 mg/kg/day i.v. was

CLINICAL PHARMACOLOGICAL STUDIES WITH FORMYL-LEUROSIN

Table 5 DOSAGE OF F-LEUROSINE IN PHASE I TRIAL

/EVALUATION PERIOD/

Dose /Lv_ / NO of Antitumour Side mg/kg patients effect effect

0,05 x 9-12 7 + -0,08 x 9-12 7 + -

ttt ttl ttt ttt Days 1-3 8-10 15-17 22-24

given 5 consecutive days as initial dose. This dose was gradually increased up to 0,05 mg/kg/day i.v. 6 consecutive days. At this level striking anti tumour activity was revealed without signs of toxicity.

455

In the evaluation period patients received F-Leu­rosine as demonstrated above. In addition,S patients were added to the group treated on the basis of this schedule.

Table 6

Phase I. trial with F-Leurosine. Therapeutic Results.

No.of Complete Partial No patients remission remission response

AML 1 1 ALL 1 1 AUL 1 1 AMoL 1 1 CLL 3 3 Hodgkin's d. 2 2 Lsc 5 4 1 Rsc 2 1 1 Multiple mye-

loma 6 4 2

Total 22 2 14 6

456 S. ECKHARDT,I. HINDY, AND E. FARKAS

Therapeutic results obtained by F-Leurosine in leukemias and haemoblastoses are listed in Table 6.

F-Leurosine showed striking anti tumour activity in all types of leukemia. The most striking antileu­kemic effect was found in one acute myelogenous and in one acute lymphatic leukaemia, both in adults. One un­differentiated cell and one monocytic leukaemia, both with hyperacute course showed favourable response as well. Three chronic lymphocytic leukemias were mode­rately sensitive to the treatment. Therapeutic benefit was revealed in the treatment of lymphosarcoma and reticular sarcoma. Patients with multiple myeloma res­ponded with a partial remission. Two patients with far-advanced Hodgkin's disease showed no response.

Side-effects were rarely seen. In two cases fun­gal infections occurred, while in two others pneumo­nia developed. Whether these conditions are due to the therapy or might be explained by the impaired immune response caused by the disease, is still to be answered. Preliminary investigations indicate that F-Leurosine decreases both T and B lymphocyte levels.

On the basis of the Phase I trial we do believe that F-Leurosine is a promising agent in the therapy of malignant diseases of haemopoietic organs. Having similar anti tumour activity to Vincristine, but not showing neurotoxicity even in continous administration in much higher doses it seems to be a candidate drug of malignant diseases. Its use in solid tumour therapy as well its applicability as a synchroniser agent is not Jret proved. Its optimum dose also should be fur­ther explored. Nevertheless, results so far achieved justify the initiation of Phase II investigations.

Clinical Investigations with F-Leurosine

Dr.E.Farkas & Dr.S.Eckhardt

National Institute of Oncology

Budapest

Earlier at this conference Professor Eckhardt had a paper on the chemical structure and pharmacological properties of the new Hungarian Vinca derivative F-Leurosine. My paper is concerned with results ob­tained by F-Leurosine treatment of the 3 main groups of malignant haemato10gical disorders, such as leukaemias, lymphomas and multiple myeloma.

Three schedules were applied as shown in table 1 Schedules A and B represent the two types of periodic treatment, whereas schedule C represents the continuous form of treatment. The total dose was 1,0-1,2 mgfkg in each schedule.

TABLE 1

DOSAGE OF F-LEUROSINE

SCHEDULES

A 0,08 MG/KG/DAY X 3 + 4 DAYS PAUSE / X4

B 0,1 MG/KG/DAY X 4 + 3 DAYS PAUSE X3

C 0,1 MG/KG/DAY / X 8-10

457

458 E. FARKAS AND S. ECKHARDT

The drug's similarity to Vinoristine /Onoovin/ suggested us treating two poor risk patients of aoute leukaemia, not suitable for polychemotherapy beoause of having thrombopenia. It was a pleasant surprise that oomplete haematologioal remission was achieved even in these two oases. This initial sucoess promp­ted us to extend the olinical screening to other haematological malignancies.

TABLE 2

EFFECT OF F-LEUROSINE ON LEUKAEMIAS

PATIENT DIAGNOSIS SCHEDULE EFFECT

HAEMATOL. CLINICAL

SZ.L. 71 WOMAN CLL A - + T. B. 74 MAN CLL A - + F. I. 46 MAN CLL A - +

F. M. 52 WOMAN AML A CR -sz. I. 31 MAN ALL C CR + SZ.A. 67 WOMAN AMoL C IR >50% + B. I. 57 WOMAN AUL C IR ".50% +

Table 2 shows the effeot of F-Leurosine on different types of leukaemia. In contrast to the drugs benefi­cial effect in acute leukaemias, in oases of chronic lymphocytic leukaemia no haematological remission could be induced, but some clinical improvement was achieved in all the J CLL patients. In the 1st oase /Sz.L./ splenomegaly diminished, in the 2nd one /T.B./ enlargement of pathological lymphnodes became less pronounced, in the Jrd case both the lymph nodes and the spleen decreased in volume.

On the bottom of the table you see the most im­portant data related to the 4 cases of acute leukaemia of 4 different cell types. Complete haematologioal re­mission was achieved in 2 cases, one of the patients suffered from ALL, the other from AML. Clinical im­provement, parallel with the haematological one, was observed only in the case of the ALL patient. The AML patient died one month after the end of the treatment.

CLINICAL INVESTIGATIONS WITH F·LEUROSINE

.3 lIBC/1IM

80.000

70.000

60.000

50.000

40.000

30.000

20.000

10.000

1\ 1\ I \ I \ I \ I \ I \ I \ I ,

.\: . .•.. ~. . .... ,

\ "

, , , , , , , , \ \ \

sz. I. 32 YEARS MAN, ALL

TOTAL WBe IMM3

LBC /MM 3

t 0,08 MG/KG F-LEUROSINE

F.M. 52 YEARS WOMAN, AML

TOTAL WBC /MM3

\ \

t

\ \ \ \ \ \ \

LBC/MM 3

0,08 MG/KG F-LEUROSINE

'",. o ~,--~.!.--...:..' --...... _'....!'":,-' -~., ~---'----'--- DAYS

10

t t t t t t t t t t t t t t t t t t t . . . . . .

Figure 1

459

Figure I shows the changes in total WBC count and absolute LBC count for these two patients. Unbro­ken solid curves represent total WBC counts, broken curves show the count of LBC-s per mikroliter each. The red curves represent the ALL, the blue ones the AML patient. Red and blue arrows show the days of treatment. The total '\'iBC and LBC counts run nearly parallel in each case. In case of the ALL patient complete haematological remission developed by the lOth day after the beginning of treatment, and lasted 2months. Our AML patient got into remission on the

8th day of treatment but 14 days after finishing it she fell into relapse which was untreatable because of serious thrombopenia and consequent bleeding.

Our patient of acute monoblastic leukaemia was previously successfully treated with VP-162l3. Two month after the end of treatment relapse developed and F-Leurosine was chosen as antileukaemic drug. Before the treatment the patient had a WBC count of 76.000, ratio of monoblasts was 10%. After an 8 days' treatment total WBe count fell to 20.000, ratio of blast cells ~ecreased to 1%, but ratio of mature mono­cytes was 50% in the peripheral blood.

460 E. FARKAS AND S. ECKHARDT

Although it is debatable to judge a drug's effec­tiveness on the basis on one case, it is necessary to emphasize that acute monoblastic leukaemia is a very drug-insensitive malignant disease, and we hope that F-Leurosine's effectiveness in its treatment will be comparable to that of VP-162l3.

In case of our patient with acute undifferentia­ted leukaemia partial remission was achieved. The total peripheral blast cell count decreased to 1/10 of the initial level. Total WEe fell from 22.000 to 9.600.

'rABLE 3

EFFECT OF F-LEUROSINE ON LYMPHOMAS

PATIENT DIAGNOSIS SCHEDULE EFFECT

H. I. 34 WOMAN HODGKIN DIS. A -ZS.A. 30 WOMAN HODGKIN SC. C IR < 50%

P.D. 42 MAN/II LYSC. A IR > 50%

P.D. I II I LYSC. B IR < 50%

T. I. 70 MAN LYSC. A IR < 50%

D.N. 78 MAN LYSC. C IR > 50%

G.J. 42 MAN LYSC. A -B.B. 72 WOMAN RSC. C IR < 50%

Cs.A. 27 WOMAN RSC. A -

Data of patients suffering from different types o~ lymphoma are shown in table 3. Both of our Hodgkin patients belonged to the poor risk oategory. The prog­nosis of Hodgkin's disease -owing to the recent benefioial effeot of polyohemotherapy- has ohanged very favourably. Therefore -regarding the ethioal view-point­we did not treat patients who were treatable with aooep­ted' polychemotherapy- schedules. One of our poor risk patients /Zs.A./ proved to be sensitive to F-Leurosine treatment. Before the treatment she sufferred from fever whioh could not be influenoed by using antibiotios. After an 8 day's treatment her temperature became normal.

4 patients of lymphosarcoma were treated, one of them, P.D. has got two courses. Patient P.D. had en­larged pathological lymph nodes in all peripheral regions. After finishing the 2nd oourse of treatment,

CLINICAL INVESTIGATIONS WITH F-LEUROSINE 461

all but one palpable lymph nodes disappeared. The westergren value, 75 mm/hour before the treatment, fell to 12 rom/hour. Patient n.N. was also success­fully treated. The majority of his palpable lymph nodes disappeared. A moderate remission was developed by one patient IT.r.l, the only non responder lympho­sarcoma patient !G.J.! belonged to the poor risk ca­tegory.

Out of two reticulosarcoma patients one showed a moderate improvement after therapy: some of her lymph nodes diminished in volume, became softer and more mobile.

The drug's effectiveness in nrultiple myeloma is debatable. Vie have had no patient whose clinical im­provement could be supported by regression of osteo­lytic areas on the X-ray picture of the bones. Despite this failure, 3 of our patients whose capability of movement was very bad, showed a remarkable improvement after the treatment.

TABLE 4

EFFECT OF F-LEUROSINE ON MULTIPLE MYELOMA

EFFECT PATIENT SCHEDULE CLINICAL MORPHOLOGICAL

T, A. 53 WOMAN A - -B. G. 61 WOMAN A + -J. S. 64 MANIII A + -J. S. IIII B + -K. Z. 63 WOMAN A + -S. I. 54 MAN A - -

One of the 5 patients, J.S., could not move his lower extremities at all. After the second course he could stand up without help. Until now no relationship has been found between paraprotein structure and thera­peutic sensitivity.

Side-effects caused by F-Leurosine treatment are almost negligible 2 patients have got thrust Isoorl, bacterial infection occurred in one case during the

462 E. FARKAS AND S. ECKHARDT

treatment.It is important to emphasize that neuro­toxicity developed in no case.

The drug has a well tolerable toxicity on haemo­pOietic organs. The platelet count decreased in 5 cases, decrease in count of mature granulocytes occurred in three cases, remarkable lymphopenia in one case. These Side effects developed only in patients having been previously treated with high doses of cy­tostatic drugs. All the side effects proved to be temporary, the longest leucopenia lasted 14 days.

Summary

Clinical trial with a new Vinca derivative /Formyl-Leurosine/ was carried out on patients suffe­ring from different malignant disorders of the haemopoietic organs. Complete remission was achieved in patients of acute leukaemia, improvement /partial remission/ was seen in cases of malignant lymphomas, such as Hodgkin's disease lymphosarcoma and reticulo­sarcoma, and those of multiple myeloma. The drug has a well tolerable toxicity on the haemopoietic organs, but transitional inhibitory effect on mature cells of granulopoiesis may occur. In comparison to Vincristine /Oncovin/ the most important adventage is lack of neurotoxicity.

CLINICAL INVESTIGATIONS OF DIBROMODULCITOL IN THE TREATMENT

OF MALIGNANT DISEASES

Dr. J. Hindy and Dr. J. Szanto

National Institute of Oncology

Budapest, Hungary

DBD (1 ,6,Dibromo-1 ,6,DideoxydulcitoQ is a cytostatic sugar-alcohol derivative. It was synthetized by Horvath and InsHtoris in the Chinoin Pharmaceutical Factory in Hungary. DBD is a stereoisomeric variant of another Hungarian drug, Dibromomannitol or Myelobromol. The position of the hydroxyl-groups is the di fference berween the two compounds.

According to the pre-clinical investigations, both drugs have a myelotoxic effect, but DBD is able to inhibit the growth of solid tumours too.

DBD is water-insoluble, it can therefore only be given in a tablet form. They contain 50 mg or 250 mg of active substance.

On the basis of the pre-clinical investigations, two forms of admin-istration were devised: I, every day therapy and 2, two forms of "push­therapy". In general the total dose was about 100 mg/kg body weight in a treatment course for each form of therapy I and after an interval of four or six weeks the treatment could be repeated.

The pati ents were treated if they met the precondi ti ons shown on fi gure 3. Diagnosis was verified by cytology or histology in all cases except 14 patients with lung cancer. In these cases diagnosis was established only by X-ray examination before treatment, but later it was confirmed at autopsy. Haem­atology was done once a week during treatment.

On the basis of our experiences indications for DBD therapy are summarised in figure 4.

463

464 I. HINDY AND J. SZANTO

Chemical structure

OH- -H

OH- -H

l,6-dibromo-1,6-dideoxydu1citol

Single dose

5 mg/kg/day

10 mg/kg/5.day

15 mg/kg/7.day

/OBO/ Figure 1

Dosage

Figure 2

Total dose

100 mg/kg

100 mg/kg

100 mg/kg

CLINICAL INVESTIGATIONS OF DIBROMODULCITOL

Criteria of DBD-Therapy

Hgb.: 10 , 5 g% < Whc.: 4.000 < Platelets: 100.000 <

A four week interval is needed before

DBD administration. Figure 3

Clinical Application of DBD

A. Absolute indications

Chronic myeloproliferative diseases Polycythemia vera Solid tumors

Squamouscell carcinomas

- cavity of mouth - superior respiratory tracts - lung cancer - uterus cancer

Bladder cancer Breast cancer

B. Relative indications

Acute myeloproliferative diseases Lymphogranulomatosis Hypernephroma Lymphosarcoma Melanoblastoma

Figure 4

465

466 I. HINDY AND J. SZANTO

NUllber of ... i •• ion St.tic Unvalullb1e D1agnos1s ICarcin""",s' P.ti.nt. obj.ctive .ubj.ctive di .....

Lunq 160 29 32 70 Head and neck 157 30 24 103 Breast 37 4 7 26 Bladder 11 3 4 4 Uterus 31 17 5 9 Struma maligna 1 - 1 -Osteoqen1e Be. 6 - 2 « Other maliqnant tumors 56 - - 56

Total: 459 83 75 272

118,0 " 116,4 " 159 "

Figure 5. Effect of OLD on Solid Tumors

Diagnosis ICareinomali1 Squamous cell Adena ca. Other types

Lung 75/17 12/1 16/3

Head and neck 147/25 1012

Breast 14/3

Uterus 24/17

Urinary bladder 713

Total: 253/62 2614 26/5 124,5 %1 115,3 %1 119,2 "

Figure 6. Responders Treated by DBD According to Histological Classification

29

-------

29

16,3 "

CLINICAL INVESTIGATIONS OF DIBROMODULCITOL 467

Side-effects

1459 casesl

Haematological

Leukopenia 27 15,8 %1 Thrombocytopenia 20 14,3 %1

Gastrointestinal 6 11,3 %1

Allergy 1 10,2 %1

Figure 7

In patients with chronic granulocytic leukaemia the drug was administered by everyday therapy. Both the dai Iy dose of the drug and the duration of the treatment depended on the clinical and haematological state of the patients.

In general the inductive phase of the treatment - 5 mg/kg day for 14-15 days, after which the therapy is continued with a lower dose of the drug as maintenance therapy. In this group and by this method of the administration 80% objective remission was achieved. In polycythemia vera the levery-dayl therapy was also given. Generally after a period of six weeks of treatment remission could be observed and it lasted 5-15 months without any maintenance therapy. The proporti on of the objective remission was 60% in this group.

It seems to us, that the results of the DBD therapy have not reached the results of the DBM; neither in the chronic granulocytic leukaemias nor in polycythemia vera, however DBD was administered to patients already having a resistance against DBM therapy.

For the treatment of solid tumours we prefer the two forms of the push­therapy mentioned earlier.

Figure 5 shows the results of the DBD therapy according to the localization of the primary tumour and figure 6 shows the results according to the histolog­ical type of the tumours.

As may be seen we have achieved the best results in patients having squamous cell cancer.

Recently American authors have also obtained good results in cases of adenocarcinoma of the lung and in cases of breast cancer of different histolog­ical type.

468 I. HINDY AND J. SZANTO

Investigations with labelled DBD have shown that the drug is able to pass into the cerebro-spinal fluid too. Side-effects of DBD treatment were mild and transitory. See figure 7.

The leukopenia and or thrombopenia could be restored to normal with Prednisolone and blood transfusion within 14-18 days in all cases except one. In some cases the development of leukopenia 10-14 days after the end of the treatment was seen.

There was no correlation between the result of treatment and the haemat­ological side-effects.

Immunosuppressi ve acti on as a resul t of DBD therapy was not observed.

I n summary the effects of D BD treatment are: 1. 80% objective remission in chronic granulocytic leukaemia; 60% in poly-

cythemia vera and 34% in squamous cell cancer. 2. Haematological side effects were mild and transitory. 3. The drug has no immunosuppressive acti on. 4. Since treatment is oral it can be given to out-patients.

ORAL ESTRACYT® (ESTRAMUSTINE PHOSPHATE) IN THE TREATMENT OF

ADVANCED CARCINOMA OF THE PROSTATE

Anders Nillius and Imrey KBnyves

Department of Surgery I Central Hospital l Halmstad and Department of Medicine l AB LEO I Helsingborgl Sweden

Estramustine phosphate consists of nor-nitrogen mustard linked to estradiol-17~-phosphate as a carbamate. The drug has weak estrogeni c properti es i its uterotropic effect is about 100 times weaker than that of estradiol (1) .

Estracyt has a low toxicity when adminstered intravenously or orally. The LDso values in rats are 390 mg,/kg and more than 1700 mg/kg l respectively (2) •

The intravenous preparati on of Estracyt has previ ously been tested at the Department of Surgery at Central Hospital in Halmstad l Sweden (3) .

CI - CH - CH 0 2 2, 1/

N-C-O /

CI - CH - CH 2 2

OH I

0- P= 0 I

OH

Fi gure 1. Structural formula of Estramusti ne Phosphate (Estracyt~)

469

470 A. NILLIUSAND I. KONYVES

Between February 1971 and December 1974, oral Estracyt was tried in rapidly progressing prostatic carcinoma at the same department.

The clinical material consisted of 20 consecutive patients, all in clinical stage 4 according to the system of Classification suggested by the Veterans Administration Co-operative Urological Research Group (4) .

All tumours had been verified cytologically. The tumour was well differentiated in 5 patients, moderately so in 5 and poorly differentiated in 9. In one patient the degree of differentiation was not determined. Nineteen patients had skeletal metastases; 3 had also demonstrable lymph-node metastases; 3 had metastases in the lungs or pleura; and one had metastases in the brain.

The acid phosphatases were elevated in 16 patients. Four patients had a catheter d demeure. In 3 pati ents the local tumour was so extensi ve that it caused marked difficulties in defecation. In 2 patients it disturbed ureteral flow, with uraemia as a result.

Thirteen patients had severe pain and were in such a poor general condition that they were bedridden. In 7 patients the pain was judged as being of medium severity. These patients were ambulant, but they required continuous use of analgesi cs.

All the patients had previously been treated with estrogens for 3 months to 7 years. Sixteen had initially responded well to estrogen therapy, but the di sease had progressed afterwards. In the other pati ents, estrogen therapy had been tried for 3 months but without any effect. One patient had received estrogen in combination with an orchiect.omy, and two had received X-ray treatment of skeletal metastases with temporary palliation.

Table 1. Some Therapeutic Effects of Estracyt-Treatment

Symptoms and si gns before treatment Patients Results Patients

Metastases 19 Regressi on of Metastases 3

" Catheter A Demeure 4 Removal of Catheter A Demeure 2

Pain Pain Relief

Bedridden with pain 13 Bedridden with pain 5 Moderate pain 7 Moderate pain 6

TREATMENT OF PROSTATIC CARCINOMA WITH ESTRACYT 471

All other treatment of the prostatic carcinoma was discontinued during the

treatment with Estracyt. The treatment was started with 600 mg of Estracyt

daily supplied in capsular form. In 4 patients that did not respond to this

dosage, the dose was increased to 1200 mg a day. In one of these pati ents

the larger dose was effecti ve. If the pati ents responded well to the treatment,

600 mg was continued as long as it produced an effect.

The patients were followed up regularly during treatment. During the first

month of treatment examinations of the blood picture, liver function, acid

phosphatases and creatinine were made once or twice weekly. X-rayexam­

inations of the skeleton were made every third week. Cytologic studies were

performed before and 3 weeks after the beginning of the treatment.

The result was deemed to be good in 11 patients and poor or doubtful in 9

patients. In those patients in which the treatment was successful, the

subjective effects were observed within 1 week. In 11 patients palliation was

obtained in the form of reduction or cessation of pain. At the same time, there

was a definite improvement in the general condition. In 3 of these patients,

roentgenologic regression of the metastases was verified: in 1 patient of lung

metastases and in 2 pati ents of skel etal metastases.

In 2 patients the catheter could be removed after 4-6 weeks of treatment.

Table II. Clinical Response in Relation to Change of Acid Phosphatase

Activity

Acid Phosphatase Activity Clinical Response

Before Treatment Pati ents After Treatment Patients Good None

Increased 16 Decreased 9 8 1 Unchanged 4 0 4 Increased 3 0 3

Normal 4 Normal 4 3 1

Total 20 20 11 9

The tartrate-inhibited acid phosphatases, which had been elevated in 16

patients, decreased in 9 patients during the treatment. In 8 of these patients,

the therapeuti c effect was deemed as good.

Table III shows how the results are distributed on the basis of differentiation

of the cancer. The best effect was obtained in highly differentiated cancer,

472 A. NILLIUSAND I. KONYVES

but the material is too small for any definitive conclusions.

Table III Clinical Response in Relation to Degree of Differentiation

Degree of Differentiation Patients Effect of Treatment

Good None

High 5 4 1 Moderate 5 2 3 Low 9 4 5 Unknown 1 1 0

Total 20 11 9

Seventeen patients died during the observational period. In 13 patients the cause of death was prostatic carcinoma or complications caused by the cancer. In 4 patients the cause of death was cardiovascular disease. Eight patients died during the first 6 months, and in 6 of these the treatment had no effect.

Table IV Survival time of 20 patients treated with Estracyt capsules

Deaths: 8 patients died within 6 months 2 II II between 6-12 II

3 II II II 12-18 II

3 II " II 18-24 II

1 " " II 24-36 II

Total 17 patients died within 3 years

Cause of Death: Prostatic carcinoma: Cordi ovascular Disease:

13 patients 4 patients

In 2 patients, gastro-intestinal disturbances, with nausea and emesis, appeared during treatment. One of these patients tolerated 600 mg of Estracyt daily but experienced difficulties when the dose was increased to 1200 mg daily. In this patient the medication was withdrawn partly owing to the side­effect and partly to the absence of a therapeutic effect. The other patient did not tolerate 600 mg daily, but it was possible to give 600 mg daily after temporarily reducing the dose to 300 mg. On the contrary, in one patient 1200 mg was given daily for 10 weeks without eliciting this side effect. No

TREATMENT OF PROSTATIC CARCINOMA WITH ESTRACYT

significant effect on the blood picture or liver function occurred in any of these twenty pati ents.

473

Estracyt has proved in earlier trials to be effective in metastasizing prostatic carcinoma. Administration by intravenous injection often gave rise to thrombophlebitis that necessitated the discontinuation of treatment or the establishment of arteri ovenous shunts. The present trial of Estracyt has demonstrated that the oral preparation is also tolerated well when given in a high dosage over a long periOd of time. Oral Estracyt has proved to be effective against the painful state in metastasizing prostatic carcinoma in half of the patients in our material. In some of the pateints a regression of metastases was verified. In all the patients where a good palliation was obtained, a simulataneous reduction of elevated values of tartrate-inhibited acid phosphatases was noted. The side-effects were few and reversible.

REFERENCES

1. Fredholm, B. et al. (1974). Acta Pharmacologica et Toxicologica, 35, Suppl. 1, 28.

2. Estrocyt Brochure (1974). AB Leo, Helsingborg, Sweden. 3. Lindberg, B. (1972). The Journal of Urology, 108, 303. 4. The Veterans Administration Co-operative Urological Research Group

(1964). The Journal of Urology, ~, 590.

TREATMENT OF PROSTATIC CARCINOMA WITH ESTRACYT (ESTRAMUSTINE

PHOSPHATE)

F. Balogh, Z. Szendroi, L. Kisbenedek, I. Konyves and I. Szendi

Semmelweis Medical University, Budapest, Hungary and Dept. of Medicine, AB Leo, Helsingborg, Sweden

A long symptom-free period may be achieved with the therapeutic suppression of the malignant process in prostatic cancer. Nevertheless, primary resistance to hormonal treatment may occur in 5 to 20 per cent, and secondary resistance has been observed to develop in some patients after a varying period of hormone therapy. To date, no standards for the conservati ve treatment of these resistant cases have been elaborated. Instead of estrogens which are no longer effective, other anti-androgenic substances may be given combined with agents acting on the pituitary and adrenals as well as with cytostatic drugs and i rradi ati on.

This communication reports the therapeutic results obtained with estramustine phosphate.

OH I

0- P= 0 I

OH

Figure 1. Structural formula of Estramustine Phosphate (EstracytR)

475

476 F. BALOGH ET AL.

This drug is an estradiol-17~-phosphate, which is esterified with nor­nitrogen mustard as a carbamate in the 3-position of the steroid. The study is based on the data from 130 patients at four institutes. The results from 66 of these 130 pati ents have been presented previ ously (1,2,3) .

The patients were grouped according to the mode of administration of the drug as follows:

Table 1. The mode of Estracyt administration

Administration No. of pati ents

Intravenous 70 Oral 32 Intravenous followed by oral 28

Total: 130

All the pati ents had recei ved heterosexual hormone treatment for 2 to 10 years and an orchiectomy had been performed in all but 26 of the patients. The tumour diagnosis was invariably confirmed histologically (Travenol's needle biopsy or Franzen's aspiration biopsy) .

The total dose of Estracyt was 6 to 7 g admi nistered over a peri od of 3 weeks. For the subsequent 2 to 3 months weekly maintenance doses of 300 to 600 mg once or twice were given.Acid phosphatase, red blood cell count, ESR, as well as bone marrow and hepatic function were followed up regularly combined with physical and radiological examination.

The results have been assessed in terms of improvement of the primary tumour, of residual urine, .the results of various laboratory tests, the behaviour of metastases, subjective symptoms and general health.

Table II. Results

Improvement No change Deteri orati on

Prostati c tumour 69 40 21 Residual urine 67 41 22 Laboratory fi ndi ngs 77 37 16 Metastases from 41 patients/subjective 3 28 10

symptoms jgeneral condition 98 11 21

TREATMENT OF PROSTATIC CARCINOMA WITH ESTRACYT 477

Table II shows the changes observed in response to therapy in all three groups, since no significant difference was found between the individual groups. The effect of Estracyt was best reflected by the improvement in subjective symptoms, followed by decrease in the tumour size and amount of residual urine. In three of 41 patients with demonstrable metastases, X-ray regressions of these were verified. Analysis of the late results showed improvement to be transient in half of the patients in which relapse ensued sooner or later. Twenty-fi-ve of the 130 pati ents di ed withi n 6 months. The causes of death were stroke in one instance, pyelonephritis and uraemia as a result of diss­emination of the primary tumour in 5 patients, and cachexia with distant metastases in 15. Eighty patients were alive at the time of registration of this lecture. The survival time for these 80 patients is between 6 and 36 months.

Table III. Side-effects.

Thrombophlebitis 6 Disturbance of hepatic function 10 Gastro-i ntesti na I symptoms 8 Bone-marrow depression 10 Perineal complaints 5 Gynaecomastia 4

The side-effects observed in the course of Estracyt treatment are shown in Table III. Thrombophlebitis occurred in 6 patients, but it was transient and not extensive. Treatment had to be discontinued owing to recurrent thrombophlebitis in two patients and because of thrombocytopenia in three. Signs of bone-marrow depression were found in 10 patients, mostly in the form of reversible thrombocytopenia. In another 10 patients, disturbed hepatic function was observed; mild gastro-intestinal symptoms occurred in eight others. We noted perineal complaints in five patients and slight gynaecomastia in four.

In the group consisting of patients in stage 4 according to the classification of the Veteran Administration Co-operative Urological Research Group, there was transient improvement in about 50 per cent initially as regards the primary tumour. Relapse, however, occurred sooner or later.

In the group consisting of patients in good general health and without demonstrable metastases, subjective and objective improvement was observed in about 70 per cent of the pati ents, and conti nuous regressi on was confi rmed by biopsy.

478 F. BALOGH ET Al.

Figure 2. The histological picture of patient 1.

In conclusion we would like to show some histological preparations from two patients with prostatic carcinoma. In figure 2 a typical prostatic adeno­carcinoma can be seen on the left side before Estracyt treatment. The right side of this figure shows the general atrophy of the tumour after treatment.

In figure 3. the left side shows a medullary prostatic cancer before Estracyt treatment. After the treatment - right side - severe atrophy and nuclear clumping can be seen.

This type of change in the histological picture was observed relatively frequently in the prostate of patients treated with Estracyt.

TREATMENT OF PROSTATIC CARCINOMA WITH ESTRACYT 479

Figure 3 . The histological picture of patient 2.

REFERENCES

1. Szendroi, Z . and Konyves, I. (1972). Verh .dtsch Ges . Urol. 24,301. 2. Balogh, F. and Kisbenedek, L. In'. Daikos, G.K . Ed . (Athen'Sl974)

Antineoplastic chemotherapy Vol. III, 434. 3 . Szendroi Z. et a!. (1974) . Int.Urol.Nephrol., ~, 101.

CLINICAL USE OF DDMP IN CANCER CHEMOTHERAPY

L. A. PRICE and BRIDGET T. HILL

Institute of Cancer Research and the Imperial Cancer Research Fund London

Introduction.

Diaminopyrimidines possess anti tumour activity in animals (1,2) and man (3,4). We have demonstrated in tissue culture that one of these agents (DDMP, 197U, 2,4-diamino-5-(3'5' dichlorophenyl)-6-methyl pyrimidine) is highly effective against MTX-resistant cells and predicted that (a) the drug is best given in the highest possible dose for the longest possible time and that the concurrent adminis­tration of folinic acid might protect normal cells against DDMP but leave its tumoricidal effect unimpaired (5). These principles have already been shown to be clinically applicable (4). This report presents the results of a further clinical study along these lines.

Patients and Methods.

DDMP was given to 33 patients with advanced malignant disease refer­red because no other treatment was available. The drug was given orally once a week in doses ranging from 1.5 - 4 mg/kg. Folinic acid (calcium Leucovorin, Lederle) was given simultaneously i.v., i.m. or orally in doses varying from 45-90 mg. Response was defined as a 50% reduction of tumour size either radiologically or on direct measurement.

Results.

16 patients were non-assessable, either because they (a) were lost to follow up, (b) other antitumour therapy was added empirically or (c) they died before adequate doses of the drug could be given (i.e. at least 4 doses of 2 mg/kg). Of the assessable patients, 7 responded and 10 did not. The results are summarised in Tables I,

481

482 L.A. PRICE AND B.T. HILL

II and III. The side effects are summarised in Table D!. The use of selective folinic acid protection largely prevents severe marrow depression. If mild marrow depression occurs, it can be corrected simply by omitting a dose of DDMP but always giving the usual dose of leucovorin. We do not know why a single dose of leucovorin should reverse mild marrow depression. Four patients developed severe marrow depression which required admission to hospital and a folinic acid "rescue" given over several days. Platelet depression is much more marked than granulocyte depression. The conclusions from this study are summarised in Table V.

TABLE I. DDMP TRIAL

TOTAL NUMBER 33 NOT ASSESSABLE 16 ASSESSABLE 17 NON RESPONDERS 10 RESPONDERS 7

TABLE II. DDMP NON-RESPONDERS (10)

Patient Sex Age Diagnosis

ll2007 F 76 Sq. cell ca. lung 086906 M 67 Ca. prostate 104913 M 70 Ca. prostate 117521 M 62 Pleural mesothelioma 110169 M 30 Anaplastic testicular teratoma 105257 M 27 Anaplastic testicular teratoma 091194 M 32 A.M.L. 106936 M 13 A.M.L. 079391 M 41 Ca. breast 118951 M 60 Pulmonary secondaries

(adenocarcinoma). Primary unknown.

CLINICAL USE OF DDMP IN CANCER CHEMOTHERAPY 483

TABLE III. DDMP - RESPONDERS (7)

Patient Sex Age Diagnosis Duration of resEonse (Months)

112067 M 62 Sq. cell ca. lung 3 114507 M 55 Sq. cell ca. lung 2t 116908 M 65 Sq. cell ca. lung 3 114127 M 66 Oat cell ca. lung 3t 109716 M 54 Hypernephroma 12 118342 F 45 Hypernephroma 6+ 091205 M 49 Ca. rectum 2+

TABLE IV. DDMP SIDE-EFFECTS (33 Eatients)

MARROW DEPRESSION REQUIRING MORE THAN 1 DOSE LEUCOVORIN 4 SKIN RASH 6 HEADACHE OR "HAZINESS" 6 MENTAL STIMUIATION 1 NAUSEA 2 CONVULSIONS 0 DRUG-INDUCED DEATHS 0

TABLE V. CONCLUSIONS

1. DDMP HAS ANTlTUMOUR ACTIVITY IN MAN.

2. The drug can be given safely in the outpatient department using the concept of folinic acid "protection". Prolonged folinic acid "rescue" is not often necessary.

3. Further studies are indicated in an attempt to identify a particular tumour group which might respond favourably.

4. If such a subgroup is identified the future use of the drug should probably be in combination with other modalities of therapy.

References

1. Sugiura, K. Cancer Research 1952, 13, 431. 2. Nichol, C .A. Advances in Enzyme Regulation 1968, .§., 316. 3. Geils, G.F. et al, Blood, 1971, ~ 131. 4. Price, L.A., Goldie, J.H. & Hill, Bridget T., Brit. Med. J.

1975, g, 20. 5. Hill, Bridget T., Goldie, J.H. & Price, L.A. Brit. J. Cancer

1973, g§" 263.

COMBINATION OF ANTICOAGULANTS AND ANTINEOPLASTIC DRUGS

IN CANCER CHEMOTHERAPY

RIECHE, KLAUS

Central Institute for Cancer Research

1115 Berlin,Lindenberger Weg 80, G.D.R.

SUMMARY

Three clinical studies aimed at evaluating the adjuvant anticoagulation to chemotherapy of operable and inoper­able tumour-patients are presented. They revealed a safe oral or parenteral anticoagulation including defibrin­ation by Arwin in combination with mono- or polychemo­therapy. Pretherapeutic coagulation analyses are necessa­ry to minimize the complication rate. The high response rate in 8 out of 12 patients treated by Arwin and cyto­statics warrants further substantiation by randomized trials.

Pharmacological potentiation of antineoplastic drugs is one of the clinical approaches for improving therapeutic results in cancer chemotherapy. Investigation both on metastatic dissemination and haemostasis in neoplastic diseases suggested application of anticoagulants in tum­our patients (LUDWIG 1974, BRODSKY 1974, RIECHE 1969,1975 TOBELEtI et ale 1974). Several clinical studies have rev­ealed safe administration of oral or parenteral antico­agulants in tumour patients. This is especially true for dicumarols, heparin and ArVlin, respectively. Inhibitors of platelet aggregation are also going to be tested in

ihis respect. We began our studies of adjuvant anticoagulation

in chemotherapy of metastasizing tumours about 8 years ago. In the meantime tenable results were published by others who had successfully treated cancer patients with oral anticoagulants like Warfarin or heparin in combin­ation with chemotherapeutic agents (THORNES 1972,ELIAS

485

486 K. RIECHE

and BRUGAROLAS 1972, ELIAS 1973a,b). Arwin has been test­ed for treatment of tumour metastases, too (WILLIAMS and MAUGHAN 1972).

Three clinical trials were conducted to evaluate the efficacy of a combination of anticoagulation with chemo­therapy in operable and inoperable tumours. Table 1 con­tains the main data of the studies performed. In a subran­domized study involving 54 patients with operable breast cancer a continuous cyclophosphamide (100 mg/kg total do­se) treatment was compared with a combination of cyclo­phosphamide plus FalithromR (dicoumarol) over a 6-week postoperative period. From these patients 51 were evalu­able. No significant therapeutic differences were seen after this short adjuvant treatment. No adverse effects have occured. Six out of 27 patients having had cyclo­phosphamide alone showed recurrences within the 2 years of observation. Eight patients treated by cyclophospham­ide plus dicoumarol showed recurrences in the same period. The free interval (23 months) and survival rates were identical.

Table 1: Haemostasis and Tumourchemotherapy- the Three Clinical Studies

I. Operable breast cancer - subrandomized study (51 pats~

~Cyclophosphamide (100 mg/kg,27pats) Radical mastectomY.

'CyclophOsphamide plus Anticoagul­ation with dicoumarol (24 pats.)

II. Inoperable,metastasizing tumours of various origin (31 patients) 1. Heparin plus mono- or polychemotherapy (11 pats.) 2. FalithromRplus mono- or polychemotherapy (20 pts.)

Breast cancer IV stage (26 ~ats.), lung cancer (2), seminoma (1), neurosarCOma (i), melanoma (1).

III. Defibrination of blood with ArwinR (15 pats.) 1-3 U/kg infusion, 2-31 days plus mono- or polyche­motherapy in patients having lymphomas and solid tumours (12/15 patients). Hodgkin's disease IIB-IIIB (2 pats.)~l~mphosarcoma (1), lung cancer (1), breast cancer ,5), seminoma (2), melanoma (1).

ANTICOAGULANTS AND ANTINEOPLASTIC DRUGS 487

In another clinical study 31 patients with various metas­tasizing tumours have been included so far, see table 1. Heparin was given on the day of cytostatic administration in usual doses ranging from 10 000 - 30 000 IU/day. This study showed the feasibility of oombined treatment by mono- or polychemotherapy with diooumarol or heparin, re­spectively, in nearly all patients. Provided there are no contraindications, the dicoumarol treatment is the most tolerable treatment in comparison to heparin for tumour patients. The latter has a disadvantage be-cause of its antigenicity in the case of its long-term application. It would be of clinical interest to try the low dose heparin treatment as was proposed by KAKKAR (1975), for the prevention of thromboembolic complications, in combination with tumour chemotherapy. The study was not to evaluate different therapeutio effects. However, a few chemotherapeutically resistant patients showed tumour regressions when undergoing additional anticoagu­lation. This study is to be continued. The third study was aimed at potentiating tumour chemo­therapy by defibrination. Defibrination was brought about by slow administration of the thrombin-like acting snake venom derivative ArwinR kindly supplied by the Knoll-AG, Ludwigshafen. For the mode of action of Arwin, see (STOCKER and EGBERG 1973). Fibrinogen levels were maintained in ranges of 50 - 100 mg{ib for 2 to 31 days by doses of A~vin varying between 1 and 3 Twyfordunits/kg. Fifteen patients have been treated so far with Arwin. Twelve of them received a mono- or polychemotherapy. Figs. 1 and 2 illustrate therapeutic regimens. Arwin was well tolerated generally, the rate of side effects did not exceed the known extent. Additional cytostatic treatment was feasible in all but 1 patient. Eight out of 12 patients had tumour regressions, see table 3. Patients given Arwin alone did not reveal therapeutic responses. The results justify further substantiation by clinically controlled trials.

DISCUSSION Anticoagulants are receiving increasing interest for

tumour chemotherapy (urcc Symp. 1972,QUtvAL et al.1972). The three studies presented revealed safe application of anticoagulants or defibrination agents with cytostatics even in metastasizing tumours. However, a detailed eval­uation of patient's individual coagulation pattern is pre­requisite for such »urposes. The need for long lasting administration of anticoagulants in cancer patients was maintained by (THORNES 1972) and (1ITCHAELS 1974). There are various mechanisms of potentiation of cytostatics by anticoagulants. One has to envisage the possible im-

488

Ilncrod P'./us chemotherop-y"

Lf} llIB (55fj~J

J ~~.Qmp ~ fincrod

K. RIECHE

_-----.1.COPP t-------... 2 . COPP

(",10, rbI, Pr~d.,PrOCQrb.

Fig. 1 Combination regimen of the COPP-schedule and Arwin (Ancrod) in Hodgkin's disease

Table 3 Therapeutic results obtained by combination of Arwin with chemotherapy in tumour patients

Regression'> 50% ~ 50% Failures Nr. :pats. 2 6 4 Breast 1/5 2/5 2/5 Semin. 1/2 1/2 Lymph.s. 0/1 1/1 Hodgk. 2/2 Lung ca. Melanoma 1/1

1/1

ANTICOAGULANTS AND ANTINEOPLASTIC DRUGS 489

~

~ Breast Cancer .. ~ 5tod IF, <;, J;21j. ~ .0::

-Chlh,

0 1.cycle

~mp, Hnuod

~ -Chth.

10 0 segele

, {/lc/O., Vbl , I1TX, FU

~ , ,

I I - I

Chlh. I , I

I

I I

I

,'.

0 ",elide

10 31.00'1

Fig. 2: Clinical study with combination of polychemo­therapy and defibrination of blood by Arwin. Note the reaction pattern of fibrinogen ("over­shoot" reaction after cessation of Arwin). Photo­graphs below show the regression of the tumour.

provement of tumour perfusion by lowering the intra­tumoural fibrin deposition, the influence on cellular metabolism as glycolysis or DNA synthesis (KIEHL et al. 1973) by the drugs or their direct action on the surface membrane of the tumour cell. In vitro tests done by (WHEELER et ale 1974) on BURKITT-lymphoma cells did not show a potentiating effect of heparin on cytotoxic action of cytostatics. Defibrination and metastasis formation was experimentally investigated by (HAGMAR 1972). Defibrination of blood with Arwin renders it incoagul2ble and diminishes the viscosity by about 30% (EHRLY 1973). As it became knovm from successful treatment of peripheral arterial occlusion disease of the leg, defibrina~ion with Arwin might also improve tumour perfusion thus providing a better transport of drugs to the tumour cells. We prefer patients with hyperfibrinogenaemia for Arwin application.

490 K. RIECHE

From the clinical point of view, further studies using agents capable of inducing a harmless defibrination of blood in combination witb cytostatics seem worth while.

REFERENCES

1. Bell,W.;Pitney,W.R.;Oakley,C.M. (1968),Lancet I,490 2. Brodsky, I. (1974),editor: Anticoagulation in the

treatment of cane er, J. Med. 2,,1-148 3. Ehrly, A.M. (1973), VASA, Supple 1,3-27 4. Elias, E.G.;Brugarolas, A. (1972), Cancer Chemother.

Rep. part 1, 56, 783 5. Elias, E.G. (1973 a), Oncology 5,189 6. Elias,E.G. (1973 b), Proc. Am. ASS. Cancer Res. li,26 7. Hagmar,B. (1972) Europ. J. Cancer 8,17 8. Kakkar,V.V. (1975), Thromb. Diathes7 Haem. £1,87 9. ~iehl~ B.L.i stott, P.B.; Huang,A.C. and Buthala,D.A.

(1973), J. Nat. Cancer Inst. 21,705 Ludwig, H. (1974), Gynakologe, 1,204 10.

11 • 12.

13. 14.

15.

16. 17. 18.

19.

20.

21 •

Michael,L. (1974), J. Med. 2,98 Qu~val,P.;Falconet,B. et ale (1972), Bull. Chem. Th~­rap. !b 300 Rieche,K. (1969) Arch. Geschwulstforsch. ~,66 Rieche, K. (1975), Tumor-Wirtsbeziehungen aus experi­menteller und klinischer Sicht, Steinkopff,Dresden Stocker, K., Egberg,N. (1973), Thromb. Diathes. Haem. Supple !l:!i:., 361 Thomes, R.D. (1972), J. Irish ColI. Phys. Surge ~,41 Thornes,R.D. (1974), Lancet II,382 Tobelem, G. M. ; Israel, L.; Caen,J. (1974), Pathol.Biole

22,803 UICC-Symposium on Chemotherapy of Cancer Dissemination and Metastasis, Milano,Italy, May 24-26,1972 Whebler,R.H.; Bull,F.E.;Ruddon,R.W. (1974), Cancer Res. ]i,3215 Williams,R.J.B.;r~'[aughan,E. (1972), Brit. Med. J. 1, 174

ADRIAMYCIN CARDIOTOXICITY IN MAN: EFFECT OF PRETREATMENT WITH BETA-METHYLDIGOXIN. A POLIGRAPHIC STUDY

F.P. Villani, G. Beretta, A. Pagnoni and A. Guindani

Institute of Pharmacology, University of Milan National Cancer Institute of Milan Italy

Cardiac toxicity is the major factor limiting the use of Adriamycin (ADM) for long-term treatment. This toxicity includes transient electrocardiographic abnormalities and in a small number of cases cardiomyopathy. It was found that the incidence of cardiomyopathy is strictly related to the cumulative dosage and the risk increases considerably after a total dosage of 500-600 mg/m2 : for this reason ADM is usually discontinued when such a dosage is attained (1,2,3) .

Imposition of such a dosage limit, nevertheless, ignores biological variations and may deny the drug to patients who could tolerate additional therapy or may allow its continued use in patients who develop cardic toxicity at lower doses.

On the basis of these considerations we thought it could be useful to extend the cardiologic 'Control in patients treated with ADM by means of a policardiographic tracing (PCG), in order to pick up, through the alter­ations of systolic time intervals, any precocious sign of myocardial deterior­ation.

In a second investigation, having found promising results with this method, we decided to pretreat a small number of patients with digitalis before every ADM injection, with the hope of preventing its cardiotoxic action.

The fj rst part of our study i ncl uded 27 outpati ents: all had advanced neoplastic disease of various types, no one had cardiovascular disease before the treatment with ADM and no one had an abnormal ECG upon entering the

491

492 F.P. VILLANI ET AL.

study.

The therapeutic scheme was a single dose of 60-75 mg/m2 i. v. every 3 weeks. Nobody was undergoing any treatment whi ch acted on the cardi 0-

vascular system. When this treatment became necessary, they were excluded from the study.

The method of registration and calculation of the various systolic time intervals was that described by Weissler (4,5). In table 1 are summarized the results obtained before and 30-60 minutes after the first injection of ADM in 19 patients who were followed from the beginning of the therapy: it reveals the acute effect of ADM on the considered parameters. It appears from this table that a single dose of ADM does not significantly change the duration of QS2 and 5152 but induces a significant prolongation of PEP due mostly to the prolongation of ICT. In addition there is a sig­nificant shortening of LVET; consequently either LVET PEP or still more LVET ICT are significantly diminished.

All these signs are expression of impaired myocardial function as we find in cases of congestive heart failure, myocardial infarction, hypothroidism and so on.

These alterations seem to be reversible in about 3 weeks, since a new control taken in the same patients before the second injection of A9M showed systolic time intervals equal to those measured before initiating the therapy.

Table 2 shows the influence of different total doses of ADM on the various systolic time intervals. The study, as mentioned earlier, was undertaken in 27 patients, of whom 19 were followed from the beginning. Nevertheless, the basal values refer to 34 patients: in fact this group includes the other 15 patients with neoplastic disease who were studied subsequently, as we already stated, with digitalis pretreatment.

Since some patients were followed from the beginning of therapy and others after one or more cycles, data are expressed as systolic time indices accord­ing to the regression equation previously calculated.

These results were obtained 3 weeks after having attained the correspond­ing cumulative dosage.

It appears from this table that ADM induces a significant progressive prolongation of Q52: this is brought about by a gradual increasing of PEP (mainly due to prolongation of ICT) whereas LVET is not significantly modified. There is also a progressive diminution of the ratios LVET/ICT

ADRIAMYCIN CARDIOTOXICITY IN MAN 493

and LVET/PEP, statistically significant is only LVET/ICT.

Heart rate and blood pressure did not change significantly. It is there­fore possible to conclude that there is a significant relationship between total dosage of ADM and systolic time intervals, suggesting a progressive deterior­ation of myocardial contractility. The possibility that the described alter­ations could be produced by a deterioration of the cardiovascular system due to tbe underlying disease may be excluded since there was no clinical or laboratory evidence of primary heart disease before and during ADM treat­ment; moreover the neoplastic disease was improving or in remission.

It must be stressed that two patients showed clinical signs of cardio­myopathy which appeared in one patient after 400 mg/m2 and in the other after 500 mg/m2• These two pati ents are among those who had the greatest alterations of the various intervals and relative ratios, in particular just before clinical cardiomyopathy occurred the most significantly altered indices were LVETjICT and LVET/PEP: the former was reduced below 4 and the I atter below!. 5 •

In order to ascertain whether improvement in left ventricular function occurs after cessation of ADM therapy, patients who received a cumulative dose over 500-600 mg/m2 were controlled with PCG tracing at various inter­vals after completion of the therapy. From these investigations we may conclude that left ventricular function is not yet improved two months after therapy is stopped.

Preliminary data concerning two patients whom we could control after a longer period suggest that after high dosage, improvement in left ventricular contractil ity can occur, but not before 5-6 months after therapy is stopped.

After having ascertained that beta-methyldigoxin was able to prevent the negative inotropic effect of ADM in isolated guinea-pig atria, a second series of investigation was undertaken in order to determine whether pre­treatment with digitalis could prevent the cardiotoxic effect of ADM also in man.

The therapeutic scheme of pretreatment consisted of administering OAmg of beta-methyldigoxin daily for 4 days; the 5th day 0.2mg 1-2 hours before ADM injection and O.lmg a few hours afterwards. Finally patients received 0.2mg the next day. The effect of digitalis pretreatment on systolic time intervals and relative ratios is shown in table 3. In the first column are reported the mean basal values of systolic time intervals while in the second are reported the same intervals after digitalis, before ADM injection. The results agree with the notion that digitalis produces a significant reduction

494 F.P. VILLANI ET AL.

Table 1

SYSTOLIC TIME INTERVALS (mea) BEFORE AND AFTER A SINGLE DOSE (10-75 mil m2) OF ADRIAMYCIN (meen ±SD of 19 petlents)

Before 30-60 'etter Injection Injection

Electr_echenlcel IYltole (OS2) 387±7 390+7

Left ventrlculer election time (LVET) 282±6 275±6

Mechenlcel Iystole (SlS2) 313±6 325+7

Pre-election period (PEP) 104±3 114±3

Deformation tim e (DT) 74±2 71±2

Ilovolumetric contraction time (ICT) 31±1 43±2

LVET/PEP 2.731±0.094 2.419±0.092

LVET/ICT 9.S14±0.471 6.117±0.3S3

Heert rete '76 -

Blood prellure 137±4 131±3 83±2 82±2

p

n.s.

< 0.02

n.s.

< 0.01

n.s.

< 0.001

< 0.01

< 0.01

n.l.

ADRIAMYCIN CARDIOTOXICITY IN MAN

Table 2

CUIlULATIVE E~~ECT O~ ADRIAIIYCIN ON LE~T VENTRICULAR ~UNCTION AI MEAIURED BY INDEX ~ IYlTOLIC TIllE INTERVALI (_ , 10)

CUMULATIVE DOlE BAIAL 200 400 SOO F ( .. 1,.2 _, ___ a)

VALUEI ",/m2' ,..1 .. 2 ,../m2

N ...... of pallenls 34 l' 12 •

EI __ nlc.1 .,.tola (QSa) 575'2 "7'4 "3'5 "4" 3,200

Left •• ntrlcul., el_llon II ... (L VET) 41H. 418" 410 •• 413'4 0,35

..... henl ... 1 a,atole (51 Sa) 45H3 472'5 4"'5 474'4 13,8"

P, .. el_lIon period (PEP) 141'2 154'4 1"'5 158'4 7,310

D.,. ... llon II ... (DT) 100'1 10H2 101'3 85'3 0,242

18Ovol_etrlc cantr_Ilan 11_ (ICT) 31:!:1 35'2 31:!:3 43±2 17,.4.

LVETI PEP 2,71".,073 2,721,0,121 2,541'.,15' 2,518'.,133 1,111

LVETIICT 1,401:!:O,375 ','40'.,55' 7,441 :!:O,540 1,70S:!:O,.73 1',3.1

Hurt rete 77'2 77:!:2 .1±4 14'4

BI __ re .!!!!!. ~ ill!! !!!!! 12'2 13'3 1214 13'3

495

P

< 0,05

n.a.

< 0,01

< 0,01

n.l.

< 0,01

n.l.

< 0,01

496 F.P. VILLANI ET AL.

Table 3

SYSTOLIC TIME INTERVALS (msec.) BEFORE AND AFTER A SINGLE DOSE (60-75 mgl m2) OF ADRIAMYCIN IN DIGITALIZED PATIENTS (me.n • SO of 15 P.)

Baa. I If/:. methyldlgoxln 30-60 min.

v.'ue. 0.4 mgt d.ily / .ft.r ADM 4 contec:. day. injection

Electromechanical systole (052) (376±6) 357±9 356±10

Left ventricular ejection time (LVET) (270±6) 211±1 263~9

Mechanical systole (5 152) (304±7) 211±9 2Il±10

Pre-ejection period (PEP) (106±3) 95±3 95+3

Deformation time (DT) (73±2) 69±2 67±2

Isovolumetric contraction time (ICT) (32±1) 21±2 28±2

LVET/PEP 2.593±0.114 2.782±0.13O 2.819!0.109

LVET/ICT 8.784±0.573 10.650±0.755 10.201+0.778

Heart rate (83"3) 81:4 78.4

Blood pressure 133'~ 13916 138-7 80-3 80'3 81'3

Dollerence 'rom values obtained on ~- methyldigoxin treatment.

p"

n.l.

n •••

n.l.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.S.

ADRIAMYCIN CARDIOTOXICITY IN MAN 497

either of PEP or LVET, with consequent reduction of Q$2, and an increase of LVET/ICT and LVET/PEP. These modifi cati ons are the consequence of an increased myocardial contractility induced by digitalis: digitalis in fact increases the rate of development of isovolumetric intraventricular tension and the velocity of myocardial fibres shortening during the ejection period. In the third column are reported values of systolic time intervals and ratios, obtained 30-60 minutes after ADM injection. It is clearly shown that, in contrast to that observed in pati ents wi thout pretreatment (see tabl e 1) , ADM is without effect on the duration of various intervals and ratios in patients pre-treated with digitalis.

We then studied the effect of the pretreatment with digitalis on the modifications of systolic time intervals and ratios induced by different cumulative dosage. At the moment the study is still going on and we have not yet a sufficient number of patients to draw definitive conclusions; in any case the results are very promising.

Figures 1 and 2 show the effect of the pretreatment with digitalis on the duration of PEP and ICT. It seems that in patients pretreated with digitalis, there are no modi fi cati ons of these parameters lin contrast .to those observed in non pretreated patients. Similarly LVET/ICT (see figure 3)

EFFECT OF PRETREATMENT WITH /1- METHYLDIGOXIN ON PRE-EJECTION PERIOD (PEP) IN PATIENTS TREATED WITH ADRIAMYCIN

PEP INDEX

170

160

150

140

PEP

(9) (12)

T. t 100 200 300 400 5~O 'mg/M2

BASAL VALUES

- IN BRACKETS THE NUMBER OF PATIENTS

--___ e-_e PATIENTS WITHOUT PRETREATMENT

PATIENTS PRETREATED WITH DIGITALIS

Figure 1

body surface area

498 F.P. VILLANI ET AL.

EFFECT OF PRETREATMENT ,. METHYLDIGOXIN ON THE ISOVOLUMETRIC CONTRACTION TIME (lCT) IN PATIENTS TREATED WITH ADRIAMYCIN

ICT

msee

40

30 ----- -f ;I; ;;; r ------~;;-

20

T~! ____ ~! ____ ~! ____ ~I ____ ~! ____ ~I ____ ~!

t 100 200 300 400 500 1.", m2

BASAL body surface are.

VALUES

• IN BRACKETS THE NUMBER OF PATIENTS

• • • PATIENTS WITHOUT PRETREATMENT --- ---- PATIENTS PRETREATED WITH DIGITALIS

Figure 2

which has a negative correlation with the total dosage in patients treated with only ADM, is apparently not modified in patients pretreated with digitalis.

SUMMARY

Data obtained in our investigation show that a single dose of Adriamycin (60-75 mg m2) temporarily reduces myocardial contractility.

Data obtained after different cumulative doses of Adriamycin show a progressive deterioration of myocardial function, related to the total dosage.

Improvement in left ventricular function, after a cumulative dosage of 500-600 mg m2, may occur after therapy is stopped but not before 2 months.

Our study suggests that careful monitoring of systolic time intervals provides a valuable means in detecting precocious cardiotoxicity and for a

ADRIAMYCIN CARDIOTOXICITY IN MAN

EFFECT OF PRETREATMENT WITH'- METHYLDIGOl«N ON THE RATIO OF THE LEFT VENTRICULAR EJECTION TIME TO ISOVOLUMETRIC CONTRACTION TIME

(LVET/ICT) IN PATIENTS TREATED WITH ADRIAMYCIN

10 (7)

"" -------~t---(12)

BASAL 100 200 300 400 500 mgl m2 VALUES body surface area

- IN BRACKETS THE NUMBER OF PATIENTS

~e - ____ e-_e PATIENTS WITHOUT PRETREATMENT

- - -_. ---- PATIENTS PRETREATED WITH DIGITALIS

Figure 3

more rational administration of ADM allowing maximum dosage without excessive risk for cardiac toxi city.

Finally, data obtained with beta-methyldigoxin pretreatment suggest that digitalis may prevent the cardiotoxic effect brought about by ADM at I east as far as the acute cardi otoxi city is concerned.

REFERENCES

1. Lefrak, E.A., Pitha, J., Rosenhein, S. and Gottlieb, J.A. Cancer, 32, 302 (1973) .

499

2. Gottlieb, J.i::, Lefrak, E.A. and Q'Bryan, R.M. Proc. Am. Ass. Cancer Res., 14, 88 (1973)

3. Blum, R.H. and Carter, S.K. Ann. Int. Med., 80, 249 (1974) 4. Weissler, A.M., Harris, W.S. and Schoenfeld, C~. Circulation,

37, 149 (1968) . 5. WeisSi'er, A.M., Harris W.S. and Schoenfeld, C.D. Am. J.

Cardiol. 23, 577 (1969) .

COMBINATION OF ADRIAMYCIN AND BLEOMYCIN IN THE TREATMENT

OF ADVANCED CERVICAL CANCER

Nicole NATALE, Costantino MANGIONI and Giorgio BOllS

I Clinica Ostetrica e Ginecologica University of Milan

Squamous cell carcinoma of the cervix has been seldom treated by chemotherapy and has usually had a low response rate with short duration. Among the drugs that gave the best results in the treatment of this cancer are Adria­mycin (ADM) and Bleomycin (BlM). The present investigation has been undertaken to determine if the combination of the two drugs could get a better result than the administration of each single drug.

ADM has been ad~inistered intravenously (i.v.~ at a dose level of 75mg/m on day 1, BlM i.v. at 15mg/m on day 1 and 8; the cycle is repeated on day 21. Doses of ADM are reduced to 75% in irradiated patients. A minimal treatment of two courses is required to evaluate tumour response, then it is discontinued for progressive tumour; when improvement is obtained, cour~es are continued to a maximum dose of ADM 460-500 mg/m , in order to avoid card~otoxicity which becomes evident at the dose of 550 mg/m .

41 patients have been treated by this combination, 24 of whom can be evaluated (Table 1). In 3 cases thera­py was discontinued for severe toxicity after the first cycle, in 6 for rapidly progressive tumour, which did not allow a second course and in two cases the therapy was refused by the patient after one and two cycles respect­ively. 6 more cases had not been evaluated for the absen­ce of clinical masses; in these cases deep lymphatic metastases were found at laparotomy, not evident to lym-

501

502 N. NATALE, C. MANGIONI, AND G. BOllS

TABLE I

Treated patients 41 Evaluated patients 24 Not evaluated patients 17

early severe toxicity 3 progressive tumour 6 blind therapy 6 treatment refused 2

phography. Patients' age ranged from 34 to 75 years with a median of 55. All the patients had been irradiated; 14 of them had surgery associated, usually simple hysterec­tomy with bilateral salpingooophorectomy and partial colpectomy; in one case a radical hysterectomy with lym­phadenectomy was followed by irradiation because of me­tastases to iliac lymphnodes. In two cases chemotherapy was administered after irradiation and surgery and in one case after irradiation alone with no effect (Table 2). At the beginning of the treatment every patient had at least one measurable mass.

Response was evaluated as follows; CR: disappearance of all clinical tumor PR: decrease of more than 50% mass with no

increase of the other localizations for two or more months

01: 25-50% decrease of two perpendicular diameters of at least one mass with no increase of the others for two or more months

NR: no change or progression of the disease

The evaluation was performed twice a month with clinical therapy preceeding combined chemotherapy examination,

TABLE 2

Irradiation 9 Irradiation plus surgery 11 Surgery plus irradiation 1 Irradiation plus chemotherapy 1 Irradiation plus surgery plus

chemotherapy 2

TOTAL 24

COMBINATION OF ADRIAMYCIN AND BLEOMYCIN 503

once a month with chest Xrays, urograph and ultrasound scanning. Side effects were controlled twice a month with complete blood cell count and once a month by hepatic and renal function test (alkaline phosphatase, SGOT, SGPT, bilirubin, BUN, serum creatinine); EKG was done every month. Side effects were scored as ~ollows:

WBC : I = 3.000-2.000/mm3 2 = 2.000-1.000/mm 3 below 1.000 3

Platelets 1 = 100.000-75.000/m~ 2 = 75.000-50.000/mm 3 = below 50.000 3

RBC 1 3.000.000-2.000.000/mm3 2 2.000.000-1.500.000/mm 3 below 1.500.000

For score I the dose of ADM was reduced to 75%, for score 2 or 3 the treatment was delayed until complete recovery.

The results are summarized in Table 3. An overall response of 33.3%is obtalned. In 6 cases (25%) a partial or complete remission is achieved for 2-8 months with a median of 5. The two cases with complete remission are still free of any clinical evidence of disease after 2 and 7 months.

The tumor localization did not respond in the same way (Table 5). The best results are obtained in pulmona­ry metastases with a 55% of response in 9 cases, the least responsive seem to be lymphatic metastases (29%). The different response rate may be due to the different blood supply to tumor localization. This fits also with the 31% of response of vaginal and pelvic localization, in which blood supply is reduced by post-radiation fibrosis.

Survival does not seem to be improved in responding

TABLE 3

CASES DURATION MEDIAN RANGE

NO RESPONSE 16 66.7% OBJECTIVE IMPROVEMENT 2 8.3% 2 2 PARTIAL REMISSION 4 16.7% 5 ~-8 COMPLETE REMISSION 2 8.3% 3+-7+ 3 -7+

PARTIAL + COMPLETE REMISSION 6 25.0% 5 2-8

Response of patients to Adriamycin and Bleomycin treatment

504 N. NATALE, C. MANGIONI, AND G. BOllS

TABLE 4

CASES SURVIVAL MEDIAN RANGE

NO RESPONSE 16 66.7% 5 ~-12 OBJECTIVE IMPROVEMENT 2 8.3% 8+-10 8 -10 PARTIAL RESPONSE 4 16.7% 5 ~-l~ COMPLETE RESPONSE 2 8.3% 3+-7+ 3 -7

PARTIAL+COMPLETE RESPONSE 6 25.0% 5 3+-12

Survival in months of treated patients.

patients in front of not responding ones.

Side effects are present in 83.3% of the evaluated patientso All these patients complained chills and fever up to 40 C after BLM administration. The temperature lasts few hours and remittance is spontaneous. If we exclude this aspect of toxicity, the side effects rate decreasesto 62.5% (Table 6). The main toxicity is repre­sented by alopecia (54.2%), myelodepression (45.9%), nausea and vomiting (45.9%). In two patients mild roen­tgenographic signs of pulmonary fibrosis developed. Skin toxicity was represented by typical brown stria in five patients. In no case cardiotoxicity appeared, pro­bably because the maximum tolerated dose of ADM has never been reached.

In the present investigation the association of ADM and BLM was well tolerated by approximatelyl/3 of the patients. Except the three cases in which it was suspended for severe toxicity due to myelosuppression, we dis­covered 7 cases of marked leukopenia and 3 cases of

TABLE 5

PELVIC AND VAGINAL SITES PULMONARY SITES LYMPHATIC SITES HEPATIC SITES

5/16 5/9 2/7 1/1

31% 55% 29%

100%

Response to chemotherapy in relation to the site of tumor masses. Number of response/number of cases.

COMBINATION OF ADRIAMYCIN AND BLEOMYCIN

IJBC PLATELETS RBC ALOPECIA CUTANEOUS STRIA PULMONARY FIBROSIS NAUSEA AND VOMITING PHLEB IT IS

NO SIDE EFFECTS ~EVER AND CHILLS

TABLE 6

GRADING 123

2 2

4 3

7 3 1

505

TOTAL

11 45.9% 8 33.4% 3 12.5%

13 54.2% 5 20.8% 2 8.3%

11 45.9% 1 4.2%

9 37.5% 20 83.4%

Toxicity in evaluated patients treated with ADM and BLM. For description of grading see text.

marked thrombocytopenia, that recovered and did not appear wi th continuing treatment. No death was due to toxic effects. This association is nevertheless quite well tolerated, also considering that every patient had a full course of pelvic irradiation, which is known to affect myelofunction as well as general conditions. Another point to outline is that therapy has always been admin­istered on an outpatient basis. The response rate of 33.3% is quite good, if we consider the results obtained in cervical carcinoma. The literature reports for the single agents we used in association a percentage of remission from 0% to 90% in squamous cell carcinoma from different origin. In the treatment of cervix uteri the best results are those of De Palo (1973) and Bryan (1973) with 30% of response. It is of interest that the dura­tion of the remission appear to be as high as the reduc­tion of the tumor. In spite of the satisfactory response the survival is not influenced by chemotherapy, both the patients responding and those in which no improvement of the tumor was obtained have the same survival.

In conclusion we can say that this association also with its limited therapeutic effect may play an useful role in the chemotherapy of squamous cell carcinoma of the cervix uteri.

TREATMENT OF CHEMOTHERAPY RESISTANT NONSEMINOMATOUS

TESTICULAR TUMORS WITH DDP (NSC-119875)

R. OSieka, U. Bruntsch, W.M. Gallmeier, S. Seeber and C.G. Schmidt Innere Universitatsklinik und Poliklinik (Tumorforschung) D-4300 Essen 1, Hufelandstr. 55, West Germany

SUMMARY

During the last years three consecutive trials in combination chemotherapy of disseminated nonseminomatous testicular cancer were conducted. A four-drug regimen (actinomycin D, methotrexate, vinblastine, cyclophospha­mide) yielded a 42 % response rate, a three-drug regimen (adriamycin, vincristine, bleomycin) 55 %, and another three-drug regimen (adriamycin, vincristine, and bleo­mycin by continuous infusion) yielded a 70 % response. Patients refractory to these programs were entered on a ~rotocol which called for DDP ,ois-~iamminedichloride platinum (II), (NSC-119875) 20 mg/m daily x 5 q. 4 weeks i.v. push injections. Results for 15 evaluable patients were: 1 CR, 7 PR, 3 NC, 4 PD.

INTRODUCTION

Combination chemotherapy has yielded an increasing number of objective responses in the treatment of dis­seminated nonseminomatous testicular cancer. This trend is reflected in the results obtained during the past years in the Essen Oncology Center.

From 1968 to 1973, 51 consecutive patients were treated with a four-drug chemotherapy regimen oonsisting of:

507

508

actinomycin D vinblastine methotrexate cyclophosphamide

R. OSIEKA ET Al.

0.5 - 1.0 mg i.v. daily x 5 10 mg i.v. on day 1 10 mg i.v. on days 1 and 2 100 to 200 mg daily p.o.

q 4 - 6 weeks

An objective response rate of 42 % was achieved, however, only two complete remissions were observed. Median duration of remission for partial responders was 4.5 months (Hoffken et ale 1974).

Following the introduction of adriamycin into the treatment of solid tumors, more emphasis was put on an efficient use of the so-called tumor antibiotics. The encouraging results of Gottlieb and associates in using adriamycin in combination with vincristine and bleomycin in 1974, gave rise to a similar study in Essen. The ex­cellent results of Gottlieb, however, could not quite be reduplicated (Burgess et ale 1975). Although application of projected doses of cytostatic agents was achieved in all cases, the response rate was only 55 %. The percen­tage of complete responses was similar (5/16), as was the median duration of remission for partial responders. The lower response rate probably calls for a more detailed comparison of initial stage or initial tumor cell burden and histologic subclassification (Seeber et ale 1975). Also,toxicity of the "ABO"-regimen was not higher than the toxicity of the previously used four-drug regimen.

Although dose schedule dependency relates to all cytostatic agents,there has been a particular uncer~ainty about the optimal dose schedule for bleomycin (J~rgensen 1972). The question whether bleomycin acts more efficiently on resting or actively proliferating cells, still is not settled in the literature, nor has its time of action during the mitotic cycle been conclusively de­termined (Twentyman and Bleehen 1973, Hahn et al. 1973, Barranco et ale 1975). Dose fractionation experiments also have been inconclusive with regard to treatment choices for human neoplasia.

Still, when continuous intravenous infusion of bleo­mycin was found to be superior to intravenous push in­jections (Krakoff 1974), a regimen of continuous intra­venous application of bleomycin over 48 hours in addition to adriamycin and vinblastine was adopted:

TREATMENT OF CHEMOTHERAPY-RESISTANT TUMORS WITH DDP

adriamycin vinblastine bleomycin

40 mg/m~ 10 mg/m2 30 mg/m

i.v. push on day 1 i.v. push on day 1 continuous infusion 48 hrs, days 1 & 2.

509

In this study an initial response rate of 70 % was observed, but median duration of remission seems to be less than four months. Correspondingly, a low number of complete remissions was observed.

In a review on chemotherapy for nonseminomatous testicular tumors, prepared by Smithers in 1972, the alkylating agents were ranked into a B category vs. an A category ranking for tumor antibiotics, antimetabolites and vinca alkaloids. Today, the role of alkylating agents in the treatment of such tumors has to be reassessed: thus, high dose intermittent cytoxan treatment yielded an impressive response rate (8/9) in testicular nonseminoma­tous cancer (Buckner et al. 1974), as did a combination of bleomycin and CCNU (Sonntag et al. 1975).

The introduction of platinum compounds, the action of which is tentatively described as alkylating through some metabolic product (Higby et al. 1974), was a major breakthrough in the treatment of testicular neoplasms.

RESULTS

It is the purpose of this communication to report on cis-diamminedichloride platinum (NSC-119875) as a second line drug after previous treatment with chemotherapy has failed. The compound NSC-119875 was generously supplied by the National Cancer Institute, Division of Cancer Treatment, Bethesda, Md., USA.

Patients who were defined as refractory to previous treatment when progressive disease was evident after two courses of multiple drug therapy, became eligible for this treatment protocol only in the presence of normal liver, kidney, and bone marrow function. The treatment schedule called for intravenous push ~njections of the platinum compound NSC-119875, 20 mg/m daily x 5, which was to be repeated every four weeks. Drug toxicity was to be monitored by biweekly controls of serum creatinine, transaminase and electrolyte levels, and WBC and thrombo­cyte counts. Audiograms were to be performed every other month.

510 R. OSIEKA ET AL.

The results of treatment are summarized in the following table. Response to previous multiple drug chemotherapy is indicated but the number of patients in each category seems to betoo small to draw any conclu­sions:

response to previous therapy response to DDP-therapy

progressive disease: 5

no change: 2

partial response: 8

partial remissions: 3

progressive disease: 2

no change: 1

progressive disease: 1

complete remissions: 1 partial remissions: 4 no change: 2 progressive disease: 1

total: 15 pats.: 1 OR, 7 PR, 3 NO, and 4 PD.

In all cases where an objective response to plati­num therapy occurred, this became evident very briefly after initiation of therapy. Maximum reduction of tumor size was observed on chest plates during the second or third week of the treatment cycle, with regrowth of pul­monary lesions occurring in some cases already in the fourth week. The following types of response were ob­served: rapid and complete disappearance of pulmonary lesions with no recurrence, partial response with diminuition of small pulmonary lesions and no evidence of regrowth, partial response of very advanced lesions with eventual regrowth.

Only in patients who had received more than two courses of platinum therapy, serum creatinine-clearance was reduced. 4/15 patients experienced such an impairment of renal functions that platinum therapy had to be dis­continued. Nephrotoxicity was only partially reversible in such patients but never was the immediate cause of death. No ototoxicity or profound myelosuppressive effects were observed until now.

TREATMENT OF CHEMOTHERAPY-RESISTANT TUMORS WITH DDP 511

DISCUSSION

Despite the high initial response rates that have been achieved in the treatment of nonseminomatous testi­cular cancer by multiple drug chemotherapy, the short median duration of response in most series makes an im­pressive increase in overall survival unlikely. The pla­tinum compound NSC-119875 is another rapidly effective remission inducing agent, the usefulness of which is limited by nephrotoxicity and rapid recurrence of lesions.

The solid tumors are composited of cell lines with various degrees of differentiation, which probably differ in their innate sensitivity to chemotherapeutic agents and furthermore are spread over the various stages of the mitotic cell cycle. Rapid decrease of tumor size by vari­ous treatment regimens may well be induced by reduction of only one or two proliferative compartments of such cell line and none in others. The very rapid emergence of resistance e'Ten to polychemotherapy, may call for se­quential or alternate use of all hitherto effective regimens in a predetermined fashion. This does not obviate the necessity of determining experimentally and in controlled studies the possibilities of therapeu­tic synergism between new drugs, such as the platinum compound, and other effective agents. Also, ways to cir­cumvent the toxicity ceilings on many useful agents should be explored, as has been achieved in the reduction of nephrotoxicity of platinum compounds by concurrent ad­ministration of penicillamine derivatives (Ohnuma et ale 1975, Cvitkovic et ale 1975, and Higby et ale 1975).

The results of such trials could be incorporated into therapy regimens which by rapid and predetermined use of drug combinations could possibly eliminate the various clonogenic cells before they become refractory to therapy.

References

Barranco, S.C., Novak, <T.K.,Humphrey, R.M. (1975), Cancer Res., 35, 1194. - Buckner, C.D., Clift, R.A., Fefer, A., Funk, D.D., Glucksberg, H., Neiman, P.E., Paulsen, A., Storb, R., and Thomas, E.D. (1974)t Cancer Chemother. Rep. Pt. 1, 58, 709. - Burgess, M.A., Einhorn, L.H. and Gottlieb, J.A. (1975), Proc. Amer. Ass. Cancer Res. & Amer. Soc. clin. Oncol. 16, 244, Abstr. No. 1094. -Cvitkovic, E., Currie, V., Krakoff, I.H. and Golbey, R. (1975), Proc. Amer. Ass. Cancer Res. & Amer. Soc. clin. Oncol. 16, 273, Abstr. No. 1208. - Hahn, G.M., Ray, G.R.,

512 R. OSIEKA ET AL.

Gordon, L.F. and Kallman, R.F. (1973), J. nat. Cancer Inst., 50, 529. - Higby, D.J., Wallace, H.J., Jr., Albert, D.J. and Holland, J.F. (1974), Cancer, 33, 1219. - Higby, D.J., Wallace, H.J., Jr. and Bekesi, J.G. (1975), Proc. Amer. Ass. Cancer Res., 16, 131, Abstr. No. 523. - Hoffken, K., Tingelhoff, J., Hornung, G. and Schmidt, C.G. (1974), Z. Krebsforsch., 82, 307. -J~rgensen, S.J. (1972), Europ. J. Cancer, 8, 93. -Krakoff, I.H. (1974), in: Abstracts, 11th Int. Cancer Congr., Florence, 20-26 Oct., 1974. Vol. 1, 367. -Ohnuma, Holland, J.F. and Vogl, S. (1975), Proc. Amer. Ass. Cancer Res. & Amer. Soc. clin. Oncol., 16, 272, Abstr. No. 1205. - Seeber, S., Gallmeier, W.M., Hoffken, K., OSieka, R., Bruntsch, U. and Schmidt, C.G. (1975), Dtsch. med. Wschr., 100, 1319. - Smithers, D.W. (1972), Brit. J. Urol., 44, 217. - Sonnta~, R.W., Brunner, K.W., Batz, K. and Ryssel, H.J. (1975), Cancer Chemother. Rep. Pt. 1, 59, 429. - Twentyman, P.R. and Bleehen, N.M. (1973), Brit. J. Cancer, 28, 500.

COMBINATION CHEMOTHERAPY OF ADVANCED HODGKIN'S DISEASE

WITH ADRIAMYCIN, DTIC, CCNU AND BLEOMYCIN

R. OSieka, U. Bruntsch, W.M. Gallmeier, S. Seeber and C.G. Schmidt Innere Universitatsklinik und Poliklinik (Tumorforschung) D-4300 Essen 1, Hufelandstr. 55, West Germany

SUMMARY

Eighteen patients with advanced Hodgkin's disease who were refractory to previous MOPP-therapy were entered into a new treatment protocol, consisting of adriamycin, DTIC, bleomycin and CCNU. Three patients who never had responded to previous chemotherapy did not respond to the new regimen. Two complete remissions and 11 partial remissions were achieved in a group of 15 patients with prior response to MOPP-therapy, and only 2 had progressive disease.

INTRODUCTION

The MOPP-regimen, as first described by DeVita and coworkers, by now has earned general acclaim as the op­timal regimen for the treatment of advanced Hodgkin's disease (Goldsmith and Carter 1974, Bonadonna et al. 1975). Attempts to improve the results obtained with the original protocol either by addition of new cytostatic agents, maintenance therapy or by the combined modality approach did not yield superior results. The complete remission (CR) rate of 81 % achieved by DeVita's group surpasses the response rates of many other trials with the same regimen. Thus, Frei achieved only 64 %, Mubashir 54 %, and Jacquillat 47 % (Frei et al. 1973, Mubashir et al. 1973, Jacquillat et al. 1975). Converse­ly, the percentage of induction failures was between 5 % and 25 %, and the median survival of such patients

513

514 R. OSIEKA ET AL.

ranges between 4 to 6 months. These patients designated as primary induction failures present many problems of intensive medical care to the oncologist but, fortunate­ly, are rare among any group of Hodgkin's disease patients.

Patients that become refractory to MOPP-therapy after previous response to such therapy, represent a more ill-defined group. Objective tumor size measure­ments are often obscured after previous therapy, and only after new cytostatic agents were introduced into the treatment of Hodgkin's disease, the physician's willingness to make use of all diagnostic tools, inclu­ding repeated biopsies, has increased. Since all of the patients present with evidence of noncontiguous extra­lymphatic spread of the disease, stage is not of value as a prognostic factor, nor is histological subclassifi­cation of the initial biopsy. Thus, in the authors' institution the following histologic subclassifications of initial biopsies from patients who were refractory to MOPP-treatment have been observed:

lymphocyte predominance: 6 nodular sclerosis: 7 mixed cellularity: 5 lymphocyte depletion: 6 unclassified: 8.

The search for an effective treatment of this patient group resulted in the design of a program in which drugs were combined with a mode of action inde­pendent of those used in the MOPP-regimen.

PATIENTS AND TREATMENT

Adriamycin in combination with DTIC (NSC-45388) has a well-documented effectiveness in Hodgkin's disease. Since resistance to adriamycin sometimes develops within a few months and t~e generally accepted total dose limit is around 500 mg/m , it was attempted to stretch treat­ment cycles by spacing other effective drugs in between. Bleomycin and CCNU (NSC-79037) were selected, not only because of their proven effectiveness in phase I trials but also because they seem to act more efficiently on resting cells than on actively proliferating cells (Twentyman and Bleehen 1973). Thus, a treatment protocol designated "Post-MOPP" was instituted:

COMBINATION CHEMOTHERAPY OF HODGKIN'S DISEASE 515

week 1: adriamycin 100 mg i.v. on day DTIC 250 mg i.v. daily x 5

week 3,5,9,11: bleomycin 15 mg i.v. week 7: CCNU 140 mg p.o. on day 1

Dosage was not adjusted to body surface area but previous impairment of bone marrow function was taken into account individually.

RESULTS

1

Of 18 evaluable patients only three had never responded to previous MOPP-therapy. None of these patients did respond to the new drug regimen, and all quickly died. Of the remaining 1? patients who had achieved partial remissions under prior MOPP-therapy, two patients achieved a complete remission, 11 had a partial remission, and only two had progressive disease. In three patients with partial response to the new regi­men there was some regrowth of pulmonary lesions during treatment intervals, indicating either resistance to some of the drugs in the regimen or inadequate dose scheduling.

Toxicity was acceptable; a comparison of WBC fre­quency distributions under previous MOPP-therapy and under the "Post-MOPP"-regimen showed only a small shift to lower values. Statistical significance tests were not carried out because the sampling was not similar. However, the incidence of severe thrombocytopenia (less than 50.000/cmm) was increased under the "Post-MOPP"­regimen.

DISCUSSION

Similar effectiveness of new drug combinations, completely different from the MOPP-regimen, in the induction treatment of advanced Hodgkin's disease has definitely been established by Bonq,donna. Our "Post­MOPP"-regimen was designed only to treat patients who were refractory to MOPP-treatment, yet the results seem to be satisfactory. An analysis of survival patterns in comparison with historical controls cannot be presented at the time of reporting. Although three of our patients remained refractory to any cytostatic agent used, we feel encouraged to use the "Post-MOPP"-regimen in pa­tients who have become refractory to MOPP-therapy after prior response.

516 R. OSIEKA ET AL.

References

Bonadonna, G., Uslenghi, C. and Zucali, R. (1975), Eur. J. Cancer, 11, 251. - Frei, E. III, Luce, J.K., Gamble, J.F., Coltman, C.A., Jr., Costanzi, J.J., Talley, R.W., Monto, R.W., Wilson, H.E., Hewlett, J.S., Delaney, F.C. and Gehan, E.A. (1973), Ann. intern. Med., 79, 376. - Goldsmith, M.A. and Carter, S.K. (1974), Cancer, 33, 1. - Jacquillat, C., Weil, M., Auclerc, G., Delbrlick, H.~ Chastang, C., Chelloul, N., Boiron, M. and Bernard, J. ,1975), Dtsch. med. Wschr., 100, 785. -Mubashir, B.A., Shullenberger, C.C. and Gamble, J.F. (1973), 8th. med. J., 66, 779. - Twentyman, P.R. and Bleehen, N.M. (1973), Brit. J. Cancer, 28, 500.

PROTEOLYTIC ENZYMES IN THE TREATMENT OF MALIGNANT PLEURAL

EFFUSIONS AND SOLID METASTASES

O. Kokron, M. Micksche, C. Cerni, R. Titscher and H. Wrba

Institute for Cancer Research, University of Vienna Austria and Ludwig-Boltzmann-Institute for Clinical Oncology, Austria

In the last decades, a change in the etiology of pleural effusions has occurred. At the present, more than 40 per cent of these are based on malignant disease.

In the period from 1968 to 1973, we have observed in our department 4?7 patients with malignant pleural effusions; of these 412 cases were due to met­astases to the pleura whereas 15 cases were rare primary tumours of the pleura. The patients are as follows according to the site of the primary tumour: 183 patients had verified lung cancer, whereas 39 were not verified; 134 breast cancer i 16 ovari an carc i noma and 13 mesothel i oma • The rest were tumours of different origin and 14 cases of unknown localization.

These pati ents were treated as follows: one group was treated by radi 0-

gold install~~bon into the pleural cavity, the exudate was sucked off and 50 to 100 mCi of Au was given for the following 8 days. Due to positive influences of Proresid (a podophylline derivative) on the primary and metastatic mammary carcinoma, this drug was used in the treatment of 28 patients. It was applied intrapleurally every 4 and 8 days. 21 patients were treated by local admin­istration of cyclophosphamide in a dose of 200 mg/week.

A group of 236 patients did not receive any local drug application.

The last group consists of patients who were treated locally with proteolytic enzymes (Wobe-MugosR) .

The differences accordi ng to the total number of pati ents are due to the

517

518 O. KOKRON ET AL.

fact that in the beginning of this study a few patients were given more than one local treatment.

The proteolytic enzymes are a mixture of fractionated hydrolysates obtained from calf thymus, bovine pancreas, pisum sativum and lens esculenta plus papo­yotin and mannitol. These substances are supplied in Iyophilised form and are reconstituted by Lidocam immediately before application. The treatment is as follows: after puncture of the pleural cavity the exudate is sucked off. In some cases the cavity is flushed with sterile physiological saline solution and after this the first dose of 100mg Wobe Mugos is applied intrapleurally. Increasi ng amounts are then gi ven twi ce a week up to the maximal dose of 10 ampules (1000mg per session) •

Toxic side effects with this treatment were observed very rarely and, if at all, they were mostly an increase in body temperature and in some cases chills for several hours. If this drug is given intraperitoneally, shock symptoms might occur, but we did not observe this by the route of administration we used.

The results of the local therapy in malignant pleural effusions are given in figure 1 .

A therapeutic success was defined as no re-accumulation of exudate, ie dryness of the pleural cavity. This was observed in 11 per cent of the patients with radiogold-, in 18 percent of the patients with proresid-, in 14 per cent with cyclophosphamide therapy compared with 47 percent of the patients who

LOCAL TREA!I!'IENT OF HALIGNANT PLFTJRAL El!'FUSIONS

Treatment-regimen

Radio-gold (198Au )

Proresid

Cytoxan

\Jobe-Mugos

no drug

Fi gure 1

Success Number of patients

5 / 44 (11 %)

5 / 28 (18 %)

3 / 21 (14 %)

57 /119 (47 %)

42 /236 (19 ~)

TREATMENT WITH PROTEOLYTIC ENZYMES

LOCAL TREATMENT OF MALIGNANT PLEURAL EFFUSIONS BY PROTEOLYTIC ENZYMES ('M)BE MUGOSR )

Primary Success no Success

bronchogenic ca 3 9 (4 +)

mammary ca 61 4 (1 Ifo)

other {ovary etc.} 6 3 ( 11f')

total number of patients 70 16 (61ft)

Figure 2

519

were treated wi th proteolyti c enzymes. Pati ents who did not recei ve local drug treatment showed success in 19 percent of the cases.

This clearly demonstrates that the treatment of malignant pleural effusions with proteolytic enzymes is superior to other treatment regimes.

Therefore we are using this drug as the only treatment alternative in malignant secondary tumours located in ·the pleural cavity.

In the last two years 86 patients, 65 with mammary carcinoma and 12 with bronchogenic carcinoma and 9 with different primaries were treated by local enzyme application for malignant pleural effusions. 70 of these 86 patients responded well to this treatment and were considered a success, whereas a failure was observed in 16 cases. The type of histology of the successfully treated patients was in 93 percent adenocarcinoma. This is very important considering some possible mechanisms of the drug. It is our impression that proteolyti c enzymes have a good effect in the treatment of adenocarci nomas.

In addi ti on to local treatment, patients were further treated by i nfusi ons of proresid, and in some cases by polychemotherapy and also by systemic applications of Wobe Mugos. The therapeutic effectiveness was in all cases evaluated according to the effect of local treatment.

In 12 cases treated by local enzyme apptication, we investigated bio­chemical parameters, ie lactatedehydrogenase, muramidase, glucose, lactase and the protei n content of the harvested exudates and have tri ed to correlate

520 O. KOKRON ET AL.

LOCAL TREATMENT OF MALIGNANT PLEURAL EFFUSIONS BY PROTEOLYTIC ENZYMES (\NOBEMUGOSR)

Histologic type Patients %

adenocarcinoma 65 93

scirrhus 4 1 7 squamous cell 1 J

70 100

Figure 3

these data with the clinical picture: there was no change in the protein content in all samples, but in 7 patients, after application of a dose of 500mg there was an increase in the concentrati on of lactate and a decrease of gl ucose concentration. At the same time,there was a rise in the activity of lactate­dehydrogenase and also of muramidase, tbus pointing out that the proteolytic enzymes may have lead to a massive cell death. Whether this increase was due to a release from tumour cells or leucocytes, which are always present in the exudate, could not be clarified.

In a few cases the elevation of lactate was not accompanied by a simul­taneous change of the other parameters, since these tumours were all classified as mesotheliomas or squamous cell carcinomas, the increase might be explained by a penetrati on of these metabol i tes across the cell membrane rather than by disintegration of tumour cells.

There are several reasons for the systemic treatment of cancer patients by proteolytic enzymes alone or in combination with other treatment regimen.

It is well documented that especially cancer patients with advanced disease show an increase in fibrinogen, and a reduction in the fibrinolytic and pro­teolytic potential. An increase of the protease inhibitors 01 antitrypsin and 02 macroglobulin has been demonstrated in lung cancer patients. The stickiness of cancer cells might influence the appearance of metastases. Blocking serum factors have been found to abrogate cell-mediated immune reactions in vitro and a simi lar mechanism might be invol ved in the in vivo situati on.

The possible action of systemically applied proteolytic enzymes may have

TREATMENT WITH PROTEOLYTIC ENZYMES 521

some influence on fibrinolysis, proteolysis. A decrease of the proteinase­inhibitors might be one further action, together with a possible action on the adherence of cancer cells to blood vessels. The direct cytolytic effect might be also one point that should be taken in consideration, as well as a deblocking activity, which might restore immune-cytolysis.

Considering all these points of view, we have initiated a therapy study in patients with inoperable lung cancer (histologically classified as adeno­carcinoma). These patients were treated by daily application of proteolytic enzymes in form of an enema; the route of applications is somewhat problematic, but the resorption of the active material has been demonstrated to be most effecti ve . I n very few cases we have tri ed to in j ect these enzymes into metastatic tumour-nodules especially in cases of bronchogenic cancer. In some cases an objective regression could be observed, whereas other tumours did not respond.

In conclusion it can be stated that the treatment of cancer patients -especially those with pleural effusions - by proteolytic enzymes has been quite effective in our hands and that the results achieved in patients treated by systemic application of Wobe Mugos are very encouraging.

INTRA-ARTERIAL CHEMOTHERAPY OF HEAD AND NECK SQUAMOUS CELL CARCINOMA

R. Medenica, P. Alberto, W. Lehmann, M.A. Hopf

Division of Onco-Hematology, Dept. of Internal Medecine

University Hospital, Geneva, Switzerland

Good therapeutic results in the treatment of head and neck squamous cell carcinoma as judged by rapid remission without side effects, have been obtained by several Japanese authors by the ad­ministration of high concentrations of anti-cancer drugs via the arteries.

The substances used are those with a short half-life and acti-vity which is not mediated by their metabolites. employed to localize the major irrigating artery ascertain its availability to catheterisation by neous or surgical method.

Angiography is of a tumor and to either a percuta-

Indication for treatment by this method was posed for head and neck squamous cell carcinoma too far advanced for surgery or irra­diation, as these tumors are easily and precisely evaluated for the­rapeutic efficacy. Mitomycin C, Adriamycin and Bleomycin are the agents of choice against head and neck squamous cell carcinoma, but they are extremely toxic when given parenterally. Since their anti­tumor effect is obtained direct~y, and not after metabolisation, we decided to study their effect when administered intra-arterially directly into the tumor.

Methods and Materials

24 cases of head and neck squamous cell carcinoma were included in our study (Fig. 1). The average age of the patients was 57 years. The tumors were localized as follows: 4 in the floor of the mouth, 3 in the maxillary sinus area, 3 in the tonsillar area, 6 invasive tumors of the tongue, 2 of the epiglottis, 2 of the larynx and

523

524 R. MEDENICA ET AL.

4 of the trachea. 9 patients had received no prior treatment, 15 had been treated as follows: 3 by surgery, 3 by radiotherapy, 4 by surgery and radiotherapy, 1 by chemotherapy alone, and 4 by surgery, radiotherapy and chemotherapy. All 24 case~ were inoperable and not susceptible to radiotherapy, being all beyond Stage III. The goal in these patients was to reduce tumoral mass to allow sur­gery and/or irradiation to be performed.

WJ.ft;,;.r<. Of; PATIEPJrS Z4 ~t(.A.G£. AGE. 57 P~1012.. ~"""tl..iT 15

a q

.s~.~xc. '5

R.X ~

.3UR..G~lj p., 4 ~I-«) 1 ~2.Q. U cm;HO 4

t..e::eAL.IZAI1O~ OF ~ /o..lOI--IA FL.ooR. OF T~ HOUT./-I- 4 SJ...IAX ~

TOl1.siI..LAR ~

t.i "1Gl.1.AtL • G

t:pIGLOITIS 2. LA2.~x. 2-~ 4

Fig. 1

Intra-Arterial Administration

Four methods were used: The first group of 3 patients received once weekly injections of Mitomycin C lOmg, Adriamycin 20mg and Bleomycin 15mg for 3 weeks, each time requiring catheterisation.

The second group of 9 patients were treated vja indwelling catheters for 3 days. Everyday, lOmg of Mitomycin C , 20mg of Adriamycin and 15mg of Bleomycin were injected in succession.

In the third group 5 patients were treated with indwelling catheters for one week. On day 1, lOmg of Mitomycin C were injected, on day 2, 20mg of Adriamycin, on day 3 15mg of Bleomycin, then the cycle was repeated with lOmg of Mitomycin C given on day 7.

In the last group 7 patients were treated with lOmg of Mitomy­cin C intra-arterially once a day for 5 days (Fig. 2).

Each of the patients was followed regularly with clinical and endoscopic examinations. Hematologic, digestive, cardiac, pulmonary cutaneous, urogenital and neurologic functions were controlled.

INTRA-ARTERIAL CHEMOTHERAPY

The efficacy. of the treatment was measured by endoscopy and arteriography. A regression of tumor mass greater than 75% was the criteria for partial remission.

I-I~c. LA. AL L 2:::,. } -to~~

'bet.> t. v. J> 2.0011(,

I I I I , I :t> 1 2- ~ 4 5 ID '7 ~'i'b

CAT4t cA-m t Fig. 2

Results

525

Of the first group, 3 patients catheterized once weekly, 2 cases presented partial remissions, while one case presented continued progression of the disease despite the treatment (Fig. 3).

The second group of 9 patients receiving daily injections of the 3 antibiotics for 3 days, showed 7 partial remissions and 2 cases unchanged.

The 7 day sequential treatment of 5 patients resulted in 4 partial remissions and 1 patient unchanged.

Of the 7 patients who received Mitomycin C daily for 5 days, 6 showed partial remissions and one remained unchanged.

~luigv.T/01J IAJTM-AIt"$R.IAL k10Ut>e12. e.p Me. p

O'I-llIffit.JzAllOAJ VHJLL'I ~ t. 0 '\ !> f>II.'1 Pf.orocoL CJ 7 2. 0 ~ r»H PwrOCCL :, 4 1 0 ~lTUl'lC1kJ ~ 7 , 1 0

Fig. 3

526 R. MEDENICA ET AL.

Thus, of 24 patients treated via indwelling arterial catheters, 19 presented partial remission which lasted 4 to 64 weeks (Fig. 4). 4 cases presented no change and one case progressed while under treatment. There were no deaths related to the therapy.

•. .~LJLT.s . I}JJbeTlaJ I ~e-A -AtTEfLIAL.

UUMBct. OF- AATlBJTS t.4 .iX)t.6;rIOtJ I

~tAL ~lasCtJ 1'1 !T4S 1AAS I OVeA-TiOfJ a ta(1~ ,41 WfUs 4-G4 ..... uo~ ~ '1 tW-IS I ~e.ssPO "I

J.ttr~ 0

Fig. 4

It is interesting to note that in the 9 patients who had not been previously treated, adequate diminution of tumor mass was realized so that they could be treated subsequently by surgical excision or radiotherapy. Of these nine, four were in the group treated with only Mitomycin C (Fig. 5). Three of the four were subsequently operated and two are in complete remission. Three of the nine were treated in the 7 day combined program. Two of the three underwent surgery and one is in complete remission. The re­maining two from the group of untreated patients received the com­bined therapy in 3 days; one was operated and complete remission was obtained.

i~ A;2,~iAL ilJ.JtmiaJ lO Pl2:k;ViOU5 nu;;A.T1--IE1JT

.au.t itJ is.TVrr iotJ IJOJ.l&~ n; AAritur.s ~TfJ) i.A. 1~/6: IICaJ ISS! or.>

~lAC ffJ 5D 4 !:. Z. ~J....I ~ 1-lM,c. ~w 7D ~ t. 1 ~L.~ A-~12. ~u.c HI !>D Z. 1 1

Fig. 5

INTRA-ARTERIAL CHEMOTHERAPY 527

No hematological, digestive, cardiac or pulmonary toxicity was noted in any of the groups (Fig. 6). The local cutaneous toxicity however was very important, with one case presenting an extensive cutaneous necrosis. Four patients presented moderate alopecia and four extensive alopecia_ Those patients who had previously undergone radiotherapy showed a reactivation of post-actinic dermatitis. In these patients injections had to be given slowly as the injections were found to be especially painful.

, . . TOX-ICrrY.OF 1m12A ~Tt,.RIAL Tl2,bAn--R;rur

IVOH~ Ik- ~"'TiUJrs L4

DU'r'TIOO OF ~~.sr H~~ DAYS) 12

mx.lcrry 0 I ][ l1. Ul;Kt>., TOl.OOi CAL 2.+ 0 0 0 nGST;vt; 2.4 0 0 0 ~rAe fA 0 0 0 PUL.HOlJA'R-:f 2.4 0 0 0

C.urMJ.bT H()CUS H~~ 17 G 1 0 ~~jA 1G 4 4 0 .

Fig. 6

Conclusion

This study of 24 cases of head and neck squamous cell carcinoma brings us to the conclusion that intra-arterial chemotherapy of these tumors with Mitomycin C, Adriamycin and Bleomycin is effi­cacious and quite feasible given adequate technical know-how and equipment.

In non-irradiated patients, Bleomycin in doses of 15mg given over 10 minutes is well tolerated. In the previously irradiated patients, prudence is mandatory.

Adriamycin is well tolerated in doses of 20mg given over 10 minutes. Local toxicity is not augmented after radiotherapy.

Mitomycin C, with its short half-life is indicated for intra­arterial injection. We reached doses of 50mg with only minimal side effects. A similar dose given parenterally would certainly

528 R. MEDENICA ET AL.

cause medullary aplasia.

From these results, we feel that intra-arterial chemotherapy of head and neck squamous cell carcinoma is justified in all cases beyond Stage III without metastases.

CHEMOTHERAPY OF GLIOBLASTOMA MULTIFORME:

A STATISTICAL ANALYSIS OF ITS EFFECT

Kazuo TAKEUCHI * Keiko HOSHINO ** * Dept. of Neurosurgery, Kyorin U. School of Medicine

** National Institute of Hospital Administration * 6-chome, Shinkawa, ~1itaka-shi, Tokyo

** l-chome, Toyama-cho, Shinjuku-ku, Tokyo

SUMMARY

Statistical evaluation of the effect of chemotherapy on the duration of survival in the postoperative period of 102 cases of glioblastoma multiforme resulted in the following conclusions: 1. Neither individual nor combined utilization of radiotherapy and chemotherapy have significant effect on the period of post­operative survival where glioblastoma is concerned. 2. Even though a significant effect of chemotherapy could not be estab­lished statistically, it appears certain that it is as clinically effective as radiotherapy which has received high acclaim of late. 3. Postoperative adjuvant treatment, including chemotherapy, has an e.ffect on the postoperative survival period equal in signifi­cance to such factors as the duration of preoperative symptoms and the location of the tumor.

INTRODUCTION

Correct evaluation of the effect of chemotherapy on brain tumors is quite difficult. However, it is possible to gain some idea of its effect by studying its application to glioblastoma, a disease which has remained unresponsive to all methods of treat­ment available to date and pursues a rather short clinical course, and assessing its influence on the postoperative survival period (Bering, Jr. et al. 1967).

529

530 K. TAKEUCHI AND K. HOSHINO

MATERIALS

Our series consisted of 102 patients with histologically ve­rified glioblastoma multiforme encountered during the past 16 years. They included 94 adults and 8 children, or 55 males and 47 females. The tumor was located in the supratentorial region in 93 cases, and in the infratentorial area in 9 cases. Histol­ogical findings were indicative of astrocytoma, grade 4, in 54 cases, grade 3 in 21 and grade 2 - 3 in 6 cases. In 11 cases, a shift from grade 1 - 2 in the primary stage to grade 3 - 4 was discovered either in the terminal stage or at the time of autopsy. Other findings were obtained in the remaining 10 cases.

METHODS

It is difficult to deal with the survival effect of chemo­therapy on an all-inclusive basis due to the variable character­istics of individual patients and the diversity of therapeutic measures taken. Accordingly, our present series was reduced to 71 cases through the exclusion of a total of 31 cases comprising children, patients with infratentorial involvement or with astro­cytoma of grades other than 3 or 4 non-surgical cases and those who died within one month of surgery.

There are some factors known to affect the prognosis of glio­blastoma: those intimately involved and those not. The former include age at onset, histological findings, operative method and radiation therapy, and the latter the sex of the patient, the location of the tumor, it size and extension, the surgeon and steroid therapy. Even where the same set of factors are involved, however, different results often have been obtained by different investigators. This consideration prompted us to attempt a sta­tistical analysis in terms of 37 coded factors which were seem­ingly concerned with a valid assessment of the effect of chemo­therapy, such as the characteristics of the patient and the type and features of treatment instituted. Such factors as the size of the tumor, the amount of tumor removed and the clinical and pathological effects of chemotherapy were omitted here as no ac­curate information was available.

The statistical analysis techniques used include chi-square analysis for verifying the correlation between the afore-mentioned factors and the survival period as well as quantification analysis (Hayashi) subservient to quantitative recognition of the relative relationship between the two. The T-test was carried out in order to obtain an index of the magnitude of the difference between categories for each factor.

CHEMOTHERAPY OF GLIOBLASTOMA MULTIFORME

RESULTS

Factors that determine the survival period in all conceiva­ble respects are the presence or absence of calcification of the tumor and histological findings. Next come the location of the tumor and the duration of preoperative symptoms. Various ad­juvant treatments also have been found to be of equal signifi­cance. Factors of still lesser implication but possibly involv-ed in certain respects include preoperative state, the presence

531

or absence of cyst formation of the tumor and age at onset. The category of adjuvant treatments including chemotherapy may there­fore be considered as implicative as the location of the tumor or the duration of preoperative symptoms, even if less implicative than histology or the presence or absence of calcification. In­cidentally, various treatments have each been confirmed to be in­dependent of the characteristics of the patient, the correlation coefficient between the two being low. The multiple correlation coefficient with these factors is 0.8395, so that the known factors account for 70.5% of the total. It is possible that included in the remaining unknown component (29.5%) are those factors which determine the survival period and which allow for selection from among various methods of treatment available. Statistically, how­ever, the multiple correlation coefficient is considerahle.

Studies of the various adjuvant treatments were carried out in regard to the time of their initiation, the type of treatment, the procedures involved, the chemotherapeutic agents used and the chronological relationship with other treatments. As a result, no significant difference between groups was noted in the survi­val rate at p < 0.05. For example, no substantial difference in survival effect was present between groups receiving and not re­ceiving radiation therapy during first hospitalization. On the other hand, the survival rate was lower in groups receiving che­motherapy than in those not receiving chemotherapy but there was no statistically significant difference.

The chemotherapy practiced by us consisted mainly of bleomy­cin given systemically (Takeuchi et al. 1974), antimetabolites given by continuous intracarotid infusion (Takeuchi and Atsuchi 1970) and cerebral perfusion with alkylating agents (Takeuchi et al. 1964). A study of the survival rate in groups treated and not treated with bleomycin also failed to reveal any marked dif­ference.

Similarly, an estimate was made of the survival rate through­out the whole course of hospitalization in order to highlight the difference due to chemotherapy as well as that ascribable to ra­diation therapy. In both respects, the treated group displayed a higher survival rate than the untreated group although, here again, the difference was not statistically significant at p<0.05.

532 K. TAKEUCHI AND K. HOSHINO

The same study carried out with 80 cases including those who died as early as within one month of admission or surgery in­dicated a prominent difference between groups receiving and not receiving radiation therapy, a difference which was significant at p<O.OI. The difference between groups receiving and not re­ceiving chemotherapy also was found significant at p<0.05. It is inappropriate, however, to include those patients who died so soon as not to allow postoperative adjuvant treatment.

With a view to appraising the effect of each single treat­ment, a comparison was made between groups receiving different types of treatment: 6 cases placed on chemotherapy postopera­tively, 27 cases receiving radiation therapy alone and 7 cases receiving no adjuvant treatment postoperatively. At first sight, both types of treatment appeared to be of considerable use. A comparison of these 2 treated groups with another group of 31 cases receiving combined therapy indicated the latter had the poorest survival rate. The difference, however, cannot be re­garded as significant statistically considering the number of cases concerned and their pattern of distribution. Nor can it be concluded that chemotherapy is less useful than radiation therapy. It follows that the effect of chemotherapy cannot be ignored if radiation therapy is, as has been alleged, truly ef­fective both clinically and statistically.

Incidentally, there was no specific reason for the 6 patients receiving chemotherapy alone, but they probably represented cases that followed a relatively favorable course postoperatively, re­quiring chemotherapy alone before being discharged. In all but one of these cases, therefore, various types of treatment were additionally used during the second hospitalization. In most cases receiving a combination of chemotherapy and radiation ther­apy even during the first hospitalization it is highly probable that the unsatisfactory clinical response elicited by either of the component therapies had an unfavorable influence on the sur­vival rate.

Lastly, a somewhat different attempt was made to assess the role played by chemotherapy in 6 patients who survived 5 years or more postoperatively, a period extraordinarily long for patients with glioblastoma multiforme. As a result, it was found that chemotherapy was utilized in all of these cases, presumably con­tributing more or less to their longer survival. Of these pa­tients, 2 proved to have lived a useful life for a period of about 5 years without further treatment following postoperative systemic application of bleomycin as the final therapeutic ef­fort. It is to be noted, however, that histological findings indicated a lesion of grade 3 or so in these 2 cases. It appears that more serious lesions remain beyond the reach of chemotherapy.

CHEMOTHERAPY OF GLIOBLASTOMA MULTIFORME

REFERENCES

1. Bering, E. A., Jr., Wilson, C. B. and Norrell, R. A., Jr. (1967), J. Neurosurg., 27, 1.

533

2. Takeuchi, K., Yahagi, Y., Nagai, M., Mizutani, R., Sato, F., Chigasaki, R., Asano, K., Uei, I. and Morimoto, W. (1964), Neurol. med.-chir., 6, 122.

3. Takeuchi, K. and Atsuchi, M. (1970), in "Progress in Antimicrobial and Anticancer Chemotherapy", ed. by R. Umezawa, University of Tokyo Press, Tokyo, Vol. II, p. 226.

4. Takeuchi, K., Sueyoshi, S., Rara, M. and Ogihara, R. (1974), in "Progress in Chemotherapy: Proceedings of the 8th International Congress of Chemotherapy", ed by G. K. Daikos, Athens, Vol. 3, p. 919.

1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA (BCNU) IN THE TREAT­

MENT OF PRIMARY CENTRAL NERVOUS SYSTEM TUMORS

George A. Koutras, Nicholas A. Pavlidid, Nicholas Kordiolis, Vrisiis Samaras and John Taptas Greek Anticancer Institute and Nuclear Research Center "Democritus" Athens, Greece

Three groups of patients with CNS tumors (Group A nonresected tumors, Group B partially-resected tumors, and Group C totally-resected tumors) were treated with BCNU, radiation (Group A 7/8, Group B 6/6 and Group C 4/6) and vincristine sulfate (Group A 4/8, Group B 2/6 and Grpup C 4/6). Patients received 1 to 5 courses of therapy. Rad­iation was administered during the first course of BCNU. Best results were obtained in Group C with the totally­resected tumors. Three out of 6 patients in this group are in excellent condition 6, 13 and 17 months from the initiation of therapy. However, only 1 patient of Group A is in very good condition at 6 months and 3 patients of Group B at 4 and 8 months. Dead are 6/8 of Group A, 3/6 of Group Band 2/6 of Group C. The addition of vincristine sulfate to BCNU and radiation cannot be evaluated from these results.

Since the nitrosoureas became available some 12 years ago a number of studies have been published on the results of their use in various malignancies (RaIl et al 1963, De Vita and Gold 1964). This class of antitumor agent with an as yet unknown mechanism of action, though evidence in­dicates that they probably act as alkylating agents (Wheeler and Chumley 1967), are effective against a variety of tumors, includings primary and

The authors are grateful to the National Cancer Institute, Bethesda, Maryland, U.S.A. for the supply of BCNU.

535

536 G.A. KOUTRAS ET AL.

metastatic brain tumors (Walker and Hurwitz 1970, Wilson et al. 1970).

It is the purpose of this study to report our results of treatment of primary CNS tumors with BCNU in combination withra­diation and, in half the cases, with vincristine sulfate (VCR).

MATERIALS AND METHODS

Twenty patients with various CNS malignancies were divided, according to tumor resection, into 3 groups. Group A composed of 8 patients, 6 males and 2 females, with non-resected tumors. Two of them were biopsied and were found to be astrocytomas grade II-III. On the remaining of patients the diagnosis was based on angiography and/or air encephalography. According to the findings 2 supposed to have gliomas, 2 glioblastomas, and 2 multiform glio­blastomas. Group B composed of 6 patients, 4 males and 2 females with partial tumor resection. This group included 1 glioblastoma grade IV, 2 multiform glioblastomas, 1 oligodendroglioblastoma, 1 meningiosarcoma and 1 malignant teratoma. Group C included 6 pat­ients, 3 males and 3 females, with total tumor resection. Patho­logy of those tumors revealed 2 astrocytomas, one of them grade III, 1 astroblastoma, 1 glioma grade III, 1 multiform glio­blastoma and 1 meningiosarcoma.

All patients but one were adults aged 35 to 69 with mean age 48.1 years. Freshly prepared solution of BCNU was administered i.v. at 200 mgs/m2. Each administration with or without VCR was considered as a course of therapy. Five patients received one course, 11 patients 2 courses, 3 patients 3 courses and 1 patient 5 courses. The second course was usually given 6 weeks after the first unless side effects did not permit it. All other courses were given with increasing intervals between them.

Along with BCNU VCR was given in 10 patients at a dose of 1.5 mgs per week for 6 weeks for the 1st course, reduced to 1.0 mg per week x 6 for the following courses.

Patients receiving irradiation were treated during the 1st course of chemotherapy. Two patients with recurrent tumors who had been irradiated and another with no previous irradiation were not irradiated. A 4th patient, however, with a recurrent tumor had a supplemental dose of irradiation during the first course of BCNU.

Steroids had been started before BCNU administration and con­tinued as long as necessary. Details of radiation dosage, BCNU courses, prednisone or prednisolone administration, etc. are seen

TREATMENT OF PRIMARY CENTRAL NERVOUS SYSTEM TUMORS

in Table I.

Complete blood counts, urinalysis, BUN, rBS, uric acid and liver function tests were performed once to twice weekly during each observation period.

537

The assessment of improvement was based mainly on the object­ive neurologic response of each patients and considered to be more than 50% when it was very significant.

RESULTS

Our results are summarized in Table I. Three patients of Group A showed no change and 3 more, less than 50% improvement. Only 2 patients showed significant improvement. One of those 2 patients was not irradiated. This patient, however, had a very significant response and he would have survived more than 6 months if he had not refused to return for therapy as he was instructed. He was in relapse when the 2nd course was administered. Two patients of this group are still alive at the time of writing 6 and 8 months after the initiation of therapy, while the remaining 6 died within a year. One of the 2 surviving patients is in poor condition and the other is fair.

In the same Table are seen the results obtained in Group B. In this group one patient showed no change, one less than 50% im­provement and 4 more than 50%. The first 2 patients and 1 third with significant response died within 9 months from the initiation of their therapy. The remainder of the patients, i.e. No 4,5, and 6 are alive 8 and 4 months after therapy.

Results obtained in patients of group C, with the totally­resected tumors of the brain, are seen in the same Table. Three patients with previous irradiation were treated for tumor recur­rence. One of them had a supplemental dose of irradiation, along with the first course of therapy. One patient out of the 5 of this group died at 3 months from the initiation of therapy and a 2nd patient is also considered dead because he was in very poor condi­tion at the time of discharge. Four patients are alive from 6 to 17 months after the initiation of their therapy. The longest sur­viving patient is a female with a small glioma which was irradiat­ed during the first course of BCNU. This patient hasn't had any therapy after the completion of the 3rd course of BCNU some 14 months ago. Patient No 6 is also of interest because in the past 13 months he had 5 courses of BCNU with VCR, for a recurrent as­trocytoma. This patient continues to be in excellent condition. In this group of patients only one showed no change and two less than 50% improvement.

538 G.A. KOUTRAS ET AL.

TABLE 1. Results of therapy with BCNU, radiation and VCR in central nervous system tumors.

THE RAP Y No Patient Sex Age Tumor BCNU P VCR Co6O Results

courses GROUP A: non-resected tumors 1 C.E. M 43 A II-III I 5100 N.C.Dead 50 d 2 M.T. M 50 ? G I +++ + 2700 N.C.Dead I y 3 Z.G. F 48 ? G/ma 2 ++ 5100 )50% Dead 9 mo? 4 A.K. M 69 ? m-G/ma 2 ++ 6000 N.C.Dead 7,5 mo 5 A.M. M 13 ? G 2 ++ 3850 (50% Dead 2.5 mo 6 M.Y-M. M 55 ? m-G/ma 2 +++ + No )50% Dead 6 mo 7 E.P. F 48 ? G/ma 2 +++ + 4640 (50% Alive 8 mo 8 T.K. M 40 A II-III 2 +++ + 5250 (50% OK 6 mo GROUP B: partially-resected tumors 1 A.K. F 56 G/ma IV I +++ 5800 < 50% Dead 9 mo 2 P.P. M 65 m-G/ma I + 5400 N.C.Dead 7 mo

III-IV 3 C.M. M 46 m-G/ma 2 + + 6000 )50% Dead 5 mo 4 B.K. M 44 MSa 3 +1- 5200 )50% OK 8 mo 5 M.T. F 55 m-T/ma I ++ 5000 )50% OK 4 mo 6 G.B. M 41 01-G/ma 2 ++/- + 6100 )50% OK 4 mo GROUP C: totally-resected tumors 1 C.T. M 47 AlII 2 ++ + 5400 (50% Dead 4 mo ? 2 P.K. F 48 Glp 3 +++ + 5400 )50% OK 17 mo 3 A.T. F 35 MSa * 2 No N.C. Alive 6 mo 4 A.V. M 60 Alma 2 ++ + 2550 (50% Dead 3 mo 5 V.Y. F 54 m-G/ma 3 ++ 5700 )50% OK 6 mo 6 N.V. M 45 A* 5 ++/- + No )50% OK 13 mo

A: astrocytoma, A/rna: astroblastoma, G: glioma, G/ma: glioblastoma m-G/ma: multiform glioblastoma, MSa: meningiosarcoma, m-T/ma: malig­nant teratoma, 01-G/ma: oligodendroglioblastoma, *patients with previous irradiation. P: prednisone or prednisolone up to 25 mgs (+), 50 mgs (++), or 75 mgs (+++), O.K.: patients is excellent condition. N.C.: no change in status.

Prompt objective neurologic response was characteristic in some of our patients. especially in those who improved significant­ly.

Side effects. Table II summarizes the side effects which were observed during and/or shortly after BCNU administration. Nausea and vomiting were very common side effects in our patients. Pain in the vein during drug administration was reduced or reliev­ed by slowing the rate of administration. Flusting of the face. quite common in our patients. along with headache and drowsiness were also seen. Pain at the tumor area and pain in the chest. a tightness-like of feeling. were observed in a small percentage too.

TREATMENT OF PRIMARY CENTRAL NERVOUS SYSTEM TUMORS 539

TABLE II. Side effects attributed to a~NU

No of patients % During and/or shortly after administration

Pain in the vein 12 60 Nausea 18 90 Vomiting 17 85 Flushing of the face 11 55 Headache 2 10 Drowsiness 7 35 Pain at the tumor area 1 5 Pain in the chest 1 5

Long after administration Leukopenia {(3000) 3 15 Thrombocytopenia {(100000) 3 15 Anemia (Hct fall) 10%) 15 75 Venous thrombosis 7 35 Abnormal liver function tests 3 15 Hyperuricemia 1 5 Infection 3 15 Skin pigmentation 2 10

Side effects seen some days or weeks after drug administration and attributed to BCNU are summarized in the same Table. Most common of all was anemia followed by venous thrombosis. Venous thrombo­sis, however, was seen mainly in patients receiving vincristine sulfate and only one had not received it. Leukopenia and thrombo­cytopenia were not serious and life threatening. Only 3 patients developed infections because of leukopenia. Abnormal liver funct­ion tests, in the form of elevated SGPT, SGOT, and alkaline phos­phatase were a transient finding. Hyperuricemia was also seen in one patient, and skin pigmentation in two.

COMMENT

Recent review studies by Carter et al. (1970) and Wasserman et al. (1975) give an overall response rate of about 45% for prima­ry brain tumors with BCNU alone. Somewhat better results were re­ported by Fewer et al. (1972) with BCNU alone, however, the addit­ion of VCR to BCNU decreased the response rate significantly. Ra­diation and BCNU have given significantly better results to glio­mas when combined than either one alone (Walker and Gehan 1972).

Our results show that a significant improvement (more than 50%) was achieved in 45% of our patients and a further, but lesser, re­sponse in another 30% of our cases. Only 25% of our cases showed no change. Since some of our patients are still under observation

540 G.A. KOUTRAS ET AL.

and therapy and in remission it is difficult to find out the durat­ion of median response. Moreover, this becomes more difficult because some of our patients with significant improvement have re­fused further therapy. Characteristically patients who experienced significant objective neurological response did it, some times, the very first day of ~rug administration. This was observed in both patients with complete or no tumor resection. The addition df vincristine cannot be evaluated from those results.

Our results suggest that this combination, i.e. BCNU plus si­multaneous irradiation of brain tumor and possibly vincristine gives better response rate in primary CNS tumors. However, more cases and time are necessary to evaluate it.

REFERENCES

Carter, S.K., Shabel, F.M., Jr.,Broder, L.E., and Johnston, T.P. (1972), Advances in Cancer Research, 16, 273. De Vita, V., and Gold, L. (1964), Proceedings of the American As­sociation for Cancer Research, 5, 15. Fewer, D., Wilson, C.B., Boldrey, E.B., Enot, K.J., and Powel, M.R. (1972), The Journal of the American Medical Association, 222, 549. RaIl, D.P., Ben, M., and McCarthy, D.M. (1963), Proceedings of the American Association for Cancer Research, 4, 55. Walker, M.D., and Gehan, E.A. (1972), Proceedings of the American Association for Cancer Research, 13, 67. Walker, M.D., and Hurwitz, V.S. (1970), Cancer Chemotherapy Reports, Part 1, 54, 263. Wasserman, T.H., Carter, S.K. and Carbone, P.P. (1975), Proceedings XI International Cancer Congress, Florence 1974, 5, 274. Wheeler, G. and Chumley, S. (1967), Journal of Medicinal Chemistry, 10,259. Wilson, C.B., Boldrey, E.B., and Enot, K.J. (1970), Cancer Chemo­therapy Reports, 54, 273.

BCNU AND CCNU CHEMOTHERAPY OF TUMOURS OF THE CENTRAL NERVOUS

SYSTEM*

G. Robustelli Della Cuna and P. Paolette

Sezione di Oncologia Medica della Clinica del Lavoro and Clinica Neurochirurgica, University 'of Pavia Italy *Supported in part by Grant 08090/04 of the U.S. Public Health Service.

INTRODUCTION

A variety of anticancer agents have been used in the treatment of malignant tumours of the Central Nervous System either alone or in combination with radiotherapy. Unfortunately they failed to produce evidence of consistent and long term palliation. Recently the availability of some nitrosourea compounds (BCNU, CCNU and Methyl-CCNU) stimulated interest in treatment of brain tumours. BCNU and CCNU have been effective in primary and metastatic tumours of the CNS. These agents, due to their liposolubility, enter rapidly into cerebro-spinal fluid where an effective therapeutic level of these drugs can be demonstrated after parenteral or oral administration. BCNU and CCNU have been shown to cause objective improvement approximately in 40% of patients who had malignant glioma. with a mean duration of response of 5.2 months. Several reports suggest that the combination of radiotherapy and nitro­sourceas may improve survival in patients with tumours of the CNS.

The purpose of this study is to compare two groups of randomized patients bearing primary and metastatic tumours of the eNS who were treated between May 1972 and June 1975 by surgery, radiotherapy and chemotherapy; the metastatic tumours were treated only with radiotherapy and chemotherapy.

MATERIALS AND METHODS

Eighty-one patients treated for malignant tumours of the CNS were included in this study. 60 of them were treated by surgery, post-operative radiotherapy and chemotherapy and 21 had radio-

541

542 G.R. DELLA CUNA AND P. PAOLETTE

SURGERY

FIGURE 1

THERAPEUTIC MODALITY IN C.N.S. TUMORS

R A N D 0 M

BCNU

CCNU

R A

-2"<1 3,d_- .. -BCNU D I·tcycl. I cycles

0 T H E R

_CCNU A - 2"'!.3'ot __ • 1·t cycl. P eyel.s

y

*

80 mg / m2 i.v .• 3 DAVS EVERV 8 WEEKS

130 mg/m2 p.o. EVERV 8 WEEKS

P R 0 - Support G th.rapy R E S S I 0 - Support N

th.rapy

* 5500 RA05 IN 6 WEEKS STARTING 15 DAYS AFTER THE FI"SY CYCLE OF CHEMOTHERAPY

therapy and chemotherapy with BCND or CCND. Figure 1 shows the outline of treatment for operable malignant brain tumours. Randomization of patients took place within 10 to 15 days from surgery or at the time of diagnosis in patients bearing metastatic tumours of CNS. BCND (80mg/m2/day for 3 days i.v.) and CCND (13Omg/m2 orally) were started immediately after randomization. Radiotherapy, 5,500 rads in 6 weeks, delivered with cobalt, started fifteen days after the end of the first cycle of chemotherapy. The following cycles were repeated every 8 weeks starting 15 days after the end of radiotherapy. Corticosteroids were used with equal frequency and duration in both groups of patients. Figure 2 shows the clinical and instrumental devices during chemotherapy: brain scans were performed on the 3rd, 6th, 9th and 11th cycle;

neurological tests were performed after surgery and before chemo­therapy and on the 3rd, 7th and 12th cycle. The bone marrow biopsy was perfor~ed at the start of chemotherapy and on the 3rd and 6th cycle and, if necessary, in the following cycles. Figure 3 shows the dose reduction schedule, the nadir of white blood cells and platelets was determined on the haemograms obtained on the 3rd, 5th and 6th week of treatment. Figure 4 shows the criteria of evaluation of the haematological toxicity in a combined fashion: peripheral blood and bone marrow. Figure 5 shows patient classi­fication according to the kind of tumour and type of surgery. Figure 6 shows the characteristics of patients entered into study: the total number of patients was 81, (50 were males and 31 females); median age was 50 years (51.5 years for patients treated with BCND

and 49 years for patients treated with CCND). The histopathologic diagnoses were: BCND group - Glioblastoma 16; Astrocytoma grade II 12; Metastatic 8. CCND group - Glioblastoma 18; Astrocytoma grade II 6; Metastatic 12. The sites of tumour location in each group are shown in figure 7.

BCNU AND CCNU CHEMOTHERAPY

CLINICAL AND INSTRUMENTAL DEVICES DURING CHEMOTHERAPY

CHEMO - N£UIIO- SlCULL IllAIN N£URO- O£SIoIO-

BLOOD BLAST. BONE THERAPEUTIC LOGICAL EEG RADIOl. STEROL LVIoIPH MARROW

CYCLES TESTS X-RAY SCAN TESTS TEST COUNTS TEST BIOPSV

,SI • • • • • 2nd • • • • • • 3'd • • • • • • 4th • • • • 51h • • • • • • 6th • • • • • • 7th • • • • • 8th • • • • • • 9th • • • • • • 10th • • • • • • II Ih • • • • • • • 121h • • • • • • •

• If racessary

FIGURE 2

DOSE REDUCTION SCHEDULE

Leucocytes /mm3 Platelets/mm3 Subsequent Dose (nadir)

>4000

<4000> 3000

< 3000> 2000

<2000

(nadir)

>140_000 BCNU 8Omg/m2x3 CCNU 130mg/m2

< 140.000> 80.000 BCNU 60 mg/m2x 3 CCNU 90 mg/m2

< 80.000>50.000 BCNU 40 mg/m2)( 3 CCNU 60 mg/ m2

< 50.000 Bone Marrow Biopsy Delay further treatment until: PIa telets return more than 110.000 Imm3

Leucocytes return more than 3500/mm 3

The next dose will be:

BCNU 40mg/m2 " 3 CCNU 80 mgl m2

FIGURE 3

543

544 G.R. DELLA CUNA AND P. PAOLETTE

HEMATOLOGICAL TOXICITY Combined evaluation of peripheral blood and bone

marrow changes

SCORE 4 3 2 1

PARAMETERS 100·/. 75·/. 50·/. 25·/.

BONE MARROW

TOTAL CELLS ++ +- +- +-

RED CELL SERIES ++ ++ +- +-

WHITE CELL SERIES ++ ++ +- --MEGAKARYOCYTES ++ +- +- --

PERIPHERAL BLOOD

HEMOGLOBIN ++ +- +- +-~ 14 g'/,

ERYTHROCYTES ++ +- +- +-

~4.5 x 10 mm3

LEUCOCYTES ++ +- +- --~4.5 x 10 mm3

PLATELETS ++ ++ +- --~150 x 10 mm3

CLASS of TOXICITY A B C D

A: NO TOXICITY

B: MODERATE TOXICITY

C: MARKED TOXICITY

D: SEVERE TOXICITY

FIGURE 4

PATIENT CLASSIFICATION ACCORDING TO

THE KIND OF TUMOR AND TYPE OF

SURGERY

A Glial Intracranial tumor

A1 - Tumor Removal

A2 - Decompression. Biopsy and/or Shunt

A3 - No Surgical Therapy

C Metastatic Intracranial Tumor

C3 - No Surgical Therapy

FIGURE 5

BCNU AND CCNU CHEMOTHERAPY 545

CHARACTERISTICS OF PATIENTS ENTERED INTO THE STUDY

TOTAL BCNU CCNU

No. 81 38 43

MALES 50 25 25

FEMALES 31 13 18

MEDIAN AGE 50 51,5 49

A, 31 12 19

A2 19 11 9

A3 10 7 3

C3 20 8 12

ASTROCYTOMA 17 12 5

GLIOBLASTOMA 34 16 18

MEDULLOBLASTOMA 4 - 4

OTHER C.N.S. TUMORS 6 2 4

C.N.S. METASTATIC TUMORS 20 8 12

FIGURE 6

TUMOR LOCATION IN EACH TREATMENT GROUP

SITE TOTAL No. OF TUMORS

BCNU CCNU OF PATIENTS

R. FRONTAL 7 3 4

L. FRONTAL 8 2 6

R. TEMP-PARIETAL 26 16 10

l. TEMP- PARIETAL 19 8 11

R. OCCIPITAL 1 1 0

l. OCCIPfTAL 2 2 0

POST. FOSSA 15 5 10

3rd 'VENTRICLE 3 1 2

TOTAL 81 38 43

FIGURE 7

546 G.R. DELLA CUNA AND P. PAOLETIE

BCNU IN C.N.S. TUMORS

Therapeutic Response according to the Clinical Classification of KARNOFSKY

GROUP 0 I

0-0 A-B-C A-B- C

A, 2/12 3/12 7/12

A2 0/11 3/11 8/11

A3 1/7 2/7 4/7

C3 1/8 3/8 4/8

TOTAL 4/38 11/38 23/38

FIGURE 8

CCNU IN C.N.S. TUMORS

Therapeutic Response according to the Clinical Classification of KARNOFSKY

GROUP 0 I

0-0 A-B-C A-B-C

A, 1 /19 4/19 14/19

A2 0/9 0/9 9/9

A3 2/3 0/3 1/3

C3 2/12 2/12 8/12

TOTAL 5/43 6/43 32/43

FIGURE 9

BCNU AND CCNU CHEMOTHERAPY

HEMATOPOIETIC TOXICITY DURING CHEMOTHERAPY

Cycle BCNU CCNU mean nadir mean nadir

WBC PLATS. wec PLATS.

1 2

3 4 5 6

7

5.2 173 6.9 5.0 152 5.2 4.5 127 5.0 4.3 142 4.1 4.1 130 3.3 3.8. 105 4.1

3.9 107 3.8

FIGURE 10

INCIDENCE OF HEMATOLOGICAL TOXICITV IN 81 PATIENTS BEARING C.N.S. TUMORS

Group No. of Class of Toxicity Cases A B C 0

B A, 12 3 5 2 2

C A2 11 3 2 6 0

N A3 7 2 1 1 3

U C3 8 4 0 2 2

Total 38 12(320'0) 8(21"10) 11(29"10) 7(18"10) • L-47"1o -...J

C A, 19 7 4 3 5

C A2 9 2 2 3 2

N A3 3 1 1 1 0

U C3 12 4 1 4 3

Total 43 14(32"10) 8(20"10) 11(25"10) 10(23"10) L- 48"1o---l

FIGURE 11

180 173 168 171 134 100 105

547

548 G.R. DELLA CUNA AND P. PAOLETIE

RESULTS

The therapeutic response according to the Clinical Classifica­tion of Karnofsky is shown in figure 8 for patients treated with BCNU and in figure 9 for patients treated with CCNU. Improvement {Class I of the Karnofsky Classification) was observed in 60% of patients treated with BCNU and in 74% of patients treated with CCNU. Figure 10 shows haematopoietic toxicity during chemotherapy. The incidence of leukopenia and thrombocytopenia was similar in both treatment groups and became evident starting from the 5th cycle of treatment. Figure 11 summarizes the data on haematopoietic toxicity evaluated in a combined modality as shown above: no significant difference of toxicity was observed after administration of either BCNU (47%) or CCNU (48%). In general the drugs were both well tolerated. Figure 12 shows the mean survival time of 81 patients treated with two above mentioned drugs and divided according to previous mentioned classification:

Group A 1: (Glial Intracranial tumours with surgical removal) including 3 Astrocytomas and 19 gioblastomas; the mean survival time was similar in both treatment groups 22 months versus 20.2 months.

Group A 2: (Glial Intracranial tumours with decompression, biopsy and/or shunt) including 3 Astrocytomas and 8 glioblast­omas. The mean survival time was higher in patients treated with BCNU 22.9 months versus 15.8 months.

Group A 3: (Glial Intracranial tumours without surgical therapy) including no Astrocytomas and 7 glioblastomas. The mean survival time was 9 months with BCNU and 5.3 months with CCNU.

Group C 3: (Metastatic Intracranial tumours without surgical therapy). The mean survival time was higher in patients treated with BCNU (15.2 months versus 8.8 months) .

COMMENT

Because of variation in the extent and location of the tumours and in view of the poor correlation between diagnostic tests and tumour regression, we believe that the mean survival time was the sole parameter to be analysed in our cases. In a recent study made by the Brain Study Group (U.S.A.) the mean survival time of 2099 patients with malignant glioma was: 3.4 months in patients with only support therapy; 4.8 months in those treated only with BCNU; 6.9 months with only radiotherapy and 9.3 months in those with radiotherapy plus BCNU. In our experience the overall sur­vival time of the patients treated with BCNU was longer (17.2 months) than that observed in the patients treated with CCNU (12.5 months). The mean survival time for only glioblastomas was 12.6

BCNU AND CCNU CHEMOTHERAPY 549

MEAN SURVIVAL TIME OF 81 PATIENTS BEARING C.N.S. TUMORS

EVALUABLE· MEAN SURVIVAL

PATIENTS TIME RANGE (No 0' montho)

A, 12 22 (2 -34) B

Az 11 22.9 (10 -32) C

A3 7 9 (6 - 11) N

C3 8 16.2 (4 -32) U

A, 19 20.2 (3 - 33) C

A2 9 15.8 (7 -27) C

A3 3 5.3 (1 - 12) N

C3 12 8.8 (h· 18) U

FIGURE 12

months in the BCNU group and 10 months in the CCNU group.

The difference in mean survival time suggests that the addition of nitrosourea derivatives to radiotherapy can improve the type and length of the therapeutic response of the malignant tumours of the CNS.

Our observation of a longer survival in patients treated with BCNU needs further confirmation in controlled clinical trials with a higher number of evaluable patients.

REFERENCES

Hildebrand, J., Brihaye, J., Goffin, J.C. and Staquet, M. (1973). EORTC Protocol for the Study of CCNU in the Treatment of Irradiated, Operated, Malignant Glioma of the Brain. Europ. J. Cancer, 9, 459.

Walker, M.D. (1973). NItrosoureas in Central Nervous System Tumours. Cancer Chemother. Rep., 4, 21.

Walker, M.D. (1973). Brain and Peripheral Nervous System Tumours. In: Cancer Medicine. (Holland, J.F. and Frei, E.III eds.) Philadelphia, Lea & Febiger, 1385-1401.

COMBINATION CHEMOTHERAPY AND CCNU TREATMENT OF

GLIOBLASTOMA: A CO~WARATIVE TRIAL

W.-D. Heiss, A. Kroiss, J. Kiihbook and W. Profanter Neurologische und II.Medizinische Universitatsklinik, Lazarettgasse 14 A - 1090 Vienna, Austria

SmfNARY

Unselected groups of patients suffering from glio­blastoma (astrocytoma grade III or IV) received standard surgical therapy (16 cases, mean postoperative survival time 6.27 ± ~.75 months), combination chemotherapy (27 cases: 11 treated for recurrent brain tumors, mean survival time 5.80 ± ~.20 months; 16 patients receiving the therapy after surgical intervention t mean postope­rative survival time 8.96 ± 4.97 months) or CCNU mono­therapy following the surgical intervention (24 cases, mean postoperative survival time 6.98 = 4.46 months). Only the mean survival time in the group ,."i th combi­nation chemotherapy after surgery was prolonged signi­ficantly when compared with those cases, who were ope­rated upon only while the increase in survival time ob­tained with CCNU was not significant. The results indi­cate that combination chemotherapy is more effective than CCNU monotherapy.

Trials with new modifications of chemotherapy for the treatment of malignant gliomas are justified by three points.

1) The standard treatment of malignant gliomas of the central nervous system, consisting of surgery often followed by irradiation, fails to produce satisfactory survival time (KRAYENBOHL 1959, TAVERAS 1961, JELS~~ and BUCY 1967, GRUNERT et al.1973).

551

552 W.·D. HEISS ET AL.

2) In contrast to the normal brain tissue, where only nuclei of the neuroglia shmv some mitotic activity and no indications of DNA-synthesis are evident in neu­rones, the cells of malignant brain tumors have a high proliferation rate (HOSHINO et al.1972).

1) Halignant gliomas probably do not grOl" out of one malignant cell but are the result of the transformation of the cell kinetics in many cells (\HLLIS 1960). Des-pi te these t\vO special features of gl iomas which \vould render systemic chemotherapy the most promising treat­ment, the results obtained with various antineoplastic agents were not satisfactory (BERING et al. 1967, lITLSON and HOSHINO 1969, BRODER and RALL 1972). In the search for a more effective postoper~tive treatment of gliomas, two different forms of chemotherapy were tested and the results were compared to the clinical course of glioma patients receiving standard surgical and radiation therapy.

MATERIAL AND METHODS

A total of 67 patients comprise this study, all of whom suffered from primary malignant glioblastomas of the brain (astrocytoma grade III and IV according to the classification of KERNOIlAN et a1. 1949). Without prior selection they were treated following different proce­dures.

1) In 16 patients the braintumor \vas totally or sub­totally operated; 8 of these patients received post­operative radiation therapy.

2) 27 patients received a combination chemotherapy (ISRAEL et a1. 1967). The following cytotoxic agents were given in the form of 1 to 4 consecutive 7 day­cycles: methylhydrazine (125 mg i.v.) and 6 mercapto­purine (50 mg p.o.) daily; CYCIOHhospha~de (300 - 500 mg i.v. infusion) on the 1st , 3r and 5 da~i 5-fluor­ouracil (250 mg i.v. infusion) on the 2nd , 4tn and 6th day; amethopterine (5 mg i.m.) on the 1st and 4th day; vinblastine (5 mg i.v.) on the 7th day; all patients received methylprednisolone (20 mg daily i.v.) during the course of chemotherapy and until recovery of the white blood count. In 11 cases this therapy was given when glioma recurrence was diagnosed within 2.5 to 10 months after or1ginal surgical treatment. In 11 cases the combination chemotherapy was started 10 - 20 days

CCNU TREATMENT OF GLIOBLASTOMA 553

after surgical removal of the tumor; in 5 other cases chemotherapy followed the second surgical treatment. Due to the similarity of these postoperatively-treated patients, the two groups are discussed together.

3) 2~ patients received 100 mg/m2 1-(2-chlorethyl-3-cyclohexyl-l-nitrosourea) (CCNU) in 6 week intervals, starting 2 to ~ weeks following surgical removal of the primary tumor. This therapy was continued until death or until the occurrence of toxic side effects.

RESULTS

For the evaluation of the effect of the therapy in pre­venting recurrence of the tumor, the time intervals between surgical intervention and the recurrence of the tumor and between operation and death of patient, i.e. the survival time, were used. Table 1 gives the mean values for the time intervals to the recurrences and for the survival times observed for the different groups. In group 1 the tumors recurred 1.0 to 9.0 months after the first removal; these patients died 1.0 to 11.4 months after the first surgical intervention,and these values agree with the results of GRUNERT et al. (1973). With 8 of the 11 patients of group 2 receiving combination chemotherapy as a treatment for inoperable recurrences, improvements lasting for O.R to 4 months were obtained; in 2 of these cases the remissions were characterized by remarkable functional neurologic recovery, enabling the patient to lead a normal life for a few months. In the 16 cases who received chemotherapy immediately after surgery, recurrence of the tumors was observed 2.5 to 9.8 months after surgery; the postoperative survival time was between 3.~ and 18.2 months. In all patients of the 3rd group, CCNU was given following surgical removal of the tumors. The interval until a recurrence of the tumor was 1.0 to 15.7 months; postoperative survival was 1.5 to 18.5 months.

For the statistical evaluation of the results a Student-t-test was used for comparison of the mean survi­val times. Compared to the survival time of the group ,,,ith surgical treatment only, the mean survival time of patients '''ho received postoperative combination chemo­therapy was significantly prolonged, whereas the increase in survival time of patients treated with CCNU did not rench a statistically significant level.

554 W.-D. HEISS ET AL.

Table 1: Time to recurrence and survival time (mean value ± standard deviation in months) for glio­blastoma patients receiving different treatment.

Time to N Age recurrence Survival time

(months) (months)

Controls 16 22-66 4.12 + 2.98 6.27 + 3.75 -CChTh 16 18-69 5.:19 + 2.03 8.96 + 4.97x ) -CCNU 24 35-76 5.20 + 3.87 6.98 + 4.46 - -+) Different from controls t = 1.73, P < 0.05

DISCUSSION

Several Rtudies have demonstrated that the nitrosourea derivatives BCNU (1.3-bis (2-chloroethyl)-1-nitrosourea) and CCNU are able to produce objective remissions in patients with recurrent brain tumors, whether used alone (WILSON et ale 1970, 'iALKER and HURWITZ 1970, HANSEN et al. 1971, FEWER et ale 1972 a, ROSENBLUM et ale 1973) or in conjunction with other cytotoxic agents (FEWER et a1. 1972 b, HILDEBRAND et a1. 1973). Favorable effects were also observed in patients with metastatic brain tumors. Survival time was prolonged significantly only when BCNU was given together with radiation therapy ('vALKER and GEl-lAM 1972, ARMENTROUT et a1. 1974). With the combination chemotherapy described remissions were obtained in recurrent malignant gliomas and in metastatic brain tumors (HEISS et ale 1974), but this therapy was also able to prolong sur vi val time significantly \l1hen administered following surgical intervention.

The results which show a significant prolongation of survival time with combination chemotherapy and a slight but not significant increase with CCNU, are far from being satisfactory. This modest response of malignant gliomas to chemotherapy, which is not in accordance with the hypothetical considerations mentioned above, is due to the high biological malignancy of glioblastomas and to the limited permeability of the blood brain barrier for the cytotoxic agents. The lipophilic nitrosourea derivatives are able to penetrate the barrier; the concentrations reached with dosages tolerated by the organism alter the cell cycle and lead to inhibition of cell division (~HLLSON and DUFFY, 1974), but they are probably not high enough to be lethal to all glioma cells. With combination chemotherapy similar alterations

CCNU TREATMENT OF GLIOBLASTOMA 555

in mitotic activity occur, and many monstrous poly­nuclear cells and/or necroses may be observed (JELLINGER et a1. 1975), which prove that the cytotoxic substances have penetrated the tumor tissue. But again the concen­trations reached are not sufficient to prohibit permanently recurrence of the glioma.

The results may iustify one conclusion: a combi­nation chemotherapy utilizing several agents with different actions on proliferating tumor cells without containing a specifically lipophilic compound is more effective in glioblastomas than a monotherapy applying one cytotoxic agent able to penetrate the blood brain barrier. Further search for effective chemotherapy of malignant gliomas should aim at a combination of sub­stances with specific action on the cell cycle as well as specific penetration into the brain.

REFERENCES

ARMENTROUT~S.A., FOLTZ,E., VERMOND,H., and OTIS,P.T. (1974), Cancer Chemother.Rep. 58, 841.

BERING,E.A., lHLSON,C.B., and NORRELL,H.A. (1967) J.Neurosurg. 27, 1.

BRODER,L.E., and RALL,D.P. (1972) Progr.exp.Tumor Res. 17, 373.

FEWER,D., \HLSON,C.B., BOLDREY,E.B., ENOT,K.J., and POWELL,M.R. (1972 b). J.Amer.med.Ass. 222, 549.

FEWER~ D., lHLSON, G.B., BOLDREY, E.B., and ENOT, K.J. ~1972 a). Cancer Chemother.Rep.56, 421.

GRUNERT .1 V ., JELLINGER, K., SUNDER-PLA SSMANN , H., and WOtlER,G. (1973) Hodern Aspects Neurosurg.3, 108.

HANSEN,H.H., SELAWRY,O.S., MUGGIA,F.l'-1., and WALKER,M.D. (1971), Cancer Res. 31, 223.

HEISS, W. -D., KROISS,A., KOHBOCK,J., and PROFANTER, If. ~ 1974) MUnch. med. \.Jschr. 116, 1957.

HILDEBRAN~ ,J ., BRIHA YE, J ., \'I'AGENKNECI-IT, L., NICI-IEL, J. , and KENIS,Y. (1973) Europ.J.Cnncer 9, 627.

HOSHINO,T., BARKER,M., \HLSON,C.B., BOLDREY,E.B., and FEWER,D. (1972) J.Neurosurg. 37, 15.

556 W.-D. HEISS ET AL.

ISRAEL,L., DELOBEL,J., SORS,C., and BERNARD,E (1967) Proc. 5th Internat.Congr.Chemotherapy Vol.II, 2.

JELLINGER,K., HEISS,lv.-D., and GERSTNER,L. (1975) in preparation.

KERNOHAN, J. W., HABON, R.F., SVIEN ,H.J., and ADSON ,A. tv. (1949) Proc.Mayo Clin. 24, 71.

JELSl>tA, R., and BUCY, P.C. (1967), J .Neurosurg. 27, 388.

KRAYENBUHL,H. (1959), Acta neurochir. Suppl.6, 31.

ROSENBLUM,M.L., REYNOLDS,A.F., SMITH,K.A. 1 RUMACK,B.H., and WALKER,M.D. (1973) J.Neurosurg.3~, 306.

TAVERAS,J.M. (1961) Clinical Neurosurgery Vol.7. 'Williams and lHlkins, Baltimore.

WALKER, M.D. , and HURWITZ,B.S. (1970) Cancer Chemother. Rep.54, 263.

WALKER,M.D., and GEHAr.l,E.A. (1972) Proc.63 Ann.Meet. Amer.Ass.Cancer Res.13.

WILLIS,R.A. (1960) Pathology of Tumours. Butterworths, London.

WILLSON,N., and DUFFY,P.E. (1974) Neurology (Minneap.) 24, 465.

WILSON,C.B., and HOSHINO,T. (1969), J.Neurosurg.31, 589.

WILSON,C.B., BOLDREY,E.B., and ENOT,K.J. (1970), Cancer Chemother.Rep. 54, 273.

COMBINATION OF ADRIAMYCINE, VM 26, CYCLOPHOSPHAMIDE

& PREDNISONE tAVmCP) IN CHEMOTHERAPY OF LYMPHO & RETICU­

LUM CELL SARCOMA (STAGES & TOPOGRAPHIC FORMS III & IV).

x x x J.L.MISSE~ , P. POUILLART , J.L. AMIEL X L. SCHWARZENBERGX, M. HAYATx , F. de VASSAL, M. MUSSETx , D. BELPOMMEX, C. JASMINx , C.ALBAHARYxx , R. DEPIERRExXX & G. MATHEx

x Institut de Cancerologie & d'Immunogenetique, Hopital Paul-Brousse and Service d'Hematologie Institut Gustave Roussy, 94800 Villejuif

xx Hopital de Saint-Denis, 93 Saint-Denis

xxx Departement de Pneumologie, Hopital Paul-Brousse 94800 - Villejuif

The treatment of lymphosarcoma and reticulosarcoma (LRS) has been influenced by two main factors :

a) the development of new chemotherapy compounds (1), especially VM26 or demethyl depipodophyllotoxin

thehylidene glucoside (objective result rate, 45 % -see refs. 2 and 3) and adriamycin (objective result rate; 50 to 60 % - refs. 4,5 and 6) i both compounds are par­ticularly effective in treating this type of disease.

b) Improved knowledge of the course of the illness, which depends on its histocytological type and ana­

tomical aspect (7). The latter was studied during the most complete possible topographioal inventory, inclu­ding clinical examination radiology, isotopic investiga­tions and exploratory laparotomy (7) including micros­cope examination of several biopsy specimens (7). There has thus been a considerable decline in the num­ber of Stage I or II LRS cases considered curable by radiotherapy, and a corresponding increase in the number of stage III and IV cases successively treated by maxi-

557

558 J.L. MISSET ET AL.

mal regression induction chemotherapy EORTC, sometimes completed by radiotherapy applied to certain "icebergs" in the event of large initial localized tumours (10) ; such radiotherapy may be followed by additional chemo­therapy and also by immunotherapy similar to that appli­ed to acute lymphoid leukemia ; this immunotherapy has had remarkable effects on Stage IV leukemic lymphore­ticulosarcomas (11).

The aim of the present work is to set out the first results obtained using a new drug combination composed of adriamycin, VM 26, cyclophosphamide and prednisone for remission induction in Stage III and IV LRS. The combination is based on knowledge of the effects of each drug used separately (2, 3, 4, 5, 6 and 12) and on the fact that sequential administration in the order mention -ed above enables the first of these drugs to achieve (a) partial cell synchronization, and therefore cell recruitment prior to administration of the last drugs during a more sensitive phase of the cell cycle (13), and (b) potentialization of the last drugs' cytostatic action (14).

It should be pointed out that some of the patients included in the trial had already undergone failure to other treatment, whereas others had had relapses. Such patients which had poorer results than those in the first perceptible phase of the disease reduce the frequency of favourable results.

PATIENTS & METHODS

Patients

24 Patients (12 men and 12 women) whose ages ranged from 5 to 69 wure given AVmCP treatment as described be­low. In Table I, patients are classified according to the histological type and topographical aspect of their ill­ness at the time they entered the trial. There were 14 cases of diffuse lymphosarcoma : three had reached stage or topographical form III and eleven were at Stage IV and showed histologically demonstrated visceral lesions ; two patients were suffering from nodular lymphosarcoma, one being at stage III and the other at stage IV ; eight others were reticulosarcoma cases, three of them at Stage III and five at Stage IV.

21 Patients were in the first perceptible phase of their illness, two in the second and one (after two re­lapses) in the third. These three patients had already un-

CHEMOTHERAPY OF L YMPHO AND RETICULUM CELL SARCOMA

TABLE I - CLASSIFICATION OF PATIENTS ACCORDING TO­

HISTOLOGICAL TYPE OF TOPOGRAPHICAL STAGE

Diffuse Nodular Diffuse TOTAL LS LS RS

------- ------- -------

Stage III 3 1 3 7

Stage Iv 11 1 5 17

Total. •.. 14 2 8 24

TABLE II - INTERMITTENT CYCLIC SEQUENTIAL

AVmCP CHEMOTHERAPY

Day 1 Adriamycine 40 mg/m2 LV.

Day 2 VM 26 60 mg/m2 LV.

559

Days 3 & 4 Cyclophosphamide 300 mg/m2 1.M. or I.V.

Days 3 to 7 Prednisone 40 mg/-m2

Interval of 15 to 21 days.

560 J.L. MISSET ET AL.

dergone extensive radiotherapy during a previous phase.

Chemotherapy

Treatment was intermittent and comprised cycles and intervals. Each cycle consisted of the following : Day 1 - IV injection of 40 mg/m2 adriamycin ; Day 2 - IV injec­tion od 60 mg/m2 VM26 ; Days 3 and 4 : 1M or IV injection of 300 mg/m2 cyclophosphamide and Days 3 to 7 : oral ad­ministration of prednisone (Table II).

The interval between cycles, lasting from 15 to 21 days, was designed to ensure hematological and immunolo­gical restoration. A cycle was resumed only if the blood contained at least 2 500 polynuclear 1eucocytes, and 100 000 platelets per mm3.

Administration of eight similar cycles to the same patient was easily tolerated. After the eighth cycle, the use of adrimycin was discontinued because os its po­tentially toxic effects on the myocardia.

RESULTS

Toxicity

The chief toxic effects are mentioned in Table III.

Hematological toxicity was reflected in the follow­ing : Leucopenia was constant ; the nadir was under 1500 1eucocytes/mm2 in 11 cases and less than 500 in 4. Throm­bobytopenia was also constant : the number of platelets was under 50 000 in five cases and under 20 000 in one. Hematological restoration was complete in less than 21 days in all patients.

Cytopenia complications were few : a regressive fe­verish condition in three cases, but no hemorrhage. One death occurred during medullar aplasia in the case of a 63 years old man. He was suffering from nodular lymphosar­coma which had earlier been irradiated and treated with chloramphenicol between two chemotherapy cycles, after previous cycles had been well tolerated. All severe or complex cases of cytopenia occurred in patients who had already undergone radiotherapy.

In addition to hematological toxicity, the following were observed :

a) alopecia, which was constant after several cycles ; b) major or minor digestive disturbances such as nausea,

CHEMOTHERAPY OF L YMPHO AND RETICULUM CELL SARCOMA

TABLE III - T 0 X I CIT Y

LEUCOPENIA <:1500 G.B.

THROMBOPENIA ~50 000

INFECTION : Regressive

Lethal x

ALOPECIA

NAUSEA - VOMITTING (severe)

CARDIAC TOXICITY : Clinical

Biological

Severe ASTHENIA

DIABETIC DECOMPENSATION

CISTITIS WITH HEMORRHAGE

FAILURE TO TOLERATE VM 26

11

6

3

1

20

5

o 1

2

1

1

1

x Patient previously treated with chloramphenicol (see article, p. 3).

561

TAB

LE

IV

-

Dif

fuse

L

ym

ph

osa

rco

ma

Sta

ge III

Dif

fuse

L

ym

ph

osa

rco

ma

Sta

ge

IV

No

du

lar

Ly

mp

ho

sarc

om

a

To

tal

for

LS

RE

SUL

TS

OB

TA

INE

D

DU

RIN

G

STA

GES

III

& I

V

OF

LYM

PHO

&

RE

TIC

UL

OSA

RC

OM

A,

USI

NG

A

CO

MB

INA

TIO

N

OF

AD

RIA

MY

CIN

, VM

26

PRE

DN

ISO

NE

A

ND

C

YC

LOPH

OSP

HA

MID

E

Co

mp

lete

R

em

issi

on

R

eg

ress

ion

T

ota

l

!3~!§§!~!}

_~~Q_:L_

__ ~2Q_~__ _[

~!b~

f§

TOTA

L --

----

1 2

3

7 4

11

1 1

2 --

---

----

----

----

----

--

9 4

3 1

6

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

Reti

cu

losa

rco

ma,

Sta

ge III

Reti

cu

losa

rco

ma

Sta

ge

IV

3 2

3

1 2

5

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

==

TOTA

L -

ALL

P

AT

IEN

TS

1

4

4 1

5 2

4

01

0

-~

<- r 3:

~ m

-I

m

-I » r

CHEMOTHERAPY OF LYMPHO AND RETICULUM CELL SARCOMA 563

vomitting and anorexia, which occurred in five cases but, were kept under fairly good control by symptomatic treat­ment ; c) one case of acute intolerance to VM26, which was then

replaced by vincristine ; d) no cardiac toxicity was observed attributable to adria­

mycin. In one case, however, administration of the drug was stopped after the sixth cycle because of a rise in seric phosphokinase creatine.

Overall tolerance of this pattern of treatment seemed to us excellent, and we had no difficulty in applying an eight-cycle programme of intensive treatment. Even where the patient's general condition on arrival was very poor, particularly in cases of disseminated tumors, the speed of lesion regresssion enabled treatment to be continued under satisfactory conditions.

Antitumoral Effectiveness

The treatment's effects on tumoral regression are shown in Table IV. The overall complete remission rate was 58 % and the combined rate for complete and incom­plete remissions (the latter being defined as a reduction of more than 50 % in tumour size) was 75 %.

Results were obtained very quickly - often as early as after the first cycle - and all cases of complete remis­sion reached that stage after the third chemotherapy cycle at the latest. The five cases of total failure included the death during aplasia already mentioned. Two other pa­tients, as well as a third case of slight regression (less than 50 % reduction in tumour size) had all undergone pre­vious radio and chemotherapy. It seems the treatment failed to affect the course of the disease in two cases only.

No statistical evaluation can yet be made as regards the length of the remissions obtained. Nevertheless, eight out of the 14 complete remissions recorded were still last­ing in progress when the present study was made, that is, after a lapse of two to thirteen months ; six patients had relapse between three and eight months after complete re­mission was observed.

DISCUSSION

The problem of applying initial intensive chemotherapy to disseminated lympho and reticulosarcomas is at present being studied in many departments. The large number of pat-

564 J. L. MISSET ET AL.

terns of treatment suggested proves the absence of gene­ral agreement so far on anyone type of chemotherapy. Once remission has been obtained, the problem of further treatment is perhaps even harder to solve, because inten­sive chemotherapy is too toxic to be continued for long. Most research teams admit that complete remissions are short-lived.

The treatment studied in the present work seems to us of interest because it has proved highly effective and is in general extremely well tolerated. Moreover this ex­perimental protocol was recently chosen by the OERTC's Leukemia and Hematosarcoma Group as the initial intensive treatment for dissiminated LRS. After eight cycles of AVmCP, the plan is to administer further monthly chemo­therapy cycles combining vincristine, cyclophosphamide and prednisone after prior application of radiocherapy to initially large or residual lesions.

We might of course be asked why other drugs, shown by ourselves or others to be active in cases of LRS -for instance vincristine (15), nitrosoureas (objective result rate - 28 %, 16), or bleomycin (objective result rate - 40 %, 17) - were not included in our treatment cy­cles. Obviously however we had to make a choice in order to avoid using a veritable cocktail of drugs, and this choice, as well as our treatment sequence, was based on the principles of synchronization (13) and potentiation (14). Our final combination was therefore markedly dif­ferent from those that English-speaking authors often suggest for treatment of "lymphomas", such as MOPP, for which results in lympho and reticulum cell sarcoma, have been disappointing, a combination of vincristine and cyclophosphamide or prednisone for which the complete remission rate varied from 30 to 50 % depending on the authors (19, 20 and 21), or more complex associations of these drugs with bleomycine (22), BeNU (23), adriamycin or even several of the drugs used in the experiments listed under refs. 24, 25, 26, 27 and 28. Such combina­tions do not make use of the pharmacological principles referred to above and have proved extremely toxic.

The combination described here was remarkably well tolerated by patients who had not undergone previous ex­tensive radiotherapy as long as proper intervals were observed between cycles. It proved more effective than most of the combinations reported in the literature.

CHEMOTHERAPY OF LYMPHO AND RETICULUM CELL SARCOMA 565

SUM MAR Y

24 patients with advanced lynpho or reticulosarcama (Stages III and N) were treated by intermittent cyclic ch€!1Dtherapy. The cycles CClIFrised 40 grjm2 adriamycin on Day 1, 60 ng/m2 VM26 on day 2, 300 ngjm2 cyclo~SJ:tlamide on Lays 3 and 4, and 40 ngjm2 predni­sone on Days 3 to 7. Intervals between cycles ranged from 15 to 21 days.

'!he overall response rate, including CClIFlete and incarplete remissions, was 75 % and the CXInplete remission rate 58 % (14 patients) • Maxinrurn efficacy was observed after one to three cycles, and eight cycles were easily administered over 6 nonths. Hemato­logical toxicity was m::xlerate as a rule but severe in four cases. Other toxic effects were mild or m::xlerate. Median duration of remis­sion cannot yet be estimated but scxre patients are still in remis­sion after nore than a year.

REFERENCES

1. MATHE G. & KENIS Y. La chimiotherapie des cancers (Leucenu.es, hematosarcanes et tumeurs solides). 3rd ed., Paris, 1975 -Expansion Scientifique.

2. EORK: Clinical Screening Group. Clinical screening of 4 dane­thyl-epipoCbphyllotoxin D. thenylidene glucoside (VM 26) in rralignant lyrrphcmas and solid tUlIDrs. Brit. Med. J., 1972, 2, 744. -

3. MATHE G., SCHWARZE:NBER:; L., PaJILIARI' P., WEINER R., OIDHAM R. , JASMIN C., IDSENFELD C., HAYAT M., SCHNEIDER M., AMIEL J .L. , CEOARA B., MUSSEl'-STERESm M. & de VASSAL F. Essai de traite­ment de divers herratosarClClOOs par Ie 4-derrethyl-epipodophyllo­toxine D. thenylidene glucoside (VM 26 ou EPT). Nouvelle Presse Med., 1974, 1, 447.

4. JONES S., IDSENBER:; S.A., KAPlAN H.S., KADIN M. & OORFMAN R. Non Hodgkin's lyrrphoma, single agent chenotherapy. cancer, 1972, 30, 3l.

5. BCNAlJJNNA G., M:NFERDINI S., de rENA M., FOSSATI -BELIANI F. & BERETI'A G. Phase 1 and preliminary phase 11. Evaluation of adriamycin. cancer Research, 1970, 30, 2572.

6. GOrI'LIEB J. A., GUTmR-lAN J. U., MC GREDVEJ K B., IDDRIGUEZ V., & FREI E. III. Ch€!1Dtherapy of rralignant lyrrphcma with adriamycin. cancer Research, 1973, 33, 3024.

566 J. L. MISSET ET AL.

7. MA'IHE G. Les Mnatosarcarres non OOdgkiniens. Introduction, classification et inventaire tcpographique pretherapeutique. Rev. Prat., 1975, in preparation.

8. JOHNSON R. E. Patterns of invol vem:mt in non-Hodgkin's lym­phmas and the irrplications for treatment decision naking. Brit. J. cancer, 1975, 31, 237.

9. WBIANA M., POUILlARI' P., HAYAT M., SCEILIENiER M., GERARD­MAlOIANT R., SClIIlJ.1BEl{;ER J. R., BRU;ERE J., AMIEL J. L. & MA'IHE G. Resul tats de la radiotMrapie dans les stades 1 et 11 des 1ymphosa.rcares et reticulosarcanes. Bull. cancer, 1974, 61, 93.

10. POUILlARI' P., AMIEL J. L., MA'IHE G. & WBIANA M. Le traitement des 1ynph:>reticulosarccmes de 1 'adulte (a 1 'exclusion des fOl:m:s du carrefour aerodigestif superieur). Nouv. Presse Med., 1975, ,!, 247.

11. MA'IHE G. BELPCMo1E D., DAN'IO!EV D., POUILlARI' P., SCHliMBERGER J. R., & IAFLEllR M. Leukaemic 1ynphosarcmas : res~tive prognos­sis of the three types: prolynphocytic, 1ynphoolastic (or 1ym­phob1astoid) and inmmob1astic. Blood Cells, 1975, 1:., 25-36.

12. MA'IHE G., SCE.WEISGU'IH 0., BRUIE G., SCHNEIDER M., AMIEL J. L., SCHVARZENBERG L. & CATl'AN A. Essai de traitement par 1a cyclo­phosphamide de 1a 1eucemie aigue 1ynphoide et du 1ymphosarccme. Presse Med., 1963, 71, 402.

13. PCXJTI.J:ARr P., SCHWARZENBERG L., MA'IHE G., SCHNEIDER M. JASMIN C., HAYAT M., WEINER R., de VASSAL F., AMIEL J. L., BEYER H. P. & FAJBISCM:CZ S. Essai clinique de carbinaisons chimiotMra­piques basees sur la notion de tentative de synchronisation ce11ulaire. Administration premiere d'un antimitotique suivie de 1 'awlication de p:roduit (8) cycle ou phase dependant (s). Nbuv. Presse Med., 1972, 1:., 1757.

14. POUILIJ\Rl' P., SCllWARZENBERG L., AMIEL J. L. & MA'IHE G. Potentiation of drugs using charotherapy in dissaninated solid tUIlDrs : breast, bronchial and central nervous system. cancer, 1975, in press.

15. MA'IHEG., SCl-MEISGU'IHO., BRUIEG, BREZINC., AMIELJ. L., SCH'JARZENBEJ:G L., SCHNEIDER M., CATl'AN A., JASMIN C. & 9WDA R. Essai de trai terrent par 1a 1eurocristine de 1a 1eu­cemie aigue 1ynphob1astique et du 1ynphoblastosarcane. Presse rredica1e, 1963, 10, 529.

16. CARIER S. V., SCHABEL F. M., BIDDER L. E. & JOHNS'IDN T. P. BCNJJ and others nitrosoureas in cancer treat.ment ; a review. Adv. cancer Res., 1972, 15, 273.

CHEMOTHERAPY OF LYMPHO AND RETICULUM CELL SARCOMA 567

17. EORIC Clinical Screening Group. BIEDIr!YCine in the reticuloses. Brit. Med. J., 1972, ~, 285.

18. lO'lENBRAUN C., de VI'I1I. V. & SERPICl< A. A. combination chenDthe­rapy with nitrogen mustad, vincristine, probarbazine & prednisone in lyrll'tx>sarcana. and reticulun cell sarcrna. cancer, 1970, 25, 1018. -

19. HOOGSTRATEN B., Cl'lEN A. H. & LENHARD R. E. CoIrbination charothe­rapy in lyrrpb:>sarcana. & reticulum cell-sarcana.. Blood, 1969, 33, 370.

20. BAGLEY C., de VITA V., BERARD C. & c.ANNE:LOO G. AdvancEd lynpho­sarcaras intensive cycld.cal canbination charotherapy with cycle­phos~de - vincristine and prednisone. Arm. Int. Med., 1972, 76, 227.

21. LUCE J. K., GAMBLE J. F., WILSCN H. E., MCNIO R. W., ISAACS B.L., PAlMER R. L., COL'IMAN C. A. jr, HEM.E'IT J~ S., GEHAN E. A. & FREI III E. cari:>ined cyclophosphamide, vincristine and prednisone therapy of malignant lynphana. Cancer, 1971, 28, 306.

22. LUCE J. K., DEIANEY F. C. & GEHAN E. A. I&nission induction charotherapy of disseminated malignant lynphana with coobination of bleanycine, cyclophosphamide, vincristine and prednisone. Am. Ass. cancer Research, 1973, 147 66. (Abstract 262).

23. OORANT J. R., LESSNER H. E., WEB V. & VALEZ--GAK:IA E. BCNU, Cycl0ph0s~de, vincristine and prednisone therapy far mixed and histiocytic lyrrph.crna and hodgkin' s disease. cancer Charoth. Rep., 1973, 57, 103.

24. MUKHERJI B., JAGODA A. & GETIGEN H. S. Cyclic charotherapy in lyrrphana. cancer, 1971, 28, 886.

25. LEVIT!' M., MARSH J., d e <XN1'I R., MI'ICHELL M., S:KEEL R., FAH3ER L. & DERl'IN0 J. O::rnbination sequential charotherapy on advan­ced reticulum cell sarCXJna. cancer, 1972, 29, 630.

26. BCNAOONNA G. Chemioterapia dei linfani linfocitioo istiocitico p. 269-275, in "I linfani Maligni" (P. Bucalossi, U. Ve:ronesi, G. Bonaoonna, H. Emanuelli, eds). ca.sa Editrice Anbrosiana, Milano, 1974.

27. FREI III E. canbination charotherapy with bleanycin, adriaIrlYcin, cyclop,osphamide, vincristine and prednisone in mn-Hodgkin' s lym­phorra. Brit. J. cancer, 1975, in press.

28. ~ P. P. Non-Hodgkin's lynphara.. Recent observations on natural history and intensive treat:Iren.t. cancer, 1972, 30, 1511.

o 0

COMPARISON OF THE Q).MBINATIOOS a) pm + VCR , b) PIN + VCR~~, o ___ .x 0

c) PIN + VCR-4AJ..lVl ~ ~ and d) PIN + VCR + ASP

IN INDUCrrON OF FIRST REMISSICN OF ACUl'E LYMPHOID LEUKEM[A.

,/

G. MATHE, F. de VASSAL, J. L. AMIEL, P. POUII..Il\RI', L. SCHWARZENBERG, c. JASMIN', M. HAYAT, J .L. MISSET, M. MUSSEl'

Insti tut de Cancerologie et d' Irmnmogenetique, Hospital Paul-Brousse; Service d 'Hematologie de l'Institut Gustave lbussy, 948(X)- Villejuif (France)

Since the intrcx1uction of aminopterin 27 years ago, by Farber et al. (1948) (1), a dozen cmpounds have been made available to which the cells of acute lymphoid leukemia (ALL) are sensitive (see Mathe & Kenis, 1975) (6).

These a:rtp:lllIlds have been used differently acrording to different authors and shared differently between the remission induction phase and the follcwing one, called the "maintenance cherrotherapy" phase by those who treat ALL uniquely by cherotherapy (see Mandelli et al. 1973) (2) and the "pre-im­munotherapy cell mmber reduction" phase by us, who treat

o Ireans the silnul taneous canbination of the drugs

x Ireans the sequential o:nbination of the drugs

PIN = prednisone; VCR = vincristine; ADM = adr~cine; CAR = cytosine arabinoside ; ASP = L-asparaginase.

569

570 G. MATHE ET AL.

this disease by cherot.herapy followai by active :iImuno­therapy (see Math~ et al. 1969 (3), 1976 (4) ).

Our decision to a:1minister each drug in the remission induction J;i1ase or in the cx:rrplementary chstot.herapy was baserl en the folladng considerations: a) the first treat­IIelt phase is applied to a disease cmprising a great mmiJer of neoplastic cells, many of than are not in the cycle ; b) the second to the residual inperceptible disease, c:x:rrprising only a ffM logs of cells; and c) S-dependent encostatics (6-nercaptopurine, met:b:>trexate) have been sbJwn to work better than non S-dependent drugs to maintain remission (see MaW & Kenis, 1975) (6), which let us suppose that cells in remission may enter nore often into the cycle.

Thus, it seens reasonable for these above reasons to give, for remissien induction, the cmpounds which are not S-deperrlent drugs for the cmplementary cell-reducing phase.

M:>roover, the first objective is to obtain a remission in the highest prqx>rtion of patients possible. As any of these drugs, when applied alone, induces remission in > 50% of the subjects, the obj ect is to combine t:b:>se drugs ch::>sen for remission in:luction in order to obtain the maximum. percentage of remissions.

MEl'lDDS AND MATERIAL

Methais

We have ~ the follafling 4 nodalities

a) Pramisone (pm) + Vincristine (VCR) :

~ : 40 ng/m2/day x 30 days ; VCR : 1,5 ng/m2 once week x 4

b) pm + VCR, follcwed by adriamycine (ADM) in case of failure :

ADM : 10 ng/m2/day x 4 days

c) Pm + VCR, followed by adrianoccine (ADM) in case of failure, itself follcwed, if it does not induce the so-called CXllplete remission (CR.), by VCR + cytosine arabinoside (CAR) :

VCR : 15 rrg/m2 at day 1 of the cycle ; CAR : 240 ng/m2 (in infusions of 12 hours), at day 2, 3, 4 and 5 of the cycle ; cycles can be repeated.

d) pm + VCR + ~aginase (ASP) : pm : 40 mg/It12day x 30 days; VCR : 1,5 ng/m2/cnce a week x4. ASP : 10 000 ~/m2/twice weekly, for two weeks. I

TA

BIE

I

-C

XM

?LE

'IE

RE

MIS

SIO

NS

AC

CQ

RD

lN2

'10

1\G

E

NII1

BE

R

PE

RC

EN

TAG

E

OF

CO

MP

LETE

R

EM

ISS

lOO

S

DR

UG

S

OF

CA

SE

S.

IES

S T

HA

N

15

YE

AR

S

FJO

.1 1

5 '

ill

65 Y

EA

RS

pm

+

VC

R

11

4

91

%

71

/78

66

%

2

4/3

6

pm

+ VCR~AI:M

11

4

93

%

7

3/7

8

72

%

26

/36

pm

-t

VC

R -

--+

AI:

M--

+

VC

R

+

CA

R

11

4

98

%

7

7/7

8

72

%

26

/36

PD

N

+

VC

R

+ A

SP

20

1

00

%

16

/16

1

00

%

4/4

----

AL

L

AG

FS

83

%

9

5/1

14

87

%

99

/11

4

90

%

1

03

/11

4

10

0 %

2

0/2

0

z o c (')

-I o Z

o "T1

"T1 ::c

en

-I

::c

m s:: ~ o z o "T

1 r -< s::

'"

0

::t: Q

o r m

C " m s::

5>

til ......

TAB

LE

II

-C

OM

PLET

E R

EM

ISSI

ON

A

CC

OR

DIN

G

TO

THE

CY

TO

LO

GIC

AL

T

YPE

S

INCI

DEN

CE O

F "C

CMPL

E'IE

REM

ISSI

ON

S"

ACC

ORD

JN3

'IO

'!HE

C

Y'IO

IffiI

CA

L T

YPE

S N

umbe

r

of

case

s M

lCID

-PO

OLYM

PHOC

YTIC

M

AC

ro-

PID

LYM

PHO

BIA

STIC

U

NC

IAS

SIF

IED

LY

MPH

OBI

AST

IC

LYM

PHO

BIA

STIC

PDN

+ V

CR

114

100

%

23

/23

87

%

3

4/3

9

80 %

2

1/2

6

65

%

17

/26

PDN

+ V

CR

--+A

I'M

91

89

%

35

/39

84

%

23

/26

78

%

2

0/2

6

in c

ase

fail

ure

PDN

+ V

CR

-"'A

I:M

i V

CR -

CA i

n c

ase

91

92

%

3

6/3

9

92 %

2

4/2

6

76 %

2

0/2

6

of

fail

ure

l?D

N +

VC

R +

AS

P

20

100

%

2/2

10

0%

9/9

10

0%

4/4

10

0%

3/3

10

0 %

2/2

I ,

1.11

'-I ~

G) 3:

~ ::c

m'

m

-I

}>

r

INDUCTION OF FIRST REMISSION OF LYMPHOID LEUKEMIA

Patients

The patients subnitted to this study were all subjects suffering for acute lymphoid leukaemia (AIL). They were of all ages (see table I), and they belonged to the four a::mron types of ALL we have described: the prolympOOcytic, the rnicrolyrrphoblastic, the macrolymphoblastic and the prolyrrpho­blastic (Mathe et al. 1971) (7), as indicated in table II. There were no patients with the inmunoblastic type (Mathe et al. 1974) (5) included in this study.

Tolerance

No intolerance manifestations were seen in groups a, b and c. In the group of patients wlx> received PDN + VCR + ASP, 2 cases of hyperglycemia (1,60 gil) and 2 cases' of hypofibri­nemia (0,40 gil) were observed, which were benign OW'ing to symptomatic treatrrent and cessation of ASP administration.

Results

Table I shows that the simlltaneous cx:aubination of PIN and VCR gives 83 % cooplete remission (CR), noticeably less in adults (66 %) than in children (91 %).

The sequential combination of PIN + VCR followed by ADM gave slightly better results (87 %) ; but the difference from those of the first cx:aubination is not significant.

The other sequential canbination, PIN + VDR followed by ADM, followed itself if necessary by VCR + CAR slightly increased the percentage (90 %), but this difference is not significant cxxrpared to the first and serond cx:aubinations.

The simultaneous combination of PDN + VCR + ASP has induced CR in all the 20 patients yet subnitted to it : 16 children and 4 adults. The difference between this and the other rrodali ties is not significant because the nurrber of patients subnitted to it is not great enough, but it is ranarkable that all subject entered remission.

Considering the respective incidence of the CR induced by the different chenotherapy rrodalities acrording to the different cytological types, one sees in table II that the hightest percentage of CR is obtained with the first tY.O rrodalities on the rnicrolymphoblastic and prolympOOcytic types and the lowest on the prolymphoblastics,while that of the macrolynphoblastic is intermediate. With the third rrodali ty, the incidence of CR is increased for the latter type, while that of the prolyrrphoblastic is still noticea­bly lower than that of the others.

573

574 G. MATHE ET AL.

Only the last canbination gave CR in all patients, but the nurrber of subjects with the four types of ALL, was too small to allaN any definitive conclusion to be made.

SlM1ARY

The simultaneous combination of prednisone and vincristine has induced 83 % c:x:rrplete remission (C R) in ALL patients of all ages ; this oambination follaNed by adria!T!Ycine in case of failures has given 87 % CR. This cx:robination followed by cytosine arabinoside in the case of failure has given 90 % CR, and the simultaneous combi­nation of prednisone, vincristine and asparaginase has induced cx:nplete remission in all patients sulIni tted to it.

REFERENCES

1.

2.

3.

4.

5.

6.

7.

FARBER S.,DIAMlID L.K.,MEKER R.D.,SILVESTER R.F. & vDLFF J.A., Terrporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopterocyl-glutarnic acid. New Engl.J.Med.,1948,238,787. MANIELLI F., AMAOORI S., MARIANI G. (eds) Therapy of acute leukemias" Minerva Medica, Rorre, 1974.

" MATHE G., AMIEL J. L., SCllWARZENBERG L., SClINElDER M. , CA'ITAN A., SCliLUMBERGER J. R., HAYAT M. & de VASSAL F. Active inmunotherapy for acute lynphoblastic leukaemia. Lan~t, 1969, 1,697. MATHE G., de VASSAL F., IlELGAIXl M., POu:o:..IARI' P., BEIJ>CM.1E D., JOSEPH R., SCllWARZENBERG L., AMIEL J. L., SCHNEllER M. , CA'ITAN A., MUSSET M., MISSET J .L. & JASMIN C. 1975 Evaluation of BCG active inmunotherapy results in acute lynphoid and undifferentiated leukaemias. Cancer Inmmol. IImumoth., 1976, in press. MAM G., BELPO~ D., DANTCHEV D., POu:o:..IARI' P., JASMIN C. , MISSET J. L., MUSSET M., AMIEL J. L., SCHLUMBERGER J. R. , L. SCHWARZENBERG, HAYAT M., de VASSAL F. & LAFLEUR M. Irrmunablastic acute lyn¢oid leukaemia. Biomedicine, 1974, 20, 333.

MA'l'HE G. & KENTS Y. (eds) La chimiotMrapie des cancers, leuoemies, hematosar<XllEs et turreurs solides. 3e ed. 1975,.. Expansion Scientifique. MATHE G., POUILLARr P., STERESCD M., AMIEL J .L. ,SCHWARZENBERG L. ,SClINElIER M. ,HAYAT M., de VASSAL F. ,JASMIN C. & LAFLEUR M. Subdivision of classical varieties of acute leukemia : correlation with prognosis and cure expectancy. Eur.J.Clin.Biol.Res. ,1971,16,554.

CLINICAL EVALUATION OF PEPTICHEMIO IN SOME HEMOBLASTOSES

AND SOLID TUMOURS

G. Pacilio, L. Annunziato, L. Campanella, G. Scotti

Autonomous Department of General Antineoplastic Chemotherapy, Cardarelli Hospital Naples, Italy

SUMMARY

Peptichemio, a peptide complex of m-fdi-(2-chloroethyl) -aminOJL­phenylalanine was submitted for clinical assessment in 120 patients, with hemoblastoses and solid tumours. A good antitumour effect was observed, especially in malignant lymphomas and some types of solid tumours. The tolerability was good on the whole.

Peptichemio is a recent antineoplastic drug, j?repared by De Barbieri et al. (1974), made up of a complex of peptides of m-Ldi-(2-chloroethyl) -amino] -L-phenylalanine, with amino acids both physiologic and antagonistic to L-configuration. In the Department of General Antineoplastic Chemotherapy of the Cardarelli Hospital of Naples we have been using Peptichemio for more than 2 years now and we report here the preliminary results obtained.

PATIENTS AND METHODS

120 patients were treated, 111 of them are evaluable. Ages varied from 6 to 70 years. 4 different chemotherapeutic schedul es were used: A) Peptichemio was administered initially for 4 consecutive days. After a

12-day interval, the administration was resumed twice a week. S) Peptichemio was injected initially every other day for 4-6 doses. After a

12-day interval the administration was continued once or twice a week. C) Peptichemio was administered for 3 consecutive days. After a 6-day

interval the administration wasrepeated for 3 more days. After some

575

576 G. PACILIO ET AL.

TABLE 1 - Response to the treatment of lymphomas and solid t'Wllours with Peptichemio administered by 5% glucose solution phleboclysis.

Cas e s Res p 0 n s e .-

DIAGNOSIS Only Objective Treated Evaluable None Subjec- (' 5096 >500;6 tive

HODGKIN'S 17 15 2 7 6 DISEASE

LYNPHOCITIC 8 8 1 4 3 LYMPHOMA

HISTIOCYTIC 18 16 1 1 5 9 LYMPHOMA

RHABDO- 3 ; 1 2 MYOSARCOMA

OTH}JR SOFT-TISSUE 14 14 6 6 2 SARCOMAS

EWING SARCOMA 3 3 1

OSTEOSARCOI1A 2 2 1 1 2

CHONDROSARCm1A 2 1 1

MELANOr.1A 3 3 3

TOT A L 70 65 16 2 25 22

CLINICAL EVALUATION OF PEPTICHEMIO 577

TABLE 2 - Response to the treatment of the solid tumours of the adults with Peptiohemio administered in 5% gluoose solution.

Cas e s R e s p 0 n s e

D i a g nos i s Only Objective Treated Evaluable None Subjec ~5()01o tive

RHINOPHARYNX 6 5 1 2 CARCINOMA.

ORAL CAVITY 5 5 3 2 CARCINOMA.

GASTROJilliITERIC CARCINOMA. 8 8 1 6

LUNG CANCER 8 8 3 2 3

MAMMARY CARCINOMA. 13 10 2 1 5

TID'IOUR OF THE TE8- 2 2 1 TIC1E: SEMINOMA.

EMBRYONAL CARCINOMA. 3 3 2

CHORYONEPITHELIOliiA 2 2 1 OF THE UTERUS

CARCINOMA. OF UNKNOWl' 3 3 1 1 ORIGIN

TOT A L 50 46 13 5 19

References - A. DE BARBDiTIlliI et ale Atti del Simposio sul Peptichemio, lII11an, 18 Novembre 1912. Ed. LS.lII., 11ilano 1914, p. 13.

)50%

2

1

2

1

1

1

1

9

TABL

E 3

: F

irst

to

xic

si

gn

and

p

erce

nta

ge

of

all

si

de-e

ffects

aft

er

com

ple

tio

n o

f on

e o

r m

ore

cou

rses

of

Pep

tich

emio

ad

min

iste

red

by

ph

leb

ocly

sis

(sch

edu

les

A,

B,

C,

D).

S i

g n

STO

MA

TITI

S

MAR

ROW

IN

VO

LVEM

ENT

ALO

PEC

IA

NO

TOX

ICIT

Y

I A

VER

AG

E DO

SE

OF

PEPT

ICH

EMIO

EM

PLO

YED

Fir

st

tox

ic si

gn

Sch

edu

les

Sch

edu

le

A,

B,

C

D

I S

i g

n (3

3 ca

ses)

(2

9 case

s)

53%

10

%

iALO

PEC

IA

STO

MA

TITI

S

MAR

ROW

27

%

25%

IN

VO

LVEM

ENT

To

xic

si

gn

s aft

er

t~e

com

ple

tio

n o

f tr

eatm

en

t i

jChe

dUle

s A

. B

, C

(3

3 ca

ses)

86%

80%

75%

Sch

edu

le

D

(29

case

s)

49%

44%

44%

20%

15

%

iGA

STR

O-I

NTE

STIN

AL

DIS

TUR

BA

NC

ES

32%

13

%

0%

0%

FEV

ER

18%

0%

LOCA

L N

ECR

OSI

S 8%

7%

NO

TO

XIC

ITY

0%

0%

AV

ERA

GE

DO

SE

OF

2 15

0mg/

m2

PEPT

ICH

EMIO

2

2 l3

Om

g/m

EM

PLO

YED

ON

TH

E 38

0 m

g/m

48

0mg/

m

WHO

LE

01

'.

j ex

>

G)

""0 » n r 0 m

-I

» !"""

CLINICAL EVALUATION OF PEPTICHEMIO 579

other 6 days it was resumed once or twi ce a week. D) Was analogous to schedule C, except for the length of the intervals which

was 12 days.

28 patients were treated with schedule A, 30 with schedule B, 18 with schedule C and 35 with schedule D.

The single 25 mg m2 dose was administered rapidly i.v in 5 percent glucose (20-30 minutes), followed by saline. This dose was reduced to 15 mg m2 in pre-treated pati ents or those over 65 years of age. The dosage was regulated and timed according to the haematology of patients. The total average dose ranged between 130 and 480 mg m2• The response was classified as follows: subjective response: symptomatic improvement, without any effect on the tumour maSSj objective improvement, a change in the neoplastic mass of more or I ess than 50 percent. Tabl es 1 and 2 report a breakdown of the cases treated and of the results obtained. Table 3 gives the type of toxicity encountered with the different schedules.

RESULTS

Peptichemio seems to have good antineoplastic activity in haemoblastoses and solid tumours, particularly in the treatment of hystiocytic lymphoma (III and IV stages) and other malignant lymphomas. A useful response was also obtained in several solid tumours. A remarkable effect on pain was observed, after the first administration. Tolerance was gocx:l on the whole; side effects were observed mainly in the hemopoietic system, but they were of short duration and subsided after withdrawal of the durg and were less evident with schedule D. Alopecia, gastro-enteric disturbances, venous troubles were also observed. Pepti chemi 0 has shown a gocx:l effect in the treatment of hemoblastoses and solid tumours, alone or in combination with other drugs, as demonstrated by our ~perience and by that of other researchers in Italy and abroad.

CHEMOTHERAPEUTIC MANAGEMENT OF NON-HODGKIN'S LYMPHOMA.

COMPARATIVE STUDY OF VARIOUS COMBINATIONS

NAZLI GAD-EL-MAWLA, M.D. et al

Cancer Institute, Cairo University

Kasr El-Aini Street - Cairo - EGYPT

SUMMARY

74 patients with non-Hodgkin's lymphoma attending the Medical Oncology Dept., Cancer Institute, Cairo University, received at random two regimens of combina­tion chemotherapy. The group who received MOPP achieved 60%, while the group receiving CPP achieved 56% CR.

Although malignant lymphoma patients represent 7.8% of those attending the Cairo Cancer Institute annually, yet amongst those attending the Medical Oncology Dept •• Medical and Pediatric Units, it stands the first.

Non-Hodgkin's lymphoma cases are more prevalent than Hodgkin's cases. Thus, in the year 1973, amongst 281 cases of malignant lymphoma attending the Cairo Cancer Institute, 127 were of the Non-Hodgkin's type, with the lymphocytic type predominating.

Chemotherapeutic management of Non-Hodgkin's lym­phoma still poses an international problem. For us, its management is of particular importance due to its pre­valence together with the fact that we usually deal with advanced stages I III & IV. 75% of our cases belong to stage IV. Moreover, the diffuse type is more prevalent than the nodular type.

The present study evaluates the results of combina­tion chemotherapy in patients with Non-Hodgkin's lym­phoma.

581

582 N. GAO-El-MAWlA

MATERIALS AND METHODS

The studied group of patients were those suffering from Non-Hodgkin's lymphoma. They were divided into two groups of patients by randomization. The first group comprised 33 patients, 27 males and six females. They received the classical MOPP combination chemotherapy for six consecutive courses. They were followed up for six months. The second group consisted of 41 patients, 30 males and eleven females. They received CPP, each course for four weeks with two weeks rest, for four consecutive courses, and were followed up for six months (Table 1).

Hemogram including peripheral blood counts, bone marrow examination, sedimentation rate and alkaline phosphatase were done before therapy and after fini­shing each course.

Evaluation of therapy was done clinically as well as radiologically by roentgenologic examination of the chest bones and lymphography.

DISCUSSION

The first data on combination chemotherapy in Non­Hodgkin's lymphoma were published by Hoogstraten et.al~ More than 30% of patients achieved ~ CR after CTX-VCR­PRED. Subsequently Lowenbraun et al~), reporting the results with MOPP achieved 47% CR in lymphocytic and 38% with histiocytic. Other combinations followed using ADM, BLM or both with much better results.(.3)

TABLE I

MOPP: 6 mg/m~ NH2 days 1 x 8 I. V.

VCR 1.4mg/m2 days 1 x 8 LV. PROC 100 mg/m2 days 1-14 P.O. PRED 40 mg/m days 1-14 P.O.

CPP, CTX 200 mg. I,V. e.o.d.

PROC alternating with: 100 mg P.O. e.o.d.

PRED 40 mg P.O. daily.

MANAGEMENT OF NON-HODGKIN'S LYMPHOMA 583

Results I are presented in tables II and III.

TABLE II Results of treatment of 33 patients with MOPP

Histologic Subtype NM NLPD DLWD DM WLWD H.D

TOTAL

CR 3/4 2/4 2/4 1/4 2/4

10/13 20/33

TABLE III

PR 1/4 1/4 2/4 2/4 1/4 2/13 9/33

NR 0/4 I/.4 0/4 1/4 1/4 1/13 4/33

Results of Treatment of 41 Patients with Combina­tion Chemotherapy (CPP).

Histologic Subtype CR PR NR NM 3/4 1/4 0/4 NLPD 4/9 2/9 3/9 DLWD 2/4 1/4 1/4 DM 2/6 2/6 2/6 WLWD 1/1 H.D. 11/17 4/17 2/17

TOTAL 23/41 10/41 8/41

In the present study, those receiving the MOPP achieved CR in 20/33,i.e. 60%, although most of them were of the diffuse type. In the second group on CPP,CR was achieved in 23/41 i.e 56%, most of the cases were of the diffuse type. All these patients are still in CR, follow up of 6 months.

1.

2.

3.

REFERENCES

Bonadonna,G., De Lena,M' t Lattuada,A.(1974). Br. J. Cancer. (in press).

Hoogstraten,B. (1969), Blood 33.370.

Lowenbraun, S., De Vita,V.T., Serpick,A.A.(1970). Cancer 25: 1018.

CHEMOTHERAPY FOR GASTRIC AND COLORECTAL CARCINOMA BY

INTRA-AORTIC INFUSION

Kenzo Yoshikawa & Ichiji Ito

National Cancer Center Hospital,

Tsukiji 5-1-1, Chuo-ku, Tokyo, Japan

Successful responses to chemotherapy of unresectable or recurrent cancer of the stomach, colon and rectum are unexpected. The advance of surgical technique for these carcinomas seems to be already maximal. Despite consi­derable hope surgeons are disappointed in the results of chemotherapy.

Intra-arterial infusion treatment for gastric and colorectal carcinoma has limitations because several regional arteries supply the stomach, colon and rectum. From the new approach whereby the mode of dissemination in advanced gastric and colorectal carcinoma is considered infusion treatment by regional arteries of the stomach, colon and rectum is not always satisfactory. In many cases aggressive administration of anticancer drugs, should be given throughout the entire abdominal cavity. Therefore, intra-aortic infusion treatment (1,2) has been ap~lied in cases with diffuse tumor dissemination. However, bio­logical behavior is different in each cancer. While per­itoneal dissemination frequently occurs with gastric cancer, local recurrence frequently follows rectal cancer.

MATERIALS AND METHODS

This study deals with 60 evaluable cases, of 60 un­resectable or recurrent cases with gastric cancer and 21 evaluable of 26 cases with colorectal cancer who received this infusion treatment between October 1969 and March '75 in the Department of Surgery, National Cancer Center Hositpal, Tokyo, Japan.

585

586 N. GAO-EL-MAWLA

A Teflon catheter was introduced through a branch of the femoral artery. The tip of the catheter was placed above the celiac axis in cases of gastric cancer and carcinomatous peritonitis. In a locally recurrent cancer of the rectum, it was placed above the aortic bifurcation. The Proximal portion of this catheter was brought to the outside on the subclavian part and subcutaneously through the abdominal and thoracic wall and connected to a port­able infusion pump forthe continuous infusion of anti­neoplastic agents. The reason for passing the catheter through the subcutaneous tissue is to avoid dislocation, to prevent local infection around the site of catheter introduction into the artery and for ease of the patient's movement.

Infused durgs and their daily doses are shown in Table 1. The drug chiefly administered was 5-FU, which is most appropriate in this infusion treatment, because the effectiveness of 5-FU is independent of its concentration, but depends upon its duration time of administration. Cytosine arabinoside or Mitomycin C were occasionally combined with 5-FU. Administration period was 1 to 12 months.

The effectiveness was mainly judged by Karnofsky's criteria. Though the survival time indicates one of the most important factors, it merely provided a guide because of insufficient number of the cases. Good responses means those cases with mainly improvement of symptoms for at least 1 month or more. Cases which responded transiently were judged as fair.

Based upon findings at operation or autopsy the princ­ipal modes of metastases in gastric cancer were classified

Table 1. Dosage of Antineoplastic Agent s infused

5-fluorouracil

Cytosine arabinoside

Mitomycin C

Furanidyl 5-FU

Neocarcinostatin

Adriamycin

(per 24 hours)

135-250mg

10- 20mg

1- 2mg

200-500mg

3- 6mg

10 mg

GASTRIC AND COLORECTAL CARCINOMA

Table 2. Main Modes of Metastases -Gastric Cancer-

Type I. Dissemination

Ia. Simple Dissemination

lb. Other Special Subtype except for 1a

Type II.

Type II I.

Direct Invasion

Metastases to Liver

as shown in Table 2. The first type was conveniently divided

587

into two subtypes. The one indicates the group of simple peri­toneal dissemination often associated with a large amount of ascites. The other means the group in which tumor infiltration extends into the retroperitoneum or the mesenterium possibly lymphogenously besides peritoneal dissemination and is often followed by the adhesion between the loops of small intestine or followed by the shortening of the mesenterium. The second type includes the group in which there is a continuous infiltration into adjacent visceral organs from gastric lesions and, therefore, the resection is frequently impossible. Even if combined re­section of the stomach with the pancreas, the spleen, the liver and the transverse colon was performed, the prognosis of such cases was so poor as compared with that of unresected cases in previous experiences. The third type includes the group with liver metastases mainly by the route of portal vein.

Based upon the principal modes of metastases, the cancer of the colon and rectum was also classified. Dissemination is rarely observed in cancer of the colon and rectum. On the other hand hepatic metastases are frequently observed in cancer of the colon, particularly the signoid colon. In cancer of the rectum, most of the recurrences belong to a locally recurrent type (Table 3).

Table 3. Main Modes of Metastases -Colon and Rectal Cancer-

1. Tumor Type

Locally Recurrent Type

The Other Type

II. Type of Hepatic Metastases

III. Type of Peritonitis

588 K. YOSHIKAWA AND I. ITO

Ia I

Ib

II

III

Total

Table 4. Modes of Metastases and Clinical Response

good fair

6/11 54.5'70 2/11 18.1% :3/11

0/13 3/13 23.0% 10/13

2/32 6.2'70 11/32 34.3% 19/32

0/4 1/4 25.0% 3/,1

t)/60 13.3'70 17/60 28.3% 35/60

RESULTS

no

27.2%

76.9'70

59.3%

75.0%

5~. 3'70

According to the classification by the principal modes of metastases in gastric cancer as before mentioned, 6 of 11 cases with the type of simple peritoneal dissemination showed good response. On the other hand, any fruitful results from the procedure were not available in most cases with the type of direct invasion, in all of 13 cases with the subtype Ib, or in all of 4 cases with the type of hepatic metastases (Table 4).

Most of the cases belonged to the third and the fourth type of Borrmann's classification. Three of 38 cases with the third type showed good response and 5 of 16 cases with the fourth type responded better (Table 5).

As most of the cases revealed signet ring cell carcinoma or poorly differentiated adenocarcinoma, the response is not dis­cussed by the difference in the degree of the differentiation (Table 6).

Table 5. Borrmann's Classification and Clinical Response

good fair no

llor.4 5/16 31.2% 6/16 37.5% 5/16 31.2'70

13or.3 3/3~ 7.8'70 9/38 23.6% 26/38 6~.4'70

Bor.2 0/5 2/5 40.0'70 3/5 60.0'70

Bor.l 0/1 0/1 1/1

GASTRIC AND COLORECTAL CARCINOMA 589

Table 6. Histological Type and Clinical Response

good fair no

Papillary 0/2 0/2 2/2 adenocarcinoma

Well/Moderate 0/11 3/11 27.2% 8/11 72.7"10

diff . tUb. adenoca.

Signet ring cell 5/36 13.8% 11/36 30.5% 20/36 55.5%

carcinoma

Poorly diff. 3/11 27.2% 3/11 27.2"10 5/11 45.4"10 adenocarcinoma

Table 7 shows the correlation between the principal modes of metastases and clinical response in colon and rectal cancer. Good response was obtained to a locally recurrent type of the rectal carcinoma.

DISCUSSION

Successful chemotherapy for gastric and colorectal carcinoma is still difficult at present. In this paper, the principal modes of metastases were especially classified in each cancer based upon the findings at operation or autopsy. Good responses were obtained to a type of simple peritoneal dissemination in gastric cancer and to a locally recurrent type in rectal cancer.

Gastric cancer is frequently followed by carcinomatous peritonitis. While the cases with simple dissemination type are frequently accompanied by a large amount of ascites in earlier stage, most of the cases with direct invasion type are followed by the symptoms of carcinomatous peritonitis at terminal stage.

Table 7. Modes of Metastases and Clinical Response

good fair

locally recurrent 4 1

tumor () 1

hepatic metastases 0 0

peritonitis 1 ()

no

2

10

2

0

590 K. YOSHIKAWA AND I. ITO

Therefore, the effectiveness of this infusion treatment upon carcinomatous peritonitis descended from 54.5% (6/11) to 14.2% (8/56) in wide meaning.

On the other hand, cancer of the rectum is frequently followed by a locally recurrent type from anatomical condition. However, good responses were obtained to this type in rectal cancer. I cannot understand the difference between the types of gastric and rectal cancer which responded.

SUMMARY

Over a 5-year period, 92 patients with unresectab1e or recurrent carcinoma of the stomach, colon and rectum were treated by the continuous infusion of antineoplastic agents, chiefly 5-FU, into the aorta at a level above the celiac axis or aortic bifur­cation for a period of 1 to 12 months. Eight of 66 (60 evaluable) cases with gastric carcinoma and 5 of 26 (21 evaluable) cases with colorectal carcinoma obtained good response. This procedure resulted in success with a simple peritoneal dissemination type of gastric carcinoma and with a locally recurrent type of rectal carcinoma.

REFERENCES

1. Yoshikawa, K. , et al.: Prolonged Intra-aortic Infusion Therapy with Antitumor Agents for Advanced Cancer of the Stomach, Colon and Rectum, Jap. J. Surg. 1:256-262, 1971.

2. Yoshikawa, K., et al.: Continuous Infusion Method of Anti­tumor Agents through Intra-aortic Catheterization, Jap. J. Cancer Clin. 18:133-137, 1972. (in Japanese)

EFFECT OF ADJUVANT CHEMOTHERAPY WITH MITOMYCIN C ON THE RECURRENCE

OF GASTRIC CANCER AFTER RADICAL SURGERY

Toshifusa Nakajima, Tamaki Kajitani, Atsuo Fukami and Ichiro Ohashi Surgery Department of Cancer Institute Hospital, Tokyo

1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

The administration of Mitomycin C in prolonged small dose regimens was performed on 340 cases of gastric cancer after radical surgery. All the cases were followed postoperatively for 8-12 years. The cause of death was surveyed for the deceased cases for the eval­uation of the inhibitory effect of the present method on recur­rence in five types. Local recurrence was inhibited by a mean of 13% in cases of serosal involvement (S+), infiltrating tumor type, and undifferentiated cancer. Peritoneal dissemination was suppressed by a mean of 15.2% in cases of N2-N3 lymph node metasta­sis, S(+), infiltrating type, undifferentiated cancer, and vascular invasion (V+). Hematogenous metastasis was controlled by a mean of 13.8% in cases of localized and intermediate type and differentiated cancer and tended to be inhibited in cases of (V-).

In spite of the recent progress of cancer chemotherapy, ther­apeutic results of solid cancer by chemotherapy alone are not sat­isfactory. There are not a few reports on the slight life-prolonging effect of the combination of radical surgery and various chemo­therapies. At the Surgery Department of Cancer Institute Hospital, Mitomycin C (MMC) was administered postoperatively in a prolonged small dose regimen to patients with gastric cancer after radical surgery from 1961 to 1966, whose postoperative course was observed for 8 to 12 years. We evaluated the effect of chemotherapy based on the postoperative survival rates and also the rate recurrence in five types.

Materials and Methods Of 485 patients receiving radical surgery for gastric ca~cer

in our department during 1961-1966, 145 were excluded for discon­tinuation of medication or for other reasons (other malignant

591

592 T. NAKAJIMA ET Al.

disease double cancer, etc). The patients were divided into treated and control groups in alternate fashion preoperatively. A dose of O.08mg/kg of MMC was given 10 times starting on the day of operation (twice weekly or daily). The cause of death was investigated by autopsies or other clinical findings. Recurrenceswere classified into five types; local recurrence, peritoneal dissemination, hematogenous metastasis, recurrence in the residual stomach and distant lymph node metastasis.

Constitution of Subjects Of the 340 cases, 132 were in the treated group and 208 in the control group. There was a considerable number of cases of dis­continuation and excluded cases, but there was no difference in the

clinical stages related to the lymph node metastasis and serosal involvement between the treated and control groups.

Results I. Survival after Surgery

Postoperative survival curves of treated and control groups are shown in Fig. 1. In the upper graph which includes cases dying from causes other than cancer or from unknown causes, signifi­cant difference was observed between two groups up to 9th post­operative year (the difference in the five year survival rate is 17.9%). The lower graph shows a significant difference up to the 3rd postoperative year and in the 7th year. The mark~ showed that P value is between 0.1 and 0.05. At other points there was no significance in the survival rates between two groups.

II. Recurrence rates according to the types of recurrence. In order to equalize as much as possible background factors which influence prognosis, the cases were classified into several groups according to the following factors, the extent of lymph node metastasis and serosal involvement, gross pathology, his­tology, and the presence or absence of vascular invasion of cancer cells in the primary lesions. Eight years recurrence rate was compared in the treated and control groups. 1) Recurrence rates related with lymph node metastasis no and nl group*l showed no difference in the 8 year recurrence ~~te of any kind between two groups. In the n2 and n3 groups ,the rate of peritoneal dissemination was significantly different between treated and control groups (13.9%).

*1. No lymph node metastasis and metastasis to the primary regional lymph nodes near the stomach. *2. Metastasis to secondary lymph nodes of stomach and to the re­gional lymph nodes of organs adjacent to the stomach. *3. S(-): Cancer does not involve the serosa.

RECURRENCE OF GASTRIC CANCER AFTER RADICAL SURGERY

Loc. . 1

H .·nl d l '~~"hf..u!.

I .. . ·t..,.~ t"' ':10 . ...

° r ", I..,,1I1

' ym~Jh

11fJ(J t" )o

Postoperative survival curves of the treated and control "rcups

top ,nclud.,"C de .. d c •• es due to c.uses. other t~n cMK.r bot tom •• ctud1nc de.d C.,., due to C...".5 other than c..,cer

-..'"---------___ U.,,, 4' :1"'7.1·;-.--~--~-""" ___ 'I.'"

10 II 12

• ., •• r .fl., aur •• ry

.", ...l. ~----""'---4~--____ IO.".

10 . ~~; ~I::"->--Oo_--_____ --oo 43.'" ~

10 II 12

Figure 1

Inhibi tory Effect of AdjuvMt Chemotherapy on Various Types of Recurrence.

.p OUI P a U\ [.=J 0 01 PO I

Figure 2

593

594 T. NAKAJIMA ET AL.

2) Recurrence rates related with serosal involvement S(-) group*3 showed no difference in each recu~rence rate between treated and control groups. In the S(+)*4 group, statistical difference in recurrence rates of the local recurrence and peritoneal dis­semination was observed between treated and control group (12.0% and 14.7% respectively).

3) Recurrence rates related with gross pathology. When the cases were classified into 3 groups, namely localized and intermediate tumor type, infiltrating type and superficial type, difference in the recurrence rate of hematogenous metastasis was observed in the localized and intermediate type (15.4%). Difference in local recurrence and peritoneal dissemination was observed in the infiltrating tumor group (13.7% and 16.3% respectively).

4) Recurrence rate related with histology in the differentiated cancer group, statistical difference in hematogenous metastasis was observed between treated and control groups (11.9%). In the undifferentiated cancer group, difference in local recur­rence and peritoneal dissemination was observed between both groups (13.3% and 18.9% respectively).

5) Recurrence rate related with vascular invasion positive vascular invasion group (V(+» showed a significant difference in the recurrence rate of peritoneal dissemination (12.3%). A slight difference, but not significant, in the hematogenous metastasis was observed in the negative vascular invasion group (V(-». In Fig. 2 is summarized the difference in rate of each recurrence type related with various factors. Difference in the rate of local recurrence between treated and control groups was observed in the cases of S(+), infiltrating tumor typ~ and undifferenti­ated cancer. Difference in the peritoneal dissemination was observed in the cases of N2(+)-N3(+) group, S(+), infiltrating tumor type, undifferentiated cancer and V(+) group. Difference in hematogenous metastasis was observed in the cases of local­ized and intermediate tumor type, and differentiated cancer and slight, but no significant difference in the V(-) group. Difference in the rate of recurrence in the residual stomach and in the distant lymph nodes was not evaluated because of small number of cases.

Discussion It has been reported both from animal experiments and clinical

trials that combination of surgery and chemotherapy for the solid cancer yields better therapeutic results than their inde-pendent use. In the previous reports, the evaluation of chemo-theraputic effect was based on the survival rate after surgery. We tried to evaluate it not only from survival rate but also the recurrence rate of various types of relapse whose clinical stages were in almost the same composition between the treated and control groups.

*4. S(+): Cancer involves the serosa.

RECURRENCE OF GASTRIC CANCER AFTER RADICAL SURGERY 595

Difference in the recurrence rate between both groups was attri­buted to the suppressive effect of chemotherapy with MMC on the recurrence of gastric cancer. We also reported that the admin­istration of MMC in the same regimen was ineffective for noncurative cases with gastric cancer in which cancer focus remained postoper­atively by gross observation. These findings might suggest that the suppressive effect of chemotherapy with MMC in a prolonged small dose regimen would be dependent upon the amount of tumor cells left behind the surgery.

LONG-TERM CANCER CHEMOTHERAPY FOR STAGE III TO IV GASTRIC

CANCER FOLLOWING NON-CURATIVE RESECTION

Taiko Abe, Tetsuro Kajiwara, Tetsuro Kamata & Shegeo Tsuboi Department of Surgery 2nd Hospital of Tokyo Women's Medical College Tokyo, Japan

Diagnostic technique and surgical treatment of stomach cancer has made progress in recent years. Benefit however, has been limited to early stage cancer. The poor prognosis of advanced cancer in stage III to IV remains a challenge.

In order to improve therapeutic results in this ad­vanced stage cancer, we have since 1965, been using anti­cancer chemotherapy as an adjuvant against gastric cancer after non-curative resection. The purpose of this report is to describe the effect of chemotherapy on advanced stage gastric ancer following non-curative resection.

Cases included 30 patients with stage III and 20 patients with stage IV gastric cancer following non-curative resection. The rate of curative resection in our hands was 34.2% during the same period.

As the initial treatment, a large dose of Mitomycin C (MMC)(lmg/kg of body weight) was Qiven to 23 cases and intermittent large doses (8 to lomg/week) was administered to 20 cases. In the remaining patients, M.F.C. was used instead. All the patients were placed on the long-term maintenance therapy with 5-FU in a dose of 500mg once or twice weekly.

Beneficial effects of the chemotherapy were shown by the favourable survival rates of our patients. I year survival was obtained in 50%, 2 year in 30%, 3 year in 20% and 5 year in 15% of the patients, and 38% of them were alive and well at the time of this survey. Our results could be compared favourably with those

597

598 T. ABE ET AL.

from other institutions and our survival rates over two years fol­lowing operation were better than those in other reports.

STUDY MATERIAL

The material consists of 50 cases of advanced stomach cancer after non-curative resection admitted to our service during the 10-year period from 1965 to 1974. Criteria for the evaluation of non­curative resection were based on the histopathological findings on the cancer tissue remaining at the cut end or remaining lymph node metastasis, peritoneal metastasis and hepatic metastasis, depending on inadequacy of resection.

Thirty-five patients belonged to stage III and 15 to stage IV. There were 23 males and 27 females, most of them in their fifties. During the same period, gastrectomy was performed on 315 patients of whom 115 were malignant.

Modes of administration

I. Super large dose of MMC 23 (0.8-1 mg/kg XI)

2. Intermittent large dose of MMC 20 (8-10mg/W X 4 - 5)

Study Material 3. T riagents of M.F.C. (MMC4mg+ 5FU500mg+C2OIg/W xl0) 7

Stage Total amount of MMC

Stage ill 35

Stage N 15 I. less than 50mg 35 2. 50 -100mg 13

Sex 3. more than 100mg 2 Male; Female = 23 ; 27

Age 6

Total amout of 5FU 30 - 40y 40 - 50 12 I. less than 5000mg 14

50 - 60 17 2. 5000 - 1 0000 kg 9

60 - 70 12 3. 10000 - 30000 mg 10 70 - 3 4. more than 30000 mg 14

total 50 5. 0 3

Figure 1 Figure 2

LONG-TERM CANCER CHEMOTHERAPY 599

MODES OF Am:INISTRATION

Three modes of administration were tried.

1. Super large dose of MMC of 0.8 to 1 mg/kg was given to 23 cases.

2. Intermittent large dose of MMC of 8 to 10 mg/week (1 mg/kg in total) was tried in 20 cases.

3. Tri-agent cembinatien ef M.F.C. was given to. 7 cases.

As a maintenance therapy, 5FU ef 500 mg was given ence er twice a week en leng-term basis and in case ef recurrence ef cancer, the above mede 1, 2 er 3 was repeated. The detail en the super large dose treatment was reperted already at the 9th meeting ef this Cen­gress.

The total ameunt ef MMC administered was less than 50 mg in 35 cases, 50 to. 100 mg in 13 cases and mere than 100 mg in 2 cases. 5FU given tetalled more than 30,000 mg as a long-term maintenance in 14 cases and less than 5,000 mg in 14 cases.

Histelegically, 39 had adenecarcinema, 5 carcinoma simplex, 5 scirrhesma cancer and 1 gelatineus cancer. After gastrectomy, Bil­lreth-II was dene in 37 cases and Billreth-I in 11 cases. Tetal gastrectemy was done in 2 cases.

RESULTS

Effect en survival span. Out ef 20 cases studied frem 1965 to. 1969, 50% survived fer 1 year, 30% for 2 years, 20% fer 3 years and 15% for 5 years. In stage III, the 5-year survival rate was 27.3%, which appreximates that after curative gastric resection performed in eur department during the same study peried.

Medes ef administratien and pregnesis. The super large dese administratien ef MMC has been preved effective in view of the 3- to. 4-year survival rate.

Pathelogical finding in non-curative gastrectomy and pregnesis. The mest impertant facter leading to. nen-curative gastrectemy was inadequate lymph nede resection and residual cancer tissue at the cut end follewed it. As to. prognesis, cases with remaining lymph nede metastasis and residual cancer tissue shewed relatively goed pregnosis. Macrescepically, Berrmann II shewed relatively better

600

~ Survivil hVlng Yllrs

1< 0

1-2 0

2-3 0

3-4 0

4-5 0

" 5 3

100"10

50"10

T. ABE ET AL.

Effect on survival span

death total 100,"

10 10

4 4

2 2

0 0

1 1

0 3

Figure 3

5 years passed Cases (20/50)

1965-1969

2 3 4 5y

Modes of administration and prognosis

0--0 Super large dose of MMC

Intermit tent large dose of MMC

~ M.F.C.

2 3 4 5Y

Figure 4

LONG-TERM CANCER CHEMOTHERAPY

~ finding Survlv.1

.. rs

<1

1-2

2-3

3-4

4-5

> 5

total

R?\ SurVlV" ,.." < 1

1-2

2-3

3-4

4-5

> 5

total

;?\ Sur"'I..,.1 ,.. .. <1

1-2

2-3

3-4

4-5

> 5

total

Pathological findings and prognosis

ow aw S, p N H H 3-4 H

100~

13 9 6 6 13 2 \ - ow positive

4 4 1 4 10 0 \ - S, II.

1 0 0 0 6 0 '\ - P

0 0 0 0 1 0 50~ '\ Ns _ .. .... -'\

0 0 0 0 3 0 , .. , .. ,- (owaw SPN . =,::~~ogy)

0 2 0 0 1 0 ................

18 15 7 10 34 2 5y

Figure 5

Type of cancer and prognosis (Borrmann)

I n

1 3

0 1

0 3

0 1

0 1

0 3

1 12

- ...... dlffer.l- ~ff. hit.. ... ... t". t".

12 4

7 3

5 1

0 0

1 2

1 1

23 11

m N

16 4

12 0

3 0

0 0

2 0

0 0

33 4

Pathology and prognosis

Sewr· CarCI--. --cane: ... Sonple. 100'"

4 3

1 2

1 0

1 0

0 0

1 0

8 5

Figure 6

- AdInoeIreinorM differentiated type _~~fftNntiltedt".

- SCir ........ etneer

"'"- c.rcinDml ..... x

601

602 T. ABE ET AL.

Side effect

I. Leucopenia «3000) 25

2. Thrombocytopenia « 5 X 104 ) 3 3. Anemia «300XW) 10

4. Albuminuria 13 5. Hypoprotnemia «6g/dJ) 18 6. GOT, GPT t (>I00ut) 6

Causes of death

I. Recurrence of cancer 18

2. other

Ileus 4 Fulminant hepatitis 4

iii Cerebral hemorrhage I ;., Unknown 4

(non-cancerous death 32.2",)

Figure 7

results than Borrmann III and all the four cases with Borrmann IV expired within a year after surgery and the effect of chemotherapy was not observed. This treatment was ineffective against simple cancer but effective against undifferentiated adenocarcinoma.

Side effects. Leucopenia (less than 3000) was seen in 50% of the cases, thrombocytoyenia (less than 5 x 104) in 6%, anemia (less than 300 x 104) in 20%, increase in GOT and GPT by 100 U or more in 30% and albuminuria in 36%.

Autopsy of 31 cases revealed recurrence of cancer in 18 cases, ileus in 4, fulminant hepatitis in 4, cerebral hemorrhage in 1 and other lesions in 4. Thus, non-cancerous death was seen in 10 cases (32%). This fact tells us the importance of careful clinical con­trol of the patients under this mode of treatment.

COMMENT AND CONCLUSION

1) Compared with the 5-year survival rate at about 10% in non­resected gastric cancer reported by other institutions in Japan, it can be said that the results of our treatment are relatively better. The 5-year survival rate at 27.3% closely approaches to that of cur­ative gastrectomy performed during the same period, and an adjuvant use of chemotherapy seems to be effective.

2) The super large dose administration of MMC followed by long­term maintenance with 5FU was found to be effective.

LONG-TERM CANCER CHEMOTHERAPY 603

3) As to non-curative findings, our treatment was effective in the cases with rema1n1ng lymph node metastasis and cancer tissue re­maining at the cut end.

4) From histological point of view, the therapy was effective against undifferentiated adenocarcinoma with remaining lymph node metastasis. This seems consistent with the conclusion of Nishi on extensive lymph vessel involvement in undifferentiated adenocarci­noma.

5) By reviewing the side effects and the causes of death, es­pecially bone marrow and hepatic dysfunction leading to non cancer­ous death in 32.2%, it should be said that the development of new and safer anticancer agents and improved use of now available agents are needed.

(General rules for gastric cancer study in surgery and pathol­ogy established by the Japanese Research Society for Gastric Cancer (1971) were used to classify our cases.)

CHEMOTHERAPY FOR ADVANCED OVARIAN MALIGNANCY

Umberto VillaSanta

University of Maryland School of Medicine Department of Obstetrics and Gynecology 22 South Greene Street, Baltimore, Maryland 21201

Two years ago I had the honor and the privelege to present at this society the results of treating a hundred cases of ovarian carcinoma with Thiotepa. While there was a satisfactory effect in Stages Ic through III, the results were disappointing in Stage IV patients, and there were no survivors after twenty months.

In an attempt to improve these results, a different regime has been tried in patients with Stage IV carcinoma of the ovary. Ac­cording to the latest bulletin from the Radiumhemmet, one must al­locate to this stage all cases with distant metastases, cases with pleural effusion and positive cytology, and those with parenchymal liver metastases. (Table 1)

A group of fifteen patients with Stage IV epithelial carcinoma of the ovary have been treated with a combination of drugs after initial surgery. Six patients had widespread abdominal and hepatic metastases, two had metastatic supraclavicular lymphnodes, six had pleural effusion with positive cytology, and one had metastases in the supraclavicular lymphnode and pleural effusion. (Table 2)

Stage IV

Growth involving one or both ovaries with distant metastases.

If pleural effusion is present there must be positive cytology to allot a case to Stage IV.

Parenchymal liver metastases equals Stage IV.

TABLE I

605

606 U. VILLASANTA

ADENOCARCINOMA OF OVARY STAGE IV

Hepatic metastases 6

Supraclavicular lymph-nodes metastases 2

Pleural effusion 6

Pleural effusion and supraclavicular 1. n. metastases 1

TOTAL 15

TABLE II

All patients had abdominal exploration before inclusion in this study. The operation ranged from biopsy of tumor masses, to bi­lateral salpingo-oophorectomy with or without total or subtotal hysterectomy. Ascites with positive cytology was present in all patients. (Table 3)

Between one and two weeks after surgery patients were treated with a combination of cyclophosphamide and F.U.D.R. Cyclophospha­mide was given by intravenous "push" in doses of 7-8 mg/Kg/day for 5 consecutive days, but rarely patients received more than 400 mg per day. F.U.D.R. was given via a continuous intravenous infusion in doses of 2 mg/Kg/day, diluted in 5% glucose in water, for 5 con­secutive days. (Table 4)

Patients were hospitalized during the whole treatment, but kept ambulatory. Nausea and vomiting were common, but responded to or could be prevented by phenothiazine. Stomatitis occurred occasion­ally, but was not severe and responded to mouth washes. No severe episodes of gastro-intestinal bleeding were observed. Alopecia was common, but transient with normal hair regrowth three to six months

INITIAL SURGERY

Exploratory laparotomy and biopsy 8

Bilateral salpingo-oophorectomy 1

Bil. S. O. and subtotal hysterectomy 2

Bil. S. O. and total hysterectomy 4

TABLE III

CHEMOTHERAPY FOR ADVANCED OVARIAN MALIGNANCY

CHEMOTHERAPY

Cyclophosphamide

F.U.D.R. (Floxuridine)

7-8 mg/Kg/day for 5 days I. V. "push"

1.8-2 mg/Kg/day in 5% Gl/w x 5 days continuous intravenous infusion

TABLE IV

607

after the end of treatment. There was a severe depression of the bone marrow as monitored by peripheral blood counts. The maximum effect was usually seen ten to fifteen days after the course of treatment. Leukocytes and platelets were mostly depressed, while erythrocytes and hemoglobin were not commonly effected.

At the end of the five days of chemotherapy the patients were discharged from the hospital and seen at weekly intervals. As soon as the adverse reactions had subsided (usually three to five weeks), the patients were re-admitted to the hospital for another course of chemotherapy using the same drugs.

These patients received between two and eight cycles of treat-ment.

The first and most dramatic effect noted was the reduction of pleural effusion and ascites. Three of the six patients with pleural effusion are still alive, and in one there is no evidence of disease 33 months after the initial surgery. Another patient who, in addition to pleural effusion had supraclavicular lymphnode metastases, is alive with no evidence of disease 16 months after diagnosis.

Initial Sursrery Second Look Outcome a TAll & BSO A 20 m c dis.

[: Expl. Lap. D 7 m

D 9 m 6 PI. Ef. BSO & SAH A & w 33 m

{: 20 m

BSO & TAH 8 m c dis.

1 Pl. Ef. & L. N. TAH & BSO A & W 16 m

TABLE V

608

2 Supraclavicular L. N.

Initial Surgery

[1 Expl.

11 Expl.

TABLE VI

Lap.

Lap.

U. VILLASANTA

Outcome

A 9 m c dis.

D 12 m

There was also measurable reduction of the abdominal tumors, so that in a few cases a "second look" operation succeeded in re­moving the neoplastic tissue. Three of the six patients with dif­fuse abdominal and liver metastases are still alive 15-18 and 24 months respectively after diagnosis, and in only one, there is positive peritoneal fluid. This patient has had one exploratory laparotomy which failed to reveal any disease. The peritoneal washing was also negative. Eleven months later peritoneal fluid, obtained by cul-de-sac aspiration, showed again malignant cells. The patient, however, has refused any further treatment. (Table 6)

Of two patients with supraclavicular lymphnode metastases, one died after 12 months, another one is alive nine months after an exploratory laparotomy revealed a large retroperitoneal mass. The mass, however, has not increased in size during this time and the patient is doing well, without ascites and continues to take cyclic treatments. (Table 7)

Cyclophosphamide has been extensively used for metastatic ovarian carcinoma. F.U.D.R. has been recommended for intra-arter­ial infusions only due to its toxicity. Because diffuse metasta­ses from ovarian carcinoma cannot be reached with regional intra­arterial infusion, an attempt has been made to use F.U.D.R. via the intravenous route, in doses three to twenty folds higher than those recommended for intra-arterial use (0.1-0.6 mg/Kg/day).

Initial Surgery Second Look Outcome

1 D 14 m

Expl. Lap.

lAH , 6 Hep. Metas. BSO

1 D 9 m

1 D 6 m 1 A & W 15 m

BSO Hyst. A & W 24 m

BSO & SAH ExpL Lap. A 18 m c dis.

TABLE VII

CHEMOTHERAPY FOR ADVANCED OVARIAN MALIGNANCY

%

100 ,,------,.

40

20

2 4 6 8 10

SURVIVAL

12 14 16

IDNl'HS

Figure 1

609

18 20 22 24 26

Adverse reactions to this combined therapy were expected, but not too serious, even if one patient, on one occasion, had a fall of the peripheral blood count to 600 WEe and 17,400 platelets. As for the effectiveness of the treatment, the results speak for them­selves: eight of fifteen patients are still alive, and two have survived over 24 months with no evidence of persistent or recur­rent disease. (Fig. 1)

CHANGES IN CLINICAL AND HISTOLOGICAL PATTERNS OBSERVED IN PATIENTS WITH ADVANCED CARCINOMA OF THE OVARY TREATED WITH PROGESTERONE

G.A. Paraskevas, PH. Angelakis, H. Deligeorgi­Politi 2nd Surgical Clinic Aretaion Hospital University of Athens, Greece

Progestins are now used in many centres as an adjuvant treatment in carcinoma of the Ovary. This is based on empirical observations by various investigators since 1960 (1,2,3,4,5,6). Most of them agree that 25-35% of patients with advanced ovarian carcinoma have an objective response to this treatment e.g. regression of tumour masses, reduction of ascitic fluid, improvement of general condition etc. In a previous communication (7) we reported on a prospective study which started in 1968 and included 33 such patients. Since then we have added 12 more similar cases. All 45 of them were found at operation to have carcinomas of the ovary outside the pelvis (stage IV) proven histolo­gically. Seven of them had previous treatment by radiotherapy or chemotherapy or both. The treatment regime consists of two weekly intramuscular injections of 500 mg. 17- Medroxy progesterone caproate starting from the fourth post-operative day and continued for life. In some instances of prolonged survivors this regime was switched to 200 mg medroxy progesterone acetate tablets daily.

The patients are observed frequently for a period of one to three months and if they are not responding to progesterone they are treated additionally with quintuple chemotherapy (Oncovin, Methotrexate, 5-Fluorouracil Cyclophosphamide, Prednisone), or by Radiotherapy in some cases with localized large solid masses. During the last two years we submitted 7 patients of the study to laparatomy, 3 of them because of threatened ileus and

611

612 G.A. PARASKEVAS ET AL.

in four as a reevaluating "second look" operation which seems to be in vogue lately.

OPERATION

Our aim at the second laparotomy to remove as much malignant tissue as possible or take more biopsies from various parts of the abdominal and pelvic disease. The reason for this was to compare as many sections as possible with the previous histology as ovarian carcinoma is well known to show histological differences in its various parts. Whenever it was feasible a colostomy or side to side anastomoses was performed to relieve the obstructive symptoms. In this paper we present mainly the histological findings in these patients and the changes seen which were brought about most probably by the prolonged progesterone treatment. We also pin-point some interesting clinical observations made during the course of the disease.

HISTOLOGICAL FINDINGS

The most striking feature seen in the material obtained at the second operation when it was compared with that of the first, was in the degree of differen­tiation in all 7 cases. The tumour appeared well differentiated with abundant connective stroma and sometimes with cyst formation.

The difference was more striking in three cases who previously had quite undifferentiated tumours to the extent that the original diagnosis was in some doubt and particularly as to the organ of origin. An adenocarcinoma with some differentiated areas turned to clear all carcinoma after 20 months of progesterone treatment and chemotherapy. The remaining two patients had endometreoid tumours of medium differentation. One had minimal disease 22 months later, the other even less of a tumour mass with complete differentation 18 months later.

CLINICAL OBSERVATIONS

The overall objective response was 40%. In six cases the response was impressive with almost dramatic decrease of the tumour masses and complete dissapearance. The best survivors are three patients living without

CHANGES IN CLINICAL AND HISTOLOGICAL PATTERNS 613

disease for 72, 63 and 48 months. It maybe of interest to mention that the first two developed parkinsonism after the fourth year of treatment that improved when progest erone was withdrawn. In one of them a pelvic mass appeared seven months later and when progesterone was recommenced the mass decreased and parkinsonism reappeared. In seven of the eighteen who initially responded, distant metastases appeared after the initial remission. Three died of cerebral metastases and four developed osseous lesions. Uncontrolable ascites was the cause of death in only 2 patients.

DISCUSSION

There is no doubt that large doses of progestins are beneficial to a varying percentage of patients with advanced carcinoma of the ovary. By reviewing the literature we failed to find any good explanation for this effect. The patients of ours who had a second operation had been treated by progesterone for periods varying from 13 to 22 months, and in most of them the tumour appeared well differentiated with cuboidal cells lining tubes or clefts, adequate stroma and sometimes cyst formation. Some of these changes are reminiscent of the ones seen in patients with endometrial carcinoma treated successfully with progesterone. Clearly one expects this similarity of response to the proved endometreoid carcinoma of the ovary but if this correla­tion is to be extended to all our patients who responded to this treatment, then one would suggest that many undifferentiated or pure papillary adenocarcinomas of the ovary are probably of endometreoid varieties, which are impossible to establish with the usual criteria at the advanced stages of the disease. The undifferentiated carcinoma which changed to clear cell after long progesterone treatment could be of mesonephric origin but again it could be related to the clear cell carcin­omas of the endometrium.

Obviously, it might be justified to subject many more patients to a reevaluating laparotomy and especially the very long survivors in order to draw safer conclus­ions as to the nature of this interesting hormonal action. A more elaborate model of study such as frequent measur­ing of the amounts of gonadotrophin, pregnanediol and oestrogens during progesterone treatment may also be of some value.

614 G.A. PARASKEVAS ET AL.

REFERENCES

1. Jolles, B. British J. Cancer, 16, 209, 1962. 2. Varga, A. & Henriksen, E. Obst. & Gynec. ~, 51, 1964 3. Kaufman, R.J. M.Cl. North America 22, 845, 1966. 4. Anderson, G.D. Am. JObst & Gynec. 2£, 87, 1965. 5. Ward, C.W.H. J. Obst. & Gynec Bri.Cmwlth. 12, 555,1972. 6. Kelly, R.M. & Baker, W.H. New England J. Med. 264

216, 1961 7. Paraskevas, A.G. et al. Progress in Chemotherapy,

Vol. III, 396, 1974. 8. Novak's Gynaecologic and Obstetric Pathology. W.B.

Saunders Co. 1962.

LIST OF CONTRIBUTORS

Abe, T. Aerts, F. Aiginger, P. Akiyama, M. Alberto, P. Amery, W. Andritsakis, G.P. Angelakis, P.H. Annunziato, L. Aoshima, M. Asakura, H. Athanassiou, A. Ayten, M.D.

Bach, J.F. Balogh, F. Barasoain, I. Bardos, T.J. Barry, V.C. Bartal, A. Belton, J.G. Benhamin, R.S. Beretta, G. Boasberg, P.D. Bobikov, E.V. Bolis, G. Borgers, H. Bruntsch, U. Burlakova, E.B.

Campanello, L. Cerni, C. Chandra, P. Christov, I. Cohen, Y. Comninos, A.C.

Conalty, M.L. Constantoulakis, M. Conti, G.

Dalcq, J. Davies, D.A.L. De Barbieri, A. Be Brabander, M. De Cree, J. Deligeorgi-Politi, H. Della Cuna, G.R. De Ruiter, J. Desplenter, L. Di Carlo, F. Dimitriadis, M. Donchev, T. Dudnick, T.V. Dzurillay, E.

Ebener, U. Eckhardt, S. Esaki, R. Evseenko, L.S.

Farkas, E. Fomina, M.M. Fox, B. Franz, G. Fujita, H. Fukami, A. Funahashi, K.

Gad-el-Mawla, N. Gaintseva, V.D. Gallmeier, W.M. Galt, R.M.

615

616

Gati, E. Gaur, V. Gause, G.F. Genazzani, E. Gericke, D. Geuens, G. Gorbacheva, L.B. Gorkova, S.W. Gotz, A. Guindani, A.

Haasz, R. Hall, T.C. Hardy, R.E. Heiss, W-D. Hellmann, K. Hill, B.T. Hill, J.M. Hill, N.O. Hindy, I. Hisano, G. Holczinger, L. Hopf, M.A. Hori, M. Hoshing, K. Huys, J.

Institoris, E. Ioannidou, G.B. Ishida, T. Ito, I. Iwaguchi, T.

Jansen, J. Janssen, P.A.J. J ellinghaus, W. Jeney, A. Jerusalinsky, E.L. Johnson, R.K. Jolles, G.

Kaether, M. Kajitani, T. Kajiwara, T. Kalpaktsoglou, P.K. Kamata, T. Karrer, K. Kasiananko, LV. Kawano, M.

Kawase, I. Kllan, M. D . Kllanykova, O.K. Kllwaja, T.A. Kimura, K. King, J.J. Kisbenedek, L. Kitoda, K. Knock, F.E. Kohzai, K. Kokron, O. Kolar, G.F. Kondyli, A. P. Konyves, I. Kopper, L. Kordiolis, N. Kornhuber, B. Koutras, G. Kroiss, A. Kubo, K. Kuehboeck, J. Kuhbock, J. Kukushkina, G.V.

Lapis, K. Laurent, C. Lehmann, W. Lowi tz, B. B.

MacLellan, A.S. Maddock, C.L. Maezawa, S. Maidhof, R. Maj, S. Mangioni, C. Mannes, P. Maral, R. Maurer, W. Medenica, R. Messaropoulos, T. Messer, M. Micksche, M. Minenkova, E. A. Moens, R.

Nakajima, T. Natale, N. Navashin, S.M. Nevinny, H.B.

CONTRIBUTORS

CONTRIBUTORS

Niimoto, M. Niitani, H. Nillius, A.

Odujinrin, 0.0. Oester, Y.T. Ohashi, I. Ohira, S. O'Neill, G.J. Osieka, R. O'Sullivan, J.F. Ota, K. Oyama, A.

Pagnoni, A. Pal'Mina, N.P. Paolette, P. Paraskevas, G.A. Pavlidid, N.A. Pawelski, S. Poetzi, P. Ponsinet, G. Poroshenko, G.G. Portales, A. Pozmogov, A.I. Price, L.A. Pridun, N. Profanter, W. Pujman, V.

Razis, D.V. Ridway, H. Rieche, K. Roberts, J.J. Robinson, E. Rojo, J.M. Rowland, G.F.

Saijo, N. Saito, T. Saitoh, o. Sakurai, Y. Samaras, V. Sannazzari, G.L. Sasaki, H. Sazykin, Y.O. Schieweck, K. Schlesiger, J. Schmidt, C.G.

Schmidt-Ruppin, K.H. Schultze, B. Scotti, G. Seeber, S. Semon sky , M. Serebryanyi, A.M. Sezina, N.P. Sheikh, K.M.A. Shorin, V.A. Shibata, K. Smink, T. Smolyanskaya, A.Z. Sokolov, A.B. Sokolova, I.S. Sane, S. Soulpi-Margariti, K.E. Speer, R. Street, E.W. Sugiyama, M. Sunkel, C. Suzuki, S. Swiniarski, J.K. Sylvester, R. Szanto, J. Szende, B. Szendi, I. Szendroi, Z.

Takacs, J. Takeuchi, K. Taniguchi, T. Taptas, J. Taufer, M. Terentjeva, T.G. Titscher, R. Tohge, T. Tominaga, K. Torbochkina, L.I. Tran Ba Lac, P. Tsuboi, S. Tsubura, E. Tsukagoshi, S. Tutlyte, V.S. Twomey, D.

Umezawa, H.

Van den Berg, H.W. Van de Veire, R.

617

618 CONTRIBUTORS

Van Putten, L.M. Van Vaerenbergh, P.M. Venditti, J.M. Vesela, H. Villa Santa, U.

Watanabe, K. Wodinsky, I. Wrba, H.

Yamagata, S. Yoshikawa, K.

Zangger, J.