Recent strategies for the treatment of multi-drug resistance in cancer cells

Ojima, Bounaud & OderdaRecent strategies for the treatment of multi-drug resistance in cancer cells

Monthly Focus: Oncologic, Endocrine & Metabolic

Recent strategies for the treatment ofmulti-drug resistance in cancer cells

Iwao Ojima, Pierre-Yves Bounaud & Cecilia Fumero Oderda

Department of Chemistry, State University of New York at Stony Brook, StonyBrook, NY 11794-3400, USA

The multi-drug resistance (MDR) phenotype is defined as a cross-resistanceto a wide range of structurally and mechanistically diverse anticanceragents. The emergence of the MDR phenotype in tumours seriously affectsthe outcome of cancer chemotherapy, and thus the development of strate-gies to overcome MDR in cancer cells becomes essential and of currentdemand. This review focuses on patents published in 1996 - 1997 detailingMDR modulators and alternative approaches to the treatment of MDRtumours.

Keywords:chemosensitiser, drug-resistant tumours, multi-drug resistance,multi-drug resistance-associated protein, multi-drug resistant modulator,multi-drug resistant reversal agent, P-glycoprotein

Exp. Opin. Ther. Patents (1998)8(12):1587-1598

1. Introduction

Cancer is a major worldwide health concern with an estimated 6 millionnew cases per year [1,2]. In the United States alone, it is second only to heartdisease as a cause of death, with lung cancer as the leading malignancy.Fortunately, most new cases are diagnosed at an early stage and an ablativetreatment (surgery, radiotherapy) is used to remove the malignant tumour.In the remaining cases, the cancer has spread by metastasis and the onlychance for survival resides in chemotherapy. However, only a fraction (5 -10%) of the metastasised cancers are curable because of intrinsic resistanceto the treatment or acquired resistance after repeated therapies.

The ability of tumour cells to evade the therapeutic effect of anticancerdrugs is called MDR. MDR is characterised by a decreased sensitivity oftumour cells to a wide variety of structurally and mechanistically diverseanticancer agents [3]. At least two mechanisms for cellular resistance havebeen invoked [4]. The first corresponds to changes in the expression oractivity of proteins or enzymes, e.g., the cellular pumps (P-glycoprotein[Pgp], a 170 kDa protein and the MDR protein [MRP], a 180 kDa protein),glutathione S-transferase (detoxification enzyme), DNA topoisomerase II(target for many DNA-intercalating and non-intercalating drugs), andprotein kinase C (phosphorylation enzyme). The second mechanisminvolves changes in cellular physiology, such as the structure of the plasmamembrane, or the cytosolic pH. An overexpression of the mdr1 gene,which codes the Pgp, was found to be a common characteristic in resistanttumour cells and consequently the majority of MDR research has focusedon the cellular pump Pgp. Such a model clearly validates the observed

15871998 © Ashley Publications Ltd. ISSN 1354-3776

Review

1. Introduction

2. Strategies to overcomeMDR in tumour cells

2.1 MDR modulators

2.2 Anticancer agents for thetreatment of MDR tumours

2.3 Antisense oligonucleotides

3. Structure-activityrelationship studies

4. Expert opinion

Bibliography

Patents

http://www.ashley-pub.com

Expert Opinion on Therapeutic Patents

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

decrease in intracellular concentration of the antitu-mour drugs by involving an active efflux system of thetoxins.

Over the last 17 years, a large number of drugs havebeen shown to restore the sensitivity of resistanttumour cells against anticancer drugs to a level similarto the respective sensitive tumour cells [3,5]. They canbe classified in five broad categories:

• calcium channel blockers, e.g., verapamil [6]

• calmodulin antagonists, e.g., trans-flupenthixol [7]

• cyclic peptides, e.g., cyclosporine A [8] and its po-tent derivative PSC833 (Sandoz) [9]

• steroids and hormonal homologues, e.g., meges-trol acetate [10], tamoxifen [11]

• miscellaneous compounds, e.g., amiodarone [12],the quinoline MS209 (Mitsui) [13], the acridone car-boxamide GF-120918 (Glaxo) [14] , the

triazinoaminopiperidine S-9788 (Servier) [15], dipy-ridamole [16] and its derivative BIBW-22(Boehringer Ingelheim) [17] (Figure 1)

Several of these compounds are currently in PhaseII/III clinical trials.

The goal of this article is to review the patent literatureof MDR modulators and alternative strategies toovercome MDR in tumour cells for the years 1996 and1997. This article is presented as an update of theprevious review by Ecker and Chiba [18].

2. Strategies to overcome MDR in tumourcells

2.1 MDR modulators

Verapamil (1), a calcium channel blocker, was the firstmodulator reported in the literature [6]. It works as acompetitive inhibitor of Pgp, resulting in the in vitrosensitisation of drug-resistant cell lines to a number ofantitumour drugs. However, cardiovascularside-effects at concentrations needed for effectivereversal activity limit its use in the clinical applica-tions. Other calcium channel blockers, structurallydifferent from verapamil, were found to possesspotent chemosensitising activity, especially dihydro-pyridine compounds such as nicardapine orniguldipine [3]. Three new series of dihydropyridinesare claimed by Nikken Chem. Co. Ltd. [101-103].Recovery of the sensitivity to vincristine andadriamycin against the drug-resistant VJ-300 cell linewas evaluated, with compound 2 displaying apotentiation index (PI) of 177 and 13.9 at 1 µg/ml,respectively. The PI corresponds to the IC50 value ofthe antitumour drug alone over the IC50 value of theantitumour drug in combination with the modulator.Potent in vivo activity against vincristine-resistantmurine leukaemia P388/VCR implanted in CDF1 micewas also observed, with a %T/C ratio of 172% whencompound 2 (100 mg/kg) was co-administered withvincristine.

Another calcium channel blocker with Pgp antago-nism is pentoxifylline [19]. Cell Therapeutics, Inc.claims about a hundred derivatives of pentoxifylline,as exemplified by compound 3 [104]. A 10-foldincrease in the uptake of doxorubicin in thePgp-positive murine leukaemia cell line P388/MDRwas observed at 1.3 µM concentration of 3. At thesame concentration, 3 sensitised the doxorubicin-resistant Chinese hamster cell line LZ100/DX to

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1998)8(11)

1588 Recent strategies for the treatment of multi-drug resistance in cancer cells

S-9788

BIBW-22

GF-120918

F

NH

F

N N

NN

NHCH

2

NH

CH2

N

N

N

N N

O

N

OH

CH3

CH3

OH

N

O

CH3

CH3

CH3

CH3

NH

O

OCH3

NH

N

O

O

CH3

CH3

O

Figure 1: The structures of S-9788, BIBW-22 and GF-120918.

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

paclitaxel, colchicine, and actinomycin D, as well asthe adriamycin-resistant human breast carcinoma cellline MCF7/ADR to doxorubicin with a 100-foldincrease in tumour cell death.

Amiodarone is a potassium channel blocker,commonly used as anti-arrhythmics, that was found toexert potent Pgp inhibition in vitro [12]. Severeside-effects, however, prevented its clinical use. Aclosely related series of derivatives is claimed by EliLilly & Co., which are based on the benzo[b]thiophenestructure as exemplified by compound 4 [105]. Thecompounds are claimed to have a significant effect inreversing the MRP- and the Pgp-mediated MDR,although no data are presented.

Nitrogen-containing central aromatic rings are now arecurring structural feature since the discovery of thepotent modulating activity of dipyridamole and itsderivatives BIBW-22 and S9788 [15-17]. A Pfizer patentclaims five purine derivatives, such as 5, to retainadriamycin in MDR human carcinoma cell line KBV1,but no data are presented [106]. Several series ofimidazole derivatives are independently patented bythree companies. Janssen Pharm NV claims novel

fused imidazole derivatives such as 6 [107].Compound 6 demonstrated in vivo activity against theadriamycin-resistant murine leukaemia tumourP388/ADR implanted in B6D2F1 mice. A %T/C ratio of132 - 141% was observed when 6 wasco-administered (0.63 - 20 mg/kg) with adriamycin,which corresponds to a 14 - 23% increase in survivaltime than that of the group treated only withadriamycin. Novel imidazothiazole compounds, suchas 7, are claimed by Nikken Chem. Co. Ltd. [108].Compound 7 sensitises the drug-resistant VJ-300 cellline to adriamycin (PI = 10) and vincristine (PI = 211),and a %T/C ratio of 131% was obtained withvincristine-resistant murine leukaemia P388/VCRimplanted in CDF1 mice at a modulator dose of 10mg/kg. Ontogen Corp. claims novel substitutedimidazole derivatives in two patents [109,110].Compound 8 was found to possess a cytotoxicitypotentiation value of 0.126 µM against drug-resistantCEM/VLB1000 cell line in combination with vinblas-tine, increasing the intracellular accumulation ofvinblastine (EC50 value = 2.8 µM).


Ojima, Bounaud & Oderda 1589

N

O

O

CH3

CH3

CH3

N

O

O

CH3

CH3CH

3

CH3

2

NOO

O

NH

CH3

CH3

CH3

CH3

O

N

4

OH

O

O N

NH

S CH3

O

O

S

3

N

N N

N

CH3

O

O

CH3

NH

CH3

OH

Verapamil

1

5

H-Cl

N

OO

CH3

CH3

N

N N

N

NH

O

O

CH3

CH3

6

NO

N

N

N

O

CH3

O

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

A remarkably potent MDR modulator, GF-120918,developed by Glaxo was recently reported in theliterature to have similar activity as 5 µM of verapamilwith concentration as low as 0.02 µM [14]. Byk GuldenLomberg Chemische Fabrik GmbH claims new deriva-tives with a thienoquinoline moiety [111]. Compound9 showed MDR reversal effects against thedrug-resistant CCRF-VCR1000 cell line in combinationwith vincristine, with the PI value of 211 and 505 at 3and 10 µM concentrations, respectively. Specificinhibition of Pgp was further demonstrated. XenovaLtd. continues structure-activity relationship (SAR)studies on a hybrid structure derived from GF-120918and XR-1500, claiming 51 new compounds in theirtwo new patents [112,113]. Daunorubicin withcompound 10 showed an IC50 value of 0.15 µM in adrug accumulation assay against the drug-resistantEMT6 mouse mammary carcinoma cell line AR 1.0.Compound 10 achieved a PI value of 2000 withdoxorubicin against the same cell line.

Steroidal hormones and their analogues are anotherclass of MDR modulators. Megestrol acetate, anorally-active congener of progesterone, holds great

promise as a potentially useful clinical chemosensi-tiser [10]. In the past two years, two patents on steroidderivatives were published. Xenova Ltd. claims novelpregnane steroid derivatives obtained by methanolextraction of the roots of Cynanchum otophyllum, asexemplified by compound 11 [114]. In a drug accumu-lation assay of daunorubicin with compound 11against the drug-resistant EMT6 mouse mammarycarcinoma cell line AR 1.0, EC50 values of 0.8 - 30 µMwere obtained. Batra et al. on the other hand claimnew steroid carbamates, such as compound 12 [115].EC50 values for reduction in cancer cell survivalranged from 0.11 to 0.51 µM against the HCT-15 cellline, and from 0.15 to 0.76 µM against the AT-1 cell line(the values for verapamil are 0.5 µM and 0.74 µM,respectively). Doxorubicin with compound 12 signifi-cantly reduced the HCT-15 tumour volume in SCIDmice.

Three patents on novel polyaromatic compoundswith chemosensitising activity have been published inthe past two years. Pharma Mar SA claims the use ofthe lamellarin alkaloids such as lamellarin I (13) forthe treatment of MDR [116]. The cytotoxicity of



7

N

S

N

N NH

N

O

8

OCH

3

ON

N

NH

N

O

CH3

CH3

CH3

CH3

CH3

109

N

O

O

NH

O

NH

S

CH3

O

CH3

CH3

H-ClN

O

O

NH

O

N

NH

O

OCH

3

CH3

CH3

1112

OCH

3

CH3

CH3

O

O

O

CH3

OH

3C

O

CH3

O

O

O

CH3

OCH3

O

CH3

OH3C

O

CH3

OH

CH3

O

OH

OH

O

O

N O

CH3

CH3

CH3

O

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

adriamycin when co-administered with 13 wasevaluated against two resistant cell lines, the mouselymphoma P388/SCHABEL and the Chinese hamsterovary carcinoma CHO C5. At concentrations of 13between 0.01 and 3.0 µg/ml, the PI values varied from1.0 to ≥ 400. Novel triphenylazacycloalkane deriva-tives, such as compound 14, are claimed by MerrellPharm., Inc. to reverse MDR in tumours [117]. Thenon-toxic compound 14 was tested in combinationwith vinblastine against the drug-resistant humanepidermoid carcinoma cell line KBV1. The compoundenabled the antitumour drug to inhibit cell growth by94.2% at a concentration of 0.23 µM. Eli Lilly & Co.,which previously reported the potent MDR modulatorLY335979 [20], is now claiming 70 new derivatives ofLY335979 such as 15, but no data are presented [118].

Interesting SAR studies were reported on isoprenederivatives. Nisshin Flour Milling Co. Ltd. (or NisshinSeifun KK) claims three patents on novel isoprenederivatives [119-121]. Compound 16 displayed lowcytotoxicity and was able to potentiate the cytotox-icity of adriamycin against the adriamycin-resistanthuman breast carcinoma MCF7/ADR at a concentra-tion of 50 µM with the PI value of 42 [119,120]. Closelyrelated bis(aminoethyl)ether derivatives, such as 17,also have similar activity [121]. Such long-chainpolyenes are new for MDR-reversal agents and weshould expect more compounds of this type toemerge, such as the insecticidal polyene antibioticstipiamide [21].

Another interesting area of research is the use ofamino acid derivatives as MDR modulators. HitachiChem. Co. Ltd. et al. claim that new piperazine



17

NO

N

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

O

CH3

O

CH3

14

15

16

OH

OH

O

O

O

O

CH3

CH3

N

OH

O

N

FF

CH3

CH3

CH3

CH3

CH3

NH

O

O

CH3

CH3

OCH

3

OCH

3

CH3

CH3

CH3

CH3

CH3

CH3

13

Doxorubicin and Lamellarin I

O

O

OH

OHO

CH3

O

OH

OH

O

O

NH2

OH

CH3

O

NH

OH

O

O

OOOO

CH3

CH3

CH3CH

3

CH3

O

CH3

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

derivatives containing an amino acid and an epoxymoieties, such as NCO-700 (18) are effective [122]. Ina drug accumulation assay against the drug-resistantKBV1 cell line, NCO-700 increased the accumulationof vinblastine by 5-fold at 20 µM. In in vivo testing onnude mice carrying the resistant human tumourKB-CH 8.5, co-administration of adriamycin withnon-toxic NCO-700 (LD50 value in rats of 317 mg/kg)brought a 60% decline in the tumour volume. VertexPharm., Inc. which recently reported an efficient MDRmodulator, VX-710 [22], claims three patents on thederivatives of VX-710 that are able to treat MRP- andPgp-mediated MDR tumours using doxorubicin[123-125]. Against the Pgp-positive mouse leukaemiacell line L1210VMDRC0.6, compounds 19, 20, and 21increased the activity of doxorubicin with PI values of5.5 (0.5 µM), 14 (2.5 µM), and 8.5 (2.5 µM).Compounds 19 and 21 potentiated doxorubicinactivity against the non-Pgp expressing HL60/ADRcell line with the PI values of 125 (10 µM) and > 60 (10µM), respectively (no data were presented forcompound 20).

New types of MDR modulators have also beendisclosed in the last two years. Memorial SloanKettering Cancer Center et al. claim novel derivativesof ardeemin, amauromine, and gypsetin as efficientchemosensitisers [126]. N-Acetylardeemin (22)increased the intracellular accumulation of VP-16 inthe drug-resistant human leukaemia cell lineCCRF-CEM by 66% at 30 µM concentration.Compound 22 also increased the activity ofadriamycin against P388/DX cell line and of paclitaxelagainst the drug-resistant HCT116(VM)46 cell linewith the PI values of 5.1 (2.8 µM) and 18 (2.2 µM),respectively. Tiamulin (23), a drug for the treatment ofbacterial infection in animals, is claimed to be anon-toxic chemosensitiser by Etablissement deTransfusion Sanguine de Lyon et al. [127]. Tiamulindemonstrated an increase in the drug accumulation ofdaunorubicin and further improved the activity ofdaunorubicin against drug-resistant cell lines, mouseleukaemia P388/ADR25 (IC50 = 0.31 µg/ml), rathepatocarcinoma AS30-D/Col10 (IC50 = 0.85 µg/ml),and human lymphoblastic leukaemia CEM/VLB3.6(IC50 = 0.83 µg/ml). Novel naturally-occurringtaxanes, such as 24, are claimed by MitsubishiChemical Corp. (or Mitsubishi Kagaku KK) to increasethe sensitivity of the drug-resistant tumour cells forantitumour agents [128]. Compound 24 is claimed tohave 50% of the activity of verapamil when used incombination with vincristine. Fox Chase Cancer

Center claims novel dendroamide compounds asMDR modulators [129]. Specifically, compound 25 ismore potent than verapamil in increasing the accumu-lation of [3H]-vinblastine in the drug-resistantMCF7/ADR cell line (maximum effect was observed at20 µM concentration). Compound 25 at a concentra-tion ≥ 0.6 µM, increases the sensitivity of MCF-7/ADRcells to vimblastine to the level of sensitivity observedfor parental MCF-7 cells. Chemosensitisation alsooccurs with daunomycin and actinomycin D, but notwith cisplatin. A photoaffinity labelling experimentwith a photoreactive analogue of compound 25strongly indicates direct binding of 25 to Pgp. TheMRP pump also appears to be targeted by thiscompound, since 25 sensitised non-Pgp expressingMCF7/VP cell lines against vincristine (resistance fullyreversed at 10 µM), etoposide, and doxorubicin, butnot cisplatin.

A new class of chemosensitisers is claimed byPharmacyclics, Inc. [130]. Texaphyrin 26 is anon-toxic (LD50 mice > 40 µmol/kg) chemosensitiserthat works independently of Pgp. Compound 26 (50µM) was shown to be efficient against MES-SA celllines in combination with bleomycin (PI = 7.5),paclitaxel (PI = 2), etoposide (PI = 2), and cisplatin (PI= 2). Moreover, texaphyrins are effective photosensi-tisers for their use in photodynamic therapy. The MDRmodulator can be activated wherever light is irradi-ated on the patient, thus providing a specific andlocalised treatment.

2.2 Anticancer agents for the treatment of MDRtumours

Another approach to overcome MDR in tumour cellsinvolves the design of anticancer agents that are notaffected by the MDR phenotype. Small changes in thestructure of existing therapeutic anticancer agentsmay lead to enhanced activity against MDR tumourcells. Over the past two years, several patents claimingsuch derivatives have appeared.

Rhône-Poulenc Rorer SA claims in 8 patents anextended SAR study on the taxoid structure [131-138].These novel taxoids (27) are claimed to possessantitumour activity, in particular against tumoursresistant to paclitaxel (Taxol™) and docetaxel(Taxotere™). The antitumour activity was noted invivo in mice grafted with melanoma B16 at doses of 1 -10 mg/kg, but no data are presented. The ResearchFoundation of the State University of New York claimsa series of 11 novel taxoids with a better therapeutic



Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.



18 19

20

21

2223

24

25

HSO4-

N

N

NN

O

O

CH3

CH3

O

CH3

CH3

CH2

OCH

3

CH3

OS

NCH3

CH3

O

CH3

CH3

OH

CH2

OAcCH

3

CH3

OAc

OAc OAc

CH2

OH

O

O

OCH3

O

NH

O

CH3

CH3

N

S

O N

NH NH O

O CH3

CH3

CH3

S

N

O

CH3

OCH

3

OCH

3N

N

H+

NH

O

O

O

O

CH3

CH3

OCH3

N

O

N

OCH

3

OCH

3

OCH

3 N

O

O

CH3

Cl

N

OCH

3

OCH

3

OCH

3

O

N

O

O

O

N

OCH

3

OCH

3

NO

N

O

NO

O

O

OO

CH3CH

3

CH3

27

26

R4R3

CH3

OR5OR6O

OH

O

R1 R2

O

OH

NH

COBN

NN

N

CH3

OH

CH3

CH3

CH3

OH

OO

OO

OO

CH3

OO

CH3

GdN

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

profile than paclitaxel and docetaxel [139]. Againsthuman mammary carcinoma cell line MCF7 and theadriamycin-resistant MCF7/APR, compound 28possesses IC50 values of 0.20 nM (paclitaxel, 1.7 nM)and 2.11 nM (paclitaxel, 300 nM) respectively,denoting a 2-order increase in magnitude of cytotoxicactivity against the drug-resistant cell line.

In a patent by Priebe et al., novel bisanthracyclines areclaimed to display anticancer activity equal to orgreater than that of the parent compound doxorubicin[140]. In vitro activity evaluation was conductedagainst MCF7 cells and the MRP-expressingMCF7/VP16 cells in comparison with doxorubicin.While compound 29 was less active than doxorubicinagainst the sensitive cell line, i.e., IC50 = 4.83 µM(doxorubicin, 0.9 µM), its activity against the resistantcell line was much better than that of doxorubicin, i.e.,IC50 = 2.5 µM (doxorubicin, 14.9 µM).

Additionally, five novel colchicine derivatives areclaimed by Indena SpA [141]. These compounds werecompared with colchicine and paclitaxel for theircytotoxic activity against normal and drug-resistantMCF7 breast tumour cell lines. While the ‘foldresistance’ (FR, Equation 1) values of colchicine andpaclitaxel were 25 and 59, respectively, the novelderivatives displayed FR values of 1.7 - 9.7 (FR = 1.7for compound 30).

Equation 1

Fold resistance =IC of drug in the resistant lin50 e

IC of drug in the sensitive line50

Eli Lilly & Co. et al. patented new cryptophycinderivatives, such as compound 31 [142]. Thesecompounds are claimed to be useful for treating MDRtumour cells, but no data using these type of cells arepresented. Norsk Hydro AS claims novel1-β-D-arabinofuranosylcytosine (Ara-C, also knownas cytosar, used for the treatment of acutemyelogenous leukaemia) derivatives [143], whichexhibit activity against a wider range of tumours aswell as less degradation in vivo than Ara-C. The sameactivity, independent of the MDR phenotype, wasobserved against the drug-sensitive and resistant celllines (cisplatin-resistant NHIK3025/DDP andPgp-expressing A549). Compound 32 was evaluatedfor in vivo activity using severe combined immunode-ficiency (SCID) mice injected intravenously (iv.) withRaji human B-lymphoma cells. Treatment with 32 (20mg/kg) or Ara-C (200 mg/kg) showed T/C of 123%



28

O OHCH

3

CH3

OAcOBzO

OH

O

CH3

CH3

O

O

NH

OH

O

O

CH3CH

3

CH3 O

CH3

CH3

30

31

32

O

O

CH3

O

CH3

NH

FF F

FF

SCH

3

O

O

O

O

CH3

O NHO

OCH3

CH3

O

NH

CH3

O

CH3

OCH

3

CH3

O

O

O

OH OH

N

NO

NH2

29

HCl

HCl

OH

OH

OH

O

O OCH

3

CH3

O

OO

CH3

OH

NH

NH

OCH

3

OH O

O

O

O OHCH

3

OH

CH3

OH

O

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

and 118%, respectively. Daily treatment intraperito-neally (ip.) with 32 increased T/C to 135%.

Other types of aromatic systems have been disclosed.Boehringer Mannheim Italia SpA et al. claim 13 substi-tuted isoquinolinoindazol-6(2H)-ones such as 33[144]. Against the human colon adenocarcinoma cellline, LoVo, and its doxorubicin-resistant subline,LoVo/DX, compound 33 possesses the FR value of 1.8as compared to 74 for doxorubicin and 23 formitoxantrone. Novel 2-naphthoquinone derivativessuch as 34, which are structurally related to naturally-occurring lapachones and dunniones, are claimed bythe Wisconsin Alumni Research Foundation [145]. Invitro activity assay of these compounds againstdrug-sensitive and resistant human breast carcinomaMCF7 cell lines showed IC50 values lower than 5 - 10µM, the activity being independent of the MDRphenotype. The compounds are claimed to betopoisomerase I inhibitors. A series of substitutedtrisbenzimidazole derivatives, such as compound 35,is claimed by Rutgers University to act also astopoisomerase I inhibitors [146]. The IC50 values rangefrom 0.03 to > 25 µM (35, 0.09 µM) against the humanlymphoblast RPMI8402, 0.58 to > 25 µM (35, 0.58 µM)against the camptothecin-resistant variant CPT-K5,0.01 to 0.58 µM (35, 0.58 µM) against the humanepidermoid carcinoma KB3-1, 0.01 to 0.35 µM (35,0.35 µM) against the vinblastine-selected MDR variantKBV-1. Despite the high degree of expression of Pgpin the KBV-1 cell line, compound 35 exhibits a betteractivity than that against the drug-sensitive cell line,thus suggesting that these trisbenzimidazole deriva-tives are not substrates to Pgp.

Finally, a novel compound, caribenolide I (36), isclaimed by Shimizu and Fairchild to have a potentcytotoxic activity against the human colon carcinomaHCT-116 and a MDR subline, although no data withMDR cell lines are presented [147]. The IC50 value of1.6 nM against HCT-116 cell line was reported, and anoptimal dose of 0.03 mg/kg/day (ip.) resulted in a 50%increase in the lifespan of mice implanted intraperito-neally with P388 mouse leukaemia.

2.3 Antisense oligonucleotides

The strategies to overcome MDR all assume thepresence of MDR in tumour cells and proposesolutions to circumvent it. In those cases, Pgp, theproduct of the mdr1 gene, is the general target.Instead of treating MDR, another strategy relies onantisense or ribozyme technology to prevent the

emergence of MDR in cancer cells. Two patentsdescribe new antisense oligonucleotides designed toinhibit the expression of the mdr1 gene. Hybridon,Inc. claims effective antisense oligonucleotides thatbind to two identical sites within the mdr1 gene [148].The ability of these antisense oligonucleotides toreverse MDR in vivo is reported to be assessed againstdrug-resistant cell lines, LoVo 350-1, HCT200-1,CCRF-CEM, NCI-H596, and NCI-H446, but nobiological data are presented. The University ofNebraska claims novel oligonucleotides targeting thehuman mdr1 and MRP genes [149]. At 0.2 µM concen-tration, these nucleotides were evaluated incombination with vincristine against human multiplemyeloma cell line, RPMI-8226/Dox4, showing the PIvalue > 7500 in some cases. In a patent by City ofHope, it is shown that down-regulation of the fos/jungenes will make resistant cancer cells more sensitive



34

35

36

O

OO

CH3

CH3

CH3

N

NH

N

NH

NNH

Cl

OOH

OH

OH

CH3

CH3

CH2

O

O

OH

CH3

CH3

O

O

O

33

N

N NN

CH3

CH3

NHN

CH3

CH3

O

Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

to conventional treatment [150]. An anti-fos ribozymewas developed which was able to completely restorethe sensitivity of drug-resistant A2780AD cells toactinomycin D. It is further shown that the ribozymecan reduce the expression of mdr1, c-jun, p53, andtopoisomerase I.

3. Structure-activity relationship studies

In many cases, binding to Pgp is responsible for MDRreversal activity of MDR modulators, as demonstratedby Pgp photoaffinity labelling experiments. Theinvolvement of a receptor-ligand interaction in MDRwarrants the use of SAR studies to identify keystructural elements for reversal activity and ultimatelyto design new modulators with enhanced chemosen-sitising properties. However a modulator can act as acompetitive or non-competitive agonist or antagonistof Pgp through different types of binding. Also, theinteraction of a modulator with Pgp-regulatingenzymes, such as protein kinase C, may indirectlyaffect the Pgp efflux. Accordingly, SAR studies aremost likely limited to a single pharmacological class ofmodulators.

Extensive SAR studies were conducted on thephenothiazine family, which includes the calmodulinantagonist and antipsychotic trifluoperazine [7,23].These studies led to the development of the thioxan-thene subclass and a potent derivat ive,trans-flupenthixol. Structural features such ashydrophobic and conjugated planar rings, and asubstituted, preferably cyclic, tertiary amino group areproposed to be as essential for activity. The spatialorientation between the amino group and the planarrings is of importance as well, suggesting specificstructural requirements for binding. The SAR studieson a propafenone-like propanolamines series alsoidentified the tertiary amino group, preferablyincorporated in a cyclic non-aromatic ring structure,as a key element [24]. In a similar series, a goodcorrelation between the Pgp inhibitory activity andthe lipophilicity of the compounds was observed [25].

The SAR study on a series of taxane-based reversalagents showed slightly different conclusions [26]. Theabsence of a tertiary amine group did not impede theactivity of the series, and simple lipophilicity of thecompounds was not a major factor for activity.However, small variations in the structure could bringsubstantial differences in MDR reversal activity, thussuggesting that rather strict structural requirements

ultimately dictate the MDR modulating ability of thisseries.

4. Expert opinion

The vast number of patents published on MDRmodulators shows the strong interest of the pharma-ceutical industry in circumventing the MDRphenomenon. MDR modulators have a variety ofstructures, but most importantly these agents alsopossess different mechanisms of action for thereversal of MDR. While most of these agents interactdirectly with Pgp or the other protein pump MRP,some sensit ise drug-resistant tumour cellsindependent of Pgp. The development of antisenseagents adds an alternative strategy focusing onsuppression of the mdr gene. The plurality in themechanism of action offers a possibility to combinetwo or more modulators simultaneously for a betteractivity. At the same time, this kind of therapeutic‘cocktail’ may lower the cytotoxicty or other toxicitiesof each modulator by lowering their plasma concen-tration. For that purpose, studies on possiblesynergism between existing potent MDR modulatorsshould be conducted [27].

It is likely that optimal inhibition of MDR by an MDRreversal agent may be obtained only for a specificanticancer drug. Therefore, it is important to developan extensive arsenal of MDR chemosensitisers. Aproper combination of an improved antitumour drugand a chemosensitiser holds great promise for thesuppression or eradication of MDR in drug-resistantcancer cells.

Even though the number of potential drugs to treatMDR is increasing and the in vitro and in vivo resultsare promising, their effective use in the treatment ofclinical resistance is still uncertain. Many issues haveto be addressed, such as the pharmacological profileof the chemosensitiser, its toxicity to normalPgp-expressing tissues, and the tumour classes mostefficiently affected by the drug combination. SeveralMDR modulators are in Phase II/III clinical trials. Theoutcome will tell us more on the possibilities andprospects of MDR reversal agents as an effective toolfor cancer chemotherapy.

Bibliography

Papers of special note have been highlighted as:• of interest



Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

•• of considerable interest

1. Taxane Anticancer Agents: Basic Science and CurrentStatus. Georg GI, Chen TT, Ojima I, Vyas DM (Eds.), Ameri-can Chemical Society, Washington, DC (1995).

2. Taxol: Science and Applications. Suffness M (Ed.), CRCPress, New York (1995).

3. FORD JM: Experimental reversal of P-glycoprotein-mediated multidrug resistance by pharmacologicalchemosensitizers. Eur. J. Cancer (1996) 32A:991-1001.

•• Recent review on the different classes of MDR modulatorsand their mechanisms of action.

4. SIMON SM, SCHINDLER M: Cell biological mechanismsof multidrug resistance in tumors. Proc. Natl. Acad. Sci.USA (1994) 91:3497-3504.

•• Detailed discussion on the molecular mechanisms of MDR.

5. ROBERT J: Multidrug resistance reversal agents. DrugsFuture (1997) 22:149-158.

•• Another recent review on MDR modulators with emphasison the biochemistry and the status of clinical trials.

6. TSURUO T, IIDA H, TSUKAGOSHI S, SAKURAI Y: Over-coming of vincristine resistance in P388 leukaemia invivo and in vitro through enhanced cytotoxicity of vin-cristine and vinblastine by verapamil. Cancer Res.(1981) 41:1967-1972.

7. FORD JM, BRUGGEMANN EP, PASTAN I, GOTTESMANMM, HAIT WN: Cellular and biochemical characteriza-tion of thioxanthenes for reversal of multidrug resis-tance in human and murine cell lines. Cancer Res.(1990) 50:1748-1756.

8. TWENTYMAN PR: Modification of cytotoxic drug resis-tance by non-immuno-suppressive cyclosporins. Br. J.Cancer (1988) 57:254-258.

9. JACHEZ B, NORDMANN R, LOOR F: Restoration of taxolsensitivity of multidrug resistant cells by the cyclo-sporine SDZ PSC833 and the cyclopeptolide SDZ 280-446. J. Natl. Cancer Inst. (1993) 85:478-483.

10. WANG L, YANG CPH, HORWITZ SB, TRAIL PA, CASAZZAAM: Reversal of the human and murine multidrug re-sistance phenotype with megestrol acetate. CancerChemother. Pharmacol. (1994) 34:96-102.

11. CHATTERJEE M, HARRIS AL: Reversal of acquired resis-tance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with al-pha 1 acid glycoprotein. Br. J. Cancer (1990) 62:712-717.

12. CHAUFFERT B, MARTIN M, HAMMANN A, MICHEL MF,MARTIN F: Amiodarone-induced enhancement ofdoxorubicin and 4¢-deoxydoxorubicin cytotoxicity torat colon cancer cells in vitro and in vivo. Cancer Res.(1986) 46:825-830.

13. NAKANISHI O, TSURUO T: Multidrug resistance-reversing agents for potential clinical use. In: Multi-drug Resistance in Cancer Cells. Gupta S, Tsuruo T (Eds.),John Wiley & Sons Ltd. (1996):375-384.

14. HYAFIL F, VERGELY C, DU VIGNAUD P, GRAND-PERRETT: In vitro and in vivo reversal of multidrug resistanceby GF120918, an acridonecarboxamide derivative.Cancer Res. (1993) 53:4595-4602.

15. JULIA AM, ROCHE H, BERLION M et al.: Multidrug resis-tance circumvention by a new triazinoamino-piperidine derivative S9788 in vitro: definition of theoptimal schedule and comparison with verapamil. Br.J. Cancer (1994) 69:868-874.

16. TURNER RN, CURTIN NJ: Dipyridamole increases VP16growth inhibition, accumulation and retention in pa-rental and multidrug-resistant CHO cells. Br. J. Cancer(1996) 73:856-860.

17. LIU Z, LHEUREUX F, POULIOT JF et al.: BIB22 BS, potentmultidrug resistance-reversing agent, binds directly toP-glycoprotein and accumulates in drug-resistantcells. Mol. Pharmacol. (1996) 50:482-492.

18. ECKER G, CHIBA P: Recent developments in overcom-ing tumour cell multidrug resistance. Exp. Opin. Ther.Patents (1997) 7:589-599.

• The article covers the patent literature of MDR modulatorsfor the years 1995 and 1996.

19. BREIER A, BARANCIK M, STEFANKOVA Z, UHRIK B,TRIBULOVA N: Effect of pentoxifylline on P-glycoprotein mediated vincristine resistance of L1210mouse leukemic cell line. Neoplasma (1994) 41:297-303.

20. DANTZIG AH, SHEPARD RL, CAO J et al.: Reversal of P-glycoprotein-mediated multidrug resistance by a po-tent cyclopropyldibenzosuberane modulator,LY335979. Cancer Res. (1996) 56:4171-4179.

21. ANDRUS MB, LEPORE SD: Synthesis of stipiamide and anew multidrug resistance reversal agent, 6,7-dehydrostipiamide. J. Am. Chem. Soc. (1997) 119:2327-2328.

22. GERMANN UA, SHLYAKHTER D, MASON VS et al.: Cellularand biochemical characterization of VX-710 as a che-mosensitizer: reversal of P-glycoprotein-mediatedmultidrug resistance in vitro. AntiCancer Drugs (1997)8:125-140.

23. FORD JM, PROZIALECK WC, HAIT WN: Structural fea-tures determining activity of phenothiazines and re-lated drugs for inhibition of cell growth and reversalof multidrug resistance. Mol. Pharmacol. (1989) 35:105-115.

24. CHIBA P, BURGHOFER S, RICHTER E et al.: Synthesis,pharmacologic activity, and structure-activity rela-tionships of a series of propafenone-related modula-tors of multidrug resistance. J. Med. Chem. (1995)38:2789-2793.

25. ECKER G, CHIBA P, HITZLER M et al.: Structure-activityrelationship studies on benzofuran analogs ofpropafenone-type modulators of tumor cell multidrugresistance. J. Med. Chem. (1996) 39:4767-4774.

26. OJIMA I, BOUNAUD PY, TAKEUCHI C, PERA P, BERNACKIRJ: New taxanes as highly efficient reversal agents formultidrug resistance in cancer cells. Bioorg. Med. Chem.Lett. (1998) 8:189-194.

27. STEIN W: Saturation reversal of the multidrug pumpusing many reversers in low-dose combinations. Anti-cancer drugs (1995) 6:727-35.



Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

Patents

101. NIKKEN CHEM. CO. LTD.: WO9604268 (1996).



104. CELL THERAPEUTICS, INC.: US5670507 (1997).

105. ELI LILLY & CO.: EP-773217-A (1997).

106. PFIZER, INC.: US5583137 (1996).

107. JANSSEN PHARM. NV: WO9734897 (1997).


109. ONTOGEN CORP.: EP-812829-A (1997).

110. ONTOGEN CORP.: EP-812830-A (1997).

111. BYK GULDEN LOMBERG CHEMISCHE FABRIK GMBH:WO9728166 (1997).

112. XENOVA LTD.: WO9620179 (1996).

113. XENOVA LTD.: WO9620180 (1996).

114. XENOVA LTD.: GB-2296496-A (1996).

115. BATRA S, CARLSSON J-I, FEX T: WO9727211 (1997).

116. PHARMA MAR SA: WO9701336 (1997).

117. MERREL PHARM., INC.: US5670521 (1997).

118. ELI LILLY & CO.: WO9748689 (1997).

119. NISSHIN FLOUR MILLING CO. LTD.: EP-781552 (1997).

120. NISSHIN FLOUR MILLING CO. LTD.: EP-787716 (1997).

121. NISSHIN FLOUR MILLING CO. LTD. (NISSHIN SEIFUN KK):JP9268165 (1997).

122. HITACHI CHEM. CO. LTD. et al.: WO9726881 (1997).

123. VERTEX PHARM., INC.: WO9615101 (1996).



126. MEMORIAL-SLOAN KETTERING CANCER CENTER et al.:WO9718215 (1997).

127. ETABLISSEMENT DE TRANSFUSION SANGUINE DE LYONet al.: WO9733575 (1997).

128. MITSUBISHI CHEMICAL CORP. (MITSUBISHI KAGAKUKK): JP8268965 (1996).

129. FOX CHASE CANCER CENTRE: WO9748708 (1997).

130. PHARMACYCLICS, INC.: WO9726915 (1997).

131. RHÔNE-POULENC RORER SA: WO9533736 (1995).








139. RESEARCH FDN., STATE UNIV. OF NEW YORK:WO9732578 (1997).

140. PRIEBE W, CHAIRES JB, PRZEWLOKA T et al.: WO9734612(1997).

141. INDENA SPA: WO9701570 (1997).

142. ELI LILLY & CO. et al.: WO9723211 (1997).

143. NORSK HYDRO AS: WO9705154 (1997).

144. BOEHRINGER MANNHEIM ITALIA SPA et al.: US5604246(1997).

145. WISCONSIN ALUMNI RES. FDN.: WO9731936 (1997).

146. RUTGERS UNIV.: WO9636612 (1996).

147. SHIMIZU Y, FAIRCHILD CR: US5494930 (1996).

148. HYBRIDON, INC.: WO9602556 (1996).

149. UNIV. OF NEBRASKA: WO9640715 (1996).

150. CITY OF HOPE: WO9608558 (1996).

Iwao Ojima† , Pierre-Yves Bounaud & Cecilia Fumero Oderda† Author for correspondenceDepartment of Chemistry, State University of New York at StonyBrook, Stony Brook, NY 11794-3400, USA



Exp

ert O

pin.

The

r. P

aten

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

yers

on U

nive

rsity

on

05/1

8/13

For

pers

onal

use

onl

y.

Documents

Recent strategies for the treatment of multi-drug resistance in cancer cells