Pharmacogenomics of MDR and MRP subfamilies

Transcription

1 For reprint orders, please contact: REVIEW Pharmacogenomics of MDR and MRP subfamilies Noboru Okamura 1, Toshiyuki Sakaeda 2 & Katsuhiko Okumura 2 Author for correspondence 1 Department of Clinical Evaluation of Pharmacotherapy, Kobe University Graduate School of Medicine, 1-5-6, Minatojima-minamimachi, Chuo-ku, Kobe , Japan 2 Department of Hospital Pharmacy, School of Medicine, Kobe University, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe , Japan Tel: ; Fax: ; Drug-metabolizing enzymes, drug transporters and drug targets play significant roles as determinants of drug efficacy and toxicity. Their genetic polymorphisms often affect the expression and function of their products and are expected to become surrogate markers to predict the response to drugs in individual patients. With the sequencing of the human genome, it has been estimated that approximately genes code for drug transporters and, recently, there have been significant and rapid advances in the research on the relationships between genetic polymorphisms of drug transporters and interindividual variation of drug disposition. At present, the clinical studies of multi-drug resistance protein 1 (MDR1, P-glycoprotein, ABCB1), which belongs to the ATP-binding cassette (ABC) superfamily, are the most comprehensive among the ABC transporters, but clinical investigations on other drug transporters are currently being performed around the world. MDR1 can be said to be the most important drug transporter, since clinical reports have suggested that it regulates the disposition of various types of clinically important drugs, but in vitro investigations or animal experiments have strongly suggested that the members of the multi-drug resistance-associated protein (MRP) subfamily can also become key molecules for pharmacotherapy. In addition to those, breast cancer resistance protein (BCRP, ABCG2), another ABC transporter, is well known as a key molecule of multidrug resistance to several anticancer agents. However, this review focuses on the latest information on the pharmacogenetics of the MDR and MRP subfamilies, and its impact on pharmacotherapy is discussed. Keywords: drug disposition, genetic polymorphism, MDR subfamily, MRP subfamily, personalized medicine Most drug responses are determined by the interplay of several gene products that influence the pharmacokinetics and pharmacodynamics of, for example, drug-metabolizing enzymes, drug transporters, and drug targets. Pharmacokinetics is the relationship between the dosing amount and plasma or tissue concentration, and how this is affected by drug-metabolizing enzymes and drug transporters. Pharmacodynamics, on the other hand, is the relationship between concentration and effect, which is affected by target proteins, including receptors and enzymes. A recent review by Fierz described the concept of personalized healthcare and how drug effects and/or toxicity could be distinguished by genetic traits, SNPs and haplotypes [1]. Consequently, genetic polymorphisms of these proteins are expected to become surrogate markers for drug response in order to stratify patients and individualize treatment. The 2.9-billion-nucleotide base-pair sequence of the human genome is now available and, with the sequencing of the human genome, it has been estimated that approximately genes code for drug transporters [2]. Among the present compilation of drug transporters, the major members are classified into the efflux transporters, which include the multi-drug resistance proteins (MDRs), multi-drug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP/ABCG2), and the uptake transporters, which include organic anion transporters (OATs), organic anion transporting polypeptides (OATPs), organic cation transporters (OCTs), and peptide transporters (PepTs). The molecular and biochemical features and the possible clinical relevance of these drug transporters are well documented [2-11]; however, little information is available concerning the effects of genetic polymorphisms on phenotypes, compared with drug-metabolizing enzymes and drug targets [12-16]. To date, the clinical studies of MDR1 (P-glycoprotein, ABCB1) are the most comprehensive among drug transporters [2,17-24], but clinical investigations on other drug transporters are currently being performed around the world. MDR1 can be said to be the most important drug transporter, since clinical reports have suggested that it regulates the disposition of various types of clinically important drugs, but in vitro investigations or animal / Future Medicine Ltd ISSN Personalized Med. (2004) 1(1),

2 REVIEW Okamura, Sakaeda & Okumura Table 1. List of human ABCB genes. Gene Symbol Location Expression Function ABCB1 MDR1 7q21 Liver, kidneys, adrenals, intestine, brain, and placenta Transporter ABCB2 TAP1 6p21.3 Estrogen receptor Transporter ABCB3 TAP2 6p21.3 Estrogen receptor Transporter ABCB4 MDR2, MDR3 7q21 Liver Transporter ABCB5 7p14 Ubiquitous? ABCB6 2q33-q36 Mitochondria Transporter ABCB7 Xq13.1-q13.3 Mitochondria Transporter ABCB8 7q35-q36 Mitochondria Transporter ABCB9 12q24 Heart, brain, and lysosomes? ABCB10 1q42 Mitochondria? ABCB11 BSEP 2q24 Liver Transporter ABC: ATP-binding cassette; BSEP: Bile salt export pump; MDR: Multi-drug resistance; TAP: Transporter associated with antigen processing. experiments have strongly suggested that the members of the MRP subfamily can also become key molecules for pharmacotherapy. In addition, BCRP has been reported to play an important role in drug resistance in cancer chemotherapy. These transporters belong to the ATP-binding cassettes (ABC) superfamily and generally use ATP hydrolysis energy to work as transporters, channels, and receptors. This review, however, will focus on the latest information regarding the pharmacogenetics of the MDR and MRP subfamilies, and will discuss the impact this may have on pharmacotherapy. Pharmacogenetics of the MDR subfamily and its impact on pharmacotherapy The MDR subfamily In 1976, a 170-kDa membrane glycoprotein was isolated from colchicine-resistant Chinese hamster ovary cells [25]. This glycoprotein appeared unique to sublines showing altered drug permeability, and was named P-glycoprotein. About 10 years later, a gene was isolated from multi-drug-resistant human KB carcinoma cells, which was demonstrated to encode the human P-glycoprotein and was named MDR1 [26,27]. P-glycoprotein, now known as MDR1, is a phosphorylated and glycosylated membrane protein containing 1280 amino acids. It consists of two homologous halves containing six transmembrane domains and one intracellular nucleotide-binding domain [28,29]. Structural analysis by high-resolution electron microscopy and image analysis for MDR1 isolated from Chinese hamster ovary cells revealed that MDR1 approximates a cylinder that is 10 nm in diameter and 8 nm in maximum height. It has a large aqueous pore, which opens at the extracellular face of the membrane and closes at the cytoplasmic face [30]. Upon binding nucleotides, the transmembrane domains reorganize to open the central pore along its length in a manner that allows drugs direct access from the lipid bilayer to the central pore of MDR1 [30,31]. Although MDR1 is thought to act as an efflux pump to remove substrates from the inside to the outside of the cell, another model has been proposed whereby MDR1 acts as a flippase to carry substrates from the inner leaflet of the lipid bilayer to the outer leaflet [32,33]. The findings of Rosenberg et al. support the flippase model. A homolog of MDR1, MDR3 (or MDR2), was discovered in a human liver cdna library [34], and the gene product has been elucidated to be a transporter of phospholipids [4,35]. Subsequently, various homologous genes and their products were identified, and MDR1 is now recognized as a member of a family of related proteins, named the ABCB subfamily, which consist of two members of the MDR subfamily and nine other members (Table 1). MDR1 (ABCB1) and its related proteins have been subjected to a number of investigations focusing on acquired multi-drug resistance and possible strategies to overcome this problem [36-41]. In 1987, Thiebaut et al. showed that MDR1 was expressed in normal tissue using an in situ hybridization method [42]. MDR1 has been reported to be expressed in the liver, kidneys, small and large intestines, adrenals, brain, muscle tissue, placenta, and heart [43-45]. The physiological function of MDR1 in normal tissue was unknown until 1992, when the Tanigawara and Ueda group revealed that 86 Personalized Med. (2004) 1(1)

3 Pharmacogenomics of MDR and MRP subfamilies REVIEW MDR1 was involved in the disposition of drugs and steroidal hormones [46-49]. A number of various types of structurally unrelated drugs, including anticancer drugs, antihypertensive drugs, immunosuppressants, HIV protease inhibitors and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have been suggested as substrates for MDR1 [17-19]. In 1993, Tanigawara et al. also suggested the possibility of MDR1-mediated drug interactions [48,50], and MDR1 was recently found to be a key molecule for the interaction of digoxin with various drugs, including antihypertensive drugs, HMG-CoA reductase inhibitors, and antiarrhythmic drugs [51-53]. Following the discovery of MDR, a second member of the MDR subfamily was found, MDR3 (ABCB4). MDR3 is reported to play an important role in the secretion of phospholipids into bile [54,55]. The involvement of MDR3 in pharmacotherapy is not well known, but its physiological role has been revealed; it is responsible for pumping out phospholipids, especially phosphatidylcholine, and its deficiency results in progressive familial intrahepatic cholestasis type 3. The transporter associated with antigen processing (TAP) type 1 and 2 are also classified as belonging to the ABCB subfamily and have been defined as ABCB2 and ABCB3, respectively. They have been reported to play a pivotal role in adaptive immune response by translocating peptides from the cytosol into the endoplasmic reticulum [56,57]. A third member, the bile salt export pump (BSEP, ABCB11), has been reported to work as a pump and is responsible for moving taurine- or glycine-conjugated bile acids from hepatocytes into bile. Its deficiency results in progressive familial intrahepatic cholestasis type 2 [4,58,59]. Little information is available for these three ABCB subfamily proteins in relation to drug transport, and no data of other members are available in regards to their role in pharmacotherapy, although the tissues where they are expressed have been elucidated in some cases. Pharmacogenetics of the MDR subfamily The data on the effects of genetic polymorphisms on pharmacotherapy is restricted to MDR1, and no information is available for other members of the ABCB subfamily, to date. In 1989, a genetic polymorphism of the MDR1 gene, G2677T, was first identified by Kioka et al., which causes an alanine to serine substitution at position 893 [60]. In 2000, Hoffmeyer et al. performed a systematic screening for MDR1 genetic polymorphisms [61]. All 28 exons and the core promoter region were amplified, covering the coding exons and sequences at the exon intron boundaries that are important for mrna splicing. By analyzing 188 s, they discovered a total of 15 SNPs. The report had a tremendous impact since it indicated that a synonymous polymorphism of C3435T in exon 26 was associated with the duodenal expression of MDR1 and, thereby, digoxin plasma levels after oral administration. Since then, there have been numerous studies with larger populations and, to date, more than 40 SNPs have been identified [62]. Among them, the following polymorphisms have been relatively well investigated from the viewpoint of their effects on MDR1 expression and/or function, pharmacokinetics and pharmacodynamics: T-129C in the promoter region, C1236T (silent) in exon 12, G2677A,T (Ala893Thr,Ser) in exon 21, and C3435T (silent) in exon 26 [63-65]. However, studies involving other SNPs have been limited. Table 2 summarizes the data on the association of these four genetic polymorphisms with the expression of MDR1; their effects on the pharmacokinetics of MDR1 substrates are presented in Table 3. There is no rational explanation for why the silent polymorphisms C1236T and C3435T have such effects. One explanation is that these alleles are in linkage disequilibrium (LD) with a SNP in the promoter region and/or in the exon intron boundaries that are important for mrna splicing, but no experimental data have been presented. The data presented in Tables 2 and 3 are explained in detail in recently published reviews [17-24], but, as shown in Tables 2 and 3, no consensus has yet been reached. The reason for this discrepancy is also not clear, but future clinical trials should be carefully designed to avoid misinterpretation; that is to say, the studies should be performed taking into consideration that a haplotype analysis of the MDR1 gene is needed instead of SNP detection, the pharmacokinetic parameters should be selected carefully, and the results should depend on the drugs used and the condition of the subjects. These points are further discussed in the following paragraphs. The first point considers the need for the analysis of haplotypes. It has recently been confirmed that C3435T is in significant LD with C1236T or G2677T [66-74]. In addition, Saito et al. demonstrated that T-129C is in LD with G2677A,T and C3435T [75]. Recent reports have suggested that a haplotype analysis based on T-129C, C1236T, G2677A,T and C3435T sometimes gives a higher predictability for phenotypes and 87

4 REVIEW Okamura, Sakaeda & Okumura Table 2. MDR1 genotype-related expression of MDR1 protein or mrna. SNPs Subject N Expression Tissue Results Ref. T-129C Japanese 89 Protein Placenta TT > TC [66] Japanese 13 mrna Duodenum TT = TC [164] C1236T Patients 69 mrna Intestine CC = CT = TT [165] Patients 136 mrna Bone marrow CC = CT = TT [77] Patients 24 mrna Kidneys CC = CT = TT [166] G2677A,T Japanese 89 Protein Placenta GG > GW > WW [66] C3435T G2677A,T C3435T Protein mrna Duodenum GG = GW = WW [167] Japanese 13 mrna Duodenum GG < GW or WW [164] Patients 69 mrna Intestine GG = GW = WW [165] Patients 136 mrna Bone marrow GG < GT or WW [77] Patients 24 mrna Kidneys GW = WW [166] Healthy and patients, 21 Protein Duodenum CC > CT > TT [61] 37 Protein Duodenum CC = CT = TT [167] 32 mrna Japanese 89 Protein Placenta CC = CT = TT [66] 73 mrna Placenta CC > TT [168] 31 mrna Leukocyte CC = CT = TT [169] Japanese 13 mrna Duodenum CC < CT < TT [170] Patients 69 mrna Intestine CC = CT = TT [165] Patients 85 mrna Kidneys CC > TT [171] Patients 136 mrna Bone marrow CC < CT < TT [77] Patients 24 mrna Kidneys CC = CT = TT [166] Patients 30 Protein Lymphocyte CC = CT = TT [172] 73 mrna Placenta GG 2677 /CC 3435 > [168] TT 2677 /TT 3435 W represents A or T. MDR: Multi-drug resistance. therapy outcome than a study of SNPs [72,76,77]. Although it is not clear whether the discrepancies in the reports over the effects of these SNPs on MDR1 expression or the pharmacokinetics of MDR1 substrates will be explained by interethnic variation of the haplotype, it can be said that a haplotype analysis should be performed instead of SNP detection in future investigations. The MDR1 haplotype has been defined based on the three SNPs C1236T, G2677A,T and C3435T [67], or T-129C, G2677A,T and C3435T [75], but, very recently, Kroetz et al. defined 32 haplotypes and their subtypes [62]. Table 4 lists the haplotype frequencies defined based on four SNPs in healthy s, African Americans and Asian Americans, which were tabulated by referring to the report by Kroetz et al. [62]. Data on the Japanese was also added (unpublished results). As shown in Table 4, MDR1*24, *24A or *4A (defined according to Kroetz et al. [62]) are more frequently found in Japanese than subjects, being characterized by an A allele at position 2677, and considerable interethnic variation is expected in the haplotype distribution. In addition, two reports have recently shown that novel SNPs in the promoter region of the MDR1 gene were identified and their haplotypes were associated with MDR1 mrna levels in colon [63] and placenta [64]. Woodahl et al. have indicated that G1199A (Ser400Asn) was associated with MDR1 transport activity [65]. Although clinical studies to clarify the impacts of these SNPs and/or haplotypes are needed, these may be predictors for MDR1 function. The second issue raised is the need to carefully select pharmacokinetic parameters. In most clinical reports on the effects of the MDR1 genotype on the pharmacokinetics of drugs after single or multiple oral administrations, the discussion is often based on the effects on intestinal absorption. 88 Personalized Med. (2004) 1(1)

5 Pharmacogenomics of MDR and MRP subfamilies REVIEW Table 3. MDR1 genotype-related pharmacokinetics. SNPs Subjects N Drugs Regimen* Parameters Results Ref. T-129C C1236T G2677A,T C3435T European European Japanese Dutch European/ American Japanese Japanese European European/ American Japanese American Japanese 106 Cyclosporin A Steady state C max, C ss, AUC 0-4 TT = TC [173] 106 Cyclosporin A Steady state C max, C ss, AUC 0-4 CC < CT = TT [173] 46 Tacrolimus Steady state C trough CC = CT = TT [165] 65 Irinotecan, Steady state AUC CC < TT [174] SN Fexofenadine Single AUC 0-4 GG > GT > TT [67] 15 Digoxin Single AUC 0-4 GG = GT = TT [68] 20 Fexofenadine Single AUC, C max GG = GT = TT [175] 50 Digoxin Single AUC 0-4, C max GG = GW = WW [176] 32 Digoxin Single AUC 0-24 GG = GT = TT [177] 55 Talinolol Single or AUC or AUC 0-24 GG < GW < WW [171] steady state 46 Tacrolimus Steady state C trough GG = GA = GT = TA = TT [165] 83 Tacrolimus Steady state C ss Early-phase GG < GT + TT [178] Late-phase GG = GT + TT 106 Cyclosporin A Steady state C max, C ss, AUC 0-4 GG = GW = WW [173] 8 Digoxin Single AUC CC < CT < TT [61] CC = CT = TT [179] 12 Digoxin Single AUC 0-24 C max 32 Digoxin Single AUC 0-24 CC < TT [177] 37 Fexofenadine Single AUC 0-4 CC = CT > TT [67] 15 Digoxin Single AUC 0-4 CC > CT = TT [180] 20 Fexofenadine Single AUC, C max CC = TT [175] CC = CT = TT [176] 50 Digoxin Single AUC 0-4, C max 14 Cyclosporin A Single AUC, C max CC = CT + TT [181] 11 Digoxin Single Absorption rate CC > TT [182] 8 Loperamide Single C max, AUC 0-8, T max, CNS effect * Steady state; multiple dosing, Single; single dosing. W represents A or T. AUC: Area under the time concentration curve; C: Drug concentration; MDR: Multi-drug resistance; SS: Steady state. CC = TT [183] 89

6 REVIEW Okamura, Sakaeda & Okumura Table 3. MDR1 genotype-related pharmacokinetics (continued). SNPs Subjects N Drugs Regimen* Parameters Results Ref. C3435T (continued) G2677T,A C3435T G2677T,A C3435T G2677T,A C3435T C1236T G2677T,A C3435T Asian s / African European / African Asian / Black/Middle- Eastern/ South Asian Japanese Japanese Japanese Japanese 55 Talinolol Single or AUC or AUC 0-24 CC = CT = TT [167] steady state 24 Digoxin Steady state C min, C ss, CC < CT < TT [184] 32 Docetaxel Single Clearance CC = CT = TT [185] CC = CT = TT [186] 124 Cyclosporin A Steady state Stable dose, C min, C ss 54 Nelfinavir Steady state C min, C ss CC > CT > TT [187] 83 Tacrolimus Steady state C ss Early-phase CC < CT + TT Late-phase CC = CT + TT 106 Cyclosporin A Steady state C max, C ss, AUC 0-4 CC = CT = TT [173] 19 Cyclosporin A Steady state AUC CC > CT or TT [188] 10 Cyclosporin A Steady state AUC 0-4 CC < TT [189] 180 Tacrolimus Steady state C trough CC < TT [190] 46 Tacrolimus Steady state C trough CC = CT = TT [165] 29 Tacrolimus Steady state Dose/concentration CC < CT < TT [74] 15 Digoxin Single Bioavailability GG 2677 /CC 3435 = GT 2677 /CT 3435 < TT 2677 /TT Digoxin Single Renal clearance GG 2677 /CC 3435 = GT 2677 /CT 3435 > TT 2677 /TT 3435 Healthy 21 Loperamide Single AUC GT 2677 /TT 3435 > GG 2677 /CC 3435 = GT 2677 CT 3435 = TT 2677 /TT 3435 Patients 9 Cyclosporin A Steady state AUC, C max, C min CC 1236 /GG 2677 /CC 3435 < CT 1236 /GT 2677 /CT 3435 < TT 1236 /TT 2677 /TT 3435 * Steady state; multiple dosing, Single; single dosing. W represents A or T. AUC: Area under the time concentration curve; C: Drug concentration; MDR: Multi-drug resistance; SS: Steady state. [178] [76] [76] [191] [72] However, because of the expression of MDR1 in liver and kidneys, the effects on tissue distribution and renal or biliary excretion should be considered together with intestinal absorption. The area under the concentration time curve, the maximum concentration after a single oral administration or the trough concentrations in the steady state is often used, but since the latter is defined by the extent of absorption and systemic clearance, the effects on excretion should be included when using steady state pharmacokinetic parameters other than those after a single oral 90 Personalized Med. (2004) 1(1)

7 Pharmacogenomics of MDR and MRP subfamilies REVIEW Table 4. Haplotype frequencies (%) in MDR1 at positions of T-129C, C1236T, G2677A,T and C3435T. Japanese Polymorphism positions Nomenclature Haplotype frequencies (%) s African Asian American American T T T T *13/*13A/*14/*14A/ *16 T T G C *11/*11A/*11B/* T C G C *1/*8/*8A/*9/*9A/*9B/*9C/*10/ *20/*21/*21A/*21B/*21E/*22/ *22A/*23/*23A/*25/*26/*26A/ *26B/*26D/*27/*28/*29/ *30/*31/*32 T C A C *24/*24A T T T C *15/*15A C C A C *4A T T G T *12/*12A/*18/*19A C C G C *3/*4/*4B/*5/*5A/*6/*6A/* T C G T *2/*2A//*17*21C T C T T *17A C T G C 0.3 T C A T *2B 1.0 C T G T *12B 0.5 C T T C *15B 1.6 T C T C *21D/*26C Nomenclature is based on the reports by Kroetz et al. [62]. Haplotype frequencies were calculated using the data of Kroetz et al. [62]. Healthy Japanese subjects (n = 154). : Not identified; MDR: Multi-drug resistance. administration. The maximum concentrations in the steady state are sometimes used, but it is meaningless because it is a complicated function of not only intestinal absorption but also tissue distribution and renal and biliary excretion, giving rise to confusing results. In addition, recent reports have indicated that exposure to digoxin (a typical MDR1 substrate used as a probe for clinical investigations), cisplatin and a vitamin D derivative resulted in the upregulation of MDR1 [78-81], as did exposure to rifampicin, a well-known MDR1 inducer [82-84], whereas verapamil decreased expression [78,85,86]. It is not surprising that chronic administration of a drug results in an alteration of MDR1 expression. We should, therefore, understand that the data after a single administration are different from those after multiple administrations, and it is necessary to elucidate the association of MDR1 genotypes with the up- or downregulation of MDR1. The final issue that should be addressed in designing clinical trials is the need to relate results back to the drugs used in the trial and the condition of the participants. The pharmacokinetics is determined by absorption, distribution, metabolism, and excretion, and each step is governed by numerous drug-metabolizing enzymes and drug transporters. Their relative contribution to the pharmacokinetics differs among drugs; that is to say, the genotype phenotype association should vary depending on the type of drug, which is mainly eliminated from the body via hepatic metabolism or renal excretion. In addition, a variety of endogenous and exogenous stimuli could be involved in the molecular regulation of MDR1 expression, including cytotoxic drugs, heat shock, irradiation, inflammation, and various cytokines and growth factors [87-89]. Thus, we have to pay attention to the data on the pharmacokinetics under severe pathological conditions, such as post transplantation. The effects of the genetic polymorphisms of MDR1 and related proteins, therefore, can only be elucidated by conducting well-organized clinical studies with the haplotype analysis of several genes. In addition to the impact on pharmacokinetics, several clinical studies have been conducted to clarify the effect of genetic polymorphisms of MDR1 91

8 REVIEW Okamura, Sakaeda & Okumura Table 5. List of human ABCC genes. Gene Symbol Location Expression Function ABCC1 MRP1, GS-X pump 16q13.1 Ubiquitous (basolateral membrane) Transporter ABCC2 MRP2, cmoat 10q24 Liver, kidneys and intestine (apical membrane) Transporter ABCC3 MRP3, MOAT-B 17q21.3 Liver, kidneys and intestine (basolateral membrane) Transporter ABCC4 MRP4, MOAT-C 13q32 Ubiquitous Transporter ABCC5 MRP5, MOAT-D 3q27 Ubiquitous Transporter ABCC6 MRP6, MOAT-E 16p13.1 Liver and kidneys (basolateral membrane) Transporter CFTR ABCC7 7q31.2 Lungs Channel ABCC8 SUR1 11p15.1 Pancreas Receptor ABCC9 SUR2 12p12.1 Muscle and heart Receptor ABCC10 MRP7 6p21 Low Transporter ABCC11 MRP8 16q11-q12 Low Transporter ABCC12 MRP9 16q11-q12 Low Transporter ABCC13 21q11.2 Fetal liver and spleen Non-functional transporter ABC: ATP-binding cassette; CFTR: Cystic fibrosis transmembrane conductance regulator; cmoat: Canalicular multispecific organic anion transporter; GS-X: ATP-dependent glutathione S-conjugate export; MRP: Multi-drug resistance-associated protein; SUR: Sulfonylurea receptor. on drug response. Illmer et al. have reported that the worst overall survival in patients acute myeloid leukemia was found in individuals homozygous for allele C at position 3435 [77]. Other studies on the genetic polymorphism at C3435T have been reported that show associations between the polymorphism and response to preoperative chemotherapy in advanced breast cancer [90], response to initial antiretroviral therapy in HIV [91], prognosis in acute lymphoblastic leukemia [92], and the occurrence of nortrptyline-induced postural hypotension [93]. At the same time, no association has been reported between C3435T and response to antiviral therapy [94] and prognosis of acute lymphoblastic leukemia [95], to name a few. We are now only beginning to understand the significance of their pharmacogenetics in pharmacotherapy. In regard to TAP1, TAP2 and MDR3, polymorphisms in the genes encoding these proteins have been reported to be associated with several diseases. Zhang et al. reported that a TAP2 mutation is associated with Echinococcus multilocularis infection [96]. Genetic polymorphisms of MDR3 have been reported to be associated with progressive familial intrahepatic cholestasis type 3 [97] and intrahepatic cholestasis of pregnancy [98]. However, impact that these genetic polymorphisms have on pharmacotherapy is currently unknown. Pharmacogenetics of the MRP subfamily and its impact on pharmacotherapy MRP subfamily After the cloning of the MDR1 gene, multi-drugresistant cancer cells that do not overexpress MDR1 were established, indicating the existence of alternative mechanisms for multi-drug resistance [99-101]. Immediately thereafter, a human gene was cloned from doxorubicin-resistant small cell lung cancer H69 cells, and its product was named as MRP [102]. In 1996, Taniguchi et al. isolated another human gene as a homolog of the rat canalicular multispecific organic anion transporter from the cisplatin-resistant human cancer cell line KB/KCP4 and assigned the name canalicular multispecific organic anion transporter (cmoat) to the product [103]. Subsequently, MRP and cmoat were referred to as MRP1 and MRP2, respectively, and 13 types of related genes/proteins have been cloned, to date. The group is known as the ABCC subfamily, and members of the family are listed in Table 5. Nine members can be further categorized as belonging to the MRP subfamily, which are characterized by their functions as transporters. It has been noted that the members of the ABCC subfamily all have ATP-binding cassettes in common, and are capable of acting as channels or receptors in addition to roles as transporters. Similar to the MDR subfamily, the MRP subfamily has attracted a great deal of attention in studies focusing on the mechanisms of acquired multi-drug resistance in cancer cells. Furthermore, their role in the pharmacokinetics of drugs other than anticancer agents was investigated after a member of the family, cmoat (MRP2) [103], was found to have a great impact on the biliary secretion of drugs in hyperbilirubinemia rats [104]. MRP1 (ABCC1) is a 190-kDa membrane protein with 17 membrane-spanning domains and 92 Personalized Med. (2004) 1(1)

9 Pharmacogenomics of MDR and MRP subfamilies REVIEW Table 6A. Representative substrates for MRP1/Mrp1. Substrate Ref. Substrate Ref. Unconjugated bilirubin [192] Ritonavir [208] Bilirubin glucuronide [193] Saquinavir [208,209] Leukotriene C 4 [111,194] Mitoxantrone [210] Leukotriene D 4 [111] Folate [211] Leukotoriene E 4 [111] 15-Deoxy- 12,14 prostaglandin J 2 [212] Oxidized glutathione (GSSG) [195] Flutamide [213] Chlorambucil glutathione conjugate [196] Hydroxyflutamide [213] Melphalan glutathione conjugate [196] Methoxychlor [214] Prostaglandin A 1 glutathione conjugate [197] Phloridzin [215] Prostaglandin A 2 glutathione conjugate [197] Doxorubicin* [ ] 2,4-Dinitrophenyl-S-glutathione [111,194] Paclitaxel* [217,218] 4-Hydroxynoneal glutathione conjugate [198] Epirubicin* [218] 17β-Estradiol-17(β-D-glucuronide) [199] Vinblastine* [216,218] 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide [200] Colchicine* [218] Estrone-3-sulfate [201] Actinomycin D* [216] Vincristine [202,203] Arsenite* [218,219] Daunorubicin [204] Arsenate* [218,219] Irinotecan [205] Antimony* [218] SN-38 (active form of irinotecan) [205] Mercury* [219] Etoposide [202] N-Acetyl-leucyl-leucyl-norleucinal* [220] Methotrexate [206,207] *Assessed by sensitivity to drug in expressed cells. MRP: Multi-drug resistance-associated protein amino acids. Five transmembrane domains are connected to a core, like MDR1, which consists of two homologous halves containing six transmembrane domains and a nucleotide-binding domain [ ]. Structural analysis by electron microscopy for crystallized MRP1 isolated from the lung cancer cell line H69A suggested that the MRP1 monomer is approximately 8 10nm in size and works as a dimer [109]. MRP1 is expressed ubiquitously throughout the body, including the kidneys, lungs, and peripheral blood mononuclear cells [102,110]. In vitro studies have suggested that MRP1 acts as an efflux transporter for leukotrienes, glutathione, glucuronic acid and sulfate conjugates using ATP hydrolysis [ ]. Representative substrates for MRP1 and/or rat Mrp1 are listed in Table 6A. In addition, MK-571, which is a leukotriene D 4 receptor antagonist, was found to be an inhibitor of MRP1 function [111]. MRP2 (ABCC2), which is structurally similar to MRP1, is a 190-kDa membrane protein consisting of 17 transmembrane domains and 2 nucleotide-binding domains. MRP2 is expressed in the liver, kidneys, and intestines [103,112,113]. It is understood that MRP2 located in the biliary canalicular membranes of hepatocytes secretes bilirubin glucuronide into bile, and a deficiency of MRP2 results in Dubin-Johnson syndrome, causing hyperbilirubinemia [114,115]. Based on these findings, it has been deducted that MRP2 in the liver secretes many unchanged xenobiotics or their glucuronide, glutathione or sulfate conjugates into bile [108,116]. Representative substrates for MRP2 and/or rat Mrp2 are listed in Table 6B. The genes encoding MRP3 (ABCC3), MRP4 (ABCC4) and MRP5 (ABCC5) were discovered by Allikmets et al. using an expression sequence tag database; Kool et al. identified the genes as being homologous to MRP1 from a human cdna library [117,118]. MRP3 is structurally similar to MRP1 and MRP2 [119], and is expressed in the liver, small intestine, and colon [120]. Interestingly, Mrp3 was found to be expressed at low levels in normal conditions, but was induced under cholestatic conditions with a loss of function of Bsep and Mrp2 in rats, which are responsible for exporting bile acids into the bile [ ]. Furthermore, it was reported that MRP3 expression levels 93

10 REVIEW Okamura, Sakaeda & Okumura Table 6B. Representative substrates for MRP2/Mrp2. Substrate Ref. Substrate Ref. Bilirubin glucuronide [193] 5-Methylhydroxyfolate [240] Leukotriene C 4 [221] Para-aminohippuric acid [241] 2,4-Dinitrophenyl-S-glutathione [222] Camptothecin [242] Prostaglandin A 1 glutathione conjugate [222] Etoposide [243] Chlorambucil glutathione conjugate [223] Irinotecan [233,244] Acetaminophen glutathione conjugate [224] SN-38 (active form of irinotecan) [233] Lithocholate glucuronide [225] Vinblastine [245] Cholate glucuronide [225] Arsenite [246] Chrysin glucuronide [226] Pravastatin [247] Grepafloxacin glucuronide [227] Saquinavir [209,248] Ethinylestradiol glucuronide [228] Ritonavir [248] Genistein glucuronide [229] Indinavir [248] Nafenopin glucuronide [230] Phloridzin [215] E3040 glucuronide [231,232] Temocaprilat [249] SN-38 glucuronide [233] Phenytoin [250] Indomethacin glucuronide [234] BQ-123 [251] 17β-Estradiol-17(β-D-glucuronide) [235] Sulfinpyrazone [245] Telmisaltan glucuronide [236] 2-Amino-1-methyl-6-phenylimidazo[4,5-β]pyridine [252] Acetaminophen glucuronide [237] J [253] Chrysin sulfate [226] Z-335 [254] Taurolithocholate sulfate [238] Cisplatin* [235,255] Taurochenodeoxycholate sulfate [238] Doxorubicin* [235,255] Quercetin glucoside [239] Epirubicin* [235] Methotrexate [207] Vincristine* [235,255] *Assessed by sensitivity to drug in expressed cells. MRP: Multi-drug resistance-associated protein. are associated with the prognosis of prednisone response in patients with acute lymphoblastic leukemia [124]. Zeng et al. suggested that the substrates for MRP3 overlap with those for MRP1 and MRP2, albeit with less affinity for conjugates [119]. In contrast, MRP4 and MRP5 are structurally different from other members of the MRP subfamily. They have 12 transmembrane domains and 2 nucleotide-binding domains, although a core structure like MDR1 is maintained [125]. MRP4 and MRP5 are expressed mainly in the liver [118]. MRP4 has the ability to export nucleoside monophosphate derivatives, including reverse transcriptase inhibitors for HIV infection [126]. MRP4 is also reported to export the purine analogs methotrexate, leucovorin and folic acid, and it has been suggested that the protein confers the resistance that has been seen in acute lymphoblastic leukemia [ ]. Moreover, it was reported that MRP4 exports prostaglandin E 1 and E 2, and the efflux is inhibited by non-steroidal anti-inflammatory drugs (NSAIDs), suggesting that MRP4 acts as a physiological pump releasing prostaglandins, and the NSAIDs affect MRP4- mediated release in addition to the synthesis of prostaglandins [130]. MRP5 was reported to efflux cyclic nucleotides, as well as MRP4 [131,132]. Moreover, Dazert et al. suggested that MRP5 expressed in the heart plays an important role in the regulation of cgmp levels in cardiac or cardiovascular myocytes [133]. The MRP6 (ABCC6) gene was also identified in an anthracycline-resistant leukemia cell line, CEM [134]. The structure of MRP6 is similar to that of MRP1; it contains 17 membrane domains and 2 nucleotide-binding domains. Furthermore, MRP6 is exclusively expressed in the kidneys and liver [135,136]. MRP6 can efflux leukotriene C 4, 2,4-dinitrophenyl-S-glutathione and BQ123, as well as other MRPs, but not 17β-estradiol- 17(β-D-glucuronide) and methotrexate. MRP6- overexpressing cells exhibited resistance to etoposide, teniposide, daunorubicin, cisplatin, and actinomycin D, suggesting that MRP6 acts as 94 Personalized Med. (2004) 1(1)

11 Pharmacogenomics of MDR and MRP subfamilies REVIEW their transporter to confer resistance [137]. The MRP7 (ABCC10) gene was identified by Hopperet al. and it has been shown that the product MRP7 has 17 transmembrane domains and 2 nucleotide-binding domains [138]. MRP7 is expressed ubiquitously throughout the body, including in the kidneys, colon, testis, and skin [138]. MRP7 exhibits the ability to transport 17β-estradiol-17(β-D-glucuronide) and leukotriene C 4, but not 2,4-dinitrophenyl-S-glutathione, glucocholic acid, methotrexate, folic acid, and cyclic mononucleotides [139]. MRP7-overexpressing cells exhibit an acquired resistance to docetaxel, vincristine, vinblastine, and paclitaxel [140]. To date, MRP8 (ABCC11), MRP9 (ABCC12) and ABCC13 have also been discovered from a cdna library using an expression sequence tag database [ ]. MRP8 and MRP9 are expressed in the liver, lungs, and kidneys [141, ], and ABCC13 is expressed in the colon and fetal liver and spleen [142]. However, their functions and typical substrates are still unclear. Pharmacogenetics of the MRP subfamily Genetic polymorphisms of eight isoforms of the ABCC subfamily were extensively screened in 48 healthy Japanese subjects by Saito et al. [146]. They identified 95 genetic variations, including insertion and deletion polymorphisms, for MRP1, 41 for MRP2, 35 for MRP3, 257 for MRP4, and 85 for MRP5. Moreover, numerous genetic variants are listed in public databases (e.g., the NCBI SNP database [dbsnp] [301] and the Japanese SNP database [JSNP] [302]). Genetic variations of some members of the MRP subfamily have been reported to be associated with inherited disease; for example, Dubin- Johnson syndrome for MRP2 and pseudoxanthoma elasticum for MRP6 [ ]. These observations have suggested the possibility of the altered biological fate of substrates, depending on the genotypes, but few reports have been published on the genotype-related kinetics in humans for the MRP subfamily. Several in vitro studies have been conducted in regard with the relationships between genetic polymorphisms and expression and/or transport activity. In a study using transfected human embryonic kidney cells, Conrad et al. demonstrated that the MRP1 C1303A (Arg433Ser) polymorphism results in less transport activity for leukotriene C 4 and estrone-3-sulfate, but not for 17β-estradiol-17(β-D-glucuronide) [152,153]. Keitel et al. showed that a two-amino acid deletion and the A3517T (Ile1173Phe) polymorphism in MRP2 cause deficient maturation and impaired sorting [154,155]. Mor-Cohen et al. reported that the MRP2 A3517T (Ile1173Phe) variant causes a deficiency in maturation and impaired sorting, and that the G3449A (Arg1150His) polymorphism results in no transport activity [156]. Hashimoto et al. revealed in MRP2, the C2302T (Arg768Trp) variant causes a deficiency in maturation and impaired sorting, and the A4145G (Gln1382Arg) variant is linked to a loss of transport activity [157]. Hirouchi et al. have reported that the C2366T (Ser789Phe) and G4348A (Ala1450Thr) polymorphisms in MRP2 are associated with the change of cellular localization, but have no effects on transport activity [158]. A number of experiments in vitro suggest that drug disposition could be altered by these genetic polymorphisms. Further clinical studies are needed to confirm the impact on pharmacotherapy. Outlook With recent progress in the area of pharmacogenetics, it has been well-recognized that the genotype of drug transporters will become a surrogate marker to predict the response to pharmacotherapy in each patient. A number of SNPs have been identified in the genes encoding drug transporters, and clinical investigations on MDR1 have suggested the importance of a haplotype analysis of the genes. Consequently, it is necessary to develop high-throughput and cost-effective methods to diagnose several SNPs simultaneously, as well as to develop information technology that is capable of dealing with the data. Personalized medicine should be established, especially for cancer chemotherapy, since serious and irreversible adverse events often occur without any therapeutic benefit. Rational methods to select the best anticancer drugs and their dosing regimens have not yet been established. Multidrug resistance is one of the most serious causes of the failure of chemotherapy, and the biological factors in multi-drug resistance have been investigated, including the downregulation of uptake or induction of efflux systems, such as MDR1; induction of inactivation enzymes; alteration of targeted enzymes; changes in DNA repair processes; and alteration of apoptotic signaling. The expression of MDR and MRP subfamilies is one of the best characterized of these mechanisms. If the genotypes of the MDR and MRP subfamilies are found to affect their expression and function in tumor tissues, they must contribute to the optimization of cancer chemotherapy. In fact, 95

12 REVIEW Okamura, Sakaeda & Okumura Highlights Drug-metabolizing enzymes, drug transporters and drug targets play significant roles as determinants of drug efficacy and toxicity. Their genetic polymorphisms could be used as surrogate markers to predict the response to pharmacotherapy in individual patients. The clinical investigations on MDR1 have suggested the importance of haplotype analysis of the genes encoding MDR proteins. Consequently, it is necessary to develop high-throughput and cost-effective methods to diagnose several SNPs simultaneously, and to develop information technology that is capable of dealing with the data. MDR1 genotype- or haplotype-related pharmacokinetics and pharmacodynamics should be considered when developing clinical protocols in regards to, for example, the probe, dose regimen, amd regulation of MDR1 expression. The impact of genetic polymorphism of the MRP subfamily on pharmacokinetics and pharmacodynamics should be revealed by in vitro functional analysis and clinical investigations. These data could provide important information for personalized medicine. Recently, MDR1 has been revealed to protect against apoptosis. Future studies focusing on the physiological role of MDR1, in addition to its role as a transporter, may allow us to reconsider the role of MDR1 in cancer chemotherapy. Illmer et al. have shown that genetic polymorphisms of the MDR1 gene was associated with overall survival and relapse after therapy in acute myeloid leukemia patients [77]. There is little data at present, and studies on the usefulness of genotyping are currently underway. Recent investigations have challenged the notion that MDR1 has evolved merely to facilitate the efflux of xenobiotics and raised the possibility that MDR1 plays a fundamental role in regulating apoptosis and immunology. Although little information is available concerning the role of MDR1 in the system, Smyth and Johnstone et al. suggested that MDR1 protected cells against caspase-dependent apoptosis induced by cytotoxic drugs, Fas ligation, tumor necrosis factor, and ultraviolet irradiation [ ]. In addition, we found that MDR1 expression was upregulated by apoptotic stimuli and suppressed caspase-dependent apoptotic signaling, presumably via a mitochondrial pathway [80]. The role of MDR1 in apoptosis and immunological reactions has been discussed from the viewpoint of the sphingomyelin ceramide pathway, acidification of the intracellular space, cholesterol esterification, and cytokine release from lymphocytes. These observations suggest that the pharmacodynamics are related to the MDR1 genotype, independent of effects on pharmacokinetics, and the polymorphisms of the MDR1 gene might be a risk factor for a certain class of disease, especially in cases where a MDR1-related etiology is demonstrated. Acknowledgments The authors would like to thank Dr Tsutomu Nakamura and Mses. Chiho Komoto and Yuka Moriya from Kobe University Hospital, Japan, for their helpful and constructive discussion. Bibliography Papers of special note have been highlighted as either of interest ( ) or of considerable interest ( ) to readers. 1. Fiertz W: Challenge of personalized health care: to what extent is medicine already individualized and what are the future trends? Med. Sci. Monit. 10(5), RA111-RA123 (2004). 2. Tirona RG, Kim RB: Pharmacogenomics of drug transporters. In: Pharmacogenomics. The Search for Individualized Therapies. Licinio J, Wong ML (Eds), Wiley-VCH Verlag GmbH, Weinheim, Germany (2002). 3. Lin JH, Yamazaki M: Role of P-glycoprotein in pharmacokinetics. Clin. Pharmacokinet. 42(1), (2003). 4. Borst P, Zelcer N, van Helvoort A: ABC transporters in lipid transport. Biochim. Biophys. Acta 1486(1), (2000). 5. Sarkadi B, Ozvegy-Laczka C, Nemet K, Varadi A: ABCG2 a transporter for all seasons. FEBS Lett. 567(1), (2004). 6. Plasschaert SL, Van Der Kolk DM, De Bont ES, Vellenga E, Kamps WA, De Vries EG: Breast cancer resistance protein (BCRP) in acute leukemia. Leuk. Lymphoma 45(4), (2004). 7. Haimeur A, Conseil G, Deeley RG, Cole SP: The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Curr. Drug Metab. 5(1), (2004). 8. Ayrton A, Morgan P: Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31(8-9), (2001). 9. Dresser MJ, Leabman MK, Giacomini KM: Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters. J. Pharm. Sci. 90(4), (2001). 10. Terada T, Inui K: Peptide transporters: structure, function, regulation and application for drug delivery. Curr. Drug Metab. 5(1), (2004). 96 Personalized Med. (2004) 1(1)

13 Pharmacogenomics of MDR and MRP subfamilies REVIEW 11. Mizuno N, Sugiyama Y: Drug transporters: their role and importance in the selection and development of new drugs. Drug Metab. Pharmacokinet. 17(7), (2002). 12. Ingelman-Sunberg M, Oscarson M, McLellan RA: Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharmacol. Sci. 20, (1999). 13. Eichelbaum M, Burk O: CYP3A genetics in drug metabolism. Nat. Med. 7(3), (2001). 14. Nagata K, Yamazoe Y: Genetic polymorphism of human cytochrome P450 involved in drug metabolism. Drug Metab. Pharmacokinet. 17(3), (2002). 15. Lamba JK, Lin YS, Schuetz EG, Thummel KE: Genetic contribution to variable human CYP-3A-mediated metabolism. Adv. Drug Deliv. Rev. 54(10), (2002). 16. Tayeb MT, Clark C, Haites NE, Sharp L, Murray GI, McLeod HL: CYP3A4 and VDR gene polymorphisms and the risk of prostate cancer in men with benign prostate hyperplasia. Br. J. Cancer 88(6), (2003). 17. Sakaeda T, Nakamura T, Okumura K: MDR1 genotype-related pharmacokinetics and pharmacodynamics. Biol. Pharm. Bull. 25(11), (2002). 18. Sakaeda T, Nakamura T, Okumura K: Pharmacogenetics of MDR1 and its impact on pharmacokinetics and pharmacodynamics of drugs. Pharmacogenetics 4(4), (2003). 19. Sakaeda T, Nakamura T, Okumura K: Pharmacogenetics of drug transporters and its impact on the pharmacotherapy. Curr. Topics Med. Chem. 4(13), (2004). 20. Fromm MF: The influence of MDR1 polymorphisms on P-glycoprotein expression and function in humans. Adv. Drug Deliv. Rev. 54(10), (2002). 21. Schwab M, Eichelbaum M, Fromm MF: Genetic polymorphisms of the human MDR1 drug transporter. Annu. Rev. Pharmacol. Toxicol. 43, (2003). 22. Sparreboom A, Denesi R, Ando Y, Chan J, Figg WD: Pharmacogenomics of ABC transporters and its role in cancer chemotherapy. Drug Resist. Update 6(2), (2003). 23. Marzolini C, Paus E, Buclin T, Kim RB: Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance. Clin. Pharmacol. Ther. 75(1), (2004). 24. Ieiri I, Takane H, Otsubo K: The MDR1 (ABCB1) gene polymorphism and its clinical implications. Clin. Pharmacokinet. 43(9), (2004). 25. Juliano RL, Ling V: A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta 455(1), (1976). 26. Roninson IB, Chin JE, Choi KG et al.: Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells. Proc. Natl. Acad. Sci. USA 83(12), (1986). The first report of the cloning of the MDR1 gene. 27. Chen CJ, Chin JE, Ueda K et al.: Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrugresistant human cells. Cell 47(3), (1986). The first report of the cloning of the MDR1 gene. 28. Endicott JA, Ling V: The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Biochem. 58, (1989). 29. Gottesman MM, Pastan I: The multidrug transporter, a double-edged sword. J. Biol. Chem. 263(25), (1988). 30. Rosenberg MF, Callaghan R, Ford RC, Higgins CF: Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J. Biol. Chem. 272(16), (1997). Demonstrates the three-dimensional structure of MDR Rosenberg MF, Kamis AB, Callaghan R, Higgins CF, Ford RC: Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding. J. Biol. Chem. 278(10), (2003). 32. Higgins CF, Gottesman MM: Is the multidrug transporter a flippase? Trends Biochem. Sci. 17(1), (1992). 33. van Helvoort A, Smith AJ, Sprong H et al.: MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 87(3), (1996). 34. van der Bliek AM, Baas F, Ten Houte de Lange T, Kooiman PM, Van der Velde-Koerts T, Borst P: The human mdr3 gene encodes a novel P-glycoprotein homologue and gives rise to alternatively spliced mrnas in liver. EMBO J. 6(11), (1987). 35. Elferink RO, Groen AK: Genetic defects in hepatobiliary transport. Biochim. Biophys. Acta 1586(2), (2002). 36. Garraway LA, Chabner B: MDR1 inhibition: less resistance or less relevance? Eur. J. Cancer 38(18), (2002). 37. Gottesman MM, Fojo T, Bates SE: Multidrug resistance in cancer: role of ATPdependent transporters. Nat. Rev. Cancer 2(1), (2002). 38. Stein U, Lage H, Jordan A et al.: Impact of BCRP/MXR, MRP1 and MDR1/P-Glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells. Int. J. Cancer 97(6), (2002). 39. Mattern J: Drug resistance in cancer: a multifactorial problem. Anticancer Res. 23(2C), (2003). 40. Thomas H, Coley HM: Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting P-glycoprotein. Cancer Control 10(2), (2003). 41. Takara K, Sakaeda T, Okumura K: An update on overcoming MDR1-mediated multidrug resistance in cancer. Curr. Pharm. Des. (In press). 42. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC: Cellular localization of the multidrugresistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA 84(21), (1987). The first report showing the expression of MDR1 in normal tissue. 43. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I: Expression of a multidrug-resistance gene in human tumors and tissues. Proc. Natl. Acad. Sci. USA 84(1), (1987). 44. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC: Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein. J. Histochem. Cytochem. 37(2), (1989). 45. Cordon-Cardo C, O'Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR: Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J. Histochem. Cytochem. 38(9), (1990). 97