Antitumor Principles Derived from Vinca roseaLinn

I. Vincaleukoblastine and Leurosine

IRVINGS. JOHNSON,HOWARDF. WRIGHT, GORDONH. SVOBODA,ANDJANETVLANTIS

(Lilly Research and Organic Development Laboratories, Indianapolis, Indiana)

SUMMARY

The experimental activity of a new clinically confirmed antitumor compound,Vincaleukoblastine (C46H58O9N4),(VLB) as the sulfate has been described. Greatestactivity was seen against the P-1534 acute lymphocytic leukemia in DBA/2 mice.Late as well as early stages of this leukemia were significantly affected by this compound. No synergistic or additive effects have been observed in combination therapywith other antitumor compounds.

A second indole-indoline alkaloid, leurosine, isomerie with VLB, has also been obtained from Vinca rosea Linn, with similar demonstrable experimental antitumoractivity.

Two other alkaloids, vin doline (CssHasOz) and catharan thine (C2iH2402N2), alsoobtained from Vinca rosea, were devoid of antitumor activity singly or in equimolarconcentrations, but have been postulated as the biogenetic precursors of VLB andleurosine.

Preliminary studies in vitro demonstrated that certain compounds were capableof reversing the growth-inhibitory activity of VLB against human monocytic leukemia cells. These compounds were coenzyme A, aspartic acid, tryptophan, a-keto-glutaric acid, ornithine, citrulline, arginine, and glutamic acid.

VLB and leurosine are representatives of a new class of clinically active antitumorcompounds which may interfere with the cellular metabolic pathways leading fromglutamic acid to urea, and from glutamic acid to the citric acid cycle.

Concomitant with the preparation of clinically plasm. Shortly after these compounds were ob-useful compounds from ancient plant remedies tained Noble, Beer, and Cutts1 reported obtainingthere has been an increased interest in studying another new alkaloid as a sulfate from Vincathe pharmacological action of plant alkaloids. In rosea. They suggested the name Vincaleukoblas-the course of investigating Vinca rosea Linn, cer- tine (VLB) for this compound. The most strikingtain crude fractions of the plant were submitted biological action of VLB was its leukopenic actionfor evaluation in our cancer screening program, in normal rats, which was used for its bioassayThe fractions were discovered to give interesting (4, 21). It was subsequently demonstrated in bothand, in some cases, profound activity against the Collip (20) and Lilly Laboratories (13) thatthe P-1534 leukemia, an acute lymphocytic leu- VLB also markedly inhibited the P-1534 leukemia,kemia transplanted in DBA/2 mice. These find- It is the purpose of this report to describe someings led to a prompt phytochemical investiga- of the experimental results obtained with thesetion in these laboratories which resulted in the compounds and to suggest a possible hypothesisobtaining of three new alkaloids: leurosine, viro- for the mechanism of action of this example ofsine, and perivine (24). Leurosine was found to a new class of antineoplastic agents,have some activity against this experimental neo- 1Dept of Med j^ CollipLaboratorieS)Universityof

Received for publication January 25, 1960. Western Ontario, London, Ontario.

1016

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JOHNSON et al.—Antitumor Principles from Vinca rosea. / 1017

MATERIALS AND METHODSThe P-1534 leukemia was implanted in DBA/2

mice with 0.5 ml. of a 10~4dilution of spleen cells

in medium 199 given intraperitoneally. This tumoris widely dispersed within 24 hours. Treatmentstarted 24 hours after implantation, except wherestated otherwise, and consisted of the daily administration of 0.5 ml. of the test compounds inphysiological saline for 10 days, except when thetest compound was not water-soluble. In suchcases 0.125 per cent carboxymethylcellulose wasadded as a dispersant. Groups of five animalswere used per compound, and treatment of alltumor-bearing animals was intraperitoneal unlessotherwise indicated. Effects of a drug are in termsof increase in average survival times over thatof saline-treated control groups. At the inoculum

Briefly, the method involved the mixing of defatted ground whole plant with an aqueous solution of2 per cent tartaric acid, followed by extraction withbenzene. After partial purification by acid-basetreatment, the crude alkaloidal mixture was chro-matographed on alumina. In a typical Chromatographie column, a benzene solution of weak baseswas chromatographed on Alcoa Grade F-20 activated alumina which had been previously treatedwith 10 per cent acetic acid.

RESULTSThe activities observed with the initial total

extracts of the pknt and with the first crudefractions, including some total alkaloidal fractions,are seen in Table 1. In all these early experimentsthere were a certain number of animals which sur-

TABLE 1

ACTIVITYOFORIGINALEXTRACTOFWHOLEPLANTANDCRUDEFRACTIONSAGAINSTP-1534LEUKEMIA

MaterialDefatted

wholeplant*FractionA*FractionB*Total

alkaloidsf"u"KDosage

(mg/kg/day)120.0.530.06.07.515.015

075(Oral)Av.

wt. chg.(gm.)T/C+0.1/+2.7-0.6/+1.2-1.2/+1.2+0.8/+2.94-0.2/+0.6-2.3/+0.6+0.3/+0.5-1.5/+0.6Av.

surv.time(days)25.6/14.826.5/19.224.0/19.227.7/17.229.8/13.420.3/13.430.0/13.020.6/13.4Per

centincreaseinsurv.

time733825611225113053Per

centindefinitesurv.020402006000

*For chemical description, see reference (25).

t Total ethanol extracts, defatted and extracted with benzene and 2 per cent tartaric acid.

of malignant cells used, the average survival timeof the controls usually varied from 13 to 16days after implantation. An increase in survivaltime of 20 per cent, or more, over the controlswas considered significant. All solid tumors wereimplanted subcutaneously by trocar, and groupsof ten animals were used per compound. Inhibitions were in terms of mean tumor diameters derived from two-dimensional caliper measurementsand compared with the diameters of control groupstreated with physiological saline. An inhibitionof 30 per cent, or more, was considered significant.

Tissue culture studies involved the plantingof replicate cultures in Medium 199 for routinedrug evaluation. Growth was determined by measuring the original inoculum and the volume ofcells produced after a 1-week growth period in aVan Allen hematocrit as described by Waymouth(11). All tests were done in duplicate.

The procedures used to obtain the alkaloids ofVinca rosea have been described elsewhere (25).

vived "indefinitely." These experiments were usually terminated after 90-120 days. All such animalswere still susceptible to the P-1534 leukemia whenre-implanted.

A number of alkaloids have been obtained fromVinca rosea Linn in these and other laboratories(Table 2). It can be seen that one, leurosine, obtained in our laboratories, has the same empiricalformula as VLB. It has been shown that the twoalkaloids are isomerie and represent a new classof dimeric alkaloids containing both indole anddihydroindole moieties. The chemical and physicalproperties have been reported elsewhere (7, 19).Biologically, leurosine has also been shown tohave demonstrable activity against the P-1534leukemia (Table 3). In our experience leurosineas a free base has not been as active or as consistentan inhibitor of the P-1534 leukemia as has VLB.Consequently, the major portion of this studywas devoted to work with VLB.

A dose-response effect could be demonstrated

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1018 Cancer Research Vol. 20, August 1960

in the P-1534 leukemia (Table 3) with VLB."Indefinite" survivors observed with the crude

preparations were never observed following administration of the pure alkaloids reported here.A study was made of VLB in combination withother antitumor agents at levels which have givenantitumor effects hi this laboratory in tumor systems sensitive to the other drugs studied. Thiswas done with the hope that some synergisticeffect might be noted which would yield a clueto possible mechanism of action (Table 4). None

TABLE2

ALKALOIDSPREVIOUSLYREPORTEDFROMVinca rosea

NameAjmalacine

TetrahydroalstonineSerpentineLochnerineReserpine*Akuammine*Empirical

formulaC2iH24OsN2

C21H24O3N2C21H22Q,N2C20H2402N2CsjHioOsNîC22H2604N2M.P.re.)253-254

230-231156-157202-203264-265258-260

NEWALKALOIDSREPORTEDFROMLILLYANDOTHERLABORATORIES

VindolineVindolinine-2 HC1LochnericineVincaleukobla stine

C2iH24-6O2N2-2 HC1C2iH24O2N2

NEWALKALOIDSREPORTEDFROMLILLYLABORATORIES

LeurosineCatharanthinePeri vineVirosine

C46H5809N<C21H2402N2

C22H2604N2

154-155210-212190-193211-216

202-205126-128180-181258-264

* Not encountered in the studies reported here.

was observed, which indicated that the mechanismof action of VLB was probably different from thatof any of the other compounds studied.

An important consideration in the experimentalevaluation of a potentially useful therapeutic agentfor the treatment of neoplastic disease is the abilityof the agent to retard terminal phases of theexperimental malignancy. VLB did significantlyprolong the life span of animals given implantsof the P-1534 leukemia (Table 5).

Experimentally, VLB has not been a "broadspectrum" antitumor agent, although limited

effects against some other transplantable neoplasms have been obtained. They include activityagainst the Walker carcinosarcoma 256 in ratsand a mammary adenocarcinoma of DBA/1 mice,which were inhibited 64 and 65 per cent after 10days' treatment at 0.3 and 0.6 mg/kg/day, re

spectively. Three ascites tumors—the Ehrlich ad

enocarcinoma, an ascitic form of Sarcoma 180, and

the undifferentiated, chemically induced Freundtumor—were completely inhibited with respect

to growth of ascitic cells following 10 days oftreatment, but did develop occasional solid tumorsat the site of inoculation. The L1210 leukemia inDBA/2 mice; the C-1498 leukemia, the E 0771and 755 mammary adenocarcinomas in C57BL/6;the Mecca lymphosarcoma and Ridgway osteo-genic sarcoma in AKR mice; and solid Sarcoma180 in Swiss mice were unaffected. Dosage levelsin these studies were usually in the range of 0.3-0.45 mg/kg, but perhaps if these were increasedthere might have been some effect. Noble, Beer,and Cutts have also reported some carcinostaticeffects with VLB against a transplanted mammary

TABLE 3

RESULTSOBTAINEDWITHLEUROSINEAND VLB TREATMENTOFTHEP-1534LEUKEMIA

MaterialLeurosineuuuVLBuuCiu"ItuilDosage(mg/kg/day)7.53.06.0ISO(Oral).05.1.3.45.45.6*.6*.6*1.5(Oral)Av.

wt. chg.(gm.)T/C+0.2/+1.9+1.4/

0.0+1.4/+1.3-1.2/+0.6+2.7/+0.7+1.7/+0.7+0.6/+0.9+0.3/+0.3+0.8/4-0.6-1.7/-1.6-2.0/-1.6+0.3/-1.6-1.2/+0.7Av.

aurv.time (days)T/C22.0/14.627.3/13.026.4/17.219.6/13.423.0/16.224.8/16.222.8/13.427.0/13.630.6/13.228.6/11.426.0/11.424.6/11.427.6/16.2Per

centincrease

in surv.time5011053464153709813115012811570

* Three different lots of VLB-SO4.

tumor in DBA mice, a transplantable rat sarcoma,and more recently the L1210 and P-1534 leuke-mias in BDF1 hybrid mice, IRC 741/139B leukemia in rats, and Sarcoma 180 in Swiss (20, 21). Theineffectiveness of VLB in our studies in L1210 andSarcoma 180 may have been due to differences indosage and strains of host animals used. Increased"curability" of transplantable tumors where somehost histo-incompatibility exists is well known(14).

Mechanism studies.—The mechanism of actionof VLB is not yet known, although some preliminary observations may indicate the directionwhich such studies should pursue. Tissue culturestudies have indicated a selective inhibition ofgrowth of certain cells of malignant origin. Anexample is the J96 cell derived from peripheralblood of human monocytic leukemia, and an em-

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TABLE4—RESULTSOBTAINEDBYCOMBININGVLBTHERAPYOFP-1534LEUKEMIAWITHOTHERANTITUMORDRUGS

DRUG6-Mercaptopurine0

-Diazoacetylserine6-Diazo-5-oxo-L-

norleucineSalineHN2Triethylene

melamine8-AzaguanineCortisoneColchicine2,6-Diaminopurine6

-MercaptopurineTESPAfMyleranîEpoxypropidine§Methotrexate#6-Diazo-S-oxo-L-

norleucineSalineDOSAGE

(MG/KG/WY)Other VLBdrug0.90.9

0.4560

30 0.45600.96

3 0.4560.90.6

0.3 0.450.60.9.450.6

0.3 .2250.6.450.3

0.15 .2250.3.4560.0

30.0 .22560.0.4560.0

30.0 .22560.0.450.6

0.3 .2250.6.4560.0

30.0 .22560.0.453.0

1.5 .2253.0.451.2

0.6 .2251.2.4515.07.5

.22515.0.454.5

2.25 .2254.5.451.80.9

.2251.8.450.6

0.3 .2250.6 .45Av.

WT.CHG.(OM.)+0.3+0.2

+0.7-1.6

-1.3+1.1+0.6

+ 1.7-0.3+0.3

+0.3X0.7+0.3+0.5-2.0

-1.3-2.1-1.4

0.0-0.8+1.8

+2.4+0.5+0.1

+0.3+0.1+2.1

+0.2+0.1+0.3

+ 1-6-0.9+4.6

+1.7+0.2+0.6

+1.5-0.2+0.2

+0.2+0.9+0.2

0.0+0.8+

1.9+0.8

+0.8+3.0

+2.7+1.3+0.6FBEQ.X10X

5*xioX10

XIOX5*xio

xioX5*XIO

XIOX5*xioXIOxio

xioX5*xio

xioX5*XIO

XIOX5*XIOxioX

5*XIO

XIOX5*XIO

XIOX5*XIO

XIOX5*XIO

XIOX5*XIOXIOX

5*xioXIO

X5*xioXIO

X5*XIOxioX

5*XIOAv.

8ÃœRV.(DATS)27.0

26.021.615.022.4

23.217.6

17.025.615.2

20.820.213.630.615

019.8

19.818.8

19.021.818.0

18.023.616.0

23.621.220.8

25.422.213.6

21.221.014.8

22.022.214.622.2

27.814.2

24.222.021.422.2

25.216.0

18.620.414.2

19.820.013.2PER

CENTINCREASEIN8ÃœRV.

TIME98

915810

647029

25881052

4813150

5042

436436

367821

786057

926860

5966

6868

11083

6662

689021

405450

51

* Each compound given once daily on alternate days for 5 days each, as compared with simultaneousadministration daily of the lower levels of the drugs for 10 days. Total dosage for each group is the same.

t TESPA = N-N'-lV'-triethylenethiophosphoramide. I Myleran = 1,4-dimethanesulfonoxybutane.

§Epoxypropidine = l,l-di(2,3-epoxypropyl)-4,4-bipiperidine.# Methotrexate = 4-amino-N'°methylfolie acid.

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1020 Cancer Research Vol. 20, August 1960

bryonic connective tissue cell (LLC-Hej) isolatedin our laboratories. A TCD/50 for the J96 cellhas been 0.00083 jug/ml and, for the LLC-Hei,0.002380 Mg/ml in the same experiment. A TCD/50 is interpreted as the dose/ml of tissue culturemedium which causes a 50 per cent inhibition ofgrowth compared with that of untreated replicatecontrol cultures during a 1-week period of growth.This figure will vary with and is dependent uponthe size of the cell inoculum. This type of studyhas been used to study reversal of the cell inhibitory activity of VLB. The results obtained in atypical experiment are seen in Table 6. Glutamicacid completely reversed the growth inhibitionof VLB in the concentrations used. Tryptophan

and coenzyme A gave partial reversal of thiseffect. Of the materials tested in these preliminarystudies, glutamic acid has been the most consistent. Since this is a delicate system, its reliabilitymay be open to question. However, we havepursued this approach, since VLB has no easilydemonstrable microbiological activity. Reversalstudies in vivowith VLB and the P-1534 leukemiaare in progress, based upon these preliminarytissue culture studies with VLB as well as withleurosine. Partial reversal of the antileukemic activity of VLB has been confirmed in vivowith tryp-tophan and glutamic acid. A summary of thesubstances studied by the in vitro system is seenin Table 7. All these compounds have been tested

TABLE5RESULTSOBTAINEDWITHTREATMENTDELAYEDFORVARIOUSTIMES

AFTERIMPLANTATIONOFTHEP-1534LEUKEMIA

DranVLBSaline6-Mercaptopurine

MethotrexateEpoxypropidine

VLBSalineDOSAGE(MG/KO/DAY)FREQ.0.6

X30.45X70.6

X10.45X80.45X100.45X103.0

X100.9X104.5X100.45X10Av.

BURY.TIME(DAYS)WITHTREATMENTSTARTINGONDAY1>5218.417.6

20.0>38.5

>38.016.0324.719.0

21.624.8

37.0625.218.4

21.423.2

31.8819.418.2

19.030.4

28.81018.4

18.619.6

25.2

TABLE 6

EXAMPLEOFEFFECTSOBTAINEDWITHVARIOUSAGENTSUPONTHEINHIBITIONOFJ96 CELLSBY

VLBTISSUECULTURE

AgentaControlsVLBGlutamine+VLBCoenzyme

A+VLBFolieacid+VLBCapantothenate+VLBPhenylalanine+VLBGlutamic

acid+VLBTryptophan+VLBHematocrit

readings(H.H.)1.6,

1.80.9,0.90.7,0.81.3,

1.60.7,1.00.9,1.00.9,1.01.9,1.61.6,

1.4Average

H.R.1.700.900.751.450.850.950.951.751.50Percent

inhibition475615*504545ot12*

( AU'eompounds including VLB were in T. C. medium 199 atconcentrations of .005 /ig/ml. Hematocrit reading of originalcell inoculum was 0.3.

* Partial reversal.

f Complete reversal.

against a single TCD/50 level of VLB while fourlevels of the test compound were utilized, usuallyat .05, 0.25, .005, and .0025 yug/mlof tissue culturemedium. These preliminary observations suggestan interference with the metabolic pathways leading from glutamic acid to urea via ornithine,arginine, etc., and from glutamic acid to the citricacid cycle via a-ketoglutaric acid. A more complete report of these studies in vivo and in vitrowill be presented elsewhere.

The J96 cell has also been used to study somecytological aspects of the mechanism of actionof VLB. In one series of experiments it wasfound that VLB caused an arrested metaphase.2

This has been similarly observed in tissue culturesinoculated with urine from VLB-treated patients,

1C. G. Palmer, D. Livengood, A. Warren, P. J. Simpson,and I. S. Johnson. The Action of Vincaleukoblastine on MitosisIn Vitro (unpublished manuscript).

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JOHNSONet al.—Antitumor Principles from Vinca rosea. / 1021

indicating excretion of VLB in the urine.3 Thetype of arrest caused was that which is termedC-mitosis, which indicates an effect upon themitotic spindle. At the same concentration nopostmetaphase stages were observed with VLB-treated J96 cells, but were observed in VLB-treated LLC-Hei cells. In similar studies withEhrlich ascites cells in vivo Cutts has seen cellsin anaphase showing bridging and multipolar divisions.4

DISCUSSIONThe plant Vinca rosea Linn (periwinkle) is

an apocynaceous, ever-blooming, pubescent herbor sub-shrub which has been shown to be a sourceof many alkaloids. It has enjoyed a popular reputation in indigenous medicine in various partsof the world. For example, Peckolt, in 1910, described the use in Brazil of an infusion of theleaves to control hemorrhage and scurvy, as amouthwash for toothache, and for the healingand cleaning of chronic wounds (22).

In Europe related species have been used forthe proprietary suppression of the flow of milk(23). In the British West Indies it has been usedto treat diabetic ulcer (21) and in the Philippineshas been reported as being an effective oral hypoglycémieagent (5). More recently, Chopra et al.(3) have reported that the total alkaloids possessa limited antibacterial activity as well as a significant and sustained hypo tensive action. Thehypoglycémieand antibacterial activities havenot been confirmed, although one of the alkaloidsisolated from this plant, ajmalicine, has beenreported to possess transient depressor action onarterial blood pressure (16).

The detection of activity against the P-1534leukemia was considered particularly significant,owing to the fact that this tumor system has detected other clinically useful antitumor agentsin our laboratory (12) and has been sensitiveenough to study structure-activity relationshipsof active compounds which correlated with theclinical activity (6, 15, 17, 18). VLB has also beenclinically confirmed (9). The lack of a so-called"broad spectrum" of activity against a battery

of tran splantable murine neoplasms seems of littleimportance in the face of clinical activity andsuggests that broader use and evaluation of lesscommonly used tumor systems might offer a potential advantage in the search for clinically usefulcompounds.

3Personal communication from Drs. Thomas H. Weller andJames B. Hanshaw, Harvard University School of PublicHealth, Boston, Massachusetts.

4Personal communication.

Of the alkaloids studied, one isomerie with VLB—leurosine—hasalso shown a demonstrable retardation of the P-1534 leukemia. It has generallybeen of a lower order of activity than VLB, andless consistent. Of theoretical interest are theindole alkaloid, catharanthine, and the dihydroin-dole alkaloid, vindoline (8). Vindoline-like andcatharanthine-like molecules each approximateone-half of the leurosine and VLB molecules.A solution containing equimolar proportions ofthese two alkaloids has an infrared absorptionspectrum which approximates those of VLB andleurosine (5). These compounds either singly orin an equimolar solution have been devoid ofany anti-P-1534 activity, as have all other purealkaloids from this plant which have been tested.

It would seem possible on biogenetic, chemical,and physical grounds to speculate that vindoline

TABLE7COMPOUNDSTESTEDFORVLBREVERSAL

INTISSUECULTURE

NoreversalSerotonin

Nicotinic acidSerine GlutathioneTPNDPNCysteine

Folie acidGlutamine PhenylalanineKynurenine Ca pantothenate

Indole acetic acidPartialTryptophan

Coenzyme ACompleteGlutamicacid

Aspartic acida-Ketoglutaric

acidOrnithineCitrullineArginine

and catharanthine may represent precursors ofthe biologically active VLB and leurosine molecules. The interesting chemical and biological relationships prompt a continuing study of theseand other compounds on a structure-activity basis.

The mechanism of action of VLB is of considerable practical importance. Metaphase arrest ofthe type referred to as C-mitosis has been observed in vitro in human cells of malignant origin,but not all cells examined were equally susceptible.The classical example of an agent with C-mitoticactivity is the alkaloid colchicine. It is the authors'

opinion that this may be only coincidental insofaras usefulness of VLB is concerned. Alkaloids aresubstances of widely divergent structures whosechief similarity are their natural origin and basicnature. Biogenetically, the origin of colchicineremains obscure, and no relationship with anyother groups of alkaloids has been detected (1).The biologically active antitumor compounds described here represent a new and previously unknown class of indole-indoline alkaloids. Indolealkaloids are believed to be derived from trypto-phan (1). Previously described indole alkaloids

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1022 Cancer Research Vol. 20, August 1960

include the ergot alkaloids, the harmala alkaloids of the yohimbine and physostigmine groups,Strychnos alkaloids which include strychnine, andthe Rauwolfia alkaloids. In our experience, colchi-cine has not had the selective effects seen withVLB. Arrested metaphases of the C-mitotic typehave also been demonstrated by such diverseagents as other alkaloids, sulfhydryl reagents,antifolics, purines, certain amino acid analogs,quiñones,phenols, and polyanions (2). The preliminary tissue culture studies seem to indicatean antimetabolite action, perhaps connected withglutamic acid. An additional observation whichis suggestive of antimetabolite action is the clinicalremissions obtained by Hertz, Lipsett, and Mayin women with Methotrexate-resistant choriocar-cinoma.5 The clinical results of Kodes, Hohn,and Bond indicate that VLB not only affectsa different clinical spectrum of malignancies thanis affected by colchicine, but also has fewer toxicor side-effects (9, 10). VLB and related activecompounds, regardless of mechanism of action,are examples of clinically active substances ofnew chemical compositions which provide a newand heretofore unknown lead in cancer chemotherapy.

ACKNOWLEDGMENTSWe are indebted to N. Neuss, Ph.D., and M. Gorman, Ph.D.,

for certain of the biologically inactive alkaloids obtained fromVinca rosea and to Mr. H. B. Metcalf, Sr., for technical assistance.

REFERENCES1. BENTLEY,K. W. The Alkaloids, pp. 146-82 and 210-28.

1st ed. New York: Interscience Publishers, Inc., 1957.2. BIESELE,J. J. Mitotic Poisons and the Cancer Problem,

pp. 108-64.1st ed. Houston: Elsevier Publishing Co., 1958.8. CHOPRA,I. C.; JAMWAL,K. S.; CHOPRA,C. L.; NAIB,

C. P. N.; and PILLAY,P. P. Preliminary PharmacologicalInvestigations of Total Alkaloids of Lochnera rosea Reichb(Rattanjot). Ind. J. Med. Research, 47(l):39-46, 1959.

4. CUTTS,J. H. Biological Effects of Extracts of Vinca rosea.Proc. Am. Assoc. Cancer Research, 2(4):289, 1958.

5. GARCIA,F. A Botany, Symposium on Medicinal Plants.In: Proceedings of the Eighth Pacific Science Congress ofthe Pacific Science Association, 4:182, 1953. Published bythe National Research Council of the Philippines, 1954.

6. GERZON,K.; COCHRAN,J. E., JR.; WHITE,L. A.; MONE-HAN,R.; KRUMKALNS,E. V.; SCROGGS,R. E.; and MILLS,J. Structure-Activity Relationship of Some Diamine Bis-Epoxides in Mouse Leukemia. J. Med. Pharm. Chem.,1(3): 228-43, 1959.

7. GORMAN,M.; NEÃœSS,N.; and SVOBODA,G. H. Vinca Al-

5R. Hertz, M. B. Lipsett, and R. H. Moy, Therapy ofMethotrexate-Resistant Choriocarcinoma in Women withVincaleukoblastine. Proc. Am. Assoc. Cancer Research, 3:118,1960.

kaloids IV. Structural Features of Leurosine and Vincaleukoblastine, Representatives of a New Type of Indole-in-doline Alkaloids. J. Am. Chem. Soc., 81:4745-46, 1959.

8. GORMAN,M.; NEUSS,N.; SVOBODA,G. H.; BARNES,A. J.,JR.; and CONE,N. J. A Note on the Alkaloids of Vincarosea Linn (Catharanthus roseus G. Don) II. Catharan-thine, Lochnericine, Vindolinine, and Vindoline. J. Am.Pharm. Assoc., Se. Ed., 48(4): 256-57, 1959.

9. HODES,M. E.; ROHN,R. J.; and BOND,W. E. Effects of aPlant Alkaloid, Vincaleukoblastine in Human Beings.Proc. Cen. Soc. Clin. Invest. (1959). J. Lab. & Clin. Med.,54:826-27, 1959.

10. . Vincaleukoblastine. I. Preliminary ClinicalStudies. Cancer Research, 20:1041-49, 1960.

11. JOHNSON,I. S. Discussion of Part II: Normal and Malignant Cells in Tissue Culture. Ann. N.Y. Acad. Sc., 76(3):542-46, 1958.

12. JOHNSON,I. S., and WRIGHT,H. F. Therapeutic Activityof Basic Bis-Epoxides in Murine Leukemia and OtherTransplantable Murine Neoplasms. Acta Unió.Internat,contra Concrum (in press).

13. JOHNSON,I. S.; WRIGHT,H. F.; and SVOBODA,G. H. Experimental Basis for Clinical Evaluation of Anti-tumorPrinciples Derives from Vinca rosea Linn. Proc. Cen. Soc.Clin. Invest. (1959); J. Lab. Clin. Med., 64:880, 1959.

14. KLEIN, G. The Usefulness and Limitations of TumorTransplantation in Cancer Research: A Review. CancerResearch, 19:343-58, 1959.

15. KHAKOFF,I. H.; MILLER,D. G.; KARNOFSKY,D. A.; DIAMOND,H. D.; BURCHNENAL,J. H.; and CRAVEH,L. F. Clinical Observations of l,4-Bis-(2,3-epoxypropyl)-piperazine.One of a New Class of Alkylating Agents. Acta UnióInternat, contra cancrum (in press).

16. KRONEBERG,G.; GRAF,K.; and OVERBECK,W. Kombinationswirkungen der Rauwolfia-Alkaloids Reserpine andRaubasin. Arch. Exper. Path. u. Pharmakal., 228:169,1956.

17. MILLER,D. G.; DIAMOND,H. D.; and CRAVER,L. F. TheClinical Use of Epoxypiperazine, a New Alkylating Agent,in the Treatment of Lymphomas and Other Neoplasms.Cancer Research, 19(11): 1204-9, 1959.

18. . The Clinical Use of an Epoxide Alkylating Agent,Epoxypropidine In Neoplastic Disease. Clin. Pharm. &Therap., 1:31-38, 1960.

19. NEUSS,N.; GORMAN,M.; SVOBODA,G. H.; MACIAK,G.;and BEER, C. T. Vinca Alkaloids. III. Characterization ofLeurosine and Vincaleukoblastine, New Alkaloids fromVinca rosea Linn. 3. Am. Chem. Soc., 81:4754-55, 1959.

20. NOBLE,R. L.; BEER,C. T.; and CUTTS,J. H. Further Biological Activities of Vincaleukoblastine—an Alkaloid fromVincarosea (L.). Biochem. Pharm., 1:347-18, 1958.

21. . Role of Chance Observations in Chemotherapy.Ann. N.Y. Acad. Sc., 76:882-94, 1958.

22. PECKOLT,T. Heil-und Nutzpflanzen Braziliens. Ber.Deutsch. Pharm. Ges., 20:36-58, 1910.

28. PERHOT,E. Matièrespremièresuselles du règnevegetal,p. 1783. 1st éd.Paris: Masson et eie, 1943-1944.

24. SVOBODA,G. H. A Note on Several New Alkaloids fromVinca rosea Linn. I. Leurosine, Virosine, Perivine. J. Am.Pharm. Assoc., 47:834, 1958.

25. SVOBODA,G. H.; NEUSS,N.; and GORMAN,M. Alkaloids ofVinca rosea Linn (Catharanthsus roseus G. Don). V.Preparation and Characterization of Alkaloids. J. Am.Pharm. Assoc., Se. Ed., 48:659-66, 1959.

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1960;20:1016-1022. Cancer Res   Irving S. Johnson, Howard F. Wright, Gordon H. Svoboda, et al.   Vincaleukoblastine and Leurosine

Linn: I.Vinca roseaAntitumor Principles Derived from

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