Am J Cancer 2006; 5 (5): 285-297 1175-6357/06/0005-0285/$39.95/0

CURRENT OPINION

© 2006 Adis Data Information BV. All rights reserved.

Reversal of ABC Transporter-Dependent Multidrug Resistance in Cancer A Realistic Option? Ulrike Stein and Wolfgang Walther ¨ ¨ Department of Surgery and Surgical Oncology, Max-Delbruck-Center for Molecular Medicine, and Robert-Rossle-Clinic, Charit´e Campus Buch, Berlin, Germany

Abstract

Multidrug resistance (MDR) remains the major cause of failure of chemotherapeutic treatment in cancer therapy. Thus, reversal of MDR in cancer is a desperately needed clinical requirement and a scientific challenge. The MDR phenotype can be mediated by adenosine triphosphate (ATP)-binding cassette (ABC) transporter molecules, which lower the intracellular accumulation of an entire panel of structurally and functionally unrelated anticancer drugs. Detailed knowledge of the transporter molecules, their sequence polymorphisms, their regulation at the transcriptional, translational, and post-translational levels, as well as their functional characteristics for substrate binding and transport, represent the basis for specific and, thus, successful intervention strategies for reversal of ABC transporter-dependent MDR in the clinic. Initial data obtained with third-generation inhibitors of ABC transporter-dependent MDR in clinical trials, and new developments with tools that interfere with expression of MDR genes, thereby leading to restored chemosensitization, are very promising attempts that provide grounds for cautious optimism. This article presents an overview of different strategies for overcoming ABC transporter-dependent MDR. Approaches targeting the function as well as the expression of MDR-related ABC transporters are summarized. Clinical trials evaluating the impact of compounds for reversal of ABC transporter-dependent MDR are discussed. The question remains, is reversal of MDR in cancer truly an achievable goal, given the different tumor entities and tumor heterogeneity, different tumor resistance profiles, and different therapeutic regimens? If so, MDR reversal will probably be successful only when tailored to an individual patient with an individual tumor, resistance profile and response prediction, i.e. individual prevention, circumvention, and reversal of MDR. Tailor-made MDR reversal strategies are the ultimate goal – and probably a realistic option.

1. Cancer, Multidrug Resistance (MDR), and Adenosine Triphosphate (ATP)-Binding Cassette (ABC) Transporters To date, chemotherapy is the most effective treatment for advanced and metastatic tumors. However, the success and efficacy of chemotherapy in the clinic vary from patient to patient. Some patients experience complete responses, while others respond partially and/or transiently. Furthermore, some patients treated with multiple anticancer drugs develop cross-resistance to many other chemotherapeutic agents to which they have never been exposed; consequently, the possibility of curing these patients with chemotherapy is dramatically reduced.[1,2]

Although multiple mechanisms of drug resistance may occur in parallel or sequentially, the phenomenon of multidrug resistance (MDR) is recognized as one of the most frequent causes of drug resistance in cancer. MDR is defined as the simultaneous resistance to structurally and functionally unrelated natural product anticancer drugs. MDR reflects the ability of a tumor cell to resist otherwise lethal or sublethal doses of multiple (usually cytotoxic) drugs and represents the major cause of failure of cancer chemotherapy.[1-4] Development of MDR is mainly dependent on the expression of MDR-associated genes encoding adenosine triphosphate (ATP)binding cassette (ABC) transporter proteins, which belong to the largest family of energy-dependent transmembrane transporter

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proteins. To date, 49 human ABC transporters have been described and classified into seven subfamilies based on the assignments derived from Allikmets et al.[5-7] ABC transporters act as drug efflux pumps. Their physiologic functions are to protect epithelial cells (e.g. in the gastrointestinal tract, liver, kidney, and the capillaries of the brain) against uptake of xenobiotics and to promote excretion of the latter in the bile and urine. They also transport multiple classes of anticancer drugs out of the cell, making the cancer multidrug-resistant.[8-13] The most prominent MDR-related ABC transporters are: the MDR gene 1 (MDR1; ABCB1) encoding P-glycoprotein and thereby causing the classical, P-glycoprotein-mediated MDR, the MDR-associated proteins 1, 2, and 3 (encoded by MRP1-3; ABCC1-3), and the breast cancer resistance protein (encoded by BCRP; ABC transporter in placenta, ABCP; mitoxantrone-resistant gene, MXR; ABCG2), which leads to the atypical, non-Pglycoprotein-mediated MDR. A causal role in the generation of the MDR phenotype has been demonstrated in transfection experiments for several ABC transporters, such as MDR1, MRP1-6, and BCRP; other ABC transporters have demonstrated an ability to transport multiple cytotoxic compounds.[4] Although the drug spectra of single MDR-associated ABC transporters are overlapping, they are not identical. Additionally, their substrate specificities might vary as a result of defined point mutations within their genes. 1.1 P-Glycoprotein

The ABCB subfamily of ABC transporters has 12 members, including the most important member, the MDR1/P-glycoprotein, which was first described about 3 decades ago.[14,15] P-glycoprotein was the first cloned human ABC transporter for which the generation of the MDR phenotype was shown directly by gene transduction.[16,17] It is a drug efflux pump, with two nucleotide binding sites and two transmembrane domains, each consisting of 12 membrane-spanning α-helices, which are believed to determine the substrate specificity of the drug transported. P-glycoprotein binds a wide spectrum of hydrophobic, neutral or positively charged substrates, such as anthracyclines, vinca alkaloids, epipodophyllotoxins, and taxanes.[11,12] MDR1 expression has been analyzed in a large number of studies. High levels of P-glycoprotein have been detected in normal tissues with excretory or secretory function.[18,19] P-glycoprotein is inherently overexpressed in tumors of the colon and kidney, and in adrenocortical and hepatocellular cancers, making them primarily resistant towards a wide range of anticancer drugs.[20-23] © 2006 Adis Data Information BV. All rights reserved.

Components of multimodal cancer therapy such as cytostatics (including cytotoxic agents), heat or radiation, are able to induce MDR1 expression in vitro.[24-28] Drug- and heat-responsive elements have been identified in the MDR1 promoter, with subsequent conversion of therapy-induced stress signals into transcriptional activation.[29] Single nucleotide polymorphisms, either of the MDR1 promoter[30,31] or of the MDR1 gene itself,[32] may alter basal expression levels of P-glycoprotein as well as drug specificity. Chemotherapy- and hyperthermia-induced MDR1 elevations have also been detected in patients. In approximately 50% of all treated tumors, induction of MDR1 expression has been observed.[33] Chemosensitive tumors such as myeloma and breast, ovarian, and cervical carcinomas show low or intermediate basal MDR1 expression, but upregulation of MDR1 expression in these tumors following chemotherapy results in development of the acquired type of MDR.[34-40] In patients with sarcoma with unresectable pulmonary metastases who underwent isolated single lung perfusion with doxorubicin, MDR1 messenger RNA (mRNA) expression increased significantly 50 minutes after drug administration.[41] Hyperthermia may also induce MDR1 gene expression.[42] However, because of the mild temperatures applied, hyperthermic isolated limb perfusion in patients with sarcoma or melanoma did not lead to induction of ABC transporters, but rather to elevated expression of the MDR-related major vault protein.[43] Reproducible correlations between MDR1 levels, treatment-related inductions, and clinical outcome have been demonstrated for hematological cancers.[44] Correlation of clinical outcome parameters with MDR1 expression has also been shown for sarcomas[45-47] and breast cancer.[48,49] Although the value of Pglycoprotein as a marker for poor prognosis has been suggested,[50] the prognostic implications of MDR1 expression are still controversial. 1.2 Multidrug Resistance Protein Family

The ABCC subfamily harbors the multidrug resistance protein (MRP)-related genes (MRP1-6, ABCC1-6; and MRP7-9, ABCC10-12) and consists of 12 or 13 members, respectively.[51,52] MRP1, the most studied member, was identified more than a decade ago in non-P-glycoprotein expressing, but multidrug resistant, human tumor cell lines.[53] MRP1 is structurally similar to Pglycoprotein, with an amino-terminal extension of five membranespanning α-helices. Like MRP1, MRP2, MRP3, and MRP6 also harbor these extra amino-terminal transmembrane domains. The MRP1 spectrum of transported hydrophobic anticancer drugs overlaps with those of P-glycoprotein, including anthracyclines, vinca alkaloids and epipodophyllotoxins. The MRPs transport Am J Cancer 2006; 5 (5)

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anionic and neutral drugs conjugated to acidic ligands, or by cotransport with free glutathione.[54-56] MRP4 and MRP5, like MRP1, 2, and 3, are also organic anion transporters; they have been identified as transporters of nucleotide analogs. The causal role for MRP1 in conferring the MDR phenotype to previously chemosensitive cells has been shown by gene transduction.[57,58]

context of response to chemotherapy and survival remains to be elucidated.

Basal expression of MRP1 has been found to be ubiquitous in human tissues, including almost all malignant tissues, for example, myeloma, sarcoma, and breast, lung, and ovarian cancer.[34,59-63] Correlations of high MRP1 expression with response to chemotherapy and disease-free survival have been demonstrated for breast, lung, and ovarian carcinomas, as well as for leukemias; increases in MRP1 have also been observed in refractory hematological malignancies.[64] Prognostic significance for MRP1 expression has been reported for primary breast cancer and neuroblastomas.[65,66] However, the clinical importance of MRP1 is still a matter of discussion, since studies have both confirmed and refuted a correlation between MRP1 expression level and outcome.[67-70]

Knowledge of the expression and function of genes and proteins related to drug resistance represents an essential prerequisite for selecting patients for an appropriate treatment regimen from which they may benefit. However, different mechanisms and genes contribute to the intrinsic and/or acquired drug resistance phenotype, and single individual markers have been shown to be of limited predictive value. Thus, simultaneous analysis of a panel of resistance-associated genes is desired. To date, there are no clinical tests available for predicting response to cancer chemotherapy. However, expression profiles of MDR genes for the prediction of chemosensitivity have been generated and published in several reports. Expression profiles of 38 ABC transporters have been generated by using low density microarrays combined with quantitative real time-polymerase chain reaction (RT-PCR) for three human cell lines.[82] For the 60 cell line panel of the National Cancer Institute (NCI), expression profiles of 48 ABC transporters have been created by means of quantitative RT-PCR.[83] Moreover, sensitivity to doxorubicin was predicted in human breast tumors on the basis of microarraygenerated and quantitative RT-PCR-validated expression profiles, and was correlated with patient survival.[84] These approaches show the significance of experimental data and may serve for the development of diagnostic methods for cancer response prediction in clinical samples. However, they also demonstrate the need for standardized diagnostic tools applicable in the clinical setting. Resistance profiling represents the basis for the selection of a tailor-made treatment regimen that may prevent induction of acquired MDR or circumvent intrinsic MDR. More importantly, these resistance profiles would also provide the basis for choosing tailor-made reversal strategies.

1.3 Breast Cancer Resistance Protein

Breast cancer resistance protein (BCRP) was originally identified in mitoxantrone-resistant, but P-glycoprotein and MRP1-negative, human carcinoma cell lines.[71-73] It is the most prominent member of the ABC subfamily G (ABCG) of ABC transporters, which also harbors four additional members. In contrast to the fulltransporters, this so-called half-transporter harbors only one nucleotide binding site and only one transmembrane domain with six membrane spanning α-helices, suggesting BCRP homodimerization to gain full transport activity.[72] Transduction of BCRP complementary DNA caused the resistance phenotype.[72] The drug spectrum of BCRP considerably overlaps with that of Pglycoprotein and includes mitoxantrone, topotecan, and doxorubicin, as well as tyrosine kinase inhibitors such as imatinib and gefitinib, albeit with differing efficiencies. Other BCRP substrates are methotrexate and flavopiridol.[74-78] Vincristine and taxanes, which are typical P-glycoprotein substrates, are not transported. Defined point mutations leading to amino acid substitutions alter substrate specificity and transport efficiency.[79,80] BCRP has been detected mainly in the intestine, colon, breast, liver canaliculi and renal tubules, and is highly expressed in the placenta and blood-brain barrier.[1,2,21] Basal BCRP expression has been frequently observed in human tumors of different entities. High BCRP levels have been detected for carcinomas of the digestive tract, lung carcinomas and melanomas.[81] The clinical relevance of high and/or therapy-induced BCRP expression in the © 2006 Adis Data Information BV. All rights reserved.

2. Resistance Profiling for Prediction of Chemosensitivity and Treatment Response – an Essential Prerequisite for Reversal Strategies

3. Reversal Strategies for MDR Correlations of P-glycoprotein expression with clinical outcome parameters has been repeatedly described for several tumor entities by different laboratories around the world. Primarily, failure of cancer chemotherapy has been linked to intrinsic and/or induced expression of P-glycoprotein, the most studied MDRassociated protein. These early observations gave rise to the optimistic assumption that if a single protein confers resistance to a whole array of structurally unrelated anticancer drugs, reversal of MDR might be a realistic option. As a consequence, different Am J Cancer 2006; 5 (5)

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strategies for reversing the MDR phenotype have been extensively studied for more than 2 decades. These strategies were originally aimed at inhibition of P-glycoprotein but, subsequently, several reversal approaches targeting different MDR-associated genes have also been developed. Tools to influence the function or expression of MDR genes and proteins include employment of specific antibodies, inhibition of drug transport, introduction of antisense oligonucleotides and ribozymes, and (one of the newest developments) transduction of small interference RNA molecules. However, the most promising attempts to reverse the MDR phenotype have involved inhibitory compounds that target P-glycoprotein function.

Table I. Selected experimental approaches for reversal of adenosine triphosphate (ATP)-binding cassette (ABC) transporter-dependent multidrug resistance (MDR) targeting the function of MDR-associated ABC transporters First-, second-, and third-generation inhibitors First-generation inhibitors verapamil cyclosporine (ciclosporin) quinidine quinine nifedipine Second-generation inhibitors valspodar (PSC833)

3.1 Reversal Strategies Targeting the Function of MDR-Associated ABC Transporters

biricodar (VX710) CBT-1 (NSC-77037) Third-generation inhibitors

3.1.1 First-, Second-, and Third-Generation Inhibitors

Numerous chemosensitizers have been identified over the last decade. These compounds have been shown to restore chemosensitization towards anticancer drugs such as anthracyclines, vinca alkaloids, epipodophyllotoxins, and taxanes in ABC transporter overexpressing cells. Although these chemosensitizers belong to diverse chemical classes, they share hydrophobicity as a common feature. Structure-activity relationship studies show that these agents act by binding to membrane spanning domains of the ABC transporters, thereby inhibiting the function of the drug efflux pump.[85] A variety of approaches were evaluated with the so-called firstgeneration inhibitors of P-glycoprotein (table I). These antagonists, such as the calcium channel antagonist verapamil and the immunosuppressive agent cyclosporine (ciclosporin), have already been used for other indications. First-generation inhibitors are themselves substrates for P-glycoprotein, and compete with the cytotoxic drug for binding and efflux by the P-glycoprotein pump, thereby limiting efflux of the drug. Intracellular drug concentrations increase, leading to enhanced cytotoxicity. The cell becomes chemosensitive and the MDR phenotype is reversed. These inhibitors were very effective at overcoming MDR in cultured cells. However, they produced disappointing results in clinical settings, and the optimism of the early years declined because of different intrinsic and/or therapy-induced expressions of single or multiple MDR genes in several tumor entities. Moreover, varying correlations of expression and clinical response in certain cancers following chemotherapy were observed. Both might have been due to use of non-standardized techniques for expression analysis. In addition, sequence polymorphisms may also have led to altered drug transport. Furthermore, extremely high inhibitor concentrations were needed for MDR reversal, leading to unacceptable toxicities. © 2006 Adis Data Information BV. All rights reserved.

tariquidar (XR9576) zosuquidar (LY 335979) laniquidar (R101933) elacridar (GF120918) LY475776 V-104 ONT-093 (OC-144-093) Tyrosine kinase inhibitors Gefinitib Erlotinib Imatinib Immunologic approaches Whole antibodies Immunotoxins scFv antibody fragments Synthetic peptide inhibitors

Moreover, these inhibitors are not exclusively transported by Pglycoprotein, which results in unpredictable pharmacokinetic interactions when they are co-administered with chemotherapy. Besides the major player P-glycoprotein, a variety of other MDRassociated genes/proteins have been identified in recent years, for example, MRP1 and its family members, BCRP, and relatives of P-glycoprotein itself.[86] Taken together, these findings showed that the MDR phenotype could no longer be attributed to a sole, well characterized protein, but rather had to be understood as the net effect of an entire panel of resistance genes controlling alternative resistance mechanisms. Second-generation P-glycoprotein inhibitors were created specifically to overcome P-glycoprotein-mediated resistance (table I). Similar to first-generation inhibitors, they act as competitive subAm J Cancer 2006; 5 (5)

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strates, but with less toxicity and greater potency for P-glycoprotein inhibition. The second-generation inhibitors valspodar (PSC833), a non-immunosuppressive cyclosporine analog, and biricodar (VX710) were designed to restore the effectiveness of chemotherapeutic agents in multidrug resistant tumors. Administration of second-generation inhibitors, in combination with chemotherapy, may lead to reversal of the MDR phenotype (see section 3.1.2). However, as seen with earlier inhibitors, some limitations of second-generation inhibitors, such as unacceptable toxicity and interactions with additional transporter molecules, remained unresolved. The development of third-generation P-glycoprotein inhibitors (table I) was based on structure-activity relationships.[87-89] These newly generated compounds inhibit P-glycoprotein more specifically and with greater potency than previous generations of inhibitors. In contrast to first- and second-generation inhibitors, thirdgeneration inhibitors are not substrates for a defined ABC transporter themselves. Following binding to membrane spanning domains of the pump, they cause conformational changes of the transporter protein, thereby hindering ATP hydrolysis.[88,89] Consequently, drug transport out of the cell is prevented or reduced, leading to increased intracellular concentration of the cytotoxic drug and enhanced cytotoxicity. The specificity of third-generation inhibitors with respect to P-glycoprotein is demonstrated for the chemosensitizer zosuquidar (LY335979). Zosuquidar has been shown to reverse MDR specifically in P-glycoprotein overexpressing cells, partially reversed mitoxantrone resistance and completely reversed vinorelbine resistance. In MRP1- or BCRP-overexpressing cells, however, drug resistance was not modulated.[90] To date, tariquidar (XR9576) has been shown to be one of the most potent MDR reversing agents. Treatment with tariquidar led to complete MDR reversal at low concentrations, with a long duration of activity in a panel of resistant tumor cells.[91] The effects of tariquidar are now under evaluation in several clinical trials (see section 3.1.2). Although P-glycoprotein plays a central role in the context of MDR, the above-mentioned P-glycoprotein inhibitors may also target additional MDR-related ABC transporters with different affinities, for example, biricodar inhibits MRP and BCRP, and tariquidar inhibits BCRP in addition to P-glycoprotein.[92,93] Specific inhibitors, such as MK-571 for MRP1[78,94] or fumitremorgin C for BCRP,[78,95] have been evaluated in experimental studies and shown to work successfully in terms of the respective transporter. However, their ability to cause an increase in accumulation of anticancer drugs associated with MRP1- or BCRP-mediated MDR needs to be demonstrated in clinical trials. © 2006 Adis Data Information BV. All rights reserved.

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3.1.2 Clinical Trials

Although a variety of randomized clinical studies have been conducted to evaluate the impact of first- and second-generation inhibitors on MDR-related ABC transporters, only a minority of the phase III clinical trials thereof showed statistically significant differences in overall survival.[4,85] In NSCLC patients, verapamil treatment led to improved overall survival with vindesine and ifosfamide chemotherapy;[96] in acute myelogenous leukemia (AML) patients, longer overall survival was observed with use of cyclosporine with daunorubicin and cytarabine;[97] and in breast cancer patients, increased overall survival and response rates were observed with use of verapamil in association with chemotherapy with vindesine and fluorouracil.[98] Data for clinical trials using second-generation inhibitors are mainly available for valspodar and biricodar. In particular, the non-immunosuppressive cyclosporine analog valspodar was thought to represent a promising MDR reversal compound. Phase I and II clinical trials reported response rates of about 50% in AML and ovarian cancer patients and demonstrated the need for dose reduction of the respective chemotherapeutic drug.[34] However, phase III clinical trials showed rather disappointing results.[4] Recently published phase III clinical trials demonstrated that addition of valspodar to vincristine, doxorubicin and dexamethasone did not improve treatment outcome in patients with resistant or relapsing chronic lymphatic leukemia, or in those with recurring or refractory multiple myeloma.[99,100] In addition, no benefit for valspodar in addition to mitoxantrone, etoposide, and cytarabine was reported in patients with relapsed or refractory AML and high-risk myelodysplastic syndrome.[101] Furthermore, although elevated P-glycoprotein function and expression did correlate with low complete remission rates and overall survival in patients with untreated AML, no benefit of valspodar in addition to daunorubicin and cytarabine was determined.[102] Biricodar, another second-generation inhibitor, is able to inhibit P-glycoprotein and MRP1, which might extend the application of this inhibitory compound to additional tumor entities. Several safety and efficacy studies have been published for biricodar in solid tumors, for example, with mitoxantrone and prednisone in hormone-refractory prostate cancer, with paclitaxel in women with advanced ovarian cancer refractory to paclitaxel therapy, with paclitaxel for advanced breast cancer refractory to paclitaxel, and with doxorubicin in patients with anthracycline-resistant advanced soft tissue sarcoma.[103-106] However, clinical trials with secondgeneration inhibitors – although still emerging – have mostly reported limited success. The disappointing results were mainly due to the need for dose reduction of the chemotherapeutic drug in the context of the inhibitor, high toxicity and unpredictable Am J Cancer 2006; 5 (5)

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pharmacokinetic interactions, and additional resistance mechanisms besides P-glycoprotein (and MRP1)-dependent MDR. Based on experiences with first- and second-generation inhibitors, the potent and specific third-generation inhibitors were a source of new hope for efficient reversal of P-glycoprotein dependent MDR. Clinical trials evaluating the reversal capability of these third-generation inhibitors have already been conducted, and are summarized for tariquidar (table II) and zosuquidar (table III). A phase I clinical trial of tariquidar demonstrated sustained inhibition of P-glycoprotein in CD56+ lymphocytes by rhodamine 123 flow cytometry.[111] Moreover, tariquidar treatment led to increased Tc-99m sestamibi accumulation in normal liver and drug-resistant tumors, demonstrating both the ability of this thirdgeneration inhibitor to affect the function of P-glycoprotein and the usefulness of sestamibi for monitoring P-glycoprotein function.[110] One phase II clinical trial has demonstrated limited clinical activity of tariquidar to restore sensitivity to anthracycline or taxane chemotherapy in advanced breast carcinoma.[109] Recruitment of patients for several phase I and II clinical trials is currently underway to further evaluate the impact of tariquidar in the treatment of solid tumors, such as brain tumors, neuroblastomas, Ewing sarcomas, and rhabdomyosarcoma in children, in adrenal cortex neoplasms, and in lung, ovarian, and cervical

neoplasms, when used in association with the anticancer drugs doxorubicin, vinorelbine, vincristine, docetaxel, and etoposide.[112] In addition, two phase III trials evaluating tariquidar in NSCLC have been terminated. These trials were: “A double-blind, randomized, placebo-controlled, multicenter, phase III study of tariquidar + vinorelbine as first-line therapy in non-small cell lung cancer (NSCLC),” and “A double-blind, randomized, placebocontrolled, multicenter, phase III study of tariquidar + paclitaxel/ carboplatin as first-line therapy in non-small cell lung cancer (NSCLC).”[112] Both trials used the P-glycoprotein inhibitor tariquidar as first-line therapy in combination with chemotherapy for NSCLC, a disease with short median survival times. However, the frequency and level of P-glycoprotein expression in NSCLC as a prerequisite for use of P-glycoprotein-based inhibitors is still a matter of discussion. Perhaps patients benefit from these inhibitors because the few tumor cells with low P-glycoprotein expression levels are destroyed, which in turn prevents development of drug resistance. The results of these studies will provide data that can help answer these questions. The results of several phase I clinical trials of zosuquidar, another important third-generation P-glycoprotein inhibitor, have been published.[107,108,113-115] These trials have demonstrated the specificity of zosuquidar towards P-glycoprotein in solid and hematological cancers. Patient recruitment is underway for two

Table II. Selected clinical trials with the third-generation inhibitor tariquidar (XR9576) Type of cancer

Chemotherapeutic drug(s)

Healthy volunteers

Patients Phase

Status

Outcome

30

I

Completed

Tariquidar sustained inhibition of Pglycoprotein in CD56+ lymphocytes[104] Tariquidar inhibited sestamibi efflux in drug-resistant tumors[104]

Study reference/ identifier

Drug-resistant tumors

Vinorelbine

26

I

Completed

Brain tumors, Ewing sarcoma, neuroblastoma, rhabdomyosarcoma (children)

Doxorubicin, vinorelbine, docetaxel

36a

I

Recruiting, started Feb 2001

Chemotherapy-resistant advanced breast cancer

Doxorubicin, taxane

17

II

Completed

Lung, ovarian, cervical cancer

Docetaxel

40a

II

Recruiting, started Sep 2003

NCT00072202

Adrenocortical cancer

Doxorubicin, vincristine, etoposide

47a

II

Recruiting, started Oct 2003

NCT00073996

NSCLC

Vinorelbine

490a

III

Terminated

Pending

NCT00042315

NSCLC

Paclitaxel/ carboplatin

540a

III

Terminated

Pending

NCT00042302

a

NCT00001944 NCT00020514

Five patients with increased sestamibi uptake following tariquidar; 1 PR[105]

NCT00048633

Intention to treat.

PR = partial response. © 2006 Adis Data Information BV. All rights reserved.

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Table III. Selected clinical trials with the third-generation inhibitor zosuquidar (LY335979) Type of cancer

Chemotherapeutic drug(s)

Patients Phase

Status

Outcome

Study reference/ identifier

Advanced nonhematological Doxorubicin malignancies

38

I

Completed

Coadministration of zosuquidar with doxorubicin had little effect on doxorubicin toxicity[107]

Advanced malignancies

Doxorubicin

40

I

Completed

Safe coadministration of zosuquidar with doxorubicin; P-glycoprotein inhibition in natural killer cells[108]

AML

Daunorubicin, cytarabine

16

I

Completed

11 CR, 1 PR; P-glycoprotein inhibition in CD56+ cells in all patients[109]

Resistant solid malignancies Docetaxel

41

I

Completed

Zosuquidar minimally alters pharmacokinetics of docetaxel; 1 PR, 14 SD[110]

Advanced solid tumors

Vinorelbine

21

I

Completed

Zosuquidar may inhibit vinorelbine clearance; 8 SD[106]

AML, patients aged 55–75 years

Daunorubicin, cytarabine

70a

I and II

Recruiting, started Aug 2005

NCT00129168

AML, patients aged 55–75 years

Gentuzumab, ozogamicin

70a

I and II

Recruiting, started Oct 2005

NCT00233909

AML, patients aged >60 years

Daunorubicin, cytarabine

450a

III

No longer recruiting

NCT00046930

a

Intention to treat.

AML = acute myelogenous leukemia;. CR = complete response; PR = partial response; SD = stable disease.

additional phase I and II clinical trials evaluating the impact of zosuquidar in combination with daunorubicin and cytarabine or gemtuzumab ozogamicin in patients aged 55–75 years with newly diagnosed AML or CD33+ AML in first relapse, respectively.[112] One phase III clinical trial evaluating zosuquidar is also underway, but is no longer recruiting patients. This trial is entitled: “Daunorubicin and cytarabine with or without zosuquidar trihydrochloride in treating older patients with newly diagnosed acute myeloid leukemia or refractory anemia.”[112] All of these ongoing trials are analyzing the impact of zosuquidar on hematological cancers in older patients, a reflection of the fact that P-glycoprotein is expressed in higher levels in the leukocytes of older patients compared with those of younger patients. Thus, the results of these studies will provide insights in the potency of zosuquidar as a specific P-glycoprotein inhibitor for MDR reversal in AML.

tion, can be evaluated. It has been observed that treatment with the inhibitors valspodar, biricodar, and tariquidar leads to increased uptake of Tc-99m sestamibi in normal liver and kidney.[110,116,117] Correlation of Tc-99m liver imaging with P-glycoprotein expression has also been reported in hepatocellular carcinoma.[118] In metastatic cancers, it has been reported that treatment with tariquidar leads to increased Tc-99m sestamibi accumulation.[110] Furthermore, Tc-99m was used to predict response to paclitaxelbased chemotherapy on the basis of P-glycoprotein detection, as demonstrated for NSCLC by chest imaging.[119] Moreover, the inhibitor concentrations achieved at the tumor site are sufficient to modulate P-glycoprotein function and thus the MDR phenotype.[85,120] Taken together, these findings confirm the successful application of MDR inhibitors in P-glycoprotein-expressing normal and tumor tissues of patients.

Tc-99m sestamibi is used to evaluate P-glycoprotein levels in human tumors. Tc-99m sestamibi has two main advantages in this context: it is a radionucleotide imaging agent, already in clinical use for determining cardiac dysfunction, and it is a substrate of and thus transported by P-glycoprotein. High P-glycoprotein expression can be identified in normal tissues and tumor areas through use of this imaging agent. Furthermore, the impact of P-glycoprotein inhibitors, measured as increased intratumoral drug accumula-

Patients are currently being recruited to a phase II clinical trial to evaluate the use of sestamibi for imaging drug resistance in solid tumors.[112] Furthermore, clinical trials are ongoing to evaluate the impact of third-generation P-glycoprotein inhibitors in combination with chemotherapy, and, in these trials, Tc-99m sestamibi is being used to monitor P-glycoprotein expression and function. Recently, a correlation between Tc-99m imaging with expression of an additional ABC transporter, MRP1, was shown in

© 2006 Adis Data Information BV. All rights reserved.

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patients with hepatocellular carcinoma, possibly extending the use of imaging agents to monitor MRP1-related drug resistance.[121] 3.1.3 Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors belong to a panel of new molecular cancer therapeutics which have been developed in recent years (table I). These compounds are known to inhibit malignant cell growth and metastasis. However, their therapeutic potential depends on their access to the intracellular target. Gefitinib and erlotinib, both epidermal growth factor receptor tyrosine kinase inhibitors, and imatinib, a BCR-Abl, platelet-derived growth factor receptor and c-kit tyrosine kinase inhibitor, are examples of these recently developed agents. Interestingly, interactions of both gefitinib and imatinib with MDR-associated ABC transporters have been demonstrated.[122,123] It has been suggested that these agents interact with MDR1 and also with BCRP as competitive ATP binding site inhibitors by binding to the nucleotide binding domain.[85] Recently, treatment of chronic myelogenous leukemia cells with imatinib has been reported to result in increased intracellular doxorubicin accumulation, and to MDR reversal.[123] Thus, it is very probable that a combination of imatinib with anti-leukemic drugs might have a beneficial effect in terms of improved action of MDR-related drugs.[123] Furthermore, treatment with gefitinib resulted in reversal of chemoresistance in breast and lung carcinoma cells overexpressing P-glycoprotein or BCRP.[122] Recently, it was reported that gefitinib also modulated the function of several ABC transporters in vivo.[124] Although the clinical success of gefitinib and erlotinib with respect to reversal of drug resistance remains to be shown, one might expect a benefit for patients with tumors that overexpress P-glycoprotein and/or BCRP and who are treated with anticancer drugs which are substrates for these ABC pumps (e.g. paclitaxel, doxorubicin, topotecan).[122] 3.1.4 Antibodies, Immunotoxins, Peptides

Antibody-directed approaches are another possibility for interacting with MDR-related ABC-transporters, thereby altering their function and possibly reversing MDR (table I). Monoclonal Pglycoprotein-specific antibodies have been shown to reduce the proliferation of P-glycoprotein-expressing tumor cells.[125-127] Remarkably, best chemosensitizing results have been achieved when the monoclonal antibodies were applied in combination with inhibitors.[128,129] One of the first strategies for MDR reversal employed immunotoxins, which consist of monoclonal antibodies coupled to cytotoxic agents. After binding to the antigen, they are internalized. Their cytotoxic activity towards P-glycoprotein-expressing cells has been demonstrated.[130,131] However, the clinical use of antibodies for MDR modulation is complicated. Problems arising from the immunogenic activity of animal antibodies in humans have yet to be solved. Furthermore, © 2006 Adis Data Information BV. All rights reserved.

effective antibody concentrations must be reached in solid tumors. Moreover, the large size as well as the long half-life and retention time of these whole antibodies in the circulation may have toxic effects on normal tissues that physiologically express P-glycoprotein. One possibility might be the utilization of single-chain variable fragments of antibodies, which specifically inhibit the activity of P-glycoprotein. The potential advantages of these small fragments are better tissue penetration and tumor biodistribution, low toxicity and faster clearance from the circulation.[132] A further option for reversal of P-glycoprotein-dependent MDR is utilization of synthetic P-glycoprotein-derived peptides. Peptide inhibitors are based on the structure of transmembrane domains of P-glycoprotein and may act by disrupting the correct assembly of the transporter. In vitro, chemosensitization towards doxorubicin has been reported with use of peptide inhibitors.[133] Synthetic murine P-glycoprotein-derived peptides have been employed in the immunization of mice, leading to an increase in the efficacy of chemotherapy with doxorubicin and vinblastine, without any autoimmune symptoms.[134] These promising approaches might point to a potential clinical application in the context of chemotherapy. 3.2 Reversal Strategies Targeting Expression of MDR-Associated ABC Transporters 3.2.1 Transcription

Alternative interventional strategies have been developed to interfere with the expression of MDR-associated genes (table IV), particularly with respect to transcription control of the MDR1 gene. Several approaches have been examined that target MDR1 transcription factors. Cytostatics have been used that are able to block the induced but not the constitutive MDR1 expression. For instance, binding of the transcription factor NF-Y to the MDR1 promoter has been targeted using the antitumor agent ET-743 or HMN-176, with resultant abrogation of transcriptional activation.[135,136] In addition, the influence of c-Jun NH2-terminal kinase on P-glycoprotein has been shown.[137] Moreover, MDR1-specific transcriptional repressors have been designed for the selective inhibition of P-glycoprotein expression.[138] All of these studies reported successful findings in vitro, but the benefits of transcription interventional strategies still need to be demonstrated in the clinical setting. 3.2.2 Translation

Other approaches interfere with translational expression by targeting MDR1 mRNA (table IV). One very early developed intervention strategy used complementary oligodeoxyribonucleotides to form complexes with the target mRNA, so-called antisense RNAs.[139] The efficiency of this reversal approach was dependent Am J Cancer 2006; 5 (5)

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Table IV. Selected experimental approaches for reversal of adenosine triphosphate (ATP)-binding cassette (ABC) transporter-dependent multidrug resistance (MDR) targeting the expression of MDR-associated ABC transporters Transcription Targeting MDR1 transcription factors MDR1-specific transcriptional repressors Translation Oligodeoxynucleotides antisense oligodeoxynucleotides triplex-forming oligodeoxynucleotides Ribozymes hammerhead ribozymes multi-target ribozymes

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One of the most interesting recent approaches is the employment of RNA-interfering technologies, also known as post-transcriptional gene silencing. Very promising initial reports demonstrated specific inhibition of expression of MDR-associated genes by degradation of complementary mRNA using small interfering RNAs (siRNAs)[152,153] and short hairpin RNA (shRNA) encoding vectors.[154,155] Down-regulation of expression and function of MDR1/P-glycoprotein as well as chemosensitization in vitro and in vivo have been demonstrated.[152,154,155] Down-regulation of BCRP expression following specific siRNA transfection has also been shown, leading to reversal of an atypical MDR phenotype.[153] Although these approaches need to be examined in clinical trials, the results obtained in vivo are grounds for cautious optimism.

RNA interfering small interfering RNA short hairpin RNA Post-translation Glycosylation inhibitors Kinase inhibitors

on the design of the respective antisense oligodeoxynucleotides with respect to MDR1 mRNA target region. Best results were achieved with antisense oligodeoxynucleotides that target the 5′ region and the translation initiation region of the MDR1 mRNA.[140] Reversal of MDR with MDR1-specific antisense oligonucleotides has been demonstrated in several experimental systems.[141-143] Furthermore, the MRP1-mediated MDR phenotype could be also affected, since MRP1-specific antisense oligodeoxynucleotides have been reported to cause chemosensitization towards etoposide.[144] Transfection of BCRP-specific antisense oligodeoxynucleotides also resulted in reduction of BCRP expression in lung cancer cells, which was associated with restored SN-38 chemosensitivity.[145] In addition, triplex-forming oligonucleotides have been shown to interfere with MDR1 amplification and expression.[146,147] Catalytic RNAs (so-called ribozymes) hybridize to a complementary mRNA, thereby catalyzing site-specific cleavage of the substrate. The efficacy of ribozymes is dependent on the choice of the target region; best results have been seen when the promoter region of the respective gene is targeted.[148] A panel of ribozymes has been developed specifically for several ABC transporters, such as MDR1, MRP1, and BCRP. Hammerhead ribozymes are ribozymes with a high catalytic activity. They have been shown to reduce expression of the respective MDR protein and reverse MDR in vitro.[149-151] © 2006 Adis Data Information BV. All rights reserved.

3.2.3 Post-Translation

P-glycoprotein as well as other MDR-associated ABC transporters undergo post-translational modifications such as Nglycosylation and phosphorylation (table IV). Consequently, use of glycosylation inhibitors such as tunicamycin has been evaluated. It has been shown that N-glycosylation may contribute to correct folding and/or stabilization of P-glycoprotein without affecting MDR transport function.[156] An essential prerequisite for the transporter function of P-glycoprotein is its phosphorylation. Thus, protein kinases represent a potential target for overcoming MDR. Since protein kinase C, for example, is known to phosphorylate and thereby activate P-glycoprotein, inhibitors of this enzyme have been used to reverse MDR.[157] Additionally, selective inhibitors directly targeting P-glycoprotein might improve this approach.[158] Although these expression-based intervention strategies work well in experimental settings, there are no data available relating to their clinical application. 4. Conclusion MDR remains the major cause of chemoresistance in cancer patients. The MDR phenotype can be mediated by ABC transporter molecules, which lower the intracellular accumulation of an entire panel of anticancer drugs. Forty-nine ABC transporters have been identified within the Human Genome Project: 16 with known functions, 14 of which have been discussed in the context of human diseases. However, development and reversal of MDR must be further elucidated in the context of the known MDRassociated ABC transporters that have been described. Detailed knowledge of the transporter molecules, their sequence polymorphisms, their regulation at the transcriptional, translational, and post-translational levels, and their functional characteristics of substrate binding and transport, represent the basis for specific Am J Cancer 2006; 5 (5)

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and thus successful intervention strategies for MDR reversal in the clinic. Individual patient resistance profiling through the use of standardized methodology, prior to any treatment, and if possible, during and post-treatment, is desired. This would provide the scientific basis for selection of specific chemotherapeutic drugs in defined therapy regimens. Thereby, prevention of MDR might be achieved in tumors with intrinsically low expression of MDR genes. Moreover, circumvention of MDR might also be possible through the selection of alternative drugs. Furthermore, resistance profiling also represents the rationale and essential prerequisite for tailor-made approaches to overcoming drug resistance by including specific inhibitors or specific expression-interfering tools in treatment protocols. Based on initial data obtained with thirdgeneration inhibitors in clinical trials, MDR of cancer cells might represent a potentially surmountable obstacle to effective chemotherapy. Moreover, new developments utilizing tools that interfere with expression of MDR genes, thereby leading to restored chemosensitization, are very promising. If the mechanisms of resistance can be overcome, the spectrum of traditional agents and of treatable tumor entities will certainly be extended. Reversal of MDR in cancer is a scientific challenge and a desperately needed clinical requirement. However, the decisive question – i.e. whether patients will really benefit from reversal strategies targeting ABC transporter-dependent MDR – has not yet been answered. MDR reversal will probably be successful when tailored to an individual patient with an individual tumor, resistance profile and response prediction; this approach provides the basis for individual prevention, circumvention and reversal of MDR. Tailor-made MDR reversal strategies are the ultimate goal – and probably a realistic option. Acknowledgments No funding was used in the preparation of this article and the authors have no conflicts of interest that are of direct relevance to its contents.

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Correspondence and offprints: Dr Ulrike Stein, Department of Surgery and ¨ Surgical Oncology, Max-Delbruck-Center for Molecular Medicine, and ¨ ¨ Robert-Rossle-Clinic, Charit´e Campus Buch, Robert-Rossle-Str. 10, Berlin, 13092, Germany. E-mail: [email protected] Am J Cancer 2006; 5 (5)