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The Oncologist, Vol. 8, No. 5, 411–424, October 2003
© 2003 AlphaMed Press

ORIGINAL PAPER
Cancer Biology

The Role of ABC Transporters in Clinical Practice

Gregory D. Leonard, Tito Fojo, Susan E. Bates

Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

Correspondence: Susan E. Bates, M.D., Molecular Therapeutics Section, Cancer Therapeutics Branch, Building 10 Room 12N226, 9000 Rockville Pike, Bethesda, Maryland 20892, USA. Telephone: 301-402-1357; Fax: 301-402-1608; e-mail: sebates{at}helix.nih.gov


   LEARNING OBJECTIVES
Top
 Learning Objectives
Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
After completing this course, the reader will be able to:

  1. Discuss the function of ABC transporters.
  2. Outline the levels of expression of MDR in tumors.
  3. Determine the role of P-gp inhibitors in clinical practice.

Access and take the CME test online and receive one hour of AMA PRA category 1 credit at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
Drug resistance remains one of the primary causes of suboptimal outcomes in cancer therapy. ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via ATP-dependent drug efflux pumps. P-glycoprotein (P-gp), encoded by the MDR1 gene, is an ABC transporter normally involved in the excretion of toxins from cells. It also confers resistance to certain chemotherapeutic agents. P-gp is overexpressed at baseline in chemotherapy-resistant tumors, such as colon and kidney cancers, and is upregulated after disease progression following chemotherapy in malignancies such as leukemia and breast cancer. Other transporter proteins mediating drug resistance include those in the multidrug-resistance-associated protein (MRP) family, notably MRP1, and ABCG2. These transporters are also involved in normal physiologic functions. The expressions of MRP family members and ABCG2 have not been well worked out in cancer.

Increased drug accumulation and drug resistance reversal with P-gp inhibitors have been well documented in vitro, but only suggested in clinical trials. Limitations in the design of early resistance reversal trials contributed to disappointing results. Despite this, three randomized trials have shown statistically significant benefits with the use of a P-gp inhibitor in combination with chemotherapy. Improved diagnostic techniques aimed at the selection of patients with tumors that express P-gp should result in more successful outcomes. Further optimism is warranted with the advent of potent, nontoxic inhibitors and new treatment strategies, including the combination of new targeted therapies with therapies aimed at the prevention of drug resistance.

Key Words. Cancer • ABC transporter • Multidrug resistance • Chemotherapy • Clinical trial


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
Recent advances in medicine and science have provided us with multiple agents for use in the struggle against cancer. We now have new and improved chemotherapy drugs and targeted therapies that act at a number of sites and by a variety of mechanisms to limit cancer cell proliferation. Yet while some cancers have particularly benefited, the most recent example being chronic myelocytic leukemia with imatinib [1], others, such as non-small cell lung cancer (NSCLC), have survival figures that have remained unchanged in recent decades [2]. We are faced with a spectrum of diseases, ranging from the eminently curable, such as testicular cancer, to those that are resistant to most forms of therapy, such as pancreatic cancer. Even more frustrating are those that are exquisitely chemoradiosensitive on presentation, like small cell lung cancer (SCLC), but that invariably relapse and become resistant to therapy over a short period of time. Drug resistance undoubtedly has a significant role in this heterogeneity and understanding it could answer many of the questions and complexities of cancer treatment.


   DRUG RESISTANCE
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
Drug resistance in cancer mimics that of antibiotic resistance [3]. Limitations in drug delivery via poor absorption, excessive metabolism, environmental changes, or poor penetration to certain sites are recognized, and measures to counteract these have been explored. For example, the use of prodrugs or pegylated chemotherapy, organ-specific administration, such as hepatic arterial infusion or intrathecal therapy, hyperoxygenation, and hyperthermia are all strategies aimed at increasing drug delivery. Cellular resistance, which occurs at a molecular level, may be more difficult to overcome. For example, resistance to imatinib can be associated with a single point mutation in bcr-abl [4]. These mutations alter essential amino acids involved in the binding of imatinib to the kinase domain. Alterations in protein or enzymatic expression may also contribute to drug resistance [5]. For example, reduced levels of plasma membrane uptake proteins can alter the accumulation of cisplatin and methotrexate [6]. Bcl-2 overexpression is known to inhibit apoptosis [7]. Accentuated DNA repair occurs with topoisomerase inhibitors [8], and glutathione-associated enzymes can lead to increased elimination of compounds such as alkylating agents [9]. The diversity of these cellular resistance mechanisms provides evidence of the complexity of drug resistance, and of the difficulty entailed in trying to overcome it.

Drug resistance was first documented experimentally in mouse leukemic cells that acquired resistance to 4-amino-N10-methyl-pteroylglutamic acid in a laboratory model in 1950 [10]. In 1973, Dano discovered active outward transport of daunomycin by drug-resistant cells that were crossresistant to other chemotherapeutic agents, such as vinca alkaloids and other anthracyclines [11]. This multidrug resistance (MDR) phenotype was developed by other authors who noted the consistent overexpression of a 170-kDa cell membrane protein termed P-glycoprotein (P-gp) [12]. The gene encoding P-gp was cloned and identified as MDR1 [13]. It was noted that this ATP-dependent drug efflux pump was part of a family of transporter genes, titled ATP-binding cassette (ABC) transporters, which is the largest family of transporter proteins.


   ABC TRANSPORTERS
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
To date, 48 human ABC genes have been identified [14]. High sequence homology in the ATP-binding domains known as nucleotide-binding folds (NBFs) allows identification and classification of members of the ABC transporter family. The functional protein usually is comprised of two NBFs and two transmembrane (TM) domains. There are seven subfamilies classified as ABC transporters (ABCA through ABCG) that are expressed in both normal and malignant cells. They are involved in the transport of many substances, including the excretion of toxins from the liver, kidneys, and gastrointestinal tract, and they limit permeation of toxins to vital structures, such as the brain, placenta, and testis. Mutations in the genes encoding these transporter proteins can induce a multitude of defects, presenting as autosomal recessive conditions. The best example of this is the cystic fibrosis transmembrane regulator protein (CFTR), a member of the ABCC (multidrug-resistance-associated protein [MRP]) subfamily that is composed largely of organic anion transporters. A mutation in the encoding gene on chromosome 7 impairs the synthesis of the CFTR protein, resulting in cystic fibrosis, the often-fatal childhood illness characterized by impaired bronchial and pancreatic secretions.

Most of our knowledge regarding ABC transporters and their involvement in MDR is based on studies of P-gp, an organic cation pump that is the product of the ABCB1 (MDR1) gene. It is a full transporter comprised of 12 transmembrane segments divided into two TM domains, each linked with an ATP-binding domain (Fig. 1Go). Binding of a substrate to the high-affinity binding site results in ATP hydrolysis, causing a conformational change that shifts the substrate to a lower affinity binding site and then releases it into the extracellular space or outer leaflet of the membrane [15]. A return to the conformation able to bind drug again is achieved by hydrolysis of ATP at the second binding site. P-gp is found in normal human tissues, such as the gastrointestinal tract and brain, where it prevents accumulation of, or exposure to, toxic substances. In cancer cells, P-gp is associated with the MDR phenotype, mediating resistance to anthracyclines, vinca alkaloids, colchicines, epipodophyllotoxins, and paclitaxel [16]. Competitive and noncompetitive inhibitors of drug efflux have been identified, effecting a reversal of drug resistance. A list of commonly identified substrates and inhibitors is found in Figure 1Go.



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  		Figure 1. P-gp structure with list of substrates and inhibitors.

 
The lack of expression of P-gp in some multidrug-resistant cells stimulated the search for other efflux pumps. The MRP1 (ABCC1) was cloned from a human lung carcinoma cell line in 1992 [17]. It has an additional five transmembrane segments located at the amino terminus and connected to a P-gp-like core by a linker region. This transmembrane domain zero (TMD0) region is felt to be responsible for the organic anion affinity of MRP1 [18]. The structural similarities between MRP1 and P-gp are paralleled by some overlap in their drug resistance spectra, although taxanes are a notable exception as they are poor substrates for MRP1. Substrates include organic anions such as methotrexate. Nonanionic compounds may be transported as glutathione, glucuronide, or sulfate conjugates, or may be cotransported with glutathione without conjugation [19]. This specificity for organic anions has also been found in other MRPs. The second member of the MRP (ABCC) family, MRP2 or canalicular multispecific organic anion transporter (cMOAT), has been found to be associated with bilirubin glucuronide transport, with defects resulting in the Dubin-Johnson syndrome. It is also a transporter of MRP1 substrates and cisplatin, with the potential to confer resistance to these agents [20]. MRP3 is expressed at high levels in the liver and may be involved in the efflux of organic anions from the liver to the blood in the presence of biliary obstruction [21]. MRP4 and MRP5 have been found to transport nucleoside analogs. Both confer resistance to thiopurines, and MRP4 has been found to confer resistance to antiretroviral nucleoside analogs [22, 23]. MRP6 appears to be a lipophilic anion pump with a wide spectrum of chemotherapy resistance, and mutations in this protein are linked to pseudoxanthoma elasticum [24]. To date, the only convincing evidence of a link to clinical drug resistance for the MRP family is associated with MRP1.

Mitoxantrone resistance is conferred by a half transporter that is part of the ABCG subfamily. ABCG2 is known as the mitoxantrone resistance gene (MXR), breast cancer resistance protein (BCRP), or ABC transporter in placenta (ABC-P). It confers resistance to topotecan and CPT-11 as well as to mitoxantrone [25]. Its normal function is unknown, but it is heterogeneously expressed in the intestine, found in high levels in the placenta, and may be involved in the transport of compounds in or out of the fetal blood supply. Other ABC transporter family members may also be involved in drug resistance. ABCB11, the ‘sister of P-gp’ (SPGP), or the bile salt exporter protein (BSEP) conferred limited resistance to paclitaxel in a laboratory model [26]. ABCA2 is the largest transporter reported to date and confers resistance to estramustine, an observation made following the amplification of ABC2 (ABCA2) in an ovarian cancer cell line [27]. Although frequently included in discussions of transporter-mediated resistance, lung resistance protein (LRP) is not an ABC transporter but is a major vault protein (MVP), found in the cytoplasm and nuclear membrane, that is thought to drive drugs away from the nucleus. It has been reported as a better correlate of drug resistance than P-gp [28].


   IDENTIFICATION OF P-GP EXPRESSION
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 
Having confirmed the presence of P-gp in the MDR phenotype and rationalized a mechanism through which it could impair the efficacy of chemotherapy, it was then necessary to devise reliable techniques for its detection. Experience with assaying and measuring hormone receptors and Her-2/neu has taught us that accurate quantification of a marker is vital in evaluating its impact on prognosis, predicting responsiveness to receptor inhibitors and even to chemotherapeutic agents [29]. Expression of MDR1/P-gp has been documented by reverse transcription-polymerase chain reaction (RT-PCR) for mRNA expression and by immunohistochemistry (IHC) for protein expression. Imperfections in these methods include errors due to normal tissue contamination of tumor tissue (for total RNA methods), poor sensitivity and specificity, and difficulties in quantitation (for IHC methods). Variations in the techniques applied complicate data analysis, and a uniform measurement system has yet to be agreed upon. An important goal in MDR research is to accurately detect the presence of P-gp expression in tumors, to determine whether it is functional, and to evaluate the effect of its inhibition. Sestamibi imaging, as discussed later, is a candidate for this, although difficulties in quantitation may limit its value [30].


   EXPRESSION OF ABC TRANSPORTERS IN TUMORS
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
Role of P-gp Inhibitors
 Conclusion
 References
 
Having outlined the difficulties in detecting MDR1/P-gp in cancer cells, it is not surprising that our knowledge of the levels of expression from tumor to tumor may be incomplete. Despite this, cancers considered as primarily chemoresistant, such as renal cell, adrenocortical, colon, and hepatocellular cancers, have been shown to consistently demonstrate expression of MDR1 [31]. However, the broad intractability of these tumors indicates that resistance cannot be solely explained by MDR1. Tumors with low levels of expression at baseline, such as leukemia, breast cancer, and SCLC, develop upregulation of these markers on relapse, having previously been exposed to chemotherapy.

The most reproducible studies on expression in human cancers involve leukemic cells (Table 1Go). P-gp is expressed in acute myelocytic leukemia (AML) cells in approximately 30% of patients at diagnosis, but in over 50% at relapse [32]. Whether this represents selection and repopulation of resistant clones or upregulation due to cytotoxic therapy exposure is unclear. A lower expression rate (17%) of P-gp is found in leukemic cells from patients less than 35 years of age, compared with rates of expression in the elderly (39%), and this may partly explain the better response to therapy seen in younger patients [33]. The suggestion that P-gp is a marker of poor prognosis due to its mediation of drug resistance in affected cells is supported by studies showing that the prognostic value of P-gp can be mitigated if treatment consists of agents that are not substrates for P-gp-induced drug efflux [34]. ABCG2, MRP1, and LRP are also found in AML [33], but MRP1 is more frequently found in chronic lymphocytic and prolymphocytic leukemia [35, 36]. Controversy exists as to their prognostic implications [28, 33, 37].


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  		Table 1. Expression of P-gp and MRP in selected tumors
 
Studies in solid tumors are more heterogeneous. Table 1Go outlines selected studies examining tumor expression levels of ABC transporters. For the most part, studies were chosen where data were available on pre-and-post treatment expression levels of both P-gp and MRP. A marked variation in the reported detection rates can be seen, depending on the method of detection, previous therapy, the type of tumor, and the tumor grade. A breast cancer meta-analysis concluded that P-gp expression could be detected in 41% of patients with breast cancer, with increased levels posttherapy [38]. However, an incidence range of 0%–80% for expression of P-gp was reported in the assembled studies. Most interpretable is the finding of an increase in expression in treated populations relative to baseline [39, 40]. MRP expression in breast cancer is common, but may represent tumor contamination due to its presence in normal tissue. High levels of MRP were found in lung cancer, with incidences of approximately 80% and 100% in SCLC and NSCLC, respectively. MDR1 expression was found in 25% of lung cancer samples [41]. A low rate of expression of P-gp was found in ovarian cancer, which could explain the apparent ineffectiveness of P-gp inhibitors in current studies in those tumors [42]. Detection of MDR has also been found in childhood tumors, and encouraging results have been observed in the treatment of rhabdomyosarcomas with chemotherapy in combination with the P-gp inhibitor cyclosporine [43].

Several studies have evaluated MDR1 expression in bladder cancer. In one study, MDR1 was highly expressed in half of all normal urothelial samples. Surprisingly, low-grade tumors expressed MDR1 infrequently, while high-grade tumors expressed MDR1 in 27% of cases, with a mean MDR1 mRNA level twice that of low-grade tumors [44]. This is in contrast to MRP expression, which was frequent (55%) in low-grade bladder tumors and infrequent (8%) in high-grade tumors [45]. In paired samples, both MDR1 and MRP expression levels, measured using quantitative RT-PCR, were found to be greater postchemotherapy, with MDR expression 5.7-fold greater and MRP expression 2.4-fold greater than in untreated patients [46]. By IHC, similar posttreatment increases were found in MDR and MRP expression levels, as outlined in Table 1Go [47, 48].

Expression of P-gp in normal tissues may contribute to conflicting data because of inconsistent tissue sampling. Normal brain capillary endothelial cells express P-gp, thereby contributing to the blood-brain barrier. Endothelial cells from new capillaries formed by brain tumors were shown to express P-gp in 80% of patients, whereas actual tumor cells were positive in only 20% of patients [49].

Fewer studies have reported the expression of ABCG2 in human cancer. Table 2Go summarizes a number of studies outlining ABCG2 expression rates in human cancers. Other than in digestive tract, endometrial, and lung cancers and melanoma, tumors assayed by IHC using BXP antibody have not suggested significant levels of detection [50].


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  		Table 2. ABCG2 expression
 

   ROLE OF P-GP INHIBITORS
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
Conclusion
 References
 
Confirmation of the role of P-gp in drug resistance is well documented in laboratory models [51]; confirmation of its role in normal drug disposition has been confirmed by the altered excretion of P-gp substrates in knockout mice [52]. Still undetermined is the role of P-gp in clinical oncology. As long ago as 1981, it was discovered that drug resistance could be reversed by adding P-gp inhibitors such as verapamil and phenothiazine derivatives, already widely used in the clinic [53]. Soon after, these and other first-generation agents, such as cyclosporine, amiodarone, and tamoxifen, were being employed in clinical trials aimed at the reversal of drug resistance. The hope and expectation for convincing benefits resulted in a series of ‘home run’ trials rather than a stepwise approach that may have achieved this goal. Taken together, these trials failed to convincingly prove a role for P-gp in clinical drug resistance. Contributing to this failure were the absence of confirmation of P-gp expression in the tumors, a lack of evidence of P-gp inhibition in vivo, and P-gp inhibitor toxicity at doses administered to achieve serum concentrations comparable to those that were effective in laboratory models. A good example of the wave of enthusiasm that greeted these inhibitors and the subsequent pessimism is illustrated by myeloma data. Sonneveld et al. reported that cyclosporine could prevent multidrug resistance and allow successful retreatment with previously ineffective VAD (vincristine, doxorubicin, and dexamethasone) chemotherapy [54, 55]. A decade later, however, the same group concluded, in a phase III randomized trial, that cyclosporine did not improve outcome [56]. A phase III trial combining VAD with verapamil in myeloma reached a similar conclusion [57].

Second-generation agents were developed solely for the purpose of altering drug resistance. Some were analogs of the first-generation compounds, but were more potent and less toxic. A widely tested second-generation compound is PSC 833 (valspodar), a derivative of cyclosporin D that is 10 times more potent than cyclosporin A [58]. Results from clinical trials with this agent are still emerging in the literature. However, those results available to date are disappointing, particularly in AML trials. The most notable finding with PSC 833 was the need to reduce the dose of the anticancer agent used in combination with it. Dose reductions ranged from 25% for etoposide to 66% for paclitaxel [59]. It is now felt that those dose reductions may have compromised drug concentrations in the tumor, even with complete inhibition of P-gp. Dose reductions were required to prevent undue toxicities of the anticancer agent in combined therapy, which resulted from a reduction in the clearance of the anticancer agent.

The second-generation agent VX-710 (biricodar) restores drug sensitivity to both MDR1- and MRP1-expressing cells in vitro and may differ from PSC 833 in its drug interaction profile. Eleven percent of patients with locally advanced or metastatic paclitaxel-refractory breast cancer had an objective response when VX-710 was combined with 80 mg/m2 of paclitaxel [60]. This represented a 54% dose reduction for paclitaxel in combination with VX-710. In contrast, anthracycline-resistant soft tissue sarcomas, retreated with 60 mg/m2 of doxorubicin and VX-710, required no dose reductions [61]. There were two partial remissions in that study, and 7 of 15 patients achieved disease stabilization.

The delay in anticancer drug clearance necessitating these dose reductions resulted from pharmacokinetic interactions between the MDR inhibitor and the anticancer agent. Inhibition of P-gp in the normal liver and kidney can account for some reduction in drug clearance. Knockout mice in which the MDR orthologue has been deleted have up to 3.3-fold greater plasma levels of some anticancer agents [62]. For PSC 833, and possibly VX-710, inhibition of metabolism by cytochrome P450 is thought to cause a significant decrease in drug clearance. For PSC 833, there is also a potential contribution from impaired biliary flow, since PSC 833 has been shown to inhibit ABCB11, the bile salt exporter protein [59].

Reductions in doses of anticancer agents in combination with these inhibitors were necessary to produce levels of toxicities (and, presumably, areas under the concentration x time curve [AUCs]) comparable with those found at the maximum tolerated dose in regimens without P-gp inhibitors. In some instances, these dose reductions resulted in reduced AUCs [63]. Indeed, one possible explanation for the low response rates seen with this strategy is that the AUC may appear unchanged, due to a prolonged terminal half-life from a reduction in drug excretion, but may result in levels that are below the minimum effective dose needed to have an adequate antitumor effect. Since these modifications were required due to pharmacokinetic interactions with the chemotherapeutic agents, third-generation agents without pharmacokinetic interactions are in development. These agents are currently being studied in clinical trials.

With clinical results lacking, it has been important to develop assays that confirm the effectiveness of P-gp inhibitors in patients. Two main strategies have provided this confirmation. The CD56+ rhodamine efflux assay combines the affinity of rhodamine for P-gp and the high expression of P-gp in CD56+ natural killer cells. Circulating CD56+ cells are assayed for efflux of rhodamine. Administration of an effective P-gp inhibitor to patients inhibits rhodamine efflux from the CD56+ cells, thereby confirming the activity of the inhibitor at its target [64, 65]. However, this method does not provide information on tumor uptake. Sestamibi is a radionucleotide imaging agent already in clinical use to determine cardiac function. As a P-gp substrate, sestamibi imaging has the additional ability to outline areas of P-gp expression, including normal tissues and overexpressing tumors. Inhibition of sestamibi efflux in the liver results from P-gp inhibition and can be visualized with increased hepatic uptake on imaging with the addition of a P-gp inhibitor. Enhanced liver uptake of sestamibi has been observed following administration of PSC 833, VX-710, and XR9576 (tariquidar) [30, 66, 67]. Although this signifies the effectiveness of P-gp inhibition systemically, more relevant would be an increase in tumor uptake of sestamibi. The absence of tumor uptake may indicate P-gp expression and has been correlated with a poor response to chemotherapy [68]. Agrawal et al. demonstrated an increase in sestamibi accumulation in tumors in 13 of 17 patients with the addition of XR9576. An increase of 36%–263% was found in eight of those patients [67]. Figure 2Go depicts the increase in tumor sestamibi accumulation observed following administration of XR9576 in a patient enrolled in that trial.



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  		Figure 2. 99mTechnetium-sestamibi images before and after administration of XR9576. A 263% increase in the accumulation of 99mTechnetium-sestamibi in a previously undetected left thigh tumor after administration of the P-gp inhibitor indicates the presence of the P-gp drug efflux pump in that tumor. (Portions of this figure were in Agrawal et al. [67].)

 
The holy grail of alterations in outcome or survival figures has been more difficult to attain. Table 3Go outlines the randomized trials reported to date with a P-gp inhibitor in combination with chemotherapy. Only three randomized trials have demonstrated a statistically significant difference in overall survival with the use of a P-gp inhibitor. A Southwest Oncology Group (SWOG) study involved 226 patients with refractory, relapsed, or high-risk AML who were randomized to cytarabine and daunorubicin with or without cyclosporine [69]. The addition of cyclosporine resulted in a statistically significant improvement in relapse-free survival (34% versus 9%) and overall survival (22% versus 12%) rates. Survival and response rates were associated with higher levels of serum daunorubicin, as seen in the cyclosporine group. The maximum benefit in the cyclosporine group was observed in those with the highest levels of P-gp expression. A trial combining the first-generation P-gp inhibitor quinine with standard therapy for poor-risk AML patients supports the SWOG data, although a statistically significant result was not achieved [70]. That study demonstrated a greater response rate (52% versus 45%) in the quinine arm, although the toxicity associated with that agent may have prevented a significant benefit in survival outcome. A similar study using PSC 833 failed to duplicate those results, although benefit was suggested in a P-gp+ subset [71]. One trial each in breast cancer and lung cancer demonstrated a survival benefit for the addition of a P-gp inhibitor. In both trials, verapamil was the P-gp inhibitor administered [72, 73].


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  		Table 3. Randomized trials in P-gp reversal
 
Similar difficulties in obtaining a significant survival benefit have been demonstrated in ovarian cancer. A phase II study documented an 8.6% partial response rate in paclitaxel-refractory patients retreated with 70 mg/m2 paclitaxel and PSC 833 [74]. A phase III study, reported in abstract form, demonstrated that the combination of PSC 833 with paclitaxel and carboplatin versus chemotherapy alone failed to show a benefit in outcome for the addition of PSC 833 [42]. VX-710 has also been tested in ovarian cancer. Rowinsky et al. reported that in vivo steady-state plasma concentrations of VX-710 in combination with paclitaxel exceeded those shown to be necessary to cause MDR reversal in vitro [75]. However, a phase II trial documented only three partial responses among 45 patients treated with VX-710 and paclitaxel for ovarian cancer refractory to prior paclitaxel [76]. Stable disease and a 50%–90% reduction in serum cancer antigen 125 (CA-125) level were noted in 27% and 31% of cases, respectively. Whether those results can be converted into favorable outcomes in phase III trials, particularly given the low incidence of P-gp expression in ovarian cancer, has yet to be determined.

Often, negative results in phase III trials are not clear-cut. The phase III study with PSC 833 in AML, mentioned above, administered cytarabine, doxorubicin, and etoposide chemotherapy with or without the inhibitor to 120 previously untreated patients [71]. The PSC 833 arm was closed early due to excessive mortality. Available data demonstrated no difference in disease-free or overall survival rates. However, functional studies in the leukemic cells allowed an analysis of subsets based on P-gp expression. In patients with PSC 833-inhibitable efflux (indicating P-gp expression), the median disease-free survival was greater at 14 (versus 5) months (p = 0.07) with the addition of PSC 833. Similarly, patients treated with chemotherapy alone had a lower complete remission rate (41% versus 91%), a higher nonresponse rate (41% versus 9%), and a higher death rate (18% versus 0%) when in vitro studies exhibited PSC 833-modulated dye efflux, compared with those patients whose cells did not demonstrate efflux (p = 0.03). The findings from this subset analysis suggest that careful selection of patients is essential in facilitating positive outcomes, as those deriving benefit from MDR inhibitors may be obscured in trials failing to identify the appropriate target population. Thus, the specific elements include: appropriate selections of disease, patient population, inhibitor, anticancer agent, and trial design. Selecting diseases where P-gp-induced drug resistance is the primary mechanism of resistance, where P-gp expression is to be expected, and/or where the drug regimen contains a P-gp substrate that can be potentiated by the inhibitor being tested is critical to a successful outcome.

It could be argued that response rates with or without a P-gp inhibitor will be similar and tell us little about the efficacy of inhibitors. This is particularly true where drug combinations contain potent non-P-gp substrate drugs such as cisplatin or cytosine arabinoside (Ara-C). If chemosensitive tumors are selected for study, then P-gp inhibitors will not enhance this effect, but instead may reduce the emergence of mutant resistant clones, which would then be reflected in disease-free or survival outcomes.

One treatment paradigm that has not been fully explored is that of prevention of the emergence of resistance through the use of P-gp inhibitors. In the laboratory, PSC 833 reduced the mutation rate for doxorubicin-selected resistance in sarcoma cells by tenfold, thus reducing the likelihood of the development of resistant clones via the MDR mechanism. In those sarcoma cells treated with PSC 833, resistance was mediated by an alternative pathway with reduced expression of topoisomerase II{alpha}, the target enzyme for anthracyclines [77]. Another study examined six agents for their ability to prevent vincristine resistance in a rhabdomyosarcoma cell line. MDR modulators prevented the development of resistance, suggesting a role for the use of P-gp inhibitors prior to cytotoxic therapy [78]. The prevention of chemotherapy resistance through the prior use of a P-gp inhibitor has also been suggested in the clinic, where leukemic cells found in relapse after treatment with a P-gp inhibitor demonstrated decreased expression of MDR-1 mRNA [79]. Along with the reduced mutation rate for P-gp-related resistance, P-gp inhibitors have the potential to increase drug accumulation and limit the emergence of other resistant clones by increasing cell kill as per the concept of log-dose survival [80]. Thus, a rationale exists for early intervention with nontoxic, potent P-gp inhibitors in cancer chemotherapy.

Another potential role for P-gp inhibitors is in the modulation of oral bioavailability. It has been increasingly recognized that ABC transporters limit the absorption of many drugs from the gastrointestinal tract. Several factors would make the oral administration of the P-gp substrate paclitaxel advantageous over i.v. administration of the drug, including improved compliance, absence of a requirement for implantable devices, and a more favorable toxicity profile. In a proof-of-concept study of 14 patients with solid tumors, the combination of oral paclitaxel and the P-gp inhibitor cyclosporin A resulted in an eightfold higher bioavailability compared with paclitaxel alone (p < 0.001) [81]. For cyclosporine, the interpretation of that study is complicated by the fact that the inhibition of increased systemic exposure could also have been due to reduced metabolism of paclitaxel through inhibition of the cytochrome P450 3A4 isoenzyme by cyclosporine. A phase II trial of orally administered paclitaxel and cyclosporine in 23 patients with previously treated NSCLC resulted in an overall response rate of 26%, a median time to progression of 3.5 months, and an overall survival of 6 months, results that are in accordance with other single agent drugs given intravenously [82]. A similar trial in 24 untreated gastric cancer patients found an overall response rate of 32% and a median time to progression of 16 weeks [83]. Evidence also exists for enhanced intestinal absorption for chemotherapy drugs, such as etoposide, when administered in combination with P-gp inhibitors [84].

Data obtained from studies of single nucleotide polymorphisms (SNPs) in the MDR1 gene also support this central role for P-gp. The C3435T SNP in the MDR1 gene has been correlated with a lower expression of P-gp. It has been reported that individuals homozygous for C3435T have lower duodenal expression levels of P-gp. There is increased oral bioavailability of P-gp substrates, such as digoxin, as evidenced by greater digoxin plasma levels in those individuals [85]. Those results, implicating P-gp as playing a major role in the bioavailability of drugs, have experimental support from the MDR1 knockout mouse model. Isolated ileum from Mdr1a-negative mice demonstrated markedly greater paclitaxel and digoxin absorption and a greater regional variability than ileum obtained from wild-type mice [86]. These and other studies outline the role of P-gp in affecting drug absorption and elimination of compounds from the intestine, a process that is likely duplicated in other areas of the body, such as the placenta and the blood-brain barrier.

Other ABC transporters, including ABCG2, are also expressed in the intestine [87]. Administration of GF 120918, a coinhibitor of ABCG2 and P-gp, in combination with topotecan resulted in a greater oral bioavailability of topotecan of 97% (versus 40%) [88]. Since topotecan is a poor substrate for P-gp, this was considered good evidence for the impact of ABCG2 on the oral bioavailability of topotecan. Thus, ABC transporter inhibitors may successfully improve the oral bioavailability, and hence systemic exposure, of a number of chemotherapy agents. However, care must be exercised, as oral bioavailability, although improved, may still remain variable among patients and may not result in improved outcomes.


   CONCLUSION
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
References
 
A number of lessons have been learned from the evolution of the field of MDR. Early trials used inferior inhibitors without determining the expression of P-gp. More recent studies employed second-generation inhibitors that required the reduction of anticancer drug doses. Identifying an adequate trial design for P-gp reversal studies has proven to be difficult. Home run designs presume a clear-cut outcome and so fail to appropriately select groups or use comparative controls, omitting potentially valuable end points. Crossover designs, which await the development of resistance, risk the onset of multiple mechanisms of resistance prior to the introduction of a P-gp inhibitor, clouding the potential benefit and clinical application of the P-gp inhibitor. Randomized trials have evolved as the optimal technique, despite the requirement of a large sample size. Meticulous expression analyses, pharmacokinetic assays, and surrogate marker analyses allow accurate overall and subset analyses. Trials using these agents up front could limit the development of other mechanisms of resistance. Specific inhibitors against other ABC transporters, such as MRP1, lag behind in their development, although multispecific ABC transporter inhibitors are being tested [78]. Further refinement of our trial design and drug development will inevitably enlighten us as to the role of ABC transporters and their inhibitors in cancer cell resistance. One emerging idea is the use of P-gp inhibitors in enhancing the oral bioavailability of anticancer drugs. As molecular targets, ABC transporters may prove invaluable in facilitating the optimal use of other agents, as prognostic and predictive markers and as a therapeutic intervention.


   REFERENCES
Top
 Learning Objectives
 Abstract
 Introduction
 Drug Resistance
 ABC Transporters
 Identification of P-gp...
 Expression of ABC Transporters...
 Role of P-gp Inhibitors
 Conclusion
 References
 

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