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Review

Oncologic, Endocrine & Metabolic

Recent developments in overcoming tumour cellmulti-drug resistance

Gerhard Ecker & Peter Chiba

The development of unspecific mechanisms of resistance re-mains a serious impediment to cancer chemotherapy. The termmulti-drug resistance (MDR) is defined as resistance of cancercells to a broad variety of structurally and functionally diversechemotherapeutic drugs. On the cellular level, several mecha-nisms responsible for MDR have been identified. In addition,numerous approaches for overcoming tumour cell MDR havebeen reported in the literature. This review focuses on patentspublished in 1995 and 1996 on modulators of MDR and relatedapproaches to therapy of multi-drug resistant tumours.

Exp. Opin. Ther. Patents (1997) 7(6):589–599

1. Introduction

There are close to six million deaths caused by cancerworldwide each year [1]. About half of the patientsstricken with this disease can be cured by surgery orradiation therapy because the tumour is localised tothe site of origin. The remaining cancers includesystemic haematological malignancies and tumoursthat have metastasised. From these cancer types, atpresent only a fraction (5 - 10%) can be cured bychemotherapy; the remainder are either intrinsicallyresistant towards chemotherapy or acquire resistanceduring the course of therapy.

Clinical resistance is influenced by a wide variety offactors, such as tumour mass, stage of disease, vascu-larisation, age of the patient, pharmacokinetic and cellkinetic parameters, and the dosage and chemotherapyregimen employed. However, a correlation betweenclinical response and sensitivity of individual tumourcells can be observed. This suggests that clinicalresistance is at least in part due to biochemical resis-tance at a cellular level.

A number of specific resistance mechanisms againstnatural and semisynthetic cytotoxins as well as against

antimetabolites have been described [2]. These in-clude:

• lack of specific transport mechanisms which medi-ate cellular uptake of the compounds [3]

• decreased conversion to the biologically active me-tabolites [4]

• increased detoxification [5]

• alteration of the cellular target structure [6]

• increased excision-repair in the case of DNA-tar-geted compounds [7]

In addition, a phenotype of tumour cells has beendescribed which causes simultaneous resistanceagainst a variety of structurally and functionally diversedrugs such as anthracyclines, epipodophyllotoxins,actinomycin D, vinca alkaloids, colchicine and taxanes[8]. This phenomenon has been termed MDR [9]. Twomajor causes for MDR have been identified. The firstprecludes drugs from reaching their potential intracel-lular targets and involves active efflux at the level ofthe plasma membrane [10]. The other cause is relatedto an inability of cells to undergo programmed celldeath (apoptosis) [11].

5891997 © Ashley Publications Ltd. ISSN 1354-3776

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Active efflux is mediated by a group of ATP-dependenttransmembrane proteins, among which P-glycoprotein(PGP) [12] and the MDR associated protein (MRP) [13]are the most well characterised members. In somecases, a direct relationship between PGP expressionand chemosensitivity has been reported. These includeleukaemias and lymphomas [14-18], myeloma [19],breast cancer [20,21], oesophageal carcinoma [22],childhood sarcomas [23] and neuroblastoma [24]. Overthe last 15 years, a number of substances or substance

classes have been identified which are able to revertmulti-drug resistant cells to resemble their drug sensi-tive counterparts. These include:

• ion channel blockers such as verapamil [25]

• some dihydropyridines [26]

• amiodarone [27] and propafenone [28]

• steroids [29]

• cyclosporines [30]

• antipsychotic drugs like phenothiazines [31] andthioxanthenes [32]

• the antimalarial agent quinine [33]

• the triazinoaminopiperidine S 9788 (Servier) [34]

• the acridone carboxamide GF 120918 (Glaxo) [35]

• the pteridine derivative BIBW 22 (Boehringer Ingel-heim) [36]

Several compounds are presently used in clinical PhaseII/III in conjunction with standard chemotherapy regi-mens. This article reviews the patent literature ofMDR-modulators for the past two years. For additionalinformation on clinical, biochemical and molecularaspects of MDR, the reader is referred to the compre-hensive review of Kane [37] and references therein.

2.1 Low molecular weight multi- drug resistancemodulators

The first MDR-modulator described in the literaturewas the calcium channel blocker verapamil (1). Vera-pamil blocks the multi-drug transporter P-glycoproteinand thus leads to resensitisation of mdr1-positivetumour cells to natural product toxins. Due to its highcardiovascular potency, dexverapamil ((R)-verapamil;Knoll [BASF]), which shows only 1/100 of the cardio-vascular activity of its antipode, was used in clinicalstudies [38]. American Cyanamid Co. claims, in EP-634401-A, a series of benzylmercaptane derivatives,whereby compound 2 is specifically claimed [101].Compound 2 shows potent in vivo activity againstvincristine-resistant murine leukaemia P388/VCR im-planted ip. in CDF1 mice, thus reducing the relativetumour growth (21 days) from 10.99 to 3.35 whenco-administered (150 mg/kg/dose) with doxorubicin.

The potassium channel blocker amiodarone (3) is ahighly active PGP inhibitor in in vitro assays [39].Nevertheless, its clinical use as anti-arrhythmic agentis very limited because of its severe side-effects. Thesame situation might occur in the case of MDR-modu-lation. In EP-652004-A (Eli Lilly & Co.), a series ofderivatives are claimed, which show structural analogyto amiodarone [102]. The MDR-modulating activity of

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CH3 CH3CH3 CH3

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590 Recent developments in overcoming tumour cell multi-drug resistance - Ecker & Chiba

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents (1997) 7(6)

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raloxifene (4) and LY 139478 (5) in comparison to17α-oestradiol and tamoxifen is given. Reversal factorsin HL60/ADR cells were 7.1 and 5.7, respectively (c.f.,oestradiol and tamoxifen which showed 1.0 and 1.4,respectively). In WO9611677-A (Eli Lilly & Co.) a seriesof 53 analogues (e.g., 6) with broader structurallyvariety is claimed [103]. They are stated to show asignificant effect on PGP, although no data are pre-sented.

Two of the compounds more recently described are S9788 (Servier) [40] and BIBW 22 (Boehringer Ingel-heim) [41]. Both compounds show nitrogen containingcentral aromatic rings and seem to be specificallydesigned to act as inhibitors of the multi-drug trans-porter PGP. There were three patents published withinthe last two years claiming compounds with at leastsome structural analogy at the central aromatic moiety.Pfizer, Inc. claimed 24 derivatives (US5491234-A) witha central aminopyrimidine ring and two dimethoxy-benzene moieties (7), which seem to be characteristicfor verapamil-type MDR-modulators [104]. No biologi-cal data are presented. In a further invention fromPfizer, Inc. (US5583137) five diaminopurine analoguesare claimed (8) which also are stated to block PGPmediated transport of cytostatic drugs [105]. In analogyto the former patent, no biological data are presented.Dr Karl Thomae GmbH claimed a set of elevenpyrimido-pyrimidines (9) which show a resensitisationof adriamycin-resistant tumours [106]. Thus, cytotoxic-ity of the respective compounds was enhanced with asubtoxic concentration of 100 ng/ml adriamycin by afactor of 3 - 33.

Glaxo Wellcome, Inc. claims in WO9611007-A paren-teral pharmaceutical compositions [107] containingGF-120918A (Glaxo), an acridoncarboxamide withhigh PGP inhibitory potency [42]. Xenova Ltd. claimsa series of 58 derivatives (10; WO9620190-A) whichlook like a hybrid between GF-120918A and XR-1500(Xenova). Some of the compounds specifically claimedshow EC50 values in the nanomolar range, when testedin a daunorubicin uptake assay [108]. Generally, activi-ties of the compounds were within 0.02 - 7.0 µM.

Phenothiazines are a well established class of com-pounds with regards to MDR-modulation. There areseveral studies published which show the efficacy ofthe compounds [43]. Additionally, some SAR-studieswere undertaken, which identify three important struc-tural features, including the hydrophobicity of thetricyclic ring, the length of the alkyl bridge and thecharge on the terminal amino group [44]. St. JudeChildren’s Research Hospital claims a series of N-sub-stituted phenoxazines (11) structurally related to phe-nothiazines [109]. The compounds potentiate thecytotoxic activity of vincristine, vinblastine and

N N

NN

NH

F

F

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NH

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N

N

N N

N

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CH3

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S-9788

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CH3

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CH3

CH3

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7

8

9

Oncologic, Endocrine & Metabolic - Review 591


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etoposide with EC50 values in the range of 0.85 - 32nM.

Within the large group of MDR-modulators, steroidsare also exemplified. Progesterone is stated to exert anespecially high chemosensitising potency [45]. In thelast two years, two patents on steroid analogues werepublished. The Salk Instutite for Biological Studiesclaims a series of six synthetic steroids (12), whichenhance accumulation of antineoplastic agents such asvinca alkaloids, epipodophyllotoxins, anthracyclines,actinomycin D and plicamycin in tumour cells [110].EC50 values in CEM/VBL100 cells were between 1.1and > 8.0 µM (verapamil: 1.3 µM). Georgetown Uni-versity claims a series of progesterone analogues withthe general formula 13, which enhance cytotoxicity ofvinblastine on MDA435/LCC6 invasive, metastatic, ER-negative human breast cancer cells transduced withmdr1 cDNA [111]. Interestingly, both groups of com-pounds bear an aniline partial structure, which isunusual for steroids.

Eli Lilly & Co. claims a series of cysteinyl-leukotrienereceptor antagonists (14), which act as inhibitors ofPGP [112]. The specified 18 compounds show 25 - 73%inhibition of growth of HL60/ADR cells in combinationwith adramycin. Adriamycin alone at the tested con-centration does not usually inhibit the growth ofHL60/ADR cells. The chemical structure of the com-pounds is very interesting, because they are negativelycharged at physiological pH, but might be transportedin an uncharged form. As will be further exemplifiedin the chapter covering structure-activity relationship(SAR) studies, inhibitors of PGP are usually positivelycharged at physiological pH.

In an analogy with compound 14, the N-acetyl-S-far-nesylcysteine methyl ester (15) claimed by the DukeUniversity also seems interesting in terms of SARstudies [113]. This compound also exhibits none of thegeneral structural features of PGP-inhibitors, such asone or more aromatic rings or a positively chargednitrogen atom. Several derivatives of general structure16 were tested and showed either:

• no effect

• stimulation of the ATPase activity of PGP

• stimulation of ATPase activity at low concentrations(5 - 10 µM) and inhibition at higher concentrations(> 20 µM) (e.g., 15)

In WO9623506-A, a series of seven 5′-substituted nu-cleosides (17) is claimed which inhibit MDR in humanand mouse tumour cells [114]. Especially preferred are5-(2-bromovinyl)-2′-deoxyuridine and (E)-5-(2-bro-

movinyl)-uracil. The compounds are claimed for usein combination with alkaloids, antibiotics, antimetabo-lites and cis-platin.

At a concentration of 1 µg/ml, compound 17 enhancescytotoxicity of adriamycin in adriamycin-resistant F64cells. This seems very interesting, because antimetabo-lites and cis-platin are thought not to interact with themulti-drug transporter PGP and therefore are usuallynot affected by the MDR-phenotype. Nevertheless,Yusa et al. recently published some data which showthat azidothymidine seems to be a substrate for PGP[46].

Oguro claims 4-piperidino-piperidine (18) as an MDR-modulating agent [115]; in tests, the antitumour activityof daunomycin, adriamycin, cis-platin, mitoxantroneand methotrexate was enhanced. In analogy with theprevious compound discussed, resistance to an-timetabolites and cis-platin seems to be reversed bycompound 18. This points to additional mechanismsof resistance in the experimental model system used.

Two of the most active MDR-modulators known arethe cyclopeptides cyclosporin A [47,48] and its non-im-munosuppressive congener SDZ PSC 833, which alsolacks the renal toxicity reported for cyclosporin A [49].In WO9531474-A, a series of hydrophobic dipeptidescalled reversins are claimed which compete with cy-tostatic drugs on the multi-drug transporter PGP [116].Thus, reversin 121 (19) and reversin 205 were shownto render adriamycin cytotoxic in adriamycin-resistantK562 human erythroblastoid tumour cells in a concen-tration range of 1 - 5 µM. Adriamycin alone wasineffective in these cells.

Apart from direct interaction of modulators with PGP,several alternative mechanisms for modulation of PGPactivity are discussed in the literature. One of these isinteraction with protein kinase C (PKC), which isresponsible for phosphorylation of PGP [50]. Thus,direct inhibition of PKC [51] or interaction of catam-phiphilic drugs with the PKC substrate phosphatidyl-serine [52] could be responsible for MDR-modulation.There are several reports that the PKC-inhibitorstaurosporine is a highly active MDR-modulator [53].Ciba-Geigy AG claims a series of staurosporine ana-logues (e.g., 20b [117], 21a [118]) which potentiate thecytotoxic activity of vinblastine in PGP overexpressingKB-8511 cells (Table 1). Additionally, the Universityof Illinois claims a method for inhibition of MDR1induction and MRP expression in cancer cells usingPKC inhibitors [119]. Erbstatin, tyrphostatinA25, tyr-phostin B46, staurosporine, H7, neomycin sulfate,chelerythrine and calphostin C are specifically claimed.



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NH

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N

O

O

O

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CH3

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GF 120918XR-1500

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1213

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OH

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14

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15 16

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17

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CH3

CH3

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CH3

19

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CH3

CH3

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CH3

H

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N

NO

O

O

OCH3

CH3

CH3

CH3

CH3

CH3

CH3

H

20b 21a

Table 1: Series of staurosporin analogues from Ciba-Geigy.

Compound number Name Activity§

20a N-6-di-(methoxycarbonylmethyl)-staurosporin 92

20b N-BOC-6-methoxycarbonylmethyl-staurosporin 50

20c N-BOC-6-methoxycarbonylmethyl-staurosporin 2

21a N-BOC-6-methylstaurosporin n/a

21b N-ethoxycarbonyl-6-benzyl-staurosporin 24

21c N-(2-(tetrahydropyran-4-yloxy)-acetyl)-6-methyl-staurosporin 87

§Multi-drug resistance-modulating activity is represented as % growth of KB 8511 cells in presence of 25 ng/ml vinblastine and 0.1µM of the respective compound.



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Pharmacia & Upjohn Co. claims a novel gene for aprotein kinase associated with drug-resistance [120].The gene encodes for PKR, a novel member of theprotein kinase family. The predicted location of theprotein is the cell membrane. Analysis of the expres-sion of PKR showed higher expression in severalmulti-drug resistant cell lines compared to their sensi-tive parental cell lines. Inhibition of PKR may resensi-tise multi-drug resistant cells to chemotherapeuticdrugs.

Chugai Seiyaku KK claims a novel anticancer prepara-tion including retinoic acid as the antineoplastic agent,clotrimazol (cytochrome P-450 inhibitor) and the PGPantagonist verapamil [121]. The mixture had betterefficacy on retinoic acid-resistant HL-60 cells than thatof a mixture of retinoic acid with either the P450inhibitor or the PGP antagonist.

Another mechanism of action of MDR-modulators isunspecific interaction with membrane phospholipids[54,55]. Thus, detergents like Cremophor EL andTween 80 are also able to reverse resistance from theMDR-phenotype and to inhibit binding of the photo-affinity probe azidopine to PGP [56]. Additionally,alternative methods of cytotoxic drug delivery appearto be able to circumvent resistance. Encapsulation ofdoxorubicin in liposomes [57,58] or in polyisohexyl-cyanoacrylate nanospheres [59,60] leads to enhancedcytotoxicity against multi-drug resistant cells.Liposome encapsulated vincristine also is more toxicagainst multi-drug resistant HT-29 colon cancer cellsthan the free drug [61]. The Rush-Presbyterian-St.Lukes Medical Center claims a method for reducingMDR by co-administration of a toxin with a non-ionicamphipathic ester of fatty acids or a reverse poloxymer[122]. CRL-1605, which is commercially available fromCytRx Corp., is specifically claimed.

Liposome Co., Inc. claims pharmaceutically activeceramide/sphingomyelin liposomes, which are activeon P388/adriamycin-resistant leukaemia cells [123].The specifically claimed compound, N-hexylsphingan-ine (22), produced a 60% survival rate in mice pre-treated with the resistant leukaemia cells after elevendays. In the control group all mice died.

Georgetown University claims liposome encapsulatedtaxol for use in cancer therapy [124]. Liposome encap-sulation of taxol improves its stability and solubility,reduces anaphylactic reactions and cardiotoxicity, re-duces administration time, enhances its effectivity andmodulates MDR. The compositions contain aliposome-forming compound such as phosphatidylcholine, cardiolipin and taxol.

2.2 Antisense oligonucleotides

One approach to overcome MDR could be to preventsynthesis of the multi-drug transporters PGP and MRPvia inhibition of the corresponding mRNA. Severalgroups have reported the successful down-regulationof mdr1 mRNA and cell surface PGP using antisenseor ribozyme technology. This approach led to partialor even complete reversal of MDR in vitro [62,63]. IsisPharm., Inc. claims a series of antisense oligonu-cleotides specifically designed to hybridise to themRNA encoding for MRP [125]. These oligonucleotideshave been shown to modulate the synthesis of the MRPprotein. The size of MRP positive tumours in mice wasshown to be smaller in comparison to the control aftertreatment with the antisense oligonucleotides.

2.3 Antibodies

In addition to ‘classical’ inhibitors of drug transport,monoclonal antibodies to PGP and antibody-toxinconjugates are known to modulate MDR in vitro andin vivo [64,65]. Thus, antibodies against multi-drugtransport proteins could be a versatile tool for specifi-cally targeting toxins to multiresistant tumour cells.Immunomedics, Inc. claims, in WO9604313-A, poly-specific immunoconjugates and antibody compositesfor targeting the multi-drug resistant phenotype [126].The conjugates could carry a radioisotope or a cellkilling agent, like ricin, abrin or diphtheria toxin. Theantigenic target proteins are members of the class ofmulti-drug transporters of both human and bacterialorigin, such as multi-drug resistant Pseudomonasaeruginosa.

2.4 Toxins not affected by the multi-drugresistance phenotype

One completely different approach contrasting withthe aforementioned ones is the development of toxinswhich remain active in MDR-positive tumours. Inaddition to antimetabolites, alkylating agents and plati-num complexes, which remain unaffected by MDR, anumber of compounds structurally related to ‘classical’toxins have been developed to treat multi-drug resis-tant tumours. The most complete series has beenamongst the anthracyclines and related compounds,where discrete changes in structure lead to enhancedactivity in multi-drug resistant tumour cells [66].

Numerous compounds have been claimed within thepast two years following this approach. These include:

• naphthochinones [127,128]

• sulfonyl-anthracyclines [129,130]

• fluoro-anthracyclines [131]

• certain taxols [132]



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• colchicine derivatives [133]

• tribenzimidazoles [134]

Although, in almost all cases, the specified compoundsexhibit lower cytotoxicity in multi-drug resistant cellsthan in the analogous wild-type line, the fold resistance(FR = EC50 of the compound in the resistant line/EC50

in the corresponding WT line) is generally remarkablylower than those for the selecting toxins.

Thus, the 3′-aziridino-anthracycline 23 showed EC50

values of 13 and 22 ng/ml in the LoVo and LoVo/DOXline, respectively (FR = 1.7) [129]. The FR for doxoru-bicin in the same assay system was 6.0 (825 vs. 4975ng/ml). Similar results were obtained for the sulfonyl-anthracycline derivative 24 (FR = 5.8 vs. 52.1) [130].

A series of novel taxoids (25) were shown to exhibitactivity in adriamycin-resistant MCF7 mammary carci-noma cells with an FR value of 27.5 (docetaxel: FR =235) [132].

Additionally, a series of colchicine derivatives (26)showed remarkably enhanced cytotoxicity in multi-drug resistant MCF7 breast tumour cell lines [133]. TheFR values were between 2.2 and 5.0, whereas thosefor colchicine and taxol displayed values of 25.5 and59.0.

In contrast, Kyowa Hakko Kogyo Co. Ltd. claimed acompound (27), which shows almost identical EC50

values in lymphocytic leukaemia P388 and P388 sub-lines resistant to either adriamycin or cis-platin, cyclo-phosphamide or mitomycin (EC50 = 8.17, 15.6, 19.8,6.34 and 15.3 nM, respectively) [134].

3. Structure-activity studies

Although many excellent reviews on the phenomenoncalled MDR have been published, little is known aboutSAR studies on modulators of MDR. Recently, onereview was published dealing with first results in thisfield. Nevertheless, the discussion whether inhibitorsof PGP act via distinct receptor interaction or viaunspecific membrane interaction remains controversial[67]. Several reports show a good correlation betweenPGP inhibitory activity and interaction of the com-pounds with artificial membranes. We recently showeda good correlation between lipophilicity of the com-pounds and PGP inhibitory activity for a series ofpropafenone type MDR-modulators. A series of ben-zofurane analogues generally showed remarkablylower activity, although differential scanning cal-orimetry measurements showed membrane interactionproperties comparable to the propafenones [68].

NH

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CH3

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4. Expert opinion

Many isolates of clinical tumour samples express P-gly-coprotein. Early Phase I studies with verapamil, cy-closporin A, quinine and tamoxifen demonstratedserum concentrations of the modulators in the rangeknown to reverse drug-resistance in vitro and in animalmodels. Nevertheless, inherent pharmacological prop-erties of the modulators often precluded optimal dos-age. Early reports of success in smaller patient groupsmight in part have been due to a change in pharma-cokinetics of toxins, which share a common metabo-lism by cytochrome P450 isoenzymes with thechemosensitisers. A dose escalation mediated by de-creased catabolism mimicked a sensitising action ofmodulators [69]. In contrast, some of the secondgeneration modulators show only minor influences onP450-mediated toxin metabolism and thus do notrequire a dose reduction of the toxins in clinicalstudies. In addition, these compounds lack the doselimiting side-effects of the parent compounds.

The modulator approach seems promising especiallyin the treatment of haematological neoplasias as wellas in neuroblastoma patients.

A number of ongoing clinical Phase II and III studieshave been initiated over the past four years. We willlearn from those trials whether the concept of MDR-modulation in tumour cells will make a major contri-bution to the outcome of cancer treatment in thefuture.

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Gerhard Ecker1 † & Peter Chiba2

†Author for correspondence1Institute of Pharmaceutical Chemistry, University of Vienna, Althan-strasse 14, A-1090 Wien, Austria2Institute of Medical Chemistry, University of Vienna, Waehringer-strasse 10, A-1090 Wien, Austria



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