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ROLE OF HEPATIC TRANSPORT PROTEINS IN DRUG DISPOSITION AND DRUG-INDUCED LIVER INJURY by Jin Kyung Lee A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy. Chapel Hill 2009 Approved by: Kim L. R. Brouwer, Pharm.D., Ph.D. Gary M. Pollack, Ph.D. David Miller, Ph.D. Nita Patel, Ph.D. Mary F. Paine, Ph.D. Elaine M. Leslie, Ph.D. © 2009 Jin Kyung Lee ALL RIGHTS RESERVED ii ABSTRACT Jin Kyung Lee: Role of Hepatic Transport Proteins in Drug Disposition and Drug-induced Liver Injury (Under the direction of Kim L. R. Brouwer) Transport proteins in the liver play a crucial role in hepatic uptake and biliary excretion of drugs/metabolites. Understanding how altered hepatic transport (due to sex, genetic polymorphisms, disease states, drug interactions) influences systemic and hepatic exposure of drugs/metabolites is important from both a therapeutic and toxicologic standpoint. The objective of this dissertation research was to investigate the role of hepatic transport proteins in the hepatobiliary disposition of drugs/generated metabolites and drug-induced liver injury. Initial studies in perfused livers from male and female wild-type (WT) and breast cancer resistance protein (Bcrp)-deficient mice revealed that acetaminophen sulfate and glucuronide hepatobiliary disposition was influenced by sex-dependent Bcrp expression as well as other factors relating to conjugate formation and basolateral transport. In a second set of studies, sandwich-cultured hepatocytes (SCH), coupled with pharmacokinetic modeling/Monte Carlo simulations, were used to examine the impact of impaired biliary excretion on the hepatobiliary disposition of troglitazone (TGZ) and metabolites. Consistent with in vivo data, TGZ was extensively metabolized to TGZ-sulfate, and to a lesser extent to TGZ-glucuronide and TGZ-quinone, in both rat and human SCH. Modeling/simulation revealed that hepatocyte accumulation of metabolites was extensive when biliary excretion iii pathways were impaired; medium concentrations (analogous to systemic concentrations in vivo) may not reflect changes in hepatic exposure. Subsequent studies focused on two hepatotoxic drugs (trabectedin and sulindac) and the potential role of altered hepatic transport in drug-induced liver injury. Trabectedinmediated cytotoxicity was significantly decreased in dexamethasone-pretreated WT rat SCH and in Mrp2-deficient TR- rat SCH, compared to control WT rat SCH. These data emphasize the importance of Mrp2 and Mrp3 in hepato-protection from trabectedin-mediated cytotoxicity. Sulindac and metabolites inhibited multiple hepatic transport proteins in rat and human hepatocytes; potent inhibition of the hepatic transport of bile acids and/or drugs by sulindac sulfide and sulfone may play a previously unrecognized role in sulindac-mediated hepatotoxicity. Collectively, this dissertation research demonstrated the impact of sex-dependent transporter expression on the hepatobiliary disposition of phase II conjugates, illustrated use of an in vitro approach coupled with modeling/simulation to predict altered drug/metabolite hepatobiliary disposition, and defined the involvement of multiple transport proteins in drug-induced liver injury. iv ACKNOWLEDGEMENT First of all, I would like to thank God for His constant help and guidance. To Him be all the glory. I would like to express my sincere thanks to my advisor, Dr. Kim L. R. Brouwer, for her valuable support and guidance, but more importantly her patience druing my graduate study. I am very grateful to my dissertation committee chair, Dr. Gray M. Pollack for his outstanding teaching in pharmacokinetics, and his insightful advice for conducting pharmacokinetic modeling. I also would like to give a special thanks to my dissertation committee members, Drs. David Miller, Nita Patel, Elaine M. Leslie, and Mary F. Paine, for their scientific advice and encouragement. I am very grateful to my friends and colleagues. I want to thank Dr. Maciej J. Zamek-Gliszczynski and Dr. Xianbin Tian for their support, advice, and encouragement, as well as their training of isolated perfused liver technique. I want to thank to Dr. Koji Abe for his excellent mentorship, professional advice, but most importantly, his genuine kindness. I would like to thank to Drs. Wei Yue and Ahsan Rizwan for their unwavering friendship and enthusiastic scientific discussion. I would like to extend my thanks to my sincere friends, Tracy Marion, Brandon Swift, Dr. Kristina Wolf, Grace Yan, LaToya Griffin, Christina Won, and TaeEun Kim for their unwavering friendship and continuous support. v Most importantly of all, I would like to express my sincere thanks to my family for their unwavering love and continuous prayers over the years. Finally, I would like to express my deepest gratefulness for my husband, Changwon Lim for his endless love, friendship and support during the years of my doctoral program. vi TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………….……viii LIST OF FIGURES………………………………………………………………….……..ix LIST OF ABBREVIATIONS………………………………………………………….……xii CHAPTER 1. Introduction…………………………………………………………………………..1 2. Sex-dependent Disposition of Acetaminophen Sulfate and Glucuronide in the In Situ Perfused Mouse Liver……………………………..49 3. Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites in Rat and Human Sandwich-Cultured Hepatocytes: Use of Monte Carlo Simulations to Assess Impact of Changes in Biliary Excretion on Substrate Accumulation in Cells versus Culture Medium………………………………………………….78 4. Modulation of Trabectedin (ET-743) Hepatobiliary Disposition by Multidrug Resistance-Associated Proteins (Mrps) May Prevent Hepatotoxicity…………………………………………………………..113 5. Interactions Between Sulindac, Sulindac Sulfone, Sulindac Sulfide and Hepatic Transport Proteins………………………………………………...141 6. Conclusion………………………………………………………………………...173 APPENDIX 1. GF120918 (Elacridar) Inhibits Multidrug Resistance-Associated Protein 3 (MRP3)- and MRP4-Mediated Transport………………………..…200 2. Data Summary……………………………………………………………………220 vii LIST OF TABLES Table 1.1 Human hepatic uptake transport proteins………………………………..9 Table 1.2 Human hepatic efflux transport proteins…………………..…………....10 Table 2.1 Mouse body weights, liver weights normalized to body weight, acetaminophen extraction ratio and total recovery at 70 min in in situ single-pass perfused livers from male and female wild-type and Bcrp-deficient mice………………………………………………...…70 Table 2.2 Mean pharmacokinetic parameter estimates governing APAP, AG and AS disposition in in situ single-pass perfused livers from wild-type and Bcrp-deficient male and female mice…………………………………………………….……..71 Table 3.1 Demographics of human liver donors……………………………….…102 Table 3.2 Disposition of TGZ and generated metabolites in human SCH…………………………………………………………….103 Table 3.3 Disposition of TGZ and its generated metabolites in rat SCH…………………………………………………………………104 Table 3.4 Kinetic parameters (min-1) estimated from compartmental pharmacokinetic modeling analysis ……………………………………105 Table 3.5 Evaluation of the sensitivity of cell and medium accumulation to detect changes in biliary excretion of TS or TG utilizing data from Monte Carlo simulations…………….106 viii LIST OF FIGURES Figure 2.1 Model scheme depicting the hepatic disposition of APAP, AG and AS in the single-pass perfused mouse liver………….72 Figure 2.2 APAP concentrations (nM) in outflow perfusate from male wild-type (●) and Bcrp-deficient (○), and female wild-type (▼) and Bcrp-deficient (∆) mouse liver perfusions………….73 Figure 2.3 Cumulative biliary excretion and basolateral excretion of AG (A and B, respectively) in male and female wild-type and Bcrp-deficient perfused mouse livers…………..74 Figure 2.4 Cumulative biliary excretion and basolateral excretion of AS (A and B, respectively) in male and female wild-type and Bcrp-deficient perfused mouse livers…………..75 Figure 2.5 Representative immunoblot of Bcrp, Mrp4 and actin in crude membrane fractions from male and female wild-type and Bcrp-deficient mouse liver homogenates…..………………………76 Figure 2.6 Representative outflow perfusate rate of AG (●) and AS (■), and biliary excretion rate of AG (○) and AS (□) in male and female wild-type and Bcrp-deficient mouse livers……………………...77 Figure 3.1 Model scheme depicting the mass of TGZ, TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone (TQ) in the medium, hepatocytes, and bile of rat SCH……………………..107 Figure 3.2 Accumulation of TGZ, TS, and TG in rat SCH…………………..……108 Figure 3.3 Mass vs. time profiles of TGZ (●), TS (○), TG (▼) and TQ (∆) in the medium……………………………………..109 Figure 3.4 Mass vs. time profiles of TGZ (●), TS (○) and TG (▼) in the hepatocytes in day 4 rat SCH…………………….110 Figure 3.5 Mass vs. time profiles of TS (○) and TG (▼) in the bile canaliculi of day 4 rat SCH………………………………….111 Figure 3.6 Monte Carlo simulations with modulation of TS or TG biliary excretion………….………………………………………………..112 Figure 4.1 Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated from wild-type Wistar rats…………………………..136 ix Figure 4.2 Trabectedin-mediated cytotoxicity in SCRH from wild-type and TR- rats……………………………………………...137 Figure 4.3 Effect of BSO on trabectedin-mediated cytotoxicity…………………..138 Figure 4.4 Representative Western blot from whole cell lysates for Mrp2, 3, 4 and CYP3A1/2 in SCRH pretreated with vehicle (0.1 µM DEX), additional DEX (1 µM) or BSO (500 µM)..…………...139 Figure 4.5 Trabectedin perfusate concentrations (A) and cumulative biliary excretion (B) in isolated perfused livers from wild-type (solid circle) and TR- (white triangle) rats…………..…...….140 Figure 5.1 Chemical structures of sulindac, sulindac sulfone (S-sulfone) and sulindac sulfide (S-sulfide)…………….......165 Figure 5.2 Effect of sulindac and metabolites on hepatobiliary disposition of TC in rat SCH………………………......166 Figure 5.3 Effect of sulindac and metabolites on hepatobiliary disposition of E217G in rat SCH………………………167 Figure 5.4 Effect of sulindac and metabolites on hepatobiliary disposition of NF in rat SCH…………………………..168 Figure 5.5 Inhibition of Na+-dependent TC initial uptake in suspended rat hepatocytes by S-sulfone and S-sulfide ………...……………….169 Figure 5.6 Inhibition of Na+-independent E217G initial uptake in suspended rat hepatocytes by S-sulfone and S-sulfide……….…...170 Figure 5.7 Effect of sulindac and metabolites on hepatobiliary disposition of TC in human SCH………………………171 Figure 5.8 Inhibition of Na+-dependent TC initial uptake in suspended human hepatocytes by S-sulfone and S-sulfide ....…...172 Figure A.1 Representative Western blot of MRP3 and MRP4 in MRP3and MRP4- overexpressing membrane vesicle preparations….……216 Figure A.2 Functional activity of MRP3- or MRP4-overexpressing membrane vesicles………………………………………………………217 x Figure A.3 Inhibitory effect of GW918 and MK571 on MRP3and MRP4-mediated [3H]E217G and [3H]MTX uptake in the absence of GSH.......................................................................228 Figure A.4 Inhibitory effect of GW918 and MK571 on MRP3and MRP4-mediated [3H]E217G and [3H]MTX uptake in the presence of GSH (3 mM)………………………………….....…..229 xi LIST OF ABBREVIATIONS ABC ATP binding cassette AG acetaminophen glucuronide ALP alkaline phosphatase ALT alanine aminotransferase APAP acetaminophen AS acetaminophen sulfate BCRP breast cancer resistance protein BEI biliary excretion index BSEP bile salt export pump BSO buthionine sulfoximine CDF 5 (and 6)-carboxy-2',7'-dichlorofluorescein CYP cytochrome P450 DEX dexamethasone DHEAS dehydroepiandrosterone sulfate DILI drug-induced liver injury DMEM dulbecco’s modified eagle’s medium DPDPE [D-Pen2, D-Pen5]-enkephalin E217G estradiol-17-β-glucuronide EHBR eisai hyperbilirubinemic rats GSH reduced glutathione GST glutathione-S-transferase xii HBSS hanks’ balanced salts solution LDH lactate dehydrogenase MRP multidrug resistance-associated protein MTX methotrexate NF nitrofurantoin NSAID nonsteroidal anti-inflammatory drug NTCP sodium/taurocholate cotransporting polypeptide OATP organic anion transporting polypeptide OST organic solute transporter P-gp P-glycoprotein SCH sandwich-cultured primary hepatocytes S-sulfide sulindac sulfide S-sulfone sulindac sulfone SULT sulfotransferase TC taurocholate TGZ troglitazone TG troglitazone glucuronide TS troglitazone sulfate TQ trolitazone quinone UGT uridine diphosphoglucuronosyl transferase. xiii CHAPTER 1 Introduction This work has been published in part in Drug Transport in Liver. You, G., and Morris, M.E. (eds), In: Drug Transporters. Wiley Publishers, 2007: 359-410. Transport of xenobiotics by the liver is supported by the polarized nature of hepatocytes; the basolateral (sinusoidal and lateral) membrane domain represents the interface with sinusoidal blood, while the canalicular (apical) membrane domain faces the bile duct lumen. Basolateral transport proteins mediate the movement of compounds to and from the blood, while canalicular transport proteins mediate flux of compounds from hepatocytes to bile. Nomenclature for the human hepatic transport proteins, example substrates and relevant references are provided in Table 1.1 (uptake transport proteins) and Table 1.2 (efflux transport proteins). Hepatic Uptake Transport Proteins The Na+-taurocholate cotransporting polypeptide [NTCP, (SLC10A1)] mediates the basolateral uptake of both conjugated and unconjugated bile acids in a sodium-dependent manner with a 2:1 sodium-to-taurocholate stoichiometry. An inwardly directed Na+ gradient maintained by the activity of Na+/K+ ATPase serves as the driving force for NTCP (Meier and Stieger, 2002). Besides serving as the primary uptake mechanism for conjugated and unconjugated bile acids, NTCP also transports sulfated bile acids (e.g. chenodeoxycholate-3-sulfate, taurolithocholate-3sulfate) and steroid sulfates, as well as certain drugs (e.g. rosuvastatin, cholorambucil-taurocholate) (Trauner and Boyer, 2003; Ho et al., 2006). Ho et al. recently demonstrated ethnicity-dependent polymorphisms in the human NTCP gene resulting in decreased transport function of bile acid (Ho et al., 2004). The family of organic anion transporting polypeptides [OATPs, (SLCO)] is responsible for the sodium independent uptake of a large variety of amphipathic 2 compounds including bile acids, hormones, eicosanoids, and various drugs (Hagenbuch and Meier, 2004). Although a number of studies have revealed the substrate specificity of OATPs, the driving force behind OATP-mediated transport remains controversial. Bi-directional transport of substrates and anions (e.g. GSH and HCO3-) has been demonstrated for Oatp1a1 (Li et al., 1998; Li et al., 2000). In contrast, Briz et al. demonstrated cis-stimulation of taurocholate uptake by GSH in oocytes expressing OATP1B3, suggesting co-transport of GSH and bile acids (Briz et al., 2006). Mahagita et al. proposed that OATP1B3 functions as a bidirectional facilitated diffusion transporter rather than a bidirectional exchanger or cotransporter (Mahagita et al., 2007). Because some rodent Oatps are not orthologs of human OATP proteins, the prediction of human OATP-mediated transport from animal models is challenging (Hagenbuch and Meier, 2003). Among 11 human OATP isoforms identified, the human liver expresses OATP1A2 (previously named OATPA), OATP1B1 (previously named OATP-C), OATP1B3 (previously named OATP-8), and OATP2B1 (previously named OATP-B). OATP1A2 and OATP2B1 are widely expressed in several tissues, whereas OATP1B1 and OATP1B3 are selectively expressed in the liver (Konig et al., 2000a; Konig et al., 2000b). Despite distinct substrate affinity profiles, there are overlapping substrates for OATP1A2, 1B1 and 1B3, such as bile acids, bromosulfophthalein (BSP), dehydroepiandrosterone sulfate (DHEAS) and estrone-3-sulfate. As detailed in Table 1.1, the substrate specificity for OATP2B1 is selective, while the other three liver-expressed OATPs transport diverse compounds including organic anions, bulky cations, and neutral steroids. 3 In addition to NTCP and OATPs, organic anion transporter 2 [OAT2, (SLC22A7)] and organic cation transporter 1 and 3 [OCT, (SLC22A1 and SLC22A3, respectively)] also are expressed in the human liver and function as ion exchangers (Sun et al., 2001; Choi and Song, 2008). OAT2 is responsible for the hepatic uptake of negatively charged compounds such as methotrexate, zidovudine and taxol. While OATPs mediate transport of bulky type II cations, small cations (previously type I) are transported by OCTs. OCT1, which is expressed exclusively in liver, mediates transport of several therapeutic drugs including imatinib, metformin, and famotidine, whereas OCT3 is selective towards endogenous substrates (Table 1.1). Canalicular Efflux Transport Proteins All canalicular xenobiotic transport proteins, except the recently-identified MATE1 organic cation-H+ exchanger (Terada and Inui, 2008), belong to the ATPbinding cassette (ABC) family of proteins which mediate ATP-dependent transfer of solutes. The canalicular efflux transport system includes the multidrug resistance protein 1 [MDR1, (ABCB1)], MDR 3 (ABCB4), the bile salt export pump [BSEP, (ABCB11)], the multidrug resistance associated protein 2 [MRP2, (ABCC2)] and the ABC half-transporter protein breast cancer resistance protein [BCRP, (ABCG2)]. MDR1 (ABCB 1), generally referred to as P-glycoprotein, was first identified over two decades ago in multidrug resistant (MDR) tumor cells (Juliano and Ling, 1976); MDR1 represents the most widely studied ABC transport protein. MDR1 is responsible for biliary excretion of bulky hydrophobic and cationic substrates such as chemotherapeutic agents (e.g., daunorubicin, doxorubicin, etoposide, paclitaxel, 4 vinblastine, vincristine), cardiac glycosides (e.g., digoxin), rhodamine 123, cyclosporine A, and HIV-1 protease inhibitors (e.g., amprenavir, inidnavir, nelfinavir, ritonavir, saquinavir). MDR1 modulators have been evaluated for their ability to increase drug sensitivity during chemotherapy because overexpression of MDR1 on the surface of tumor cells causes multidrug resistance (Honma et al., 2002). MDR3 (ABCB4) is a phospholipid flippase and is involved in the biliary secretion of phospholipids and cholephilic compounds. Thus, MDR3 plays a crucial role in basic liver physiology in human and rats because phospholipids and cholesterol are responsible for the formation of micelles that solubilize bile acids in the lumen of the bile canaliculus (Carrella and Roda, 1999). Mutations in the MDR3 gene lead to progressive familial intrahepatic cholestasis type 3 (PFIC3), a disease that is characterized by increased γ-glutamyltranspeptidase levels, ductular proliferation, and inflammatory infiltrate which can progress to biliary cirrhosis. MDR3 is reported to have substrate specificity similar to MDR1, but the rate of transport of substrate is lower than for MDR1 (Evers et al., 2000). The bile salt export pump [BSEP, (ABCB11)] is the predominant bile acid efflux transport protein, and mediates excretion of conjugated and unconjugated bile acids into bile. It was reported that BSEP might also participate in transport of nonbile acid substrates such as pravastatin (Kullak-Ublick et al., 2000; Hirano et al., 2005b). Patients with progressive familial intrahepatic cholestasis type 2 (PFIC2) have a mutation in the ABCB11 gene and absence of BSEP protein expression, which leads to hepatocellular injury and necrosis caused by the increased intracellular concentration of detergent-like bile acids (Kullak-Ublick et al., 2004). 5 Inhibition of BSEP by several drugs such as troglitazone, bosentan, cyclosporine A and rifampicin has been suggested as one mechanism of drug-induced liver injury (Fattinger et al., 2001; Funk et al., 2001a; Mita et al., 2006). MRP2 (ABCC2) is a major xenobiotic efflux pump at the canalicular membrane. MRP2 plays a key role in the biliary excretion of organic anions including bilirubin-diglucuronide, glutathione conjugates, sulfated bile acids, and divalent bile acid conjugates as well as numerous drugs such as sulfopyrazone, indomethacin, penicillin, vinblastine, methotrexate and telmisartan (Table 1.2). Patients with Dubin-Jonson syndrome suffer from defective hepatic biliary excretion due to absence of MRP2. Animals with hereditary conjugated hyperbilirubinemia (Mrp2-deficient rats (GY/TR(-)) and Eisai hyperbilirubinemic rats (EHBR)) exhibit elevated MRP3 expression. This phenomenon has been explained as a compensatory mechanism to extrude MRP2 substrates into the systemic circulation under cholestatic conditions in order to reduce the burden on the liver (Konig et al., 1999b; Kuroda et al., 2004). Breast cancer resistance protein [BCRP, (ABCG2)] is highly expressed in the canalicular membrane of the liver as well as in the intestine, breast, and placenta. BCRP is a member of the ABC half-transporter protein family and forms a functional homodimer. It mediates transport of estrone-3-sulfate and various sulfated steroid compounds. It is also known to be involved in development of resistance to a variety of anticancer agents such as SN-38, mitoxantrone, topotecan and doxorubicin. Coadministration of topotecan in combination with GF120918, a potent BCRP and MDR1 inhibitor, significantly increased topotecan oral bioavailability in mice, 6 probably due to the inhibition of biliary clearance and enhanced intestinal absorption (Kruijtzer et al., 2002). Recently, several mammalian orthologues of the bacterial multidrug and toxin extrusion (MATE)-type transporters have been identified. In humans, the isoform MATE1 is localized to the bile canalicular membrane in the liver (Otsuka et al., 2005b). The driving force for organic cation extrusion by MATE1 is the inwardly directed H+ gradient (Otsuka et al., 2005b). Several organic cations (e.g. metformin, cimetidine and tetraethyl ammonium) have been identified as substrates for MATE1 (Table 1.2). The role of MATE1 in hepatobiliary disposition of drugs remains to be elucidated. Basolateral Efflux Transport Proteins The organic anion transporting polypeptides [OATPs, (SLCO)] represent the major family of basolateral uptake transport proteins for organic anions and have been hypothesized to also function as basolateral efflux transport proteins under certain conditions. However, the in vivo role of these transport proteins for basolateral egress of drugs from the hepatocyte warrants further studies (Durr et al., 2000). The ATP-dependent multidrug resistance-associated protein [MRP, (ABCC)] subfamily is a major class of efflux transport proteins on the hepatic basolateral membrane. MRP1, 3, 5, and 6 are involved in cellular transport of both hydrophobic uncharged molecules and hydrophilic anionic compounds. MRP1 (ABCC1) is responsible for efflux of various organic anions including glucuronide-, glutathione-, and sulfate-conjugated drugs. The expression of MRP1 (ABCC1) is very low on the 7 basolateral membrane in healthy liver, but MRP1 is highly inducible during severe liver injury and this induction is believed to play a role in liver protection (Wilson et al., 2003). MRP3 (ABCC3) is involved in the hepatic excretion of glucuronide conjugates (e.g., acetaminophen glucuronide), methotrexate, and estradiol-17βglucuronide (E217G). The expression level of MRP3 is markedly increased under cholestatic conditions (Hirohashi et al., 1999). MRP4 (ABCC4) and MRP5 (ABCC5) transport cyclic nucleotides such as cAMP and cGMP, as well as the purine analogs 6-mercaptopurine and 6-thioguanine (Zhou et al., 2001). MRP4 has been implicated in the transport of nucleoside-like drugs such as zidovudine, lamivudine, and stavudine, as well as non-nucleotide substrates (e.g., methotrexate), and the reverse transcriptase inhibitor azidothymidine. MRP4 also is involved in the transport of sulfate conjugates of bile acids and steroids (Zelcer et al., 2003). While the expression level of MRP5 in healthy liver is relatively low, treatment with lipopolysaccharide (LPS) results in down-regulation of MRP2 and induction of MRP5, suggesting that MRP5 may participate in the hepatic response to cholestasis (Donner et al., 2004). MRP6 is localized at the lateral side and canalicular membrane. MRP6 transports glutathione conjugates and the endothelin receptor antagonist, BQ-123 (Madon et al., 2000). Mutation of the MRP6 gene causes pseudoxanthoma elasticum (PXE), an inheritable systemic connective tissue disorder affecting the skin, eyes and blood vessels (Perdu and Germain, 2001). Clarification of the role of MRP6 in canalicular membrane transport awaits further investigation. 8 Table 1.1: Human Hepatic Uptake Transport Proteins Protein/ Gene symbol NTCP/ SLC10A1 OATP1A2/ SLCO1A2 OATP1B1/ SLCO1B1 Substrates References BSP; cholate; estrone-3-sulfate; glycocholate; taurochenodeoxycholate; tauroursodeoxycholate; taurocholate Rosuvastatin Bile acids; BQ-123; Bromosulfophthalein (BSP);DHEAS; 2,5 [D-penicillamine ]enkephalin (DPDPE); Estradiol-17βglucuronide (E217G); estrone-3-sulfate; n-methyl quinine; ouabain; prostaglandin E2; thyroid hormones (T3, T4) Deltorphin II Fexofenadine Levofloxacin Methotrexate Microcystin-LR Pitavastatin Saquinavir Arsenic (arsenite, arsenate) Atrasentan Atorvastatin; Simvastatin (Meier et al., 1997) Benzylpenicillin Bile acids; BQ-123; BSP; DHEAS; DPDPE; E217G; estrone-3-sulfate; T3, T4;leukotriene C4(LTC4); prostaglandin E2 Bilirubin, bilirubin glucuronides Bosentan Caspofungin Cerivastatin; Pravastatin Fluvastatin Irinotecan metabolite (SN-38) Methotrexate Microcystin-LR Olmesartan OATP1B3/ SLCO1B3 Pitavastatin Pravastatin Repaglinide Rifampin Rosuvastatin Troglitazone-sulfate Valsartan Amanitin Bile acids; BQ-123; BSP; Deltorphin-II; DHEAS; digoxin; DPDPE; E217G; estrone-3-sulfate; LTC4; ouabain; T3; T4 Bosentan CCK-8 Digoxin Docetaxel Enalapril Fexofenadine 9 (Ho et al., 2006) (Kullak-Ublick et al., 2001) (Gao et al., 2000) (Cvetkovic et al., 1999) (Maeda et al., 2007) (Badagnani et al., 2006) (Fischer et al., 2005) (Fujino et al., 2005) (Su et al., 2004) (Lu et al., 2006) (Katz et al., 2006) (Kameyama et al., 2005; Lau et al., 2006) (Tamai et al., 2000) (Kullak-Ublick et al., 2001) (Cui et al., 2001) (Treiber et al., 2007) (Sandhu et al., 2005) (Kameyama et al., 2005) (Kopplow et al., 2005) (Nozawa et al., 2004b) (Abe et al., 1999) (Fischer et al., 2005) (Nakagomi-Hagihara et al., 2006) (Fujino et al., 2004) (Hsiang et al., 1999) (Kajosaari et al., 2005) (Vavricka et al., 2002) (Ho et al., 2006) (Nozawa et al., 2004b) (Yamashiro et al., 2006) (Letschert et al., 2006) (Kullak-Ublick et al., 2001) (Treiber et al., 2007) (Ismair et al., 2001) (Kullak-Ublick et al., 2001) (Smith et al., 2005a) (Liu et al., 2006) (Shimizu et al., 2006) (Kopplow et al., 2005) Fluvastatin Methotrexate Microcystin-LR Monoglucuronosyl bilirubin Olmesartan OATP2B1/ SLCO2B1 OAT2/ SLC22A7 OCT1/ SLC22A1 OCT3/ SLC22A3 Paclitaxel Pitavastatin Rifampin Rosuvastatin Telmisartan Valsartan Atorvastatin Benzylpenicillin Bosentan BSP; DHEAS; estrone-3-sulfate Glibenclamide Fluvastatin Pravastatin Pitavastatin Rosuvastatin 5-Fluorouracil; Allopurinol; L-ascorbic acid; Bumetanide; DHEAS; estrone-3-sulfate; glutarate; prostaglandin E2 Erythromycin; theophylline Methotrexate Prostaglandin F2α Ranitidine Tetracycline Zidovudine Acyclovir, ganciclovir Azidoprocainamide methoiodide; n-methyl-quinidine; nmethyl-quinine; tributylmethylammonium Choline Imatinib Metformin Famotidine, ranitidine Prostaglandin E2, Prostaglandin F2α Adrenaline, noradrenaline, tyramine + Agmatine, MPP ; tetraethylammonium Atropine, etilefrine Cimetidine (Abe et al., 2001) (Fischer et al., 2005) (Cui et al., 2001) (Nakagomi-Hagihara et al., 2006) (Smith et al., 2005a) (Fujino et al., 2004) (Vavricka et al., 2002) (Ho et al., 2006) (Ishiguro et al., 2006) (Yamashiro et al., 2006) (Grube et al., 2006) (Tamai et al., 2000) (Treiber et al., 2007) (Kullak-Ublick et al., 2001) (Satoh et al., 2005) (Kopplow et al., 2005) (Nozawa et al., 2004a) (Hirano et al., 2006) (Ho et al., 2006) (Kobayashi et al., 2005a) (Kobayashi et al., 2005b) (Sun et al., 2001) (Enomoto et al., 2002) (Tahara et al., 2005) (Babu et al., 2002) (Takeda et al., 2002) (Takeda et al., 2002) (van Montfoort et al., 2001) (Grundemann et al., 1999) (Thomas et al., 2004) (Kimura et al., 2005) (Bourdet et al., 2005) (Kimura et al., 2002) (Grundemann et al., 1998) (Hayer-Zillgen et al., 2002) (Muller et al., 2005) (Grundemann et al., 1999) Table 1.2: Human Hepatic Efflux Transport Proteins Protein/ Gene symbol MRP1/ ABCC1 MRP3/ ABCC3 Substrates References Daunorubicin; Doxorubicin; etoposide; vindcristine Glutathione Methotrexate (MTX) Acetaminophen glucuronide E217G; monovalent and sulfated bile salts; MTX (Cole et al., 1994) (Madon et al., 2000) (Zeng et al., 2001) (Xiong et al., 2000) (Hirohashi et al., 1999; Zeng et al., 2001) (Madon et al., 2000) Etoposide 10 MRP4/ ABCC4 MRP5/ ABCC5 MRP6/ ABCC6 BSEP/ ABCB11 MRP2/ ABCC2 MDR1/ ABCB1 Fexofenadine (Matsushima et al., 2008) Azidothymidine cAMP; cGMP; PMEA (Schuetz et al., 1999) (Jedlitschky et al., 2000; Chen et al., 2001; Reid et al., 2003a) (Chen et al., 2002) (Reid et al., 2003a) (Jedlitschky et al., 2000) (Reid et al., 2003a) (Madon et al., 2000) MTX Prostaglandin E1; Prostaglandin E2 cAMP; cGMP Adefovir BQ-123 Verapamil; Norverapamil Phospholipids (Gerloff et al., 1998) (Matsushima et al., 2008) (Hirano et al., 2005a) (Xiong et al., 2000) (Koike et al., 1997) (Hirano et al., 2005b) (Kawabe et al., 1999) (Konig et al., 1999b) (Payen et al., 2000) (Konig et al., 1999b) (Hooijberg et al., 1999) (Hirano et al., 2005b) (Sasaki et al., 2002) (Kitamura et al., 2008) (Kim et al., 1998; Polli et al., 1999) (Bogman et al., 2001) (Ueda et al., 1992) (Marie et al., 1992) (Kim et al., 1999) (Fromm et al., 1999) (van der Sandt et al., 2000; Advani et al., 2001) (Lum et al., 1993) (Cvetkovic et al., 1999) (Yamaguchi et al., 2000) (Soldner et al., 1999) (Hirano et al., 2005b) (Kitamura et al., 2008) (Floren et al., 1997) (Spahn-Langguth et al., 1998) (Pauli-Magnus et al., 2000) (Evers et al., 2000) Albendazole sulfoxide; Oxfendazole Daunorubicin; doxorubicin; MX; sulfated conjugates Ciprofloxacin; Ofloxacin; Norfloxacin Cerivastatin Dirithromycin; Erythromycin; Rifampicin Genistein Epirubicin Irinotecan; Topotecan Imatinib (Merino et al., 2005a) (Doyle et al., 1998) (Merino et al., 2006) (Matsushima et al., 2005) (Janvilisri et al., 2005) (Imai et al., 2004) (Robey et al., 2003) (Maliepaard et al., 2001) (Burger et al., 2004) Conjugated and unconjugated bile salts; TC Fexofenadine Pravastatin Acetaminophen glucuronide; carboxydichlorofluorescein Camptothecin; doxorubicin Cerivastatin; estron-3-sulfate Cisplatin; vincristine Etoposide Glibenclamide; indomethacin; rifampin Glucuronide, glutathione, and sulfate conjugates; LTC4 MTX Pitavastatin Pravastatin Rosuvastatin Amprenavir; indinavir; nelfinavir; ritonavir; saquinavir Atorvastatin Aldosterone; corticosterone; dexamethasone; digoxin Cyclosporin A; mitoxantrone (MX) Debrisoquine; erythromycin; lovastatin; terfenadine Digoxin; quinidine Doxorubicin; paclitaxel; rhodamine 123 Etoposide Fexofenadine Levofloxacin; Grapafloxacin Losartan; vinblastine Pitavastatin Rosuvastatin Tacrolimus Talinolol MDR3/ ABCB4 BCRP/ ABCG2 11 Lamivudine Methotrexate Nitrofurantoin Pitavastatin Prazosin; rhodamine 123 Rosuvastatin Sulfasalazine MATE1/ SLC47A1 SN-38; SN-38 glucuronide Testosterone; tamoxifen; estradiol Zidovudine Tetraethylammonium (TEA) Cimetidine; metformin; procainamide; topotecan; (Wang et al., 2003) (Volk et al., 2002) (Wang and Morris, 2007) (Hirano et al., 2005b) (Ozvegy et al., 2001) (Kitamura et al., 2008) (van der Heijden et al., 2004) (Yoshikawa et al., 2004) (Janvilisri et al., 2003) (Wang et al., 2004) (Otsuka et al., 2005a) (Tanihara et al., 2007) Potential Role of Hepatic Transport Proteins in Drug-Induced Liver Injury Drug-induced liver injury has emerged as the most common reason for the withdrawal of FDA-approved drugs from the market (FDA, 2000). Drug-induced hepatotoxicity may be classified as intrinsic or idiosyncratic. Intrinsic hepatotoxicity is generally predictable, dose-dependent, and characteristic for a particular drug. In contrast, idiosyncratic toxicity is unpredictable, not dose-related, and displays no clear underlying mechanism. Drug-induced liver injury can be evaluated by measuring the activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the plasma (Benichou, 1990); liver injury is designated hepatocellular injury if the ALT level is greater than 2 times the upper limit of normal (ULN), or if a ratio of ALT/ULN and ALP/ULN is greater than 5; cholestatic injury can be characterized by elevations in ALP greater than 2 times the ULN or by a ratio of ALT/ULN and ALP/ULN that is less than 2; mixed patterns of liver injury are defined as a ratio of ALT/ULN and ALP/ULN that is greater than 2 but less than 5. Age, gender, genetic and environmental factors may affect susceptibility to drug-induced liver injury. Early studies focused on bioactivation, covalent adduct formation, immunotoxicity, and disruptions in cellular bioenergetics as mechanisms 12 underlying hepatotoxicity, but more recent data suggest that alterations in hepatic transport may be an important mechanism of toxic endpoints. Inhibition of canalicular Bsep has been reported as one mechanism of drug-induced liver injury because impaired Bsep function leads to elevated hepatic concentrations of detergent-like bile acids, which can disrupt key membrane-associated cellular functions (Spivey et al., 1993; Funk et al., 2001a). Troglitazone (Rezulin) was the first thiazolidinedione approved for the treatment of type 2 diabetes prior to its withdrawal from the market in 2000 due to idiosyncratic liver injury (Herrine and Choudhary, 1999). Several mechanisms underlying troglitazone-induced hepatotoxicity have been proposed (Smith, 2003), including Bsep inhibition by both troglitazone (KI, apparent = 1.3 µM) and its primary metabolite, troglitazone sulfate (KI, apparent = 0.23 µM) (Funk et al., 2001b). Troglitazone causes intrahepatic accumulation of bile acids, which can lead to hepatocellular necrosis and severe liver damage (Gores et al., 1998). Following troglitazone administration, Mrp2- deficient rats exhibited a marked delay in the biliary excretion and enhanced urinary excretion of troglitazone sulfate leading to elevated serum bile acid concentrations relative to wild-type rats (Kostrubsky et al., 2001). Although troglitazone has been one of the most studied examples of hepatotoxicity resulting from Bsep inhibition, other drugs, including bosentan, glibenclamide, cyclosporin, and rifampin also impair Bsep function (Stieger et al., 2000; Fattinger et al., 2001). In addition to cis-inhibition of Bsep, estradiol-17-β-glucuronide trans-inhibits Bsep following biliary excretion by Mrp2 (Stieger et al., 2000). In the absence of Mrp2, estradiol-17-β-glucuronideinduced cholestasis is not observed (Sano et al., 1993; Stieger et al., 2000). 13 Hepatic transport proteins may provide a protective role against drug-induced hepatotoxicity through changes in transporter expression (Ananthanarayanan et al., 1994). Intrahepatic and obstructive cholestasis in humans and rats result in downregulation of Oatp/Ntcp isoforms and up-regulation of Mrp isoforms (Gartung et al., 1996; Donner and Keppler, 2001; Wagner et al., 2003). In patients with hepatitis or chronic cholestasis, up-regulation of major hepatic efflux transporters such as MDR1, MRP1 and MRP3 have been reported (Wilson et al., 2003). Similarly, during liver injury caused by acetaminophen and carbon tetrachloride which are both well-known hepatic toxicants, expression of hepatic transport proteins in mice was modulated (Aleksunes et al., 2005; Moffit et al., 2006): Mrp2 protein expression was slightly increased; Mrp3 and Mrp4 expression was increased by 3.6-fold and 16-fold, respectively; Oatps and Ntcp were down-regulated. Collectively, hepatocytes may prevent accumulation of potentially toxic chemicals and protect against subsequent liver injury by limiting influx and enabling efficient efflux of harmful toxicants following down-regulation of uptake transporters and up-regulation of efflux transporters. In certain cases, impaired hepatic transport may provide protection against drug-induced liver injury. Many electrophiles that form covalent adducts with intracellular macromolecules are detoxified by conjugation with glutathione. Mrp2deficient rats do not excrete glutathione into bile, resulting in a ~2-3-fold increase in hepatic glutathione concentrations (Dietrich et al., 2001). Acetaminophen, a commonly used antipyretic and analgesic agent, induces severe liver injury at high doses when acetaminophen sulfation and glucuronidation become saturated and oxidation to N-acetyl-p-benzoquinoneimine, a potent electrophile, becomes a 14 significant metabolic pathway (Galinsky and Levy, 1981). N-acetyl-p- benzoquinoneimine may be detoxified by conjugation with glutathione, but as this co-factor becomes depleted, the electrophilic metabolite forms covalent adducts, which can lead to cellular necrosis (Dahlin et al., 1984). Administration of toxic acetaminophen doses (1 g/kg) induced the expected hepatotoxicity in wild-type rats, but had no apparent adverse effects in Mrp2-deficient rat livers (Silva et al., 2005). Elevated glutathione concentrations in Mrp2-deficient rats appear to play a protective role in acetaminophen-induced hepatotoxicity; a similar effect also would be expected for other electrophiles in Mrp2-deficient rats (Silva et al., 2005). In this dissertation project, the role of hepatic transport proteins in druginduced liver injury was assessed using troglitazone, trabectedin, and sulindac as model hepatotoxic compounds. As mentioned above, Bsep inhibition by troglitazone and troglitazone sulfate has been proposed as one mechanism for troglitazonemediated liver injury. In rats and humans, troglitazone is extensively metabolized to troglitazone sulfate (TS) which is transported by BCRP (Enokizono et al., 2007). Patients who have impaired functional activity of BCRP (e.g. due to a polymorphism or drug-drug interaction) may have increased potential for troglitazone-mediated hepatotoxicity due to higher accumulation of TS in the liver. Trabectedin is a promising anticancer agent which is currently under phase II evaluation (Schoffski et al., 2008). During phase I/II clinical trials, the most prevalent adverse effect was hepatotoxicity with elevated ALP and/or bilirubin levels (Beumer et al., 2005b). Little is known about the metabolic profiles of trabectedin in rats and humans, but multiple cytochrome P450 enzymes (3A4, 2C9, 2C19, 2D6, and 2E1) appear to be involved 15 in trabectedin metabolism. Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), has been consistently associated with liver injury among NSAIDs (Aithal and Day, 2007). Sulindac is metabolized to sulindac sulfide, which has anti-inflammatory properties, and is also transformed to sulindac sulfone, which has antiproliferative effects against colon cancer (Davies and Watson, 1997). Several investigators have demonstrated a linear correlation between sulindac sulfide concentrations and hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986). Although hepatocellular and mixed patterns of sulindac-mediated hepatotoxicity have been reported, cholestatic patterns of liver injury were most commonly observed (Tarazi et al., 1993). Modulation of Hepatic Transport Proteins by Drugs and Patient-Specific Factors As discussed above, hepatic transport proteins play an important role in the uptake and excretion of endogenous and exogenous compounds. Altered hepatic transport function (e.g., inhibition, induction, or altered translocation) may have a significant impact on the hepatobiliary disposition of drugs. Inhibition of cytochrome P450 (CYP) enzymes is an important mechanism of many toxic drug interactions, but accumulating data suggest that some interactions may be mediated, at least in part, by transport proteins. Numerous transport interactions occur at the level of hepatic uptake. Cyclosporine A increased the AUC of atovastatin, lovastatin and simvastatin ~7.4-fold, 5-fold and ~2.5-fold, respectively; these statins are transported into the hepatocyte by OATPs, and are cleared by CYP3A4-mediated metabolism and P-gp-mediated biliary excretion (Shitara and Sugiyama, 2006). The observed interaction between some statins (atorvastatin, 16 simvastatin, and lovastatin) and cyclosporine A may be caused by the combined effects on drug metabolizing enzymes and transport proteins; cyclosporine A not only inhibits CYP3A4-mediated metabolism, but also affects OATP-mediated hepatic uptake and P-gp-mediated biliary excretion. Cyclosporine A also increased the AUC of rosuvastatin ~7.1-fold (Simonson et al., 2004). This interaction may be due to impaired OATP-mediated hepatic uptake of rosuvastatin, since rosuvastain is neither a P-gp nor a CYP3A4 substrate or inhibitor (Martin et al., 2002; Cooper et al., 2003),. The interaction between atorvastatin or rosuvastatin and cyclosporine A is consistent with pharmacokinetic alterations of these statins observed in subjects with genetic polymorphisms in OATP1B1; the AUC of atorvastatin and rosuvastatin in subjects with the SLCO1B1 c.521CC genotype was 144% and 65% higher than in subjects with the SLCO1B1 c.521TT genotype (Pasanen et al., 2007). Undoubtedly, interactions in biliary excretion or basolateral efflux also occur. Although there is accumulating evidence of potential drug-drug interactions mediated by inhibition of these hepatic efflux transport proteins based on in vitro screening, clinical trials examining hepatic efflux transporter-mediated drug interactions often demonstrate an undetectable or weak interaction in humans. Impaired function of hepatic efflux transport proteins may increase hepatocelluar exposure without affecting the plasma pharmacokinetics. Drug interactions at the level of hepatic efflux can be assessed in human only via imaging techniques or quantifying biliary clearance. Alternatively, the application of a modeling/simulation approach coupled with in vitro data may address these issues as demonstrated in Chapter 3 based on Monte Carlo simulations. 17 In addition to acute inhibition, long term administration of xenobiotics can modulate transport function by affecting transcriptional, translational and posttranslational events (Hong and You, 2007). The transcriptional regulation of transporter gene expression in hepatocytes is mediated by a large variety of orphan nuclear receptors [e.g., pregnane X receptor (PXR), liver X receptor (LXR), constitutive androstane receptor (CAR) and farnesoid X receptor (FXR)] coupled with a heterodimeric partner (e.g., retinoic acid X receptor). Although many investigations have focused on the identification of regulatory mechanism(s) responsible for the induction of transport proteins (Rizzo et al., 2005; Urquhart et al., 2007), the overlapping gene targets and ligand specificity for orphan nuclear receptors make it difficult to determine how regulatory processes ultimately affect the functional pharmacokinetic outcomes of transport protein induction. Other than orphan nuclear receptors, some signaling events also can modulate transport protein expression. For example, an estrogen response element has been identified in the BCRP promoter region, and BCRP expression appears to be influenced by estrogen (E1) and/or estradiol (E2) in cancer cell lines when the E1 receptor (ER) is present (Ee et al., 2004; Imai et al., 2005). However, whether these hormones increase or inhibit BCRP expression is controversial; BCRP expression was increased by E2-mediated transcriptional up-regulation in ER-α-positive MCF-7 breast cancer cells (Zhang et al., 2006b), while another group demonstrated posttranscriptional BCRP down-regulation by E2 in the same cell line (Imai et al., 2005). In addition, BCRP expression is regulated by the phophoinositol-3 kinase (PI3K)-Akt signaling pathway via posttranscriptional/translational mechanisms in 18 BCRP-overexpressing K562 cells (Nakanishi et al., 2006). Interestingly, imatinib, a potent tyrosine kinase inhibitor, decreased BCRP expression by modulating PI3KAkt signaling pathway in the same cell line (Nakanishi et al., 2006). However, the effect of these signaling pathways on BCRP regulation in BCRP-expressing cancer or non-polarized cell lines may not be reflected in hepatocytes, or in vivo. Hepatic transport protein function can be modulated by altered translocation of transport protein(s) between the plasma membrane and intracellular vesicles. The mechanisms of intracellular trafficking of some hepatic proteins to the correct plasma membrane domain have been examined previously (Wakabayashi et al., 2006). Newly synthesized canalicular transport proteins in the Golgi apparatus may be directly trafficked to the apical membrane (e.g., P-gp), or may be stored transiently in intracellular compartments before apical delivery to the membrane (e.g., Bsep) (Kipp et al., 2001; Kipp et al., 2003); some stimuli such as taurocholate or dibutyryl (DB) cAMP are involved in Bsep and P-gp trafficking. Trafficking of Mrp2 to the apical membrane is mediated by a protein kinase Cdependent mechanism (Beuers et al., 2001). Some xenobiotics interacting with these stimuli may affect the intracellular trafficking of canalicular transport proteins, thus resulting in changes in biliary excretion. Although limited information is available for the mechanism(s) of basolateral transport protein trafficking, the cysteine residues of extracellular loop IX-X, responsible for disulfide linkages, appear to be involved in the targeting of OATP to the basolateral membrane (Hanggi et al., 2006). The current understanding of the mechanism(s) of intracellular translocation of xenobiotics is rudimentary. Interestingly, a recent 19 investigation by Munteanu et al. demonstrated the presence of functional P-gp in the Golgi and the mitochondrial membrane of doxorubicin-resistant K562 leukemia cells; doxorubicin was transported by P-gp into the mitochondria (Munteanu et al., 2006). This finding suggests that xenobiotics may accumulate within intracellular organelles via transport proteins prior to hepatic basolateral and canalicular excretion. Sex-related variations in the expression and activity of drug transport proteins may contribute to sex-related differences in therapeutic response or toxicity. P-glycoprotein has been reported to be higher in livers of men compared with women, but phenotyping with fexofenadine did not demonstrate sex differences in clearance (Schuetz et al., 1995; Kim et al., 2001). Sex-dependent expression of Bcrp/BCRP have been demonstrated in mice and humans; men have 2-3 fold higher BCRP expression than women (Merino et al., 2005b). Experimental Tools for Studying Hepatic Transport 1) Membrane Vesicles Prepared From Transfected Systems: Uptake of substrates into membrane vesicles prepared from purified basolateral and canalicular liver plasma membranes is an established technique for elucidating the kinetics of membrane transport proteins (Moseley et al., 1990; Wolters et al., 1991). Theoretically, liver plasma membrane vesicles include every transport protein that resides on the hepatocyte plasma membrane, and these can be used to assess the overall contribution of all transport proteins to the uptake and efflux of substrates. Utilization of recombinant expression systems enables the preparation of membrane 20 vesicles from cells over-expressing a single transport protein; this system has been used to determine whether a compound is an inhibitor and/or substrate for a specific transport protein. Membrane vesicles prepared from transfected cells that over- express the target transport protein have been used with either a radio-labeled compound to directly measure the uptake, or unlabeled compound to evaluate the ability of the compound to inhibit the transport of a radio-labeled probe substrate for the transport protein (Leslie et al., 2003; El-Sheikh et al., 2007). Active transport in membrane vesicles should be energy-dependent, temperature-dependent and saturable (Leier et al., 1994; Allen et al., 2002). Membrane vesicles also are a useful model system to characterize mechanisms of transport. For example, the dependence of MRP1-mediated vincristine transport on reduced glutathione (GSH) has been characterized using membrane vesicles (Allen et al., 2002). However, the utility of membrane vesicles is somewhat limited because of labor intensive, timeconsuming processes of vesicle preparation. 2) Freshly-Isolated Suspended Hepatocytes: Freshly-isolated hepatocytes in suspension are preferable to cultured hepatocytes for characterization of the mechanisms of hepatic uptake due to the technical ease of determining initial rates of uptake. To characterize uptake mechanism(s), various conditions have been applied in experiments using suspended hepatocytes, such as addition of inhibitor(s) for uptake pathways, substitution of sodium with choline, depletion of ATP and low temperature (Brock and Vore, 1984). One major disadvantage of suspended hepatocytes is that cells should be used within 6-8 h due to a rapid decrease in activity of both metabolic and transport proteins. In addition, during cell isolation, 21 hepatocytes lose polarity, and canalicular transport proteins such as P-gp and Mrp2 are internalized. Thus, use of suspended hepatocytes to determine biliary clearance is limited (Roelofsen et al., 1998; Bow et al., 2008). 3) Sandwich Cultured Hepatocytes: Hepatocytes cultured between two layers of gelled collagen in a sandwich configuration re-create the three-dimensional in vivo cellular environment and re-establish cell polarity forming canalicular membrane domains bounded by junctional complexes and bile canalicular networks (Liu et al., 1998; Liu et al., 1999c). In sandwich-cultured hepatocytes (SCH), transport proteins are expressed on the appropriate membrane domains (Hoffmaster et al., 2004a). For example, confocal microscopy revealed the co-expression of P-gp and Mrp2 on the canalicular domain, and biliary excretion of [D-penicillamine2,5]enkephalin (3HDPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) confirmed P-gp and Mrp2 function, respectively. Expression and appropriate localization of transport proteins in SCH enables the utilization of this model to investigate drug-drug interactions mediated by inhibition of hepatobiliary excretion, and to elucidate the consequences of protein induction (Annaert et al., 2001; Zamek-Gliszczynski et al., 2003; Turncliff et al., 2004). SCH exhibit down-regulation of NTCP and up- regulation of MRP3; these changes may be due to the somewhat cholestatic nature of the canalicular networks that lack normal bile drainage via the common bile duct in vivo. 4) Isolated Perfused Liver Model: The isolated perfused rat liver system was first reported by Claude Bernard in 1855 and has been used to investigate the physiology and pathophysiology of the rat liver for many years (Wolkoff et al., 1979). 22 Although it is labor intensive and time- and animal-consuming, the isolated perfused liver system has been used widely to investigate hepatobiliary transport of drugs since it maintains intact hepatic architecture, remains viable for ~ 3 hr and allows for collection of perfusate and bile. This system is applicable for use with chemical modulators (e.g., inducers or inhibitors), and livers from rodents with transport protein(s) knocked out may be perfused to determine the contribution of specific proteins to hepatobiliary disposition. For example, in situ isolated perfused mouse livers have been used to identify the role of P-gp and Bcrp in the biliary excretion of 4-methylumbelliferyl sulfate (4-MUS) using P-gp- and Bcrp-deficient mice (ZamekGliszczynski et al., 2006a); the biliary excretion of 4-MUS was obliterated in Bcrpdeficient mice while 4-MUS biliary excretion was comparable between wild-type and P-gp-deficient mice, showing the exclusive role of Bcrp in 4-MUS biliary excretion. The use of 10 µM GF120918 in isolated perfused livers from wild-type mice showed significantly reduced biliary excretion of 4-MUS, which was comparable to that in livers from Bcrp-deficient mice. 5) Animal Knockout Models: Genetic models of transport protein deficiencies also have been used in in vivo pharmacokinetic and pharmacodynamic studies to assess the role of a particular transport protein in drug disposition. Animal models with transport protein deficiencies are a valuable tool to study hepatobiliary drug transport due to intact liver physiology and preserved hepatic metabolic activity and transport processes. However, the potential disadvantage of the knockout animal is the compensatory up-regulation of other metabolic enzymes and/or transport proteins as a result of the loss of a transport system. For example, the mutational disruption of 23 mdr1 genes led to over-expression of CYP3A, 2B, and 1A proteins and increased the catalytic activity of CYP enzymes (Schuetz et al., 2000). Interestingly, this upregulation of CYP enzymes was observed only in P-gp deficient mice housed in Amsterdam, but not in P-gp deficient mice housed in the United States, suggesting that both the genetic deficiency and environmental factors regulated P450 expression in P-gp-deficient mice. Another example is the increased expression of basolateral Mrp3 in rodents with hereditary conjugated hyperbilirubinemia [Mrp2deficient rats (GY/TR-) and Eisai hyperbilirubinemic rats (EHBR)] (Kuroda et al., 2004). This compensatory up-regulation of MRP3 also is observed in patients with Dubin-Johnson syndrome who suffer from impaired biliary excretion of organic anion due to the absence of MRP2 (Tsujii et al., 1999). Another limitation of using knockout animal models is the lack of predictability of these models to humans. For example, it has been documented previously that substrate specificity between the human MDR1 and mouse Mdr1a was markedly different, suggesting species differences in P-gp-mediated transport (Yamazaki et al., 2001). Species differences in the biliary excretion of phase II conjugates between rats and mice have been demonstrated recently (Zamek-Gliszczynski et al., 2008); Bcrp1 was involved exclusively in the biliary excretion of sulfate metabolites in mice, whereas both Mrp2 and Bcrp1 were responsible for canalicular transport of sulfate metabolites in rats. 24 Specific Aims The goal of this dissertation project was to investigate the role of hepatic transport proteins in drug disposition and drug-induced liver injury. Transport proteins, individually and/or in concert, play a crucial role in hepatic uptake and excretion of drugs and metabolites. Thus, altered hepatic transport protein function (due to disease state, genetic polymorphism or drug-drug interactions) may influence systemic and/or hepatic concentrations, thereby altering therapeutic efficacy and/or toxicity. A multiexperimental approach was employed to address these key issues regarding hepatic transport mechanisms: 1) sex-related differences in the hepatobiliary disposition of phase II conjugates; 2) in vitro tools to assess altered transport function; 3) the role of transport proteins in druginduced liver injury. AIM #1. Elucidate the Mechanism of Differences in the Hepatobiliary Disposition of Acetaminophen and Metabolites in Male and Female Mice. Hypothesis: Sex-dependent hepatic Bcrp expression (male > female) results in sexdependent hepatobiliary disposition of phase II conjugates. This hypothesis was tested using an in situ mouse liver perfusion approach to compare the disposition of acetaminophen and metabolites in male and female wild-type and Bcrp-deficient mice. 1.a. Characterize the impact of sex-dependent expression of Bcrp on the hepatobiliary disposition of acetaminophen and generated metabolites [glucuronide (AG); sulfate (AS)] in male and female wild-type and Bcrpdeficient mice. 25 1.b. Use a pharmacokinetic modeling approach to estimate pharmacokinetic parameters and evaluate mechanisms by which the hepatobiliary disposition of Bcrp substrates was altered. 1.c. Compare protein expression levels of the major biliary and basolateral transport proteins among male and female wild-type and Bcrp-deficient mice. AIM #2. Assess the Utility of an Integrated in vitro Approach [Sandwichcultured Hepatocytes (SCH) and Modeling/Simulation] in Predicting Alterations in the Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites. Hypothesis: Rat and Human SCH can be used to mimic the in vivo disposition of TGZ and generated metabolites in rats and humans. Compartmental pharmacokinetic modeling and Monte Carlo simulation approaches can be applied to data from SCH to examine the consequences of canalicular transport protein modulation resulting from drug interactions or disease states, on hepatic exposure in hypothetical “populations” of hepatocytes. 2.a. Assess the metabolism of TGZ, which is governed primarily by phase II conjugation, and the hepatobiliary disposition of TGZ and generated metabolites in rat and human SCH. 2.b. Use pharmacokinetic modeling and Monte Carlo simulation approaches to predict alterations in hepatic exposure and routes of excretion of TGZ and generated metabolites. 26 AIM #3. Examine the Potential Role of Hepatic Transport Proteins in Liver Injury Mediated by Trabectedin and Sulindac. Hypothesis 1: Mrp2 as well as CYP3A1/2 play a role in the previously reported hepato-protective effect of DEX against trabectedin-mediated hepatotoxicity in rats and humans. SCH, which closely mimic the metabolic and transport capabilities of the intact liver, can predict this DEX-mediated hepato-protection. 3.a. Assess the effect of DEX and BSO on trabectedin-mediated hepatotoxicity in SCH from WT and Mrp2-deficient TR- rats. 3.b. Examine the effect of DEX and BSO on the expression levels of transport proteins in WT SCH. 3.c. Determine the role of Mrp2 in the hepatobiliary disposition of trabectedin in the rat isolated perfused liver (IPL) using WT and Mrp2-deficient TR- rats. Hypothesis 2: Interaction of sulindac and metabolites with hepatic transport proteins disrupts the hepatobiliary disposition of bile acids, bile acid conjugates and/or therapeutic drugs, thereby resulting in sulindac-mediated liver injury. 4.a. Examine the impact of acute exposure of sulindac, sulindac sulfone and sulindac sulfide on hepatobiliary disposition of nitrofurantoin (NF), taurocholate (TC) and estradiol-17-β-glucuronide (E217G) in rat SCH. 4.b. Determine the inhibitory potency of sulindac sulfone and sulindac sulfide on TC and E217G initial uptake in freshly-isolated rat hepatocytes. 4.c. Elucidate altered hepatobiliary disposition of TC by sulindac, sulindac sulfone and sulindac sulfide in human SCH, and subsequently determine the 27 inhibitory potency of sulindac metabolites on TC initial uptake in freshly-isolated human hepatocytes. 28 REFERENCES Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D, Nomura H, Unno M, Suzuki M, Naitoh T, Matsuno S and Yawo H (1999) Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J Biol Chem 274:17159-17163. Abe T, Unno M, Onogawa T, Tokui T, Kondo TN, Nakagomi R, Adachi H, Fujiwara K, Okabe M, Suzuki T, Nunoki K, Sato E, Kakyo M, Nishio T, Sugita J, Asano N, Tanemoto M, Seki M, Date F, Ono K, Kondo Y, Shiiba K, Suzuki M, Ohtani H, Shimosegawa T, Iinuma K, Nagura H, Ito S and Matsuno S (2001) LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers. Gastroenterology 120:1689-1699. Advani R, Fisher GA, Lum BL, Hausdorff J, Halsey J, Litchman M and Sikic BI (2001) A phase I trial of doxorubicin, paclitaxel, and valspodar (PSC 833), a modulator of multidrug resistance. Clin Cancer Res 7:1221-1229. Aithal GP and Day CP (2007) Nonsteroidal anti-inflammatory drug-induced hepatotoxicity. Clin Liver Dis 11:563-575, vi-vii. Aleksunes LM, Slitt AM, Cherrington NJ, Thibodeau MS, Klaassen CD and Manautou JE (2005) Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicol Sci 83:44-52. Allen JD, van Loevezijn A, Lakhai JM, van der Valk M, van Tellingen O, Reid G, Schellens JH, Koomen GJ and Schinkel AH (2002) Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 1:417-425. Ananthanarayanan M, Ng OC, Boyer JL and Suchy FJ (1994) Characterization of cloned rat liver Na(+)-bile acid cotransporter using peptide and fusion protein antibodies. Am J Physiol 267:G637-643. Annaert PP, Turncliff RZ, Booth CL, Thakker DR and Brouwer KL (2001) Pglycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes. Drug Metab Dispos 29:1277-1283. Babu E, Takeda M, Narikawa S, Kobayashi Y, Yamamoto T, Cha SH, Sekine T, Sakthisekaran D and Endou H (2002) Human organic anion transporters mediate the transport of tetracycline. Jpn J Pharmacol 88:69-76. Badagnani I, Castro RA, Taylor TR, Brett CM, Huang CC, Stryke D, Kawamoto M, Johns SJ, Ferrin TE, Carlson EJ, Burchard EG and Giacomini KM (2006) Interaction of methotrexate with organic-anion transporting polypeptide 1A2 and its genetic variants. J Pharmacol Exp Ther 318:521-529. 29 Benichou C (1990) Criteria of drug-induced liver disorders. Report of an international consensus meeting. J Hepatol 11:272-276. Beuers U, Bilzer M, Chittattu A, Kullak-Ublick GA, Keppler D, Paumgartner G and Dombrowski F (2001) Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 33:1206-1216. Beumer JH, Schellens JH and Beijnen JH (2005) Hepatotoxicity and metabolism of trabectedin: a literature review. Pharmacol Res 51:391-398. Bogman K, Peyer AK, Torok M, Kusters E and Drewe J (2001) HMG-CoA reductase inhibitors and P-glycoprotein modulation. Br J Pharmacol 132:1183-1192. Bourdet DL, Pritchard JB and Thakker DR (2005) Differential substrate and inhibitory activities of ranitidine and famotidine toward human organic cation transporter 1 (hOCT1; SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3). J Pharmacol Exp Ther 315:1288-1297. Bow DA, Perry JL, Miller DS, Pritchard JB and Brouwer KL (2008) Localization of Pgp (Abcb1) and Mrp2 (Abcc2) in freshly isolated rat hepatocytes. Drug Metab Dispos 36:198-202. Briz O, Romero MR, Martinez-Becerra P, Macias RI, Perez MJ, Jimenez F, San Martin FG and Marin JJ (2006) OATP8/1B3-mediated cotransport of bile acids and glutathione: an export pathway for organic anions from hepatocytes? J Biol Chem 281:30326-30335. Brock WJ and Vore M (1984) Characterization of uptake of steroid glucuronides into isolated male and female rat hepatocytes. J Pharmacol Exp Ther 229:175181. Burger H, van Tol H, Boersma AW, Brok M, Wiemer EA, Stoter G and Nooter K (2004) Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood 104:2940-2942. Carrella M and Roda E (1999) Evolving concepts in the pathophysiology of biliary lipid secretion. Ital J Gastroenterol Hepatol 31:643-648. Chen ZS, Lee K and Kruh GD (2001) Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6mercaptopurine and 6-thioguanine. J Biol Chem 276:33747-33754. Chen ZS, Lee K, Walther S, Raftogianis RB, Kuwano M, Zeng H and Kruh GD (2002) Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system. Cancer Res 62:3144-3150. 30 Choi MK and Song IS (2008) Organic cation transporters and their pharmacokinetic and pharmacodynamic consequences. Drug Metab Pharmacokinet 23:243253. Cole SP, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM and Deeley RG (1994) Pharmacological characterization of multidrug resistant MRPtransfected human tumor cells. Cancer Res 54:5902-5910. Cooper KJ, Martin PD, Dane AL, Warwick MJ, Raza A and Schneck DW (2003) Lack of effect of ketoconazole on the pharmacokinetics of rosuvastatin in healthy subjects. Br J Clin Pharmacol 55:94-99. Cui Y, Konig J, Leier I, Buchholz U and Keppler D (2001) Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J Biol Chem 276:9626-9630. Cvetkovic M, Leake B, Fromm MF, Wilkinson GR and Kim RB (1999) OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27:866-871. Dahlin DC, Miwa GT, Lu AY and Nelson SD (1984) N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc Natl Acad Sci U S A 81:1327-1331. Davies NM and Watson MS (1997) Clinical pharmacokinetics of sulindac. A dynamic old drug. Clin Pharmacokinet 32:437-459. Dietrich CG, Ottenhoff R, de Waart DR and Oude Elferink RP (2001) Role of MRP2 and GSH in intrahepatic cycling of toxins. Toxicology 167:73-81. Donner MG and Keppler D (2001) Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 34:351-359. Donner MG, Warskulat U, Saha N and Haussinger D (2004) Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS. Biol Chem 385:331-339. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK and Ross DD (1998) A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 95:15665-15670. Duggan DE, Hooke KF, Noll RM, Hucker HB and Van Arman CG (1978) Comparative disposition of sulindac and metabolites in five species. Biochem Pharmacol 27:2311-2320. Durr D, Stieger B, Kullak-Ublick GA, Rentsch KM, Steinert HC, Meier PJ and Fattinger K (2000) St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4. Clin Pharmacol Ther 68:598-604. 31 Ee PL, Kamalakaran S, Tonetti D, He X, Ross DD and Beck WT (2004) Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. Cancer Res 64:1247-1251. El-Sheikh AA, van den Heuvel JJ, Koenderink JB and Russel FG (2007) Interaction of nonsteroidal anti-inflammatory drugs with multidrug resistance protein (MRP) 2/ABCC2- and MRP4/ABCC4-mediated methotrexate transport. J Pharmacol Exp Ther 320:229-235. Enokizono J, Kusuhara H and Sugiyama Y (2007) Involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion and intestinal efflux of troglitazone sulfate, the major metabolite of troglitazone with a cholestatic effect. Drug Metab Dispos 35:209-214. Enomoto A, Takeda M, Shimoda M, Narikawa S, Kobayashi Y, Kobayashi Y, Yamamoto T, Sekine T, Cha SH, Niwa T and Endou H (2002) Interaction of human organic anion transporters 2 and 4 with organic anion transport inhibitors. J Pharmacol Exp Ther 301:797-802. Evers R, Kool M, Smith AJ, van Deemter L, de Haas M and Borst P (2000) Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer 83:366-374. Fattinger K, Funk C, Pantze M, Weber C, Reichen J, Stieger B and Meier PJ (2001) The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther 69:223-231. Fischer WJ, Altheimer S, Cattori V, Meier PJ, Dietrich DR and Hagenbuch B (2005) Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin. Toxicol Appl Pharmacol 203:257-263. Floren LC, Bekersky I, Benet LZ, Mekki Q, Dressler D, Lee JW, Roberts JP and Hebert MF (1997) Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clin Pharmacol Ther 62:41-49. Fromm MF, Kim RB, Stein CM, Wilkinson GR and Roden DM (1999) Inhibition of Pglycoprotein-mediated drug transport: A unifying mechanism to explain the interaction between digoxin and quinidine [seecomments]. Circulation 99:552557. Fujino H, Saito T, Ogawa S and Kojima J (2005) Transporter-mediated influx and efflux mechanisms of pitavastatin, a new inhibitor of HMG-CoA reductase. J Pharm Pharmacol 57:1305-1311. Fujino H, Saito T, Tsunenari Y, Kojima J and Sakaeda T (2004) Metabolic properties of the acid and lactone forms of HMG-CoA reductase inhibitors. Xenobiotica 34:961-971. 32 Funk C, Pantze M, Jehle L, Ponelle C, Scheuermann G, Lazendic M and Gasser R (2001a) Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167:83-98. Funk C, Ponelle C, Scheuermann G and Pantze M (2001b) Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: in vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol Pharmacol 59:627-635. Galinsky RE and Levy G (1981) Dose- and time-dependent elimination of acetaminophen in rats: pharmacokinetic implications of cosubstrate depletion. J Pharmacol Exp Ther 219:14-20. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A and Meier PJ (2000) Organic anion-transporting polypeptides mediate transport of opioid peptides across blood-brain barrier. J Pharmacol Exp Ther 294:73-79. Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, Stolz A, Suchy FJ and Boyer JL (1996) Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 110:199-209. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF and Meier PJ (1998) The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 273:10046-10050. Gores GJ, Miyoshi H, Botla R, Aguilar HI and Bronk SF (1998) Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochim Biophys Acta 1366:167-175. Grube M, Kock K, Oswald S, Draber K, Meissner K, Eckel L, Bohm M, Felix SB, Vogelgesang S, Jedlitschky G, Siegmund W, Warzok R and Kroemer HK (2006) Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clin Pharmacol Ther 80:607-620. Grundemann D, Liebich G, Kiefer N, Koster S and Schomig E (1999) Selective substrates for non-neuronal monoamine transporters. Mol Pharmacol 56:1-10. Grundemann D, Schechinger B, Rappold GA and Schomig E (1998) Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter. Nat Neurosci 1:349-351. 33 Hagenbuch B and Meier PJ (2003) The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609:1-18. Hagenbuch B and Meier PJ (2004) Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch 447:653-665. Hanggi E, Grundschober AF, Leuthold S, Meier PJ and St-Pierre MV (2006) Functional analysis of the extracellular cysteine residues in the human organic anion transporting polypeptide, OATP2B1. Mol Pharmacol 70:806817. Hayer-Zillgen M, Bruss M and Bonisch H (2002) Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3. Br J Pharmacol 136:829-836. Herrine SK and Choudhary C (1999) Severe hepatotoxicity associated with troglitazone. Ann Intern Med 130:163-164. Hirano M, Maeda K, Hayashi H, Kusuhara H and Sugiyama Y (2005a) Bile salt export pump (BSEP/ABCB11) can transport a nonbile acid substrate, pravastatin. J Pharmacol Exp Ther 314:876-882. Hirano M, Maeda K, Matsushima S, Nozaki Y, Kusuhara H and Sugiyama Y (2005b) Involvement of BCRP (ABCG2) in the biliary excretion of pitavastatin. Mol Pharmacol 68:800-807. Hirano M, Maeda K, Shitara Y and Sugiyama Y (2006) Drug-drug interaction between pitavastatin and various drugs via OATP1B1. Drug Metab Dispos 34:1229-1236. Hirohashi T, Suzuki H and Sugiyama Y (1999) Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J Biol Chem 274:15181-15185. Ho RH, Leake BF, Roberts RL, Lee W and Kim RB (2004) Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition. J Biol Chem 279:7213-7222. Ho RH, Tirona RG, Leake BF, Glaeser H, Lee W, Lemke CJ, Wang Y and Kim RB (2006) Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics. Gastroenterology 130:1793-1806. Hoffmaster KA, Turncliff RZ, LeCluyse EL, Kim RB, Meier PJ and Brouwer KL (2004) P-glycoprotein expression, localization, and function in sandwich- 34 cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm Res 21:1294-1302. Hong M and You G (2007) Regulation of Drug Transporter Activity, in Drug Transporters; Molecular Characterization and Role in Drug Disposition (You G and Morris ME eds) pp 517-531, John Wiley & Sons, Inc. Honma W, Shimada M, Sasano H, Ozawa S, Miyata M, Nagata K, Ikeda T and Yamazoe Y (2002) Phenol sulfotransferase, ST1A3, as the main enzyme catalyzing sulfation of troglitazone in human liver. Drug Metab Dispos 30:944949. Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ, Noordhuis P, Scheper RJ, Borst P, Pinedo HM and Jansen G (1999) Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. Cancer Res 59:2532-2535. Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang WP and Kirchgessner TG (1999) A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters. J Biol Chem 274:37161-37168. Imai Y, Ishikawa E, Asada S and Sugimoto Y (2005) Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2. Cancer Res 65:596-604. Imai Y, Tsukahara S, Asada S and Sugimoto Y (2004) Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer Res 64:4346-4352. Ishiguro N, Maeda K, Kishimoto W, Saito A, Harada A, Ebner T, Roth W, Igarashi T and Sugiyama Y (2006) Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans. Drug Metab Dispos 34:1109-1115. Ismair MG, Stieger B, Cattori V, Hagenbuch B, Fried M, Meier PJ and Kullak-Ublick GA (2001) Hepatic uptake of cholecystokinin octapeptide by organic aniontransporting polypeptides OATP4 and OATP8 of rat and human liver. Gastroenterology 121:1185-1190. Janvilisri T, Shahi S, Venter H, Balakrishnan L and van Veen HW (2005) Arginine482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2). Biochem J 385:419-426. 35 Janvilisri T, Venter H, Shahi S, Reuter G, Balakrishnan L and van Veen HW (2003) Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis. J Biol Chem 278:20645-20651. Jedlitschky G, Burchell B and Keppler D (2000) The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem 275:30069-30074. Juliano RL and Ling V (1976) A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455:152-162. Kajosaari LI, Niemi M, Neuvonen M, Laitila J, Neuvonen PJ and Backman JT (2005) Cyclosporine markedly raises the plasma concentrations of repaglinide. Clin Pharmacol Ther 78:388-399. Kameyama Y, Yamashita K, Kobayashi K, Hosokawa M and Chiba K (2005) Functional characterization of SLCO1B1 (OATP-C) variants, SLCO1B1*5, SLCO1B1*15 and SLCO1B1*15+C1007G, by using transient expression systems of HeLa and HEK293 cells. Pharmacogenet Genomics 15:513-522. Katz DA, Carr R, Grimm DR, Xiong H, Holley-Shanks R, Mueller T, Leake B, Wang Q, Han L, Wang PG, Edeki T, Sahelijo L, Doan T, Allen A, Spear BB and Kim RB (2006) Organic anion transporting polypeptide 1B1 activity classified by SLCO1B1 genotype influences atrasentan pharmacokinetics. Clin Pharmacol Ther 79:186-196. Kawabe T, Chen ZS, Wada M, Uchiumi T, Ono M, Akiyama S and Kuwano M (1999) Enhanced transport of anticancer agents and leukotriene C4 by the human canalicular multispecific organic anion transporter (cMOAT/MRP2). FEBS Lett 456:327-331. Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM and Wilkinson GR (1998) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest 101:289-294. Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI, Taylor A, Xie HG, McKinsey J, Zhou S, Lan LB, Schuetz JD, Schuetz EG and Wilkinson GR (2001) Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther 70:189-199. Kim RB, Wandel C, Leake B, Cvetkovic M, Fromm MF, Dempsey PJ, Roden MM, Belas F, Chaudhary AK, Roden DM, Wood AJ and Wilkinson GR (1999) Interrelationship between substrates and inhibitors of human CYP3A and Pglycoprotein. Pharm Res 16:408-414. Kimura H, Takeda M, Narikawa S, Enomoto A, Ichida K and Endou H (2002) Human organic anion transporters and human organic cation transporters mediate renal transport of prostaglandins. J Pharmacol Exp Ther 301:293-298. 36 Kimura N, Masuda S, Tanihara Y, Ueo H, Okuda M, Katsura T and Inui K (2005) Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1. Drug Metab Pharmacokinet 20:379-386. Kipp H, Khoursandi S, Scharlau D and Kinne RK (2003) More than apical: Distribution of SGLT1 in Caco-2 cells. Am J Physiol Cell Physiol 285:C737749. Kipp H, Pichetshote N and Arias IM (2001) Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver. J Biol Chem 276:7218-7224. Kitamura S, Maeda K, Wang Y and Sugiyama Y (2008) Involvement of multiple transporters in the hepatobiliary transport of rosuvastatin. Drug Metab Dispos 36:2014-2023. Kobayashi Y, Ohshiro N, Sakai R, Ohbayashi M, Kohyama N and Yamamoto T (2005a) Transport mechanism and substrate specificity of human organic anion transporter 2 (hOat2 [SLC22A7]). J Pharm Pharmacol 57:573-578. Kobayashi Y, Sakai R, Ohshiro N, Ohbayashi M, Kohyama N and Yamamoto T (2005b) Possible involvement of organic anion transporter 2 on the interaction of theophylline with erythromycin in the human liver. Drug Metab Dispos 33:619-622. Koike K, Kawabe T, Tanaka T, Toh S, Uchiumi T, Wada M, Akiyama S, Ono M and Kuwano M (1997) A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells. Cancer Res 57:5475-5479. Konig J, Cui Y, Nies AT and Keppler D (2000a) Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275:23161-23168. Konig J, Cui Y, Nies AT and Keppler D (2000b) A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 278:G156-164. Konig J, Rost D, Cui Y and Keppler D (1999) Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29:1156-1163. Kopplow K, Letschert K, Konig J, Walter B and Keppler D (2005) Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells. Mol Pharmacol 68:1031-1038. Kostrubsky VE, Vore M, Kindt E, Burliegh J, Rogers K, Peter G, Altrogge D and Sinz MW (2001) The effect of troglitazone biliary excretion on metabolite 37 distribution and cholestasis in transporter-deficient rats. Drug Metab Dispos 29:1561-1566. Kruijtzer CM, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC, Paul EM and Schellens JH (2002) Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 20:2943-2950. Kullak-Ublick GA, Ismair MG, Stieger B, Landmann L, Huber R, Pizzagalli F, Fattinger K, Meier PJ and Hagenbuch B (2001) Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology 120:525-533. Kullak-Ublick GA, Stieger B, Hagenbuch B and Meier PJ (2000) Hepatic transport of bile salts. Semin Liver Dis 20:273-292. Kullak-Ublick GA, Stieger B and Meier PJ (2004) Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126:322-342. Kuroda M, Kobayashi Y, Tanaka Y, Itani T, Mifuji R, Araki J, Kaito M and Adachi Y (2004) Increased hepatic and renal expressions of multidrug resistanceassociated protein 3 in Eisai hyperbilirubinuria rats. J Gastroenterol Hepatol 19:146-153. Laffi G, La Villa G, Pinzani M, Ciabattoni G, Patrignani P, Mannelli M, Cominelli F and Gentilini P (1986) Altered renal and platelet arachidonic acid metabolism in cirrhosis. Gastroenterology 90:274-282. Lau YY, Okochi H, Huang Y and Benet LZ (2006) Multiple Transporters Affect the Disposition of Atorvastatin and Its Two Active Hydroxy Metabolites: Application of in Vitro and ex Situ Systems. J Pharmacol Exp Ther 316:762771. Leier I, Jedlitschky G, Buchholz U, Cole SP, Deeley RG and Keppler D (1994) The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 269:27807-27810. Leslie EM, Bowers RJ, Deeley RG and Cole SP (2003) Structural requirements for functional interaction of glutathione tripeptide analogs with the human multidrug resistance protein 1 (MRP1). J Pharmacol Exp Ther 304:643-653. Letschert K, Faulstich H, Keller D and Keppler D (2006) Molecular characterization and inhibition of amanitin uptake into human hepatocytes. Toxicol Sci 91:140149. Li L, Lee TK, Meier PJ and Ballatori N (1998) Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273:16184-16191. 38 Li L, Meier PJ and Ballatori N (2000) Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol Pharmacol 58:335-340. Liu L, Cui Y, Chung AY, Shitara Y, Sugiyama Y, Keppler D and Pang KS (2006) Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers. J Pharmacol Exp Ther 318:395-402. Liu X, Brouwer KL, Gan LS, Brouwer KR, Stieger B, Meier PJ, Audus KL and LeCluyse EL (1998) Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration. Pharm Res 15:1533-1539. Liu X, LeCluyse EL, Brouwer KR, Gan LS, Lemasters JJ, Stieger B, Meier PJ and Brouwer KL (1999) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 277:G12-21. Lu WJ, Tamai I, Nezu J, Lai ML and Huang JD (2006) Organic anion transporting polypeptide-C mediates arsenic uptake in HEK-293 cells. J Biomed Sci 13:525-533. Lum BL, Fisher GA, Brophy NA, Yahanda AM, Adler KM, Kaubisch S, Halsey J and Sikic BI (1993) Clinical trials of modulation of multidrug resistance. Pharmacokinetic and pharmacodynamic considerations. Cancer 72:35023514. Madon J, Hagenbuch B, Landmann L, Meier PJ and Stieger B (2000) Transport function and hepatocellular localization of mrp6 in rat liver. Mol Pharmacol 57:634-641. Maeda T, Takahashi K, Ohtsu N, Oguma T, Ohnishi T, Atsumi R and Tamai I (2007) Identification of influx transporter for the quinolone antibacterial agent levofloxacin. Mol Pharm 4:85-94. Mahagita C, Grassl SM, Piyachaturawat P and Ballatori N (2007) Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport. Am J Physiol Gastrointest Liver Physiol 293:G271-278. Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg AC, Schinkel AH, van De Vijver MJ, Scheper RJ and Schellens JH (2001) Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 61:3458-3464. Marie JP, Helou C, Thevenin D, Delmer A and Zittoun R (1992) In vitro effect of Pglycoprotein (P-gp) modulators on drug sensitivity of leukemic progenitors (CFU-L) in acute myelogenous leukemia (AML). Exp Hematol 20:565-568. 39 Martin PD, Kemp J, Dane AL, Warwick MJ and Schneck DW (2002) No effect of rosuvastatin on the pharmacokinetics of digoxin in healthy volunteers. J Clin Pharmacol 42:1352-1357. Matsushima S, Maeda K, Hayashi H, Debori Y, Schinkel AH, Schuetz JD, Kusuhara H and Sugiyama Y (2008) Involvement of multiple efflux transporters in hepatic disposition of fexofenadine. Mol Pharmacol 73:1474-1483. Matsushima S, Maeda K, Kondo C, Hirano M, Sasaki M, Suzuki H and Sugiyama Y (2005) Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistanceassociated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein. J Pharmacol Exp Ther 314:1059-1067. Meier PJ, Eckhardt U, Schroeder A, Hagenbuch B and Stieger B (1997) Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver. Hepatology 26:1667-1677. Meier PJ and Stieger B (2002) Bile salt transporters. Annu Rev Physiol 64:635-661. Merino G, Alvarez AI, Pulido MM, Molina AJ, Schinkel AH and Prieto JG (2006) Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion. Drug Metab Dispos 34:690-695. Merino G, Jonker JW, Wagenaar E, Pulido MM, Molina AJ, Alvarez AI and Schinkel AH (2005a) Transport of anthelmintic benzimidazole drugs by breast cancer resistance protein (BCRP/ABCG2). Drug Metab Dispos 33:614-618. Merino G, van Herwaarden AE, Wagenaar E, Jonker JW and Schinkel AH (2005b) Sex-dependent expression and activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP/ABCG2) in liver. Mol Pharmacol 67:1765-1771. Mita S, Suzuki H, Akita H, Hayashi H, Onuki R, Hofmann AF and Sugiyama Y (2006) Inhibition of bile acid transport across Na+/taurocholate cotransporting polypeptide (SLC10A1) and bile salt export pump (ABCB 11)-coexpressing LLC-PK1 cells by cholestasis-inducing drugs. Drug Metab Dispos 34:15751581. Moffit JS, Aleksunes LM, Maher JM, Scheffer GL, Klaassen CD and Manautou JE (2006) Induction of hepatic transporters multidrug resistance-associated proteins (Mrp) 3 and 4 by clofibrate is regulated by peroxisome proliferatoractivated receptor alpha. J Pharmacol Exp Ther 317:537-545. 40 Moseley RH, Johnson TR and Morrissette JM (1990) Inhibition of bile acid transport by cyclosporine A in rat liver plasma membrane vesicles. J Pharmacol Exp Ther 253:974-980. Muller J, Lips KS, Metzner L, Neubert RH, Koepsell H and Brandsch M (2005) Drug specificity and intestinal membrane localization of human organic cation transporters (OCT). Biochem Pharmacol 70:1851-1860. Munteanu E, Verdier M, Grandjean-Forestier F, Stenger C, Jayat-Vignoles C, Huet S, Robert J and Ratinaud MH (2006) Mitochondrial localization and activity of Pglycoprotein in doxorubicin-resistant K562 cells. Biochem Pharmacol 71:1162-1174. Nakagomi-Hagihara R, Nakai D, Kawai K, Yoshigae Y, Tokui T, Abe T and Ikeda T (2006) OATP1B1, OATP1B3, and mrp2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin II blocker. Drug Metab Dispos 34:862-869. Nakanishi T, Shiozawa K, Hassel BA and Ross DD (2006) Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression. Blood 108:678-684. Nozawa T, Imai K, Nezu J, Tsuji A and Tamai I (2004a) Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human. J Pharmacol Exp Ther 308:438-445. Nozawa T, Sugiura S, Nakajima M, Goto A, Yokoi T, Nezu J, Tsuji A and Tamai I (2004b) Involvement of organic anion transporting polypeptides in the transport of troglitazone sulfate: implications for understanding troglitazone hepatotoxicity. Drug Metab Dispos 32:291-294. Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H and Moriyama Y (2005a) A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci U S A 102:17923-17928. Otsuka M, Yasuda M, Morita Y, Otsuka C, Tsuchiya T, Omote H and Moriyama Y (2005b) Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus. J Bacteriol 187:15521558. Ozvegy C, Litman T, Szakacs G, Nagy Z, Bates S, Varadi A and Sarkadi B (2001) Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochem Biophys Res Commun 285:111-117. Pasanen MK, Fredrikson H, Neuvonen PJ and Niemi M (2007) Different effects of SLCO1B1 polymorphism on the pharmacokinetics of atorvastatin and rosuvastatin. Clin Pharmacol Ther 82:726-733. 41 Pauli-Magnus C, von Richter O, Burk O, Ziegler A, Mettang T, Eichelbaum M and Fromm MF (2000) Characterization of the major metabolites of verapamil as substrates and inhibitors of P-glycoprotein. J Pharmacol Exp Ther 293:376382. Payen L, Courtois A, Campion JP, Guillouzo A and Fardel O (2000) Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes. Biochem Pharmacol 60:1967-1975. Perdu J and Germain DP (2001) Identification of novel polymorphisms in the pM5 and MRP1 (ABCC1) genes at locus 16p13.1 and exclusion of both genes as responsible for pseudoxanthoma elasticum. Hum Mutat 17:74-75. Polli JW, Jarrett JL, Studenberg SD, Humphreys JE, Dennis SW, Brouwer KR and Woolley JL (1999) Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharm Res 16:1206-1212. Reid G, Wielinga P, Zelcer N, De Haas M, Van Deemter L, Wijnholds J, Balzarini J and Borst P (2003) Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 63:1094-1103. Rizzo G, Renga B, Mencarelli A, Pellicciari R and Fiorucci S (2005) Role of FXR in regulating bile acid homeostasis and relevance for human diseases. Curr Drug Targets Immune Endocr Metabol Disord 5:289-303. Robey RW, Honjo Y, Morisaki K, Nadjem TA, Runge S, Risbood M, Poruchynsky MS and Bates SE (2003) Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity. Br J Cancer 89:1971-1978. Roelofsen H, Soroka CJ, Keppler D and Boyer JL (1998) Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets. J Cell Sci 111 ( Pt 8):1137-1145. Sandhu P, Lee W, Xu X, Leake BF, Yamazaki M, Stone JA, Lin JH, Pearson PG and Kim RB (2005) Hepatic uptake of the novel antifungal agent caspofungin. Drug Metab Dispos 33:676-682. Sano N, Takikawa H and Yamanaka M (1993) Estradiol-17 beta-glucuronideinduced cholestasis. Effects of ursodeoxycholate-3-O-glucuronide and 3,7disulfate. J Hepatol 17:241-246. Sasaki M, Suzuki H, Ito K, Abe T and Sugiyama Y (2002) Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2). J Biol Chem 277:6497-6503. 42 Satoh H, Yamashita F, Tsujimoto M, Murakami H, Koyabu N, Ohtani H and Sawada Y (2005) Citrus juices inhibit the function of human organic anion-transporting polypeptide OATP-B. Drug Metab Dispos 33:518-523. Schoffski P, Dumez H, Wolter P, Stefan C, Wozniak A, Jimeno J and Van Oosterom AT (2008) Clinical impact of trabectedin (ecteinascidin-743) in advanced/metastatic soft tissue sarcoma. Expert Opin Pharmacother 9:16091618. Schuetz EG, Furuya KN and Schuetz JD (1995) Interindividual variation in expression of P-glycoprotein in normal human liver and secondary hepatic neoplasms. J Pharmacol Exp Ther 275:1011-1018. Schuetz EG, Umbenhauer DR, Yasuda K, Brimer C, Nguyen L, Relling MV, Schuetz JD and Schinkel AH (2000) Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes. Mol Pharmacol 57:188-197. Schuetz JD, Connelly MC, Sun D, Paibir SG, Flynn PM, Srinivas RV, Kumar A and Fridland A (1999) MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nat Med 5:1048-1051. Shimizu M, Uno T, Sugawara K and Tateishi T (2006) Effects of itraconazole and diltiazem on the pharmacokinetics of fexofenadine, a substrate of Pglycoprotein. Br J Clin Pharmacol 61:538-544. Shitara Y and Sugiyama Y (2006) Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions. Pharmacol Ther 112:71-105. Silva VM, Thibodeau MS, Chen C and Manautou JE (2005) Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity. Biochem Pharmacol 70:1832-1839. Simonson SG, Raza A, Martin PD, Mitchell PD, Jarcho JA, Brown CD, Windass AS and Schneck DW (2004) Rosuvastatin pharmacokinetics in heart transplant recipients administered an antirejection regimen including cyclosporine. Clin Pharmacol Ther 76:167-177. Smith MT (2003) Mechanisms of troglitazone hepatotoxicity. Chem Res Toxicol 16:679-687. Smith NF, Acharya MR, Desai N, Figg WD and Sparreboom A (2005) Identification of OATP1B3 as a high-affinity hepatocellular transporter of paclitaxel. Cancer Biol Ther 4:815-818. 43 Soldner A, Christians U, Susanto M, Wacher VJ, Silverman JA and Benet LZ (1999) Grapefruit juice activates P-glycoprotein-mediated drug transport. Pharm Res 16:478-485. Spahn-Langguth H, Baktir G, Radschuweit A, Okyar A, Terhaag B, Ader P, Hanafy A and Langguth P (1998) P-glycoprotein transporters and the gastrointestinal tract: evaluation of the potential in vivo relevance of in vitro data employing talinolol as model compound. Int J Clin Pharmacol Ther 36:16-24. Spivey JR, Bronk SF and Gores GJ (1993) Glycochenodeoxycholate-induced lethal hepatocellular injury in rat hepatocytes. Role of ATP depletion and cytosolic free calcium. J Clin Invest 92:17-24. Stieger B, Fattinger K, Madon J, Kullak-Ublick GA and Meier PJ (2000) Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology 118:422-430. Su Y, Zhang X and Sinko PJ (2004) Human organic anion-transporting polypeptide OATP-A (SLC21A3) acts in concert with P-glycoprotein and multidrug resistance protein 2 in the vectorial transport of Saquinavir in Hep G2 cells. Mol Pharm 1:49-56. Sun W, Wu RR, van Poelje PD and Erion MD (2001) Isolation of a family of organic anion transporters from human liver and kidney. Biochem Biophys Res Commun 283:417-422. Tahara H, Kusuhara H, Endou H, Koepsell H, Imaoka T, Fuse E and Sugiyama Y (2005) A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters. J Pharmacol Exp Ther 315:337-345. Takeda M, Khamdang S, Narikawa S, Kimura H, Kobayashi Y, Yamamoto T, Cha SH, Sekine T and Endou H (2002) Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther 300:918-924. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M and Tsuji A (2000) Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273:251260. Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O and Inui K (2007) Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H(+)-organic cation antiporters. Biochem Pharmacol 74:359-371. Tarazi EM, Harter JG, Zimmerman HJ, Ishak KG and Eaton RA (1993) Sulindacassociated hepatic injury: analysis of 91 cases reported to the Food and Drug Administration. Gastroenterology 104:569-574. 44 Terada T and Inui K (2008) Physiological and pharmacokinetic roles of H+/organic cation antiporters (MATE/SLC47A). Biochem Pharmacol 75:1689-1696. Thomas J, Wang L, Clark RE and Pirmohamed M (2004) Active transport of imatinib into and out of cells: implications for drug resistance. Blood 104:3739-3745. Trauner M and Boyer JL (2003) Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 83:633-671. Treiber A, Schneiter R, Hausler S and Stieger B (2007) Bosentan is a substrate of human OATP1B1 and OATP1B3: inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil. Drug Metab Dispos 35:1400-1407. Tsujii H, Konig J, Rost D, Stockel B, Leuschner U and Keppler D (1999) Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin-Johnson syndrome. Gastroenterology 117:653-660. Turncliff RZ, Meier PJ and Brouwer KL (2004) Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes. Drug Metab Dispos 32:834-839. Ueda K, Okamura N, Hirai M, Tanigawara Y, Saeki T, Kioka N, Komano T and Hori R (1992) Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem 267:24248-24252. Urquhart BL, Tirona RG and Kim RB (2007) Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs. J Clin Pharmacol 47:566-578. van der Heijden J, de Jong MC, Dijkmans BA, Lems WF, Oerlemans R, Kathmann I, Scheffer GL, Scheper RJ, Assaraf YG and Jansen G (2004) Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs. Ann Rheum Dis 63:131137. van der Sandt IC, Blom-Roosemalen MC, de Boer AG and Breimer DD (2000) Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines. Eur J Pharm Sci 11:207-214. van Montfoort JE, Muller M, Groothuis GM, Meijer DK, Koepsell H and Meier PJ (2001) Comparison of "type I" and "type II" organic cation transport by organic cation transporters and organic anion-transporting polypeptides. J Pharmacol Exp Ther 298:110-115. 45 Vavricka SR, Van Montfoort J, Ha HR, Meier PJ and Fattinger K (2002) Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver. Hepatology 36:164-172. Volk EL, Farley KM, Wu Y, Li F, Robey RW and Schneider E (2002) Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance. Cancer Res 62:5035-5040. Wagner M, Fickert P, Zollner G, Fuchsbichler A, Silbert D, Tsybrovskyy O, Zatloukal K, Guo GL, Schuetz JD, Gonzalez FJ, Marschall HU, Denk H and Trauner M (2003) Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterology 125:825-838. Wakabayashi Y, Kipp H and Arias IM (2006) Transporters on demand: intracellular reservoirs and cycling of bile canalicular ABC transporters. J Biol Chem 281:27669-27673. Wang X, Furukawa T, Nitanda T, Okamoto M, Sugimoto Y, Akiyama S and Baba M (2003) Breast cancer resistance protein (BCRP/ABCG2) induces cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors. Mol Pharmacol 63:65-72. Wang X and Morris ME (2007) Effects of the flavonoid chrysin on nitrofurantoin pharmacokinetics in rats: potential involvement of ABCG2. Drug Metab Dispos 35:268-274. Wang X, Nitanda T, Shi M, Okamoto M, Furukawa T, Sugimoto Y, Akiyama S and Baba M (2004) Induction of cellular resistance to nucleoside reverse transcriptase inhibitors by the wild-type breast cancer resistance protein. Biochem Pharmacol 68:1363-1370. Wilson ZE, Rostami-Hodjegan A, Burn JL, Tooley A, Boyle J, Ellis SW and Tucker GT (2003) Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver. Br J Clin Pharmacol 56:433-440. Wolkoff AW, Goresky CA, Sellin J, Gatmaitan Z and Arias IM (1979) Role of ligandin in transfer of bilirubin from plasma into liver. Am J Physiol 236:E638-648. Wolters H, Spiering M, Gerding A, Slooff MJ, Kuipers F, Hardonk MJ and Vonk RJ (1991) Isolation and characterization of canalicular and basolateral plasma membrane fractions from human liver. Biochim Biophys Acta 1069:61-69. Xiong H, Turner KC, Ward ES, Jansen PL and Brouwer KL (2000) Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats. J Pharmacol Exp Ther 295:512-518. 46 Yamaguchi H, Yano I, Hashimoto Y and Inui KI (2000) Secretory mechanisms of grepafloxacin and levofloxacin in the human intestinal cell line caco-2. J Pharmacol Exp Ther 295:360-366. Yamashiro W, Maeda K, Hirouchi M, Adachi Y, Hu Z and Sugiyama Y (2006) Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans. Drug Metab Dispos 34:1247-1254. Yamazaki M, Neway WE, Ohe T, Chen I, Rowe JF, Hochman JH, Chiba M and Lin JH (2001) In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results. J Pharmacol Exp Ther 296:723-735. Yoshikawa M, Ikegami Y, Sano K, Yoshida H, Mitomo H, Sawada S and Ishikawa T (2004) Transport of SN-38 by the wild type of human ABC transporter ABCG2 and its inhibition by quercetin, a natural flavonoid. J Exp Ther Oncol 4:25-35. Zamek-Gliszczynski MJ, Hoffmaster KA, Humphreys JE, Tian X, Nezasa K and Brouwer KL (2006) Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat. J Pharmacol Exp Ther 319:459-467. Zamek-Gliszczynski MJ, Hoffmaster KA, Nezasa K and Brouwer KL (2008) Apparent differences in mechanisms of harmol sulfate biliary excretion in mice and rats. Drug Metab Dispos 36:2156-2158. Zamek-Gliszczynski MJ, Xiong H, Patel NJ, Turncliff RZ, Pollack GM and Brouwer KL (2003) Pharmacokinetics of 5 (and 6)-carboxy-2',7'-dichlorofluorescein and its diacetate promoiety in the liver. J Pharmacol Exp Ther 304:801-809. Zelcer N, Reid G, Wielinga P, Kuil A, van der Heijden I, Schuetz JD and Borst P (2003) Steroid and bile acid conjugates are substrates of human multidrugresistance protein (MRP) 4 (ATP-binding cassette C4). Biochem J 371:361367. Zeng H, Chen ZS, Belinsky MG, Rea PA and Kruh GD (2001) Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. Cancer Res 61:72257232. Zhang Y, Zhou G, Wang H, Zhang X, Wei F, Cai Y and Yin D (2006) Transcriptional upregulation of breast cancer resistance protein by 17beta-estradiol in ERalpha-positive MCF-7 breast cancer cells. Oncology 71:446-455. Zhou S, Schuetz JD, Bunting KD, Colapietro A-M, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H and Sorrentino BP (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a 47 molecular determinant of the side-population phenotype. Nat Med 7:10281034. 48 CHAPTER 2 Sex-dependent Disposition of Acetaminophen Sulfate and Glucuronide in the In Situ Perfused Mouse Liver This work has been presented in part at the AAPS Workshop on Drug Transporters in ADME: From the Bench to the Bedside, North Bethesda, MD, March 5-7, 2007 and has been submitted to Drug Metabolism and Disposition. ABSTRACT Breast cancer resistance protein (BCRP; ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sexdependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP), and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg2(-/-) (Bcrp-deficient) mice. Pharmacokinetic modeling was utilized to estimate parameters governing the hepatobiliary disposition of APAP, AG and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males compared to females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female compared to male mouse livers. Interestingly, Mrp4 protein was higher in female compared to male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers. 50 INTRODUCTION The breast cancer resistance protein (BCRP) belongs to the ATP-binding cassette transporter G family and was first cloned in 1998 from mitoxantroneresistant cell lines (Doyle et al., 1998). BCRP is highly expressed in the canalicular membrane of the hepatocyte as well as in the intestinal epithelia, blood-brain barrier, and placenta (Robey et al., 2008). BCRP has wide substrate specificity, including organic anions, cations, and zwitterions. BCRP mediates the transport of sulfated hormone metabolites, SN-38, mitoxantrone, topotecan, doxorubicin, flavopiridol, nitrofurantoin, various flavonoids and the cholorophyll metabolite pheophorbide A (Robey et al., 2001; Jonker et al., 2002; Nakagawa et al., 2002; Bates et al., 2004; Imai et al., 2004; Jonker et al., 2005; Merino et al., 2005a). Male-predominant expression of Bcrp has been reported in mouse liver (Merino et al., 2005b; Tanaka et al., 2005). Bcrp is influenced by sex hormones; estradiol suppresses and testosterone induces Bcrp expression (Tanaka et al., 2005). Sex-dependent differences in Bcrp expression have been shown to influence the pharmacokinetics of drugs (Merino et al., 2005b). For example, the area under the plasma concentration-time curve of nitrofurantoin in female wild-type mice was ~2-fold higher than in male mice after oral administration; the cumulative biliary excretion of nitrofurantoin was 9-fold higher in male compared to female mice. In contrast, no significant sex differences in plasma concentrations or the hepatobiliary excretion of nitrofurantoin were observed in Bcrp-deficient mice, indicating that hepatic Bcrp protein contributes to sex differences in wild-type mice (Merino et al., 2005b). 51 The role of various transport proteins in the biliary excretion of phase II metabolites has been examined using several compounds including acetaminophen, 4-methylumbelliferone and harmol, which are metabolized primarily to glucuronide and sulfate conjugates in mice (Zamek-Gliszczynski et al., 2006c). In situ singlepass liver perfusion in male mice demonstrated that Bcrp plays a predominant role in the biliary excretion of sulfate conjugates, whereas the biliary excretion of glucuronide conjugates was mediated by both Bcrp and, to a lesser extent, Mrp2. Glucuronide and sulfate conjugates of acetaminophen (AG and AS, respectively) are transported from the hepatocytes into sinusoidal blood by basolateral transport proteins; Mrp3 mediates basolateral excretion of AG whereas Mrp3, Mrp4, and other transport proteins may be involved in the basolateral excretion of AS in mice (Xiong et al., 2002; Manautou et al., 2005; Zamek-Gliszczynski et al., 2006b). In the liver, mRNA levels of Mrp4 were higher in female than in male C57BL/6 mice, but Mrp2 and Mrp3 mRNA levels did not differ based upon gender (Maher et al., 2005). Sexdependent differences in the disposition of some phase II conjugates might be anticipated, considering the important role of transport proteins in the biliary and basolateral excretion of phase II conjugates. In addition, these sex-dependent differences in hepatobiliary disposition may be of potential clinical relevance, as some sulfate conjugates are important from a physiologic [e.g., dehydroepiandrosterone sulfate (DHEAS) and estrone-sulfate] or pharmacologic (e.g., minoxidil sulfate) standpoint. The purpose of this study was to examine the influence of Bcrp and sex on the hepatobiliary disposition of phase II metabolites, using APAP as a model 52 substrate. In situ single-pass liver perfusion in male and female wild-type and Bcrpdeficient mice was utilized to examine the hepatobiliary disposition of AG and AS. Compartmental pharmacokinetic modeling was performed to recover relevant parameters governing hepatobiliary disposition of AG and AS. In light of the physiologic and pharmacologic roles that some conjugates play in vivo, it is important to investigate the potential for significant sex-dependent differences in hepatobiliary disposition of sulfate and glucuronide metabolites. 53 METHODS Materials. APAP, AG, AS, cimetidine, taurocholate and Krebs-Henseleit buffer packets were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of analytical grade and were available commercially. Mice. Male and female C57BL/6 (wild-type) and Abcg2(-/-) (Bcrp-deficient) (created by Deltagen Inc., San Mateo, CA) mice (23–30 g) were a gift from Eli Lilly and Company. Further information regarding background and breeding of these mice has been detailed previously (Nezasa et al., 2006). All animal procedures were approved by The Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. In Situ Single-pass Liver Perfusion. The abdominal cavity of each mouse was opened to expose the intestines, liver, and gallbladder. The common bile duct above the duodenum was ligated and the gallbladder was cannulated with PE-10 tubing (BD Biosciences, Franklin Lakes, NJ). A loose suture was placed around the inferior vena cava below the liver. The portal vein was cannulated with a 20-gauge catheter (B. Braun Medical, Inc., Bethlehem, PA) and the liver was perfused with oxygenated drug-free Krebs-Henseleit buffer containing 5 µM taurocholate at a flow rate of 5 ml/min. The inferior vena cava below the liver was severed immediately by an incision below the loose suture. Thereafter, the inferior vena cava above the liver was cannulated with a 20-gauge catheter. The inferior vena cava below the liver was ligated to direct all perfusate outflow to the inferior vena cava above the liver. After a 15-min pre-perfusion period to equilibrate liver temperature and stabilize bile flow, the liver was perfused with buffer containing 500 nM APAP for 40 min. Liver 54 perfusion was continued for an additional 30 min after APAP infusion with drug-free buffer. Perfusate outflow was collected at designated time points throughout the 70min experiment. Bile was collected in toto at 10-min intervals. At the end of the perfusion, livers were isolated and snap frozen. Analytical Methods. Perfusate, bile and liver homogenate samples were diluted 1:5, 1:250 and 1:10, respectively, to obtain a final sample aliquot of 500 µl. Samples were protein-precipitated by the addition of 390 µl ice-cold methanol containing the internal standard cimetidine. After the removal of protein by centrifugation at 3,000g for 15 min at 4ºC, the supernatants were analyzed by liquid chromatography with detection by tandem mass spectrometry (Applied Biosystems API 4000 triple quadrupole with TurboIonSpray interface; Foster City, CA). AS, AG and the internal standard (cimetidine) were eluted from an Aquasil C18 column (2.1 x 50 mm, dp = 5 µm; Thermo Electron Corporation, San Jose, CA) using a mobile phase gradient (A, 0.1% formic acid in water; B, 0.1% formic acid in methanol; 0–0.75 min hold at 5% B; 0.75–2.5 min linear gradient to 70% B; 2.5–3.5 min hold at 70% B, 3.5–3.6 min linear gradient to 5% B, 3.6–4 min hold at 5% B; flow rate, 0.75 ml/min; 0.8–4 min directed to mass spectrometer) and were detected in negative ion mode using multiple reaction monitoring: AS, 230 150 m/z; AG, 326 150 m/z; cimetidine, 251 157 m/z. Concentrations of APAP and the internal standard (cimetidine) were quantified using the chromatographic conditions detailed above, but were detected in positive ion mode using multiple reaction monitoring: acetaminophen, 152 cimetidine, 253 110 m/z; 117 m/z. Care was taken to confirm that no in-source fragmentation of the conjugates back to APAP occurred. Standard curves of each 55 analyte (0.5-1000 nM) were prepared in the appropriate matrices; inter- and intraday CVs were <15%. Western Blot Analysis. Mouse livers, perfused with phosphate-buffered saline solution, were removed promptly and frozen. Livers were homogenized with 3 volumes of Tris-HCl solution (pH 7.4) containing complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The homogenates were centrifuged at 1500×g for 10 min, and the supernatant was ultracentrifuged at 100,000×g for 30 min. The resulting pellet was suspended in Tris-HCl solution (pH 7.4) containing complete protease inhibitor cocktail, 1% sodium dodecyl sulfate, and 1 mM ethylene diamine tetraacetic acid. The protein concentration of the solution was determined with the BCA protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL). Bcrp expression was determined using the anti-BCRP primary monoclonal antibody (BXP-53; Alexis Biochemicals, Carlsbad, CA) at a 1:1000 dilution. Anti-MRP4 (M4I10; Alexis Biochemicals) was used at a 1:1000 dilution to determine the expression of Mrp4. Protein loading was normalized to β-actin, which was detected using a βactin antibody at a dilution of 1:5000 (C4; Chemicon, San Fransisco, CA). Immunoreactive protein bands were detected by chemilumiscence using a Bio-Rad VersaDoc imaging system and densitometry analysis was achieved using the Quantity One software package v.4.1 (Bio-Rad Laboratories, Hercules, CA). Pharmacokinetic Modeling. A compartmental modeling approach was utilized to describe the hepatobiliary disposition of APAP and metabolites in perfused livers from male and female wild-type and Bcrp-deficient mice, and to recover estimates of key pharmacokinetic parameters. AG and AS outflow perfusate and biliary excretion 56 rates were calculated as the product of flow (perfusate or bile) and outflow perfusate or biliary concentrations, respectively, during each collection period, and then were normalized to liver weight. Several structurally-distinct models were evaluated; standard model selection criteria including residual distribution and Akaike’s information criterion were used to identify the optimal structure. The compartmental model that best described the data is shown in Figure 2.1. Differential equations derived from the model scheme in Figure 2.1 were as follows: dX APAP = CLup × Cin − X APAP × (K perf,APAP + K gluc + K sulf ); X APAP 0 = 0; t ≤ 40min dt dX APAP = − X APAP × (K perf,APAP + K gluc + K sulf ); t > 40min dt dX AG −1 = X APAP × K gluc − X AG−1 × (K perf,AG + K g1); X AG −10 = 0 dt dX AG − 2 = X AG −1 × K g1 − X AG − 2 × K bile,AG ; X AG − 20 = 0 dt dX AS −1 = X APAP × K sulf − X AS −1 × (K perf,AS + K s1); X AS −10 = 0 dt dX AS − 2 = X AS −1 × K s1 − X AS −2 × K bile, AS ; X AS − 20 = 0 dt dXperfusate, APAP = Q × Cin − CLup × Cin + X APAP × K perf,APAP ; t ≤ 40min dt dXperfusate, APAP = X APAP × K perf,APAP ; t > 40min dt dXperfusate, AG = X AG −1 × K perf,AG dt dXperfusate, AS = X AS −1 × K perf,AS dt dXbile,AG = X AG− 2 × K bile,AG dt dXbile,AS = X AS − 2 × K bile,AS dt The final model included compartments representing hepatocellular spaces for APAP, AG and AS. To describe the prolonged biliary excretion of AG and AS after 57 APAP washout relative to basolateral excretion of AG and AS, an intervening compartment between the site of conjugate formation and the bile compartment was included in the model. This intervening compartment was required in order to describe disposition of AG in all livers, and of AS in wild-type but not in Bcrpdeficient mouse livers (AS biliary concentrations measured in Bcrp-deficient mouse livers were below the limit of quantitation of the assay). Differential equations describing substrate flux in each compartment were fit simultaneously to the perfusate and biliary excretion rate data by nonlinear least-squares regression (WinNonlin Pro version 4.2; Pharsight, Mountain View, CA) with a weighting scheme of 1/(Y predicted). The precision of parameter estimates was maximized with stepwise nonlinear least-squares regression. AG perfusate and biliary excretion rate data from male and female wild-type and Bcrp-deficient mouse livers were used to estimate Kperf, AG, Kg1 and Kbile,AG, while Clup, Kperf, APAP, Kgluc and Ksulf were held constant at the estimated values. Likewise, AS perfusate and biliary excretion rate data were used to estimate Kperf,AS, Ks1 and Kbile,AS while Clup, Kperf, APAP, Kgluc and Ksulf were held constant at the estimated values. Data Analysis. All data are expressed as mean ± SD unless otherwise indicated. The effects of Bcrp expression (wild-type or Bcrp-deficient) and sex (male or female) were assessed using a two-way analysis of variance. Data shown in Table 2.1, Figure 2.3 and 2.4 were analyzed further with Bonferroni’s post-hoc test, but no further analysis was performed for pharmacokinetic parameter estimates shown in Table 2.2, as an interaction between Bcrp expression and sex was observed for one of the estimates in the two-way analysis of variance. An unpaired Student’s t-test 58 was used to determine the statistical significance of differences in AS biliary excretion from male and female mouse livers because AS biliary excretion was negligible in Bcrp-deficient mice. Differences were considered to be statistically significant when p < 05. 59 RESULTS Statistically significant differences were observed in body weights among male and female wild-type mice, but mouse liver weights normalized for body weight were comparable between wild-type and Bcrp-deficient male and female mice (Table 2.1). Outflow perfusate concentrations of APAP reached a plateau within 20 min of continuous perfusion (Figure 2.2). The hepatic extraction ratios of APAP, calculated from inflow and outflow APAP perfusate concentrations between 30 and 40 min of perfusion, ranged from 24.8 to 35.4% among mouse groups (Table 2.1). Since APAP, AG and AS concentrations in liver homogenates after the 30-min washout phase were negligible, total recovery (% of dose) was calculated by adding recovered APAP, AG and AS in outflow perfusate and bile; in all cases, total recovery was comparable among all mouse groups (Table 2.1). Biliary excretion of AG in livers from male wild-type mice was ~2.5-fold higher than in female wild-type mouse livers, but AG biliary excretion between male and female Bcrp-deficient mice was comparable (Figure 2.3A). Relative to the corresponding wild-type mouse livers, biliary excretion of AG was decreased ~73% and ~54% in male and female Bcrp-deficient mouse livers, respectively. Cumulative AG excretion in outflow perfusate from male wild-type and Bcrp-deficient livers was ~1.6-fold higher compared to female wild-type and Bcrp-deficient livers (Figure 2.3B). Biliary excretion of AS in livers from male wild-type mice was ~2.2-fold higher than in female wild-type livers (Figure 2.4A). In Bcrp-deficient mice, AS biliary recovery was negligible. AS basolateral excretion in female wild-type and Bcrp- 60 deficient mouse livers was ~3.1-fold and ~2.4-fold higher, respectively, than in livers from their male counterparts (Figure 2.4B). Immunoblot analysis was performed with crude membrane fractions of liver homogenates from male and female wild-type and Bcrp-deficient mice to compare the levels of Bcrp and Mrp4 protein (Figure 2.5). As previously reported (Tanaka et al., 2005), densitometry analysis revealed that hepatic Bcrp protein, normalized to the β-actin signal, was 3-fold higher in male wild-type mice than in female wild-type mice. Interestingly, there was a trend toward increased hepatic Mrp4 protein in females; Mrp4 protein, normalized to β-actin signal, in female wild-type and Bcrpdeficient mouse livers was 3.5- and 1.6-fold higher than male mouse livers from wildtype and Bcrp-deficient mice, respectively. The model scheme presented in Figure 2.1 best described the current experimental data. Representative fits of the model to APAP, AG and AS biliary and basolateral excretion rate vs. time data are shown in Figure 2.6. Pharmacokinetic parameter estimates obtained by resolving the differential equations representing biliary and basolateral excretion rate vs. time data for APAP, AG and AS are presented in Table 2.2. The uptake clearance (CLup) was comparable to the perfusate flow rate (Q; 5 ml/min). The rate constant for APAP basolateral excretion (Kperf, APAP) was comparable among male and female wild-type and Bcrp-deficient mouse livers. Sex-dependent differences in metabolite formation rate constants were observed; the rate constant for AG formation (Kgluc) was ~2-fold higher in male mouse livers, and the rate constant for AS formation (Ksulf) was ~2-4-fold higher in female mouse livers. The basolateral excretion rate constant of AS (Kperf,AS), but not 61 AG (Kperf,AG), differed based on sex; the AS basolateral excretion rate constant was ~2-fold higher in female vs. male mouse livers. The AG biliary excretion rate constant (Kbile,AG) was higher in male than in female wild-type mouse livers (0.014 min-1 vs. 0.002 min-1), but was similar between male and female Bcrp-deficient mouse livers. The rate constant for movement of AS from hepatic compartment 1 to 2 (Ks1) was higher in male compared to female wild-type mouse livers. Likewise, the rate constant for AS biliary excretion (Kbile,AS) in male vs. female wild-type mouse livers was higher (0.107 min-1 vs. 0.043 min-1). 62 DISCUSSION The current studies support the hypothesis that sex-dependent hepatic Bcrp expression influences the hepatobiliary disposition of sulfate and glucuronide conjugates of the model substrate APAP during in situ single-pass mouse liver perfusion. Male-predominant Bcrp protein expression in wild-type mouse livers is consistent with ~2.5-fold higher biliary excretion of AG and AS in male compared to female wild-type mouse livers. Sex-dependent differences in biliary excretion of AG were not observed in Bcrp-deficient mice. Pharmacokinetic modeling revealed that the rate constants governing AG and AS biliary excretion were significantly higher in male compared with female wild-type mouse livers. AS biliary excretion was negligible and AG biliary excretion rates were markedly reduced in Bcrp-deficient mice, consistent with recent findings demonstrating an important role of Bcrp in AG and AS biliary excretion in male mice (Zamek-Gliszczynski et al., 2006c). Both Bcrp and Mrp2 appear to be involved in AS biliary excretion in rats (Zamek-Gliszczynski et al., 2005), however, AS biliary excretion was not decreased in Mrp2-deficient mice (Zamek-Gliszczynski et al., 2006c). This apparent discrepancy in the mechanisms of AS biliary excretion may be due to species-specific differences in the affinity of multiple hepatic efflux proteins for AS. Interestingly, sex differences were observed for basolateral excretion of AG and AS, with more extensive basolateral excretion of AG in male and more extensive AS basolateral excretion in female mouse livers (Figure 2.3B, 2.4B). One explanation for these findings may be that the rate constant governing AG formation (Kgluc) was greater in male mouse livers, whereas the rate constant for AS formation 63 (Ksulf) was greater in female mouse livers, suggesting sex-dependent differences in formation of AG and AS. Although the primary isoforms of UDP- glucuronosyltransferases (Ugt) and sulfotransferases (Sult) responsible for AG and AS formation in mice remain to be elucidated, previously published data demonstrate male-predominant mRNA expression of Ugt1a5 and female- predominant mRNA expression of Sult1a1 and Sult2a1/2 in mouse livers (Alnouti and Klaassen, 2006; Buckley and Klaassen, 2007). In human liver microsomes, protein expression of UGT1A6, a major isoform responsible for AG formation, was 50% higher in males compared to females (Court et al., 2001). Sex-dependent basolateral excretion of AG and AS also may be due to differences in the expression of transport protein(s) responsible for AG and AS basolateral excretion. As previously reported, Mrp3 plays a significant role in basolateral excretion of AG, whereas both Mrp3 and Mrp4, as well as other unidentified mechanisms, may be involved in AS basolateral excretion (Xiong et al., 2002; Manautou et al., 2005; Zamek-Gliszczynski et al., 2006b). Previous studies showed moderate expression of Mrp3 mRNA in mouse livers, but no differences in Mrp3 mRNA were noted between male and female mouse livers (Maher et al., 2005). In the current study, pharmacokinetic modeling revealed that the rate constants for AG basolateral excretion between male and female mouse livers were not different. Thus, greater AG basolateral excretion in male vs. female mouse livers appears to be due primarily to more extensive glucuronidation in male mouse livers. In contrast, the rate constant governing AS basolateral excretion (Kperf,AS) was significantly higher in female vs. male mouse livers; this finding cannot be explained by 64 increased AS formation in female mice. Interestingly, Western blot analysis demonstrated greater Mrp4 protein in crude membrane fractions from female compared to male mouse livers, consistent with previous findings that hepatic Mrp4 mRNA was increased in female compared to male wild-type mice (Maher et al., 2005). This sex-dependent increase in Mrp4 protein levels, coupled with more extensive sulfation in female mouse livers, may explain, in part, the higher AS basolateral excretion in female mouse livers since Mrp4 has been implicated in the basolateral excretion of AS (Lickteig et al., 2007). The pharmacokinetic model included an intervening compartment between the sites of conjugate formation and excretion into bile. This additional compartment was required to describe the prolonged biliary excretion of AG and AS during the washout phase (Figure 2.1). Movement of AG and AS from the site of conjugate formation to the site of biliary excretion was best described as a unidirectional rather than bidirectional process, based on markedly lower coefficients of variation (CV%) associated with this model fit. Similarly, pharmacokinetic modeling of the in vivo disposition of APAP and AG in rats has suggested the existence of a discrete cytosolic compartment prior to AG biliary excretion (Studenberg and Brouwer, 1993). In a study of hepatic intracellular distribution of several compounds, the presence of a cytosolic link between metabolite formation and subsequent biliary excretion also was proposed (Levine and Singer, 1972). Arias and coworkers demonstrated that newly synthesized canalicular transport proteins (e.g., P-gp) may be directly trafficked from the Golgi apparatus to the apical membrane, or may be stored transiently in intracellular compartments before apical delivery to the membrane (e.g., 65 Bsep)(Kipp et al., 2001); taurocholate or dibutyryl cAMP can stimulate trafficking of Bsep and P-gp. Immunohistochemical studies clearly revealed BCRP subcellular localization in folate-deprived MCF cells. In contrast, intense BCRP localization was noted at cell-to-cell contacts in parental MCF cells cultured in medium containing high concentrations of folic acid (Ifergan et al., 2005). Although subcellular localization of BCRP in hepatocytes remains to be elucidated, substrate binding or association with subcellularly-localized Bcrp protein prior to substrate biliary excretion may explain the observation of unidirectional flux of AG and AS (Kg1 and Ks1) suggested by the current pharmacokinetic model. In conclusion, the present study demonstrated sex-dependent hepatobiliary disposition of glucuronide and sulfate conjugates of APAP in mice due to differences in hepatic transport protein expression and conjugation. In light of the extensive use of the mouse as a model species to examine hepatobiliary drug disposition and drug-induced liver injury, such sex-dependent differences in transport proteins and phase II conjugation may explain different outcomes between male and female mice during preclinical drug development. 66 REFERENCES Alnouti Y and Klaassen CD (2006) Tissue distribution and ontogeny of sulfotransferase enzymes in mice. Toxicol Sci 93:242-255. Bates SE, Medina-Perez WY, Kohlhagen G, Antony S, Nadjem T, Robey RW and Pommier Y (2004) ABCG2 mediates differential resistance to SN-38 (7-ethyl10-hydroxycamptothecin) and homocamptothecins. J Pharmacol Exp Ther 310:836-842. Buckley DB and Klaassen CD (2007) Tissue- and gender-specific mRNA expression of UDP-glucuronosyltransferases (UGTs) in mice. Drug Metab Dispos 35:121127. Court MH, Duan SX, von Moltke LL, Greenblatt DJ, Patten CJ, Miners JO and Mackenzie PI (2001) Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms. J Pharmacol Exp Ther 299:998-1006. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK and Ross DD (1998) A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 95:15665-15670. Ifergan I, Jansen G and Assaraf YG (2005) Cytoplasmic confinement of breast cancer resistance protein (BCRP/ABCG2) as a novel mechanism of adaptation to short-term folate deprivation. Mol Pharmacol 67:1349-1359. Imai Y, Tsukahara S, Asada S and Sugimoto Y (2004) Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer Res 64:4346-4352. Jonker JW, Buitelaar M, Wagenaar E, Van Der Valk MA, Scheffer GL, Scheper RJ, Plosch T, Kuipers F, Elferink RP, Rosing H, Beijnen JH and Schinkel AH (2002) The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci U S A 99:15649-15654. Jonker JW, Merino G, Musters S, van Herwaarden AE, Bolscher E, Wagenaar E, Mesman E, Dale TC and Schinkel AH (2005) The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat Med 11:127-129. Kipp H, Pichetshote N and Arias IM (2001) Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver. J Biol Chem 276:7218-7224. 67 Levine WG and Singer RW (1972) Hepatic intracellular distribution of foreign compounds in relation to their biliary excretion. J Pharmacol Exp Ther 183:411-419. Lickteig AJ, Fisher CD, Augustine LM, Aleksunes LM, Besselsen DG, Slitt AL, Manautou JE and Cherrington NJ (2007) Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease. Drug Metab Dispos 35:1970-1978. Maher JM, Slitt AL, Cherrington NJ, Cheng X and Klaassen CD (2005) Tissue distribution and hepatic and renal ontogeny of the multidrug resistanceassociated protein (Mrp) family in mice. Drug Metab Dispos 33:947-955. Manautou JE, de Waart DR, Kunne C, Zelcer N, Goedken M, Borst P and Elferink RO (2005) Altered disposition of acetaminophen in mice with a disruption of the Mrp3 gene. Hepatology 42:1091-1098. Merino G, Jonker JW, Wagenaar E, Pulido MM, Molina AJ, Alvarez AI and Schinkel AH (2005a) Transport of anthelmintic benzimidazole drugs by breast cancer resistance protein (BCRP/ABCG2). Drug Metab Dispos 33:614-618. Merino G, van Herwaarden AE, Wagenaar E, Jonker JW and Schinkel AH (2005b) Sex-dependent expression and activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP/ABCG2) in liver. Mol Pharmacol 67:1765-1771. Nakagawa R, Hara Y, Arakawa H, Nishimura S and Komatani H (2002) ABCG2 confers resistance to indolocarbazole compounds by ATP-dependent transport. Biochem Biophys Res Commun 299:669-675. Nezasa K, Tian X, Zamek-Gliszczynski MJ, Patel NJ, Raub TJ and Brouwer KL (2006) Altered hepatobiliary disposition of 5 (and 6)-carboxy-2',7'dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice. Drug Metab Dispos 34:718-723. Robey RW, Medina-Perez WY, Nishiyama K, Lahusen T, Miyake K, Litman T, Senderowicz AM, Ross DD and Bates SE (2001) Overexpression of the ATPbinding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridolresistant human breast cancer cells. Clin Cancer Res 7:145-152. Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M and Bates SE (2008) ABCG2: A perspective. Adv Drug Deliv Rev. Studenberg SD and Brouwer KL (1993) Hepatic disposition of acetaminophen and metabolites. Pharmacokinetic modeling, protein binding and subcellular distribution. Biochem Pharmacol 46:739-746. 68 Tanaka Y, Slitt AL, Leazer TM, Maher JM and Klaassen CD (2005) Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice. Biochem Biophys Res Commun 326:181-187. Xiong H, Suzuki H, Sugiyama Y, Meier PJ, Pollack GM and Brouwer KL (2002) Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment. Drug Metab Dispos 30:962-969. Zamek-Gliszczynski MJ, Hoffmaster KA, Tian X, Zhao R, Polli JW, Humphreys JE, Webster LO, Bridges AS, Kalvass JC and Brouwer KL (2005) Multiple mechanisms are involved in the biliary excretion of acetaminophen sulfate in the rat: role of Mrp2 and Bcrp1. Drug Metab Dispos 33:1158-1165. Zamek-Gliszczynski MJ, Nezasa K, Tian X, Bridges AS, Lee K, Belinsky MG, Kruh GD and Brouwer KL (2006a) Evaluation of the role of multidrug resistanceassociated protein (Mrp) 3 and Mrp4 in hepatic basolateral excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3-/- and Abcc4-/- mice. J Pharmacol Exp Ther 319:14851491. Zamek-Gliszczynski MJ, Nezasa K, Tian X, Kalvass JC, Patel NJ, Raub TJ and Brouwer KL (2006b) The important role of bcrp (abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4methylumbelliferone, and harmol in mice. Mol Pharmacol 70:2127-2133. 69 Table 2.1. Mouse body weights, liver weights normalized to body weight, acetaminophen (APAP) extraction ratio and total recovery at 70 min in in situ singlepass perfused livers from male and female wild-type and Bcrp-deficient mice.a Wild-type Bcrp-deficient Male Female Male Female Body weight (g) 28.4 ± 0.8b 25.8 ± 1.8b 25.9 ± 1.7 24.5 ± 1.7 Liver weight/Body weight (%) 4.7 ± 0.2 4.8 ± 0.5 4.3 ± 0.2 4.4 ± 0.7 Extraction ratio (%) 35.4 ± 4.8 29.6 ± 7.1 30.8 ± 6.1 24.8 ± 1.2 Total recovery 98.4 ± 5.2 91.7 ± 8.5 93.6 ± 3.7 98.2 ± 4.8 (% of dose) Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40 min followed by an additional 30-min infusion with drug-free buffer. a mean ± SD (n = 3-4 per group) b p < 0.05 70 Table 2.2. Mean pharmacokinetic parameter estimates governing APAP, AG and AS disposition in in situ single-pass perfused livers from wild-type and Bcrp-deficient male and female mice.a Parameter Wild-type Bcrp-deficient Mean estimates (CV%) Clup (ml/min) Male 5.63 (12.1) 5.60 (13.2) Female 5.79 (4.9) 5.55 (8.8) -1 Kperf,APAP (min ) Male 0.468 (16.5) 0.588 (44.7) Female 0.525 (31.1) 0.564 (36.3) Kgluc (min-1)c Male 0.183 (36.9) 0.199 (17.3) Female 0.121 (16.5) 0.119 (8.2) Ksulf (min-1)c Male 0.007 (13.1) 0.009 (50.6) Female 0.026 (8.8) 0.018 (44.6) -1 Kperf, AG (min ) Male 0.168 (45.4) 0.247 (22.5) Female 0.217 (49.3) 0.214 (77.3) Kg1 (min-1) Male 0.044 (89.3) 0.104 (36.7) Female 0.093 (49.4) 0.092 (55.3) Kbile, AG (min-1)b,c Male 0.014 (45.4) 0.001 (66.4) Female 0.002 (68.1) 0.001 (72.1) Kperf, AS (min-1)c Male 0.379 (38.6) 0.271 (11.7) Female 0.663 (17.6) 0.634 (9.6) Ks1 (min-1)c Male 0.048 (25.3) N/A Female 0.017 (39.5) N/A Kbile, AS (min-1)c Male 0.107 (8.0) N/A Female 0.043 (72.6) N/A a n = 3-4 per group; N/A, not available; CV, coefficients of variation b p < 0.05, Wild-type vs. Bcrp-deficient mice c p < 0.05, Male vs. Female 71 Q× ×Cin-CLup×Cin Perfusate Cin K perf, AG XAG-1 CLup K gluc K perf, APAP XAPAP Kg1 K sulf Perfusate Cout K perf, AS XAS-1 Ks1 XAG-2 XAS-2 K bile, AG K bile, AS X bile, AG X bile, AS Figure 2.1. Model scheme depicting the hepatic disposition of APAP, AG and AS in the single-pass perfused mouse liver. Q, flow rate; Cin, concentration of APAP in inflow perfusate; Cout, concentration of APAP in outflow perfusate; XAPAP, amount of APAP in liver compartment; XAG-1 and XAG-2, amount of AG in liver compartments 1 and 2, respectively; XAS-1 and XAS-2, amount of AS in liver compartments 1 and 2, respectively; CLup, uptake clearance into the liver; Kperf,APAP, first-order rate constant for basolateral excretion of APAP; Kperf,AG, first-order rate constant for basolateral excretion of AG; Kperf,AS, first-order rate constant for basolateral excretion of AS; Kgluc and Ksulf, first-order rate constant for formation of AG and AS, respectively; Kg1, firstorder rate constant for movement of AG from hepatic compartment 1 to 2; Ks1, firstorder rate constant for movement of AS from hepatic compartment 1 to 2; Kbile,AG and Kbile,AS, first-order rate constants for biliary excretion of AG and AS, respectively. AS biliary excretion was lower than the limit of quantitation in Bcrp-deficient mouse livers. 72 APAP Concentration (nM) 500 400 300 200 100 0 0 20 40 60 80 Time (min) Figure 2.2. APAP concentrations (nM) in outflow perfusate from male wild-type (●) and Bcrp-deficient (○), and female wild-type (▼) and Bcrp-deficient (∆) mouse liver perfusions. Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40 min followed by an additional 30-min infusion with drug-free buffer (n = 3-4/group). 73 A AG Biliary Excretion (nmol/g liver) 2.0 1.5 * AG Basolateral Excretion (nmol/g liver) B * 1.0 0.5 0.0 Male Female Male Female Wild-type 25 * * 20 15 10 5 0 Male Female Male Female Bcrp-deficient Wild-type Bcrp-deficient Figure 2.3. Cumulative biliary excretion and basolateral excretion of AG (A and B, respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; p < 0.05). 74 A AS Biliary Excretion (nmol/g liver) 0.25 * 0.20 0.15 0.10 0.05 N.D. N.D. 0.00 AS Basolateral Excretion (nmol/g liver) B Male Female Male Female Wild-type 5 * * 4 3 2 1 0 Male Female Male Female Bcrp-deficient Wild-type Bcrp-deficient Figure 2.4. Cumulative biliary excretion and basolateral excretion of AS (A and B, respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; N.D.:not detected; p < 0.05). 75 Wild-type Male Female Bcrp-deficient Male Female Bcrp Actin Mrp4 Actin Figure. 2.5. Representative immunoblot of Bcrp, Mrp4 and actin in crude membrane fractions from male and female wild-type and Bcrp-deficient mouse liver homogenates from three independent experiments (30 µg protein/well). 76 Female Wild-type Rate (pmol/min/g liver) Rate (pmol/min/g liver) Male Wild-type 10000 1000 100 10 1 0.1 0.01 0.001 0 20 40 60 10000 1000 100 10 1 0.1 0.01 0.001 80 0 20 Time (min) Rate (pmol/min/g liver) Rate (pmol/min/g liver) 10000 1000 100 10 1 0.1 0.01 0.001 20 40 60 80 Female Bcrp-deficient Male Bcrp-deficient 0 40 Time (min) 60 80 10000 1000 100 10 1 0.1 0.01 0.001 0 Time (min) 20 40 60 80 Time (min) Figure. 2.6. Representative outflow perfusate rate (pmol/min/g liver) of AG (●) and AS (■), and biliary excretion rate (pmol/min/g liver) of AG (○) and AS (□) in male and female wild-type (solid symbols) and Bcrp-deficient (open symbols) mouse livers. Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40 min followed by an additional 30-min infusion with drug-free buffer. Curves represent the best fit of the model scheme depicted in Figure 2.1 to the data. 77 CHAPTER 3 Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites in Rat and Human Sandwich-Cultured Hepatocytes: Use of Monte Carlo Simulations to Assess Impact of Changes in Biliary Excretion on Substrate Accumulation in Cells versus Culture Medium This Chapter will be submitted to Pharmaceutical Research. ABSTRACT This study examined the hepatobiliary disposition of troglitazone (TGZ) and its derived metabolites [TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone (TQ)] over time in rat and human sandwich-cultured hepatocytes (SCH). To determine the disposition of TGZ and its metabolites generated in rat and human SCH, cells were incubated with 10 µM TGZ for 120 min. Rat and human SCH mimicked the in vivo disposition of TGZ/metabolites; TGZ was metabolized primarily to TS, and to a lesser extent to TG and TQ, in both human and rat SCH. In human SCH, the biliary excretion index (BEI) was negligible for TGZ and TQ, ~16% for TS, and ~43% for TG over the duration of the 120 min incubation; in rat SCH, BEI for TS and TG was 13% and 41%, respectively. Pharmacokinetic modeling and Monte Carlo simulations were employed to evaluate the impact of modulating the biliary excretion rate constant (Kbile) for TS or TG on metabolite accumulation in medium and hepatocytes. The required number of observations to discern statistically significant alterations in response to changes in Kbile was greater in medium than in hepatocytes, suggesting that cellular concentrations (analogous to tissue accumulation in vivo) is a more sensitive metric than medium concentrations (analogous to systemic concentrations in vivo). Simulations supported the hypothesis that altered hepatobiliary transport may not always be evident based on medium concentrations in rat SCH. Pharmacokinetic modeling/simulation coupled with data from SCH is a useful in vitro approach to predict alterations in drug/metabolite hepatobiliary disposition in humans. 79 INTRODUCTION Troglitazone (TGZ), a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, was withdrawn from the market in 2000 due to severe idiosyncratic liver injury (Graham et al., 2003; Smith, 2003). Hepatotoxicity associated with TGZ has been classified as hepatocellular liver injury (Chan et al., 2002). Several mechanisms for TGZ’s hepatotoxicity have been proposed (Smith, 2003; Masubuchi, 2006), including inhibition of the bile salt export pump (BSEP, ABCB11), the hepatic transport protein primarily responsible for biliary excretion of bile acids, by TGZ and TGZ sulfate (TS) (Funk et al., 2001a). BSEP inhibition may cause hepatic accumulation of detergent-like bile acids, thereby resulting in hepatocellular injury. Interestingly, TS is a more potent inhibitor of BSEP (IC50 = 0.4-0.6 µM) than TGZ (IC50 = 3.9 µM)(Funk et al., 2001a), emphasizing the importance of metabolitemediated hepatotoxicity, in addition to that of the parent drug. TGZ is metabolized extensively by the liver in humans as well as in rats; the in vivo metabolic profile and disposition of TGZ in rats are similar to that in humans. In humans, TGZ is metabolized extensively in the liver primarily to a sulfate conjugate mostly via sulfotransferase (SULT) 1A3 (Honma et al., 2002), and also to a glucuronide conjugate mostly via UDP-glucuronosyltransferase (UGT) 1A1 (Watanabe et al., 2002; Nakagomi-Hagihara et al., 2006); TGZ also is oxidized to a quinone derivative by cytochrome P450 isozymes CYP3A4 and CYP2C8 (Yamazaki et al., 1999). After oral administration of TGZ for 7 days in humans, the area under the plasma concentration vs. time curve at steady state (AUCss) for TS was ~7-10fold higher than that of TGZ; the plasma AUCss for TQ was comparable to that of 80 TGZ, but the AUCss for TG was only 20-40% of that for TGZ (Loi et al., 1999). Similarly, after oral administration of [14C]TGZ to male rats, the AUC of TS accounted for 89% of the AUC of total plasma radioactivity, while the AUC of TGZ and TG was only 5% and 1% of the AUC of total plasma radioactivity, respectively (Kawai et al., 1997). After intravenous administration of [14C]TGZ to male rats, the biliary excretion of TS and TG was 61% and 2% of the dose, respectively; TGZ and TQ recovery in bile accounted for less than 0.1% of the dose (Kawai et al., 1997). Since TGZ is extensively metabolized and little parent compound is excreted unchanged into urine or bile (Kawai et al., 1997), the total body clearance of TGZ is governed by its metabolic clearance (Izumi et al., 1996). Sensitive and rapid in vitro screening tools to predict hepatotoxicity are needed in the pharmaceutical industry. Primary hepatocytes remain an important tool for studying the metabolism and hepatotoxic potential of xenobiotics. Hepatocytes cultured in a single layer of collagen are suitable for cytotoxicity assays, but the use of this tool is limited due to loss of metabolic activity and short survival periods (Hewitt et al., 2007). Hepatocytes cultured in sandwich configuration between layers of extracellular matrix have overcome many of the limitations of conventional hepatocyte culture because the cells repolarize, regain their proper morphology, and maintain liver-specific metabolic activity, including stable expression of cytochrome P450 enzymes over longer times (Liu et al., 1998; Liu et al., 1999b; Liu et al., 1999d; Liu et al., 1999e; Hamilton et al., 2001; Hewitt et al., 2007; Mingoia et al., 2007). Furthermore, sandwich-cultured hepatocytes develop functional networks of bile canalicular structures between cells that are sealed by 81 tight junctions (LeCluyse et al., 1994). Measurements of biliary excretion of drugs and metabolites in humans are often indirect or require invasive procedures, but cells and canalicular networks of sandwich-cultured hepatocytes are directly accessible, allowing the quantitation of substrates and metabolites excreted into the medium, retained within the cell, or excreted into the bile (Hoffmaster et al., 2004b; Turncliff et al., 2006). The present study utilized rat and human SCH to examine the metabolism of TGZ, governed primarily by phase II conjugation, and the hepatobiliary disposition of TGZ and its generated metabolites. Moreover, the ability of this in vitro system to estimate the in vivo disposition of TGZ and metabolites previously reported in rats and humans was assessed. A pharmacokinetic model was developed to estimate key parameters governing the hepatic metabolism and disposition of TGZ and metabolites in rat SCH. Monte Carlo simulations were utilized to predict the influence of hepatic transport modulation on the hepatobiliary disposition of TGZ and metabolites in a hypothetical “population” of hepatocytes. 82 MATERIALS AND METHODS Chemicals. Penicillin-streptomycin solution, dexamethasone (DEX), Hanks’ balanced salts (HBSS), HBSS modified (HBSS without Ca2+ and Mg+), collagenase (type IV), and Triton X-100 were purchased from Sigma-Aldrich, Inc (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and MEM non-essential amino acids were purchased from Invitrogen (Carlsbad, CA). Insulin/transferrin/selenium culture supplement (ITSTM), BioCoatTM culture plates and MatrigelTM extracellular matrix were purchased from BD Biosciences Discovery Labware (Bedford, MA). TGZ was purchased from Toronto Research Chemicals Inc. (Ontario, Canada). TS, TG, and TQ were kindly provided by Daiichi-Sankyo Co., Ltd (Tokyo, Japan). All other chemicals and reagents were of analytical grade and were readily available from commercial sources. Hepatocyte Culture. Male Wistar rats (220-300 g; Charles River Laboratories, Inc., Raleigh, NC) were maintained on a 12-hr light/dark cycle with free access to water and rodent chow. The Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill approved all procedures. Rat hepatocytes were isolated using a two-step collagenase perfusion method as described previously (Liu et al., 1999c). Hepatocytes were plated at a density of 1.75 x 106 cells/well on 6-well BioCoatTM plates in Dulbecco’s modified Eagle’s medium (DMEM; 1.5 ml) containing 5% fetal bovine serum, 10 µM insulin, 1 µM DEX, 2 mM L-glutamine, 1% MEM nonessential amino acids, 100 units penicillin G sodium and 100 µg streptomycin sulfate. Hepatocytes were allowed to attach ~2 hr, and the medium was changed to remove unattached cells. Cells were overlaid ~24 hr later with BD MatrigelTM at a 83 concentration of 0.25 mg/ml in 2 ml/well ice-cold DMEM supplemented with 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, 100 units penicillin G sodium, 100 µg streptomycin, 1% ITSTM and 0.1 µM DEX. CellzDirect (Durham, NC) kindly provided human hepatocytes cultured in 6-well plates, seeded at a density of 1.5 x 106 cells/well, and overlaid with MatrigelTM. Table 3.1 shows demographics of human liver donors. Human cells were cultured in the same medium as rat hepatocytes. Both rat and human hepatocytes were cultured at 37°C i n a humidified incubator with 95% O2/5% CO2. Medium was changed daily until the experiments were conducted. Metabolism of TGZ and Accumulation of Parent and Metabolites in Medium, Cells, and Bile in Rat and Human SCH. Modulation of Ca2+ in the incubation buffer was used to manipulate the tight junctions sealing the canalicular networks between cells. In Ca2+-containing standard buffer (HBSS with 0.35 g/L sodium bicarbonate), tight junction integrity is preserved and substrates are excreted into the bile canaliculi; incubation of cells in Ca2+-free buffer (HBSS modified, with 0.35 g/L sodium bicarbonate and 0.38 g/L EGTA) disrupts the tight junctions, opens the canalicular spaces, and canalicular contents are released into the incubation medium (Liu et al., 1999e). Subtracting the amount of substrate accumulated in cells after a pre-incubation in Ca2+-free buffer from the accumulated amount in cells + bile after pre-incubation with Ca2+-containing buffer yields the amount of substrate excreted into the canalicular networks. To evaluate metabolism of TGZ, day-4 cells (rat; n=3 livers) and day-8 or day-10 cells (human; n=2 livers) were incubated at 37 °C for 10, 20, 30, 60, 90, and 120 min 84 for rat SCH, and for 30, 60, and 120 min for human SCH, with 10 µM TGZ in culture medium. Aliquots of medium were removed at each timepoint, and samples were diluted with methanol (1:2 v/v). At the designated time, cells were washed twice and incubated at 37 °C for 5 min with HBSS with Ca 2+ (cells + bile) or without Ca2+ (cells). After incubation, buffer was aspirated and cells were lysed with 1 ml of 70%:30% (v/v) methanol:water, and sonicated for 20 sec with a sonic dismembrator (model 100, Fisher Scientific, Pittsburgh, PA). Medium, and cells or cells + bile lysate samples were stored at <-70 °C until analysis. Transport of TGZ, and Preformed TS and TG Metabolites in Rat SCH. To confirm transport of TGZ and its preformed metabolites in rat SCH, media was aspirated from cells on culture day 4 and cells were rinsed twice and then preincubated for 10 min at 37°C with 2 ml of warmed HBS S+Ca2+ buffer (cells + bile) or Ca2+-free HBSS buffer (cells). Medium was aspirated and cells were treated with 5 µM TGZ, TS, or TG in 1.5 ml HBSS +Ca2+ buffer for 10 min. After incubation, the dosing medium was removed, cells were washed 3x with 2 ml of ice-cold HBSS +Ca2+ buffer. The wash buffer was aspirated, cells were lysed by adding 1 ml of 70%:30% (v/v) methanol:water, and samples were sonicated for 20 sec with a sonic dismembrator (model 100, Fisher Scientific, Pittsburgh, PA). Cells and cells + bile lysate samples were stored at <-70°C until analysis. Tre atments were performed in triplicate in n=1 experiment (TGZ) or n=3 experiments (TS and TG). Sample Analysis. Substrate accumulation was corrected for nonspecific binding to the extracellular matrix by subtracting accumulation in MatrigelTM-coated BioCoatTM plates without cells. The BCA protein assay (Pierce Biotechnology, Inc., Rockford, 85 IL) was used to quantify total protein concentration in cell lysate samples using bovine serum albumin as the reference standard, and accumulation was normalized to protein concentration. To account for the incompatibility of the protein assay with methanol, the average protein concentration for standard HBSS or Ca2+-free HBSS incubations in a representative plate from the same liver preparation was used to normalize accumulation. The medium and cell or cell + bile lysate samples were centrifuged at 12,000×g for 2 min at 4 °C, and the su pernatant was diluted 1:6 (v/v) with 79%:21% (v/v) methanol:water containing an internal standard (ethyl warfarin). A Shimadzu solvent delivery system (Columbia, MD) and a Leap HTC Pal thermostated autosampler (Carrboro, NC) connected to an Applied Biosystems API 4000 triple quadruple mass spectrometer with a TurboSpray ion source (Applied Biosystems, Foster City, CA) were used for analysis. Tuning, operation, integration and data analysis were performed in negative ionization mode using multiple reaction monitoring (Analyst software v.1.4.1, Applied Biosystems). Separation was accomplished using an Aquasil C18, 50×2.1 mm column, with a 5 µm particle size (ThermoElectron, San Jose, CA). Analysis required 15 µL of sample and a solvent flow of 0.75 mL/min. Initial gradient conditions (80% 10 mM ammonium acetate aqueous solution and 20% methanol) were held for 0.8 min. From 0.8 to 1.5 min, the mobile phase composition increased linearly to 60% methanol, and the eluent was directed to the mass spectrometer. From 1.5 to 4 min, the mobile phase composition increased linearly to 65% methanol. At 4.1 min, the methanol composition was increased to 90%. The flow was held at 90% methanol until 4.3 min. At 4.4 min, the column was equilibrated with 80% 10 mM ammonium acetate aqueous solution. The 86 total run time, including equilibration, was 5 min per injection. Eight point calibration curves (5-5000 nM) were constructed as composites of TGZ (440.0→397.1), TS (520.2→440.1), TG (616.2→440.1), and TQ (456.1→413.1) using peak area ratios of analyte and ethyl warfarin (320.8→160.9). All points on the curves backcalculated to within ± 15% of the nominal value. B-CLEAR® technology (Qualyst, Inc., Raleigh, NC) was used to calculate the biliary excretion index (BEI), which represents the percentage of accumulated substrate that is excreted into bile, using the following equation (Liu et al., 1999c): BEI (%) = Accumulationcells +bile − Accumulationcells × 100 Accumulationcells +bile Pharmacokinetic Modeling. The average rat SCH accumulation data for TGZ and metabolites, including TS, TG and TQ, were utilized in pharmacokinetic modeling analysis. Several structurally-distinct models were evaluated, and standard model selection criteria, including Akaike’s information criterion, residual distribution, and the distribution of the residual error, were used to identify the optimal structure. The model scheme shown in Figure 3.1 best described the disposition of TGZ and generated metabolites in rat SCH over 120 min in the medium (M), hepatocytes (C) and bile canaliculi (B). Differential equations derived from the model scheme in Figure 3.1 (see legend for definition of parameters) were as follows: 87 dXTGZ,M dt dXTS,M dt dXTS,M dt dXTG,M dt dXTG,M dt dXTQ,M dt = K efflux,TS × XTS,C − K uptake,TS × XTS,M; XTS,M0 = 0; t ≤ 20min = K efflux,TS × XTS,C − K uptake,TS × XTS,M + K flux,TS × XTS,B ; t > 20min = K efflux,TG × XTG,C − K uptake,TG × XTG,M; XTG,M0 = 0; t ≤ 20min = K efflux,TG × XTG,C − K uptake,TG × XTG,M + K flux,TG × XTG,B ; t > 20min = K f,TQ × XTGZ,M −K other × XTQ,M; XTQ,M0 = 0 dXTGZ,C dt dXTS,C dt dXTG,C dt dXTS,B dt dXTS,B dt dXTG,B dt dXTG,B dt = −(K uptake,TGZ + K f,TQ ) × XTGZ,M; XTGZ,M0 = 0 = K uptake,TGZ × XTGZ,M −(K f,TS + K f,TG ) × XTGZ,C; XTGZ,C0 = 0 = K f,TS × XTGZ,C + K uptake,TS × XTS,M − (K efflux,TS + K bile,TS ) × XTS,C; XTS,C0 = 0 = K f,TG × XTGZ,C + K uptake,TG × XTG,M − (K efflux,TG + K bile,TG ) × XTG,C; XTG,C0 = 0 = K bile,TS × XTS,C; XTS,B0 = 0; t ≤ 20min = K bile,TS × XTS,C − K flux,TS × XTS,B ; t > 20min = K bile,TG × XTG,C; XTG,B0 = 0; t ≤ 20min = K bile,TG × XTG,C − K flux,TG × XTG,B ; t > 20min Differential equations describing the accumulation of TGZ and generated metabolites in medium, hepatocytes and bile canaliculi were fit simultaneously by nonlinear least-squares regression (WinNonlin Pro version 5.0.1; Pharsight, Mountain View,CA) using a weighting scheme of 1/(Y predicted). Monte Carlo Simulations. Parameter estimates with associated variance (recovered by nonlinear least-squares regression of the flux data shown above) and uncontrolled error (experimental or analytical error; 15%) served as input for Monte 88 Carlo simulations. To explore hepatic TS and TG disposition in response to impaired transport in a hypothetical “population” of hepatocytes, simulations were performed by modulating the rate constant for TS or TG biliary excretion. In order to estimate the number of observations (sample size) necessary to detect a statistically significant difference in TS or TG accumulation in cells or medium due to changes in the TS or TG biliary excretion rate constant (Kbile, TS or Kbile, TG), data from Monte Carlo simulations at designated time points (10, 20, 30, 60, 90 and 120 min) were analyzed using one-way ANOVA. Based on the effect size (the square root of the ratio of the between-group to within-group variance) calculated by Cohen’s f2 (Cohen, 1988), a sample size for each time point was calculated to achieve a power of 0.80 (p<0.05)(2008). The relative power to detect a statistically significant difference in medium vs. cell at each time point was calculated as a ratio of the number of observations (sample size) necessary to detect a statistically significant difference in accumulation in medium to the number of observations necessary for accumulation in cells. 89 RESULTS Hepatobiliary disposition of TGZ and generated metabolites in human SCH. The disposition of TGZ and the generated metabolites in the medium and hepatocytes at 30, 60, and 120 min is shown in Table 3.2; cumulative recovery of TGZ and generated metabolites (medium + hepatocytes + bile) was 109-117%. At 120 min, TGZ in the medium accounted for 64% of the dose. TS was the predominant metabolite of TGZ in the medium (30% of dose) as well as hepatocytes (5.6% of dose) during the 120-min incubation; TG and TQ were recovered predominantly in the medium (3% and 2.4% of dose, respectively) with negligible cellular accumulation (less than 1% of dose). Accumulation of TGZ and TQ in bile canaliculi was negligible. The BEI values of TS (15-17 %) and TG (41-46 %) were comparable at 30, 60, and 120 min in human SCH (Table 3.2). Hepatobiliary disposition of TGZ and metabolites in rat SCH. Initial experiments confirmed that TGZ and preformed metabolites of TGZ, TS and TG, were taken up into rat SCH (Figure 3.2). Following a 10-min accumulation, average cell + bile accumulation of TGZ was only slightly greater than cellular accumulation (1600s. 1560 pmol/mg protein), consistent with a negligible BEI for TGZ of 2.3%. Average cell + bile accumulation was slightly greater than cellular accumulation for TS (2280 vs. 1940 pmol/mg protein) and TG (908 vs. 605 pmol/mg protein), consistent with more extensive BEI values of 15% and 34% for TS and TG, respectively. The disposition of TGZ and its generated metabolites in medium, hepatocytes, and bile are plotted in Figure 3.3, 3.4, and 3.5, respectively. Cumulative recovery of 90 TGZ and its metabolites from medium, hepatocytes and bile was comparable throughout the 120-min incubation period (90-104%) (Table 3.3). At 120 min, 35% of the dose remained in the medium as TGZ (Table 3.3). Among metabolites present in the medium, TS was predominant (38% of dose), followed by TG (7% of dose) and TQ (1% of dose) at 120 min. Figure 3.4. depicts the hepatocyte accumulation of TGZ and its generated metabolites over the 120-min incubation period. TS formation was rapid and extensive; cellular accumulation of TS exceeded TGZ even at 10 min (5.4% vs. 1.5% of the dose, respectively; Table 3.3). The cellular accumulation of TG was <0.4% of the dose over the 120-min incubation. TQ in the hepatocytes was negligible. The average BEI of TS and TG was 13% and 41% over the 120-min incubation time; accumulation of TGZ and TQ in bile was negligible (Figure 3.5; Table 3.3). Pharmacokinetic modeling of the hepatobiliary disposition of TGZ and its generated metabolites in rat SCH. Because data were collected at more time points in rat SCH, a compartmental pharmacokinetic model was developed using data from rat SCH. The model scheme depicted in Figure 3.1 best described the disposition of TGZ, TS, TG and TQ in rat SCH during the 120-min incubation. Representative descriptions of the best fit of the pharmacokinetic model to the TGZ/generated metabolite vs. time data are shown in Figures 3.3-3.5. Pharmacokinetic parameter estimates obtained by resolving the differential equations describing hepatocellular disposition of TGZ, TS, TG and TQ vs. time data are included in Table 3.4. The first-order rate constant for TS formation (0.382 min-1) was ~6-fold greater than that for TG formation (0.061 min-1). The 91 pharmacokinetic model incorporated a formation process for TQ from TGZ in the incubation medium, instead of cellular TQ formation, because accumulation of TQ in hepatocytes was negligible while TQ in the medium represented ~1% of the dose at 120 min. In order to explain the decreased accumulation of TS and TG in bile canaliculi after 20 min, first-order rate constants for TS and TG flux from the bile canaliculi to the incubation medium were incorporated into the pharmacokinetic model. Consistent with data from the uptake study with preformed TGZ metabolites, TS and TG (Figure 3.2), the first-order rate constant for TS uptake (Kuptake, TS; 0.005 min-1) was greater than for the TG uptake rate constant (Kuptake, TG; 0.001 min-1). The first-order rate constant for biliary excretion of TG (0.061 min-1) was ~3-fold higher for TS (0.023 min-1). The first-order rate constant for basolateral efflux of TG (0.160 min-1) was ~5-fold greater than for TS (0.031 min-1). Monte Carlo simulations Monte Carlo simulations were performed to assess the impact of modulation of the first-order rate constant for biliary excretion of TS (Kbile, TS) and TG (Kbile, TG) on the hepatobiliary disposition of TS and TG, respectively (Figure 3.6). Overall, measurements of cellular accumulation were more sensitive to changes in kinetic parameters than were measurements of substrate concentrations in medium. Cellular accumulation measurements were 1.4- to 270-times more sensitive than medium accumulation measurements for TG; cellular accumulation measurements were 2.2- to 10.2-times more sensitive than medium accumulation measurements for TS (Table 3.5). 92 DISCUSSION The present study examined the hepatobiliary disposition of TGZ and its metabolites in the SCH system; results confirmed that functional activity of phase II conjugation enzymes is preserved in rat and human SCH. In addition, pharmacokinetic modeling and simulation studies were employed to examine the impact of impaired biliary excretion on hepatobiliary disposition of TS and TG in rat SCH. In human SCH, TS was most abundant, and TQ was more abundant than TG, in the medium at 30 min; amounts of both TQ and TG were similar at 60 and 120 min in the medium. Amounts of TQ and TG in cells were also similar at all time points, but no TGZ and TQ was found in the bile at any time. Kostrubsky and colleagues treated conventionally-cultured human hepatocytes with 10 µM TGZ; at 1 hour, concentrations of TS and TQ were the highest in medium (depending on donor), followed by parent TGZ, and TG (Kostrubsky et al., 2000). Our data show that in human SCH at 1 hour, unconjugated TGZ is most abundant in the medium (88.84% of dose), followed by TS (13.37% of dose), TQ (2.23% of dose), and lastly TG (1.56% of dose). Transport of TGZ and its preformed metabolites was confirmed in rat SCH. While TGZ, TS, and TG were taken up into rat hepatocytes, it appears that TS was taken up into cells more efficiently compared to TGZ even though TS is more hydrophilic than TGZ. These data are consistent with the observation that TS is a higher affinity substrate than TGZ or TG for the uptake transporter OATP1B1, (Nozawa et al., 2004b). Further experiments revealed that in rat SCH, TS was the 93 major metabolite found in the medium, cells, and bile at all timepoints. TG was the next most abundant metabolite found in the medium and bile at all time points, while TQ was only detected in lesser amounts in the medium, but not in cells or bile. This observation may be explained by photooxidation of TGZ to TQ; thus, subsequent pharmacokinetic modeling incorporated conversion of TGZ to TQ in medium (Fu et al., 1996). Additionally, in rat and human SCH, TS and TG were transported into the bile canaliculi, while transport of TGZ into the bile canaliculi was negligible. These findings in SCH are consistent with published in vivo reports showing that little parent TGZ is recovered in rat bile, while TS and TG (68.0% and 11.6% of dose, respectively, after oral dosing of TGZ) are excreted into bile in rats (Izumi et al., 1996; Kawai et al., 1997). These medium and biliary excretion profiles resembled the plasma profiles (TS > TG) and biliary excretion (TS > TG), respectively, after oral dosing of TGZ to rats in vivo (Izumi et al., 1997; Kawai et al., 1997). The greater amount of TS compared to TG in rat SCH, and the greater value of Kf, TS compared to Kf,TG correspond to data by Izumi et al. showing that the sulfate formation clearance was 6-fold higher than glucuronide formation clearance in rats (Izumi et al., 1997). The accumulation of TS and TG in bile remained constant or decreased during the incubation time after 20 min in both human and rat SCH (Table 3.2 and Table 3.3). To account for this decrease, the pharmacokinetic model incorporated a rate constant for flux of TS and TG from the bile into the medium (Figure 3.1). The flux of other compounds from bile into the medium of SCH has been noted 94 previously. The first-order rate constant for flux of taurocholate (TC) and [Dpenicillamine2,5] enkephalin (DPDPE) from bile was reported to be 0.85 min-1 (Liu et al., 1999e) and 1.6 min-1 (Hoffmaster et al., 2005), respectively, but only 0.05 min-1 for 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) [Abe, unpublished data]. The greater rate constant for flux of TC and DPDPE compared to CDF, TS (0.17 min-1; Table 3.4) and TG (0.094 min-1; Table 3.4) may be attributed to different culture conditions: 2×106cells/60-mm dish overlaid with rat tail type 1 collagen for studies conducted with TC and DPDPE vs. 1.75×106 cells/6-well plates overlaid with MatrigelTM for studies conducted with CDF and TGZ. The accumulation of TS and TG in the medium in rat SCH (38% and 7% of the dose, respectively) was comparable to values obtained in human SCH (30% and 3% of the dose, respectively). Transport proteins responsible for the basolateral excretion of TS and TG remain to be identified. It has been documented previously that Mrp3 plays a predominant role in the basolateral excretion of glucuronide conjugates, while both Mrp3 and Mrp4 are involved in the basolateral excretion of sulfate conjugates along with additional yet-to-be-identified mechanism(s) (ZamekGliszczynski et al., 2006b). Pharmacokinetic modeling revealed a ~5-fold higher firstorder rate constant for TG than for TS basolateral efflux. This observation may reflect more efficient basolateral efflux of TG, presumably by Mrp3; Mrp3 expression was increased over time in culture in rat SCH (unpublished observation). Monte Carlo simulations revealed that cellular accumulation is a more sensitive measure of changes in the rate constant for biliary excretion of TS or TG (Kbile,TS or Kbile,TG) than medium accumulation. In practice, the number of 95 observations necessary to observe a statistically significant difference in the medium was too high (19-7271 for TG; 13-144 for TS), while the number of observations necessary to observe a statistically significant difference in hepatocytes was more reasonable (14-27 for TG; 4-27 for TS) (Table 3.5). SCH data coupled with modeling and simulation are capable of estimating the impact of altered transport function on hepatic (reflected in hepatocyte accumulation) and systemic (reflected in medium accumulation) exposure. Peak plasma concentrations or AUCplasma often have been used to examine the effect of modulation of canalicular transport proteins on drug disposition, but results of this simulation suggest that those parameters may not always reflect changes in hepatic exposure due to modulation of biliary excretory pathways. Physiologically-based pharmacokinetic modeling of pravastatin distribution also suggested that plasma concentrations are not sensitive enough to detect changes in canalicular efflux; altered canalicular efflux of pravastatin changed liver concentrations markedly, but had minimal effect on plasma concentrations (Watanabe et al., 2009). The failure of preclinical testing to predict the hepatotoxicity of TGZ emphasizes the importance of understanding the metabolism and hepatobiliary disposition of drugs in development, in both humans and in species commonly used for animal testing. One of the most efficient ways to study species differences in metabolism and hepatobiliary disposition is with the use of in vitro models. In vitro models are also an important tool for the prediction of the pharmacokinetics and pharmacodynamics of drugs in humans. One benefit of using sandwich-cultured hepatocytes is that concentrations of parent drug and metabolites in medium can be 96 measured, in addition to discriminating between accumulation within hepatocytes and bile. Efflux into medium may be analogous to efflux from the hepatocytes into plasma in vivo, while efflux into the bile may approximates biliary excretion in vivo. Because data in humans regarding intracellular concentrations in the liver of TGZ and its metabolites is lacking, as is data about efflux of parent or metabolites into bile, the sandwich-cultured model may be a useful in vitro tool to predict human in vivo hepatobiliary disposition. Herein we have shown that the sandwich-cultured hepatocyte model can be used to elucidate the hepatobiliary disposition of TGZ and metabolites in both humans and rats. In conclusion, phase II metabolites of TGZ were detected in both rat and human SCH, indicating the preservation of functional activity of sulfotransferase and UDP-glucuronosyltransferase in the SCH system. The remarkable similarity in TGZ metabolism and hepatobiliary disposition between rats and humans in vivo also was observed in rat and human SCH, respectively, suggesting that the SCH model is a useful in vitro tool to predict in vivo hepatobiliary disposition in rats and humans. Pharmacokinetic modeling and simulation, coupled with data obtained from the SCH model, may be used as an in vitro approach to determine likely alterations in drug/metabolite hepatobiliary disposition in humans. 97 REFERENCES (2008) R: A language and environment for statistical computing. R Foundation for Statistical Computing, in, R Development Core Team, Vienna, Austria. Chan YL, Chang SS, Kao KL, Liao HC, Liaw SJ, Chiu TF, Wu ML and Deng JF (2002) Button battery ingestion: an analysis of 25 cases. Chang Gung Med J 25:169-174. Cohen J (1988) Statistical Power Analysis for the Behavioral Sciences Lawrence Erlbaum Associates. Fu Y, Sheu C, Fujita T and Foote CS (1996) Photooxidation of troglitazone, a new antidiabetic drug. Photochem Photobiol 63:615-620. Funk C, Pantze M, Jehle L, Ponelle C, Scheuermann G, Lazendic M and Gasser R (2001) Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167:83-98. Graham DJ, Green L, Senior JR and Nourjah P (2003) Troglitazone-induced liver failure: a case study. Am J Med 114:299-306. Hamilton GA, Jolley SL, Gilbert D, Coon DJ, Barros S and LeCluyse EL (2001) Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions. Cell Tissue Res 306:85-99. Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM and Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159-234. Hoffmaster KA, Zamek-Gliszczynski MJ, Pollack GM and Brouwer KL (2004) Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems. J Pharmacol Exp Ther 311:1203-1210. Hoffmaster KA, Zamek-Gliszczynski MJ, Pollack GM and Brouwer KL (2005) Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5]enkephalin. Drug Metab Dispos 33:287-293. 98 Honma W, Shimada M, Sasano H, Ozawa S, Miyata M, Nagata K, Ikeda T and Yamazoe Y (2002) Phenol sulfotransferase, ST1A3, as the main enzyme catalyzing sulfation of troglitazone in human liver. Drug Metab Dispos 30:944949. Izumi T, Enomoto S, Hosiyama K, Sasahara K, Shibukawa A, Nakagawa T and Sugiyama Y (1996) Prediction of the human pharmacokinetics of troglitazone, a new and extensively metabolized antidiabetic agent, after oral administration, with an animal scale-up approach. J Pharmacol Exp Ther ka277:1630-1641. Izumi T, Hosiyama K, Enomoto S, Sasahara K and Sugiyama Y (1997) Pharmacokinetics of troglitazone, an antidiabetic agent: prediction of in vivo stereoselective sulfation and glucuronidation from in vitro data. J Pharmacol Exp Ther 280:1392-1400. Kawai K, Kawasaki-Tokui Y, Odaka T, Tsuruta F, Kazui M, Iwabuchi H, Nakamura T, Kinoshita T, Ikeda T, Yoshioka T, Komai T and Nakamura K (1997) Disposition and metabolism of the new oral antidiabetic drug troglitazone in rats, mice and dogs. Arzneimittelforschung 47:356-368. Kostrubsky VE, Sinclair JF, Ramachandran V, Venkataramanan R, Wen YH, Kindt E, Galchev V, Rose K, Sinz M and Strom SC (2000) The role of conjugation in hepatotoxicity of troglitazone in human and porcine hepatocyte cultures. Drug Metab Dispos 28:1192-1197. LeCluyse EL, Audus KL and Hochman JH (1994) Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am J Physiol 266:C1764-1774. Liu X, Brouwer KL, Gan LS, Brouwer KR, Stieger B, Meier PJ, Audus KL and LeCluyse EL (1998) Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration. Pharm Res 15:1533-1539. Liu X, Chism JP, LeCluyse EL, Brouwer KR and Brouwer KLR (1999a) Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab Dispos 27:637-644. Liu X, LeCluyse EL, Brouwer KR, Gan LS, Lemasters JJ, Stieger B, Meier PJ and Brouwer KL (1999b) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 277:G12-21. Liu X, LeCluyse EL, Brouwer KR, Gan LS, Lemasters JJ, Stieger B, Meier PJ and Brouwer KLR (1999c) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 277:G12-21. 99 Liu X, LeCluyse EL, Brouwer KR, Lightfoot RM, Lee JI and Brouwer KL (1999d) Use of Ca2+ modulation to evaluate biliary excretion in sandwich-cultured rat hepatocytes. J Pharmacol Exp Ther 289:1592-1599. Loi CM, Alvey CW, Vassos AB, Randinitis EJ, Sedman AJ and Koup JR (1999) Steady-state pharmacokinetics and dose proportionality of troglitazone and its metabolites. J Clin Pharmacol 39:920-926. Masubuchi Y (2006) Metabolic and non-metabolic factors determining troglitazone hepatotoxicity: a review. Drug Metab Pharmacokinet 21:347-356. Mingoia RT, Nabb DL, Yang CH and Han X (2007) Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening. Toxicol In Vitro 21:165-173. Nakagomi-Hagihara R, Nakai D, Kawai K, Yoshigae Y, Tokui T, Abe T and Ikeda T (2006) OATP1B1, OATP1B3, and mrp2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin II blocker. Drug Metab Dispos 34:862-869. Nozawa T, Sugiura S, Nakajima M, Goto A, Yokoi T, Nezu J, Tsuji A and Tamai I (2004) Involvement of organic anion transporting polypeptides in the transport of troglitazone sulfate: implications for understanding troglitazone hepatotoxicity. Drug Metab Dispos 32:291-294. Smith MT (2003) Mechanisms of troglitazone hepatotoxicity. Chem Res Toxicol 16:679-687. Turncliff RZ, Hoffmaster KA, Kalvass JC, Pollack GM and Brouwer KL (2006) Hepatobiliary disposition of a drug/metabolite pair: Comprehensive pharmacokinetic modeling in sandwich-cultured rat hepatocytes. J Pharmacol Exp Ther 318:881-889. Watanabe T, Kusuhara H, Maeda K, Shitara Y and Sugiyama Y (2009) Physiologically based pharmacokinetic modeling to predict transportermediated clearance and distribution of pravastatin in humans. J Pharmacol Exp Ther 328:652-662. Watanabe Y, Nakajima M and Yokoi T (2002) Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10. Drug Metab Dispos 30:1462-1469. Yamazaki H, Shibata A, Suzuki M, Nakajima M, Shimada N, Guengerich FP and Yokoi T (1999) Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes. Drug Metab Dispos 27:1260-1266. 100 Zamek-Gliszczynski MJ, Nezasa K, Tian X, Bridges AS, Lee K, Belinsky MG, Kruh GD and Brouwer KL (2006) Evaluation of the role of multidrug resistanceassociated protein (Mrp) 3 and Mrp4 in hepatic basolateral excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3-/- and Abcc4-/- mice. J Pharmacol Exp Ther 319:14851491. 101 Table 3.1. Demographics of human liver donors Donor Age Gender Ethnicity Co-medications 1 36 Female Caucasian Oral contraceptive, HCTZ 2 64 Female Caucasian Atorvastatin, Amlodipine, Benazepril, Alendronate 102 Table 3.2. Disposition of TGZ and generated metabolites in human SCH TGZ, TS, TG and TQ (expressed as % of dose) in medium, cells and bile at 30, 60 and 120 min after incubation in medium initially containing 10 µM TGZ. BEI of TS and TG was calculated as described in Materials and Methods Data represent mean of a single determination in n=2 livers (N.D, not-detectable). Time (min) 30 60 120 Time (min) 30 60 120 Time (min) 30 60 120 TGZ Medium Cells (% of dose) 93.94 3.5 88.84 2.8 64.24 2.04 TQ Medium Cells (% of dose) 2.28 0.07 2.23 0.07 2.41 0.07 Recovery (%) 112 117 109 Bile N.D N.D N.D Bile N.D N.D N.D 103 TS Medium Cells (% of dose) 3.69 6.27 13.37 6.61 30.29 5.64 TG Medium Cells (% of dose) 0.63 0.06 1.56 0.06 2.98 0.05 Bile 1.29 1.22 0.99 Bile 0.05 0.04 0.04 BEI (%) 17 17 15 BEI (%) 46 41 43 Table 3.3. Disposition of TGZ and its generated metabolites in rat SCH TGZ, TS, TG and TQ (expressed as % of dose) in medium, cells and bile at 10, 20, 30, 60, 90 and 120 min after incubation in medium initially containing 10 µM TGZ. BEI of TS and TG was calculated as described in Materials and Methods after incubation of TGZ (10 µM) in rat SCH. Data represent mean ± S.E.M (n=3 livers; N.D, non-detectable). TGZ Cells (% of dose) Time (min) Medium 10 20 30 60 90 120 94.28 ± 9.29 86.38 ± 3.42 81.10 ± 3.68 63.55 ± 4.98 46.90 ± 3.07 35.30 ± 2.87 Time (min) 1.50 ± 0.31 1.97 ± 0.21 1.57 ± 0.06 1.38 ± 0.45 0.73 ± 0.24 0.30 ± 0.13 TQ Medium Cells (% of dose) 10 20 30 60 90 120 Time (min) 1.00 ± 0.07 1.36 ± 0.23 1.27 ± 0.09 1.01 ± 0.13 0.85 ± 0.13 0.65 ± 0.14 Recovery (%) 10 20 30 60 90 120 104 103 103 99 95 90 N.D N.D N.D N.D N.D N.D Bile Medium N.D N.D N.D N.D N.D N.D 0.48 ± 0.06 2.97 ± 0.58 6.14 ± 0.57 18.61 ± 2.02 29.93 ± 2.64 37.80 ± 2.70 Bile Medium N.D N.D N.D N.D N.D N.D 0.19 ± 0.02 1.18 ± 0.10 2.41 ± 0.16 3.86 ± 0.78 5.87 ± 1.15 6.89 ± 1.47 104 TS Cells (% of dose) Bile BEI (%) 5.46 ± 0.72 7.15 ± 1.50 7.92 ± 2.17 9.20 ± 1.73 8.64 ± 1.74 7.88 ± 1.67 TG Cells (% of dose) 0.92 ± 0.47 1.56 ± 0.15 1.35 ± 0.26 1.16 ± 0.45 1.27 ± 0.34 0.75 ± 0.13 12 ± 6 19 ± 2 17 ± 5 12 ± 4 14 ± 4 10 ± 4 Bile BEI (%) 0.39 ± 0.01 0.51 ± 0.02 0.43 ± 0.04 0.31 ± 0.04 0.24 ± 0.01 0.16 ± 0.02 0.19 ± 0.02 0.33 ± 0.01 0.33 ± 0.07 0.24 ± 0.03 0.17 ± 0.09 0.13 ± 0.03 33 ± 2 40 ± 1 42 ± 5 44 ± 4 37 ± 15 45 ± 10 Table 3.4. Kinetic parameters (min-1) estimated from compartmental pharmacokinetic modeling analysis TGZ (10 µM) was incubated in day 4 rat SCH as described under Materials and Methods. Parameter estimates were generated by nonlinear regression of the mean TGZ, TS, TG and TQ data in the medium, hepatocytes and bile based on the equations described in Materials and Methods according to the model scheme depicted in Figure 3.1. Parameters Estimates (min-1) CV% Kuptake, TGZ 0.007 2 Kuptake, TS 0.005 39 Kuptake, TG 0.001 384 Kf, TS 0.382 11 Kf, TG 0.061 12 Kf, TQ 0.001 19 Kother 0.064 25 Kefflux, TS 0.031 15 Kefflux, TG 0.16 29 Kbile, TS 0.023 17 Kbile, TG 0.061 44 Kflux, TS 0.17 22 Kflux, TG 0.094 55 105 Table 3.5. Evaluation of the sensitivity of cell and medium accumulation to detect changes in biliary excretion of TS or TG utilizing data from Monte Carlo simulations. Monte Carlo simulations were performed by modulating the rate constant for biliary excretion of TS or TG by 3 different scenarios [experimentally-determined parameter estimates; 2-fold increase; 2-fold decrease]. The relative power to detect a statistically significant difference in medium vs. cell was calculated as the ratio of the number of observations (sample size) necessary to detect a statistically significant difference in TS or TG accumulation in medium to the number of observations necessary for accumulation in hepatocytes. Time TG TS Number of Observations to Detect a Statistically Significant Difference Relative Power (min) (Ncell) (Nmedium) (NMedium/NCell) 10 20 47 2.4 20 14 19 1.4 30 14 108 7.7 60 15 662 44.1 90 15 1265 84.3 120 27 7271 269.3 10 27 144 5.3 20 9 28 3.1 30 6 13 2.2 60 4 18 4.5 90 4 33 8.3 120 5 51 10.2 106 Kflux,TS Kefflux,TS XTS,M Kbile,TS XTS,C XTS,B Kuptake,TS Kf,TS Kuptake, TGZ XTGZ,M XTGZ,C Kf,TQ Kf,TG XTQ,M Kother XTG,M Kefflux,TG XTG,C Kbile,TG XTG,B Kuptake,TG Kflux,TG Figure 3.1. Model scheme depicting the mass (X) of TGZ, TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone (TQ) in the medium (M), hepatocytes (C), and bile (B) of rat SCH. Kuptake, Kefflux and Kbile represent first-order rate constants for uptake, basolateral efflux and biliary excretion, respectively. Kf, TS, Kf, TG and Kf, TQ represent first-order rate constants for formation of TS, TG and TQ, respectively; Kflux, TS and Kflux, TG represent first-order rate constant for flux of TS and TG from the bile canaliculi into the incubation medium, respectively. Dotted line represents flux from bile canaliculi to medium that occurs during incubation after the 20-min timepoint. 107 BEI (%) 2.3 15 ± 4 34 ± 2 Accumulation (pmol/mg protein) 3000 2500 2000 1500 1000 500 0 TGZ TS TG Figure 3.2. Accumulation of TGZ, TS, and TG in rat SCH. Rat SCH were treated for 10 min with 5 µM TGZ, TS, or TG and accumulation (pmol/mg protein) was measured in cells + bile (solid bars) and in cells (open bars). Values represent the mean of triplicate determinations from n=1 experiment (TGZ) or mean ± SEM of triplicate determinations in n=3 experiments (TS and TG). 108 Accumulation in Medium (pmol/well) 10000 1000 100 10 0 20 40 60 80 100 120 Time (min) Figure 3.3. Mass vs. time profiles of TGZ (●), TS (○), TG (▼) and TQ (∆) in the medium initially containing 10 µM TGZ in day 4 rat SCH (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. 109 Accumulation in Hepatocytes (pmol/well) 10000 1000 100 10 1 0 20 40 60 80 100 120 Time (min) Figure 3.4. Mass vs. time profiles of TGZ (●), TS (○) and TG (▼) in the hepatocytes in day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. 110 Accumulation in Bile (pmol/well) 1000 100 10 1 0 20 40 60 80 100 120 Time (min) Figure 3.5. Mass vs. time profiles of TS (○) and TG (▼) in the bile canaliculi of day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. 111 A B C D Figure 3.6. Monte Carlo simulations were performed to examine the impact of modulation of TS or TG biliary excretion on the disposition of TS or TG in hepatocytes (A and C, respectively) and medium (B and D, respectively). The rate constant for biliary excretion of TS (Kbile,TS) or TG (Kbile,TG) was modulated by 5 different scenarios [experimentally-determined parameter estimates (black); 2-fold increase (red); 10-fold increase (green); 2-fold decrease (yellow); 10-fold decrease (blue)]. 112 CHAPTER 4 Modulation of Trabectedin (ET-743) Hepatobiliary Disposition by Multidrug Resistance-Associated Proteins (Mrps) May Prevent Hepatotoxicity This Chapter has been published in Toxicology and Applied Pharmacology (228(1):17-23, 2008) and is presented in the style of that journal. ABSTRACT Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-hr continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 µM) exhibited a 2-3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2deficient (TR-) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR- rats was decreased by ~75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR- rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or 114 Mrp4. 115 INTRODUCTION Trabectedin (Ecteinascidin 743, YondelisTM) is a DNA alkylating agent extracted from the marine tunicate Ecteinascidia turbinate that exhibits potent antineoplastic activity against a variety of tumor cells at nanomolar concentrations (Izbicka et al., 1998). Trabectedin is currently under phase II/III evaluation in advanced gastrointestinal stromal tumors, pretreated soft tissue sarcoma, ovarian cancer and breast cancer (Laverdiere et al., 2003; Robey et al., 2004; Yovine et al., 2004; Zelek et al., 2006). Trabectedin is metabolized primarily by CYP 3A4; 2C9, 2C19, 2D6 and 2E1, and to a lesser extent conjugated by the phase II enzymes uridine diphosphoglucuronosyl transferase (UGT) and glutathione-S-transferase (GST) (Allen et al., 2002; Brandon et al., 2006). Extensive metabolism of trabectedin was demonstrated in a mass balance study conducted in patients with advanced cancer (Beumer et al., 2007b). Following i.v. [14C]trabectedin administration to patients, ~55.5% and ~5.9% of the dose was recovered in feces and urine, respectively, suggesting that trabectedin and its metabolites undergo biliary excretion (Beumer et al., 2005a). However, the involvement of transport proteins in hepatobiliary disposition of trabectedin remains to be elucidated. During phase I/II clinical trials, the most prevalent adverse effect associated with trabectedin treatment was dose-limiting hepatotoxicity secondary to increases in blood alkaline phosphatase and/or bilirubin levels (Delaloge et al., 2001; Taamma et al., 2001; Lee et al., 2005a). An in vitro cytotoxicity study using HepG2 cells showed increased trabectedin-mediated toxicity after 1-hr preincubation with 116 inhibitors of cytochrome P450 (CYP) enzymes involved in trabectedin metabolism; CYP inducers and phase II enzyme inhibitors did not significantly affect trabectedin-mediated toxicity, suggesting that trabectedin metabolites may be less toxic compared to the parent compound (Brandon et al., 2005). Pretreatment of female Fisher rats with DEX has been shown to ameliorate the hepatotoxic effects of trabectedin while coadministration of DEX with trabectedin did not reduce toxicity (Donald et al., 2003). Consistent with the protective effect of DEX on trabectedin-mediated toxicity in rats, DEX premedication markedly reduced trabectedin-mediated hepatotoxicity in patients with advanced sarcoma (Grosso et al., 2006). However, this hepato-protective effect of DEX could not be mimicked in vitro using primary rat hepatocytes cultured on a fibronectin matrix (Donald et al., 2004). Significant induction of Mrp2 mRNA and protein by DEX treatment has been reported in primary rat and mouse hepatocytes (Kast et al., 2002; Luttringer et al., 2002; Turncliff et al., 2004). Mrp2, a transport protein localized on the hepatic canalicular plasma membrane, is responsible for the biliary excretion of organic anions as well as numerous drugs including pravastatin, vincristine and etoposide (Cui et al., 1999; Kawabe et al., 1999; Konig et al., 1999a; Sasaki et al., 2002). Animals with hereditary conjugated hyperbilirubinemia [Mrp2-deficient rats (TR-) and Eisai hyperbilirubinemic rats (EHBR)] have been used extensively to examine the substrate specificity and in vivo function of Mrp2 (Westley et al., 2006; ZamekGliszczynski et al., 2006a). Patients with Dubin-Johnson syndrome lack MRP2 and hence suffer from hereditary conjugated hyperbilirubinemia (Paulusma et al., 117 1997). In the absence of Mrp2/MRP2, rats and humans exhibit increased expression of basolateral Mrp3/MRP3 as a compensatory mechanism that enables the excretion of Mrp2/MRP2 substrates into the systemic circulation, thus avoiding excessive accumulation of organic anions in the hepatocyte (Kiuchi et al., 1998; Hirohashi et al., 1999; Johnson et al., 2006; Nies and Keppler, 2007). Drug-induced liver injury has emerged as the most common reason for the withdrawal of Food and Drug Administration-approved drugs from the market (Abboud and Kaplowitz, 2007; FDA, 2007). Covalent adduct formation, immunotoxicity, and disruption of cellular bioenergetics have been considered previously as mechanisms underlying hepatotoxicity, but more recent data suggest that hepatic transport proteins may be an important site of toxic interactions (Fattinger et al., 2001; Funk et al., 2001a; Leslie et al., 2007). The objective of this study was to evaluate the capability of sandwich-cultured primary rat hepatocytes (SCRH), which more closely mimic the metabolic and transport capabilities of the intact liver, to predict the hepato-protective effect of DEX against trabectedinmediated hepatotoxicity. An additional objective was to examine the role of Mrp2 in trabectedin hepatobiliary disposition. 118 MATERIALS AND METHODS Materials. Trabectedin (Ecteinascidin 743, YondelisTM, NSC 648766) was supplied by the National Cancer Institute, Developmental Therapeutics Program Repository (http://dtp.nci.nih.gov/branches/dscb/repo_open.html) and was prepared as a 10 mM stock solution in DMSO. DEX and BSO were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), human recombinant insulin, modified essential media non-essential amino acids, L-glutamine, penicillin and streptomycin were purchased from GIBCO (Invitrogen Corporation, Grand Island, NY). ITS+ (insulin, transferin, and selenium) was purchased from BD Biosciences (Bedford, MA). Animals. Male wild-type Wistar rats (220-300 g; Charles River Laboratories, Raleigh, NC) and male TR- Wistar rats (220-300 g; in-house breeding colony originally obtained from Dr. Mary Vore, University of Kentucky, Lexington, KY) were used for hepatocyte isolation and as liver donors for isolated perfused liver experiments. Male wild-type Wistar rats (>400 g) were used as blood donors for isolated perfused liver experiments. Rats were anesthetized with ketamine/xylazine (60/12 mg/kg intraperitoneally) before surgical manipulation. The Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill approved all procedures. Hepatocyte Isolation and Culture. Hepatocytes were isolated from male Wistar or TR- rats (220-300 g) by a modification of the two-step collagenase digestion method as described previously (Liu et al., 1998). determined by trypan blue exclusion. Hepatocyte viability was >85% as Hepatocytes were plated at a density of 119 ~1.5×106 cells/well in 6-well plates coated with 0.1 ml of rat tail collagen Type I solution (1.5 mg/ml, pH 7.4) in 1.5 ml of plating medium (phenol red-free DMEM containing 5% fetal bovine serum, 0.1 mM MEM nonessential amino acids, 2 mM Lglutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 4 µg/ml human recombinant insulin and 1 µM DEX), and allowed to attach for 2-3 hr at 37 ˚C in a humidified incubator with 95%O2/5%CO2. After attachment, medium was replaced to remove dead/unattached cells. To achieve a sandwich configuration, medium was aspirated and cells were overlaid with 0.1 ml of rat tail collagen Type I solution (1.5 mg/ml, pH 7.4) at 24 hr (day 1). Collagen was allowed to gel for 1 hr in a 37 ˚C humidified incubator 95%O2/5% CO2, and then culture medium was added (1.5 ml/well; phenol red-free DMEM supplemented with 0.1 mM MEM nonessential amino acids, 2 mM Lglutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1% ITS+ and 0.1 µM DEX). Subsequently, the culture medium was changed daily until the conduct of experiments. DEX and BSO Pretreatment in SCRH. From day 2 to day4, additional DEX (1 µM) or BSO (500 µM) was added to the culture medium containing 0.1 µM DEX. On day 3, SCRH were incubated for 24 hr in culture medium containing a range of trabectedin concentrations (1-1000 nM) to compare trabectedin-mediated cytotoxicity between SCRH pretreated with vehicle (0.1 µM DEX) and additional DEX (1 µM) or BSO (500 µM). DEX and trabectedin were prepared in DMSO, and BSO was solubilized in water. Stock solutions were diluted in culture medium for treatment. The final concentration of DMSO was <0.2% and had no effect on untreated controls. 120 Determination of Cell Viability Using LDH Release. LDH leakage into the culture medium was measured at the end of the 24 hr exposure using the Cytotoxicity Detection kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. The degree of LDH release was expressed as a percentage of the maximum cellular LDH release, which was measured by adding 2% Triton X-100 to SCRH. Cytotoxici ty (% of control) = LDH sample − LDHblank LDH Triton X -100 − LDHblank Western Blot Analysis of Mrp2, 3, 4 and CYP3A1/2 Proteins. On day 4, the medium was removed and cells were washed twice with ice-cold phosphate-buffered saline (PBS). Subsequently, cells were lysed with 400 µl of lysis buffer [1 mM EDTA, 1% sodium dodecyl sulfate (SDS), pH 8, with Complete protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany)] in each well of a 6-well plate. Lysates were stored at -20 ˚C until western blot analysis. The whole cell lysates were thawed on ice and protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). Protein samples (25 µg/well) were separated by SDS-polyacrylamide gel electrophoresis, and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Blots were blocked with Trisbuffered saline with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk for 1 hr at room temperature. Subsequently, the membrane was incubated with appropriate primary antibodies for 2 hr at room temperature or over-night at 4 ˚C and then rinsed three times at 10-min intervals with TBS-T. Mrp2, 3 and 4 were detected using monoclonal antibodies M2III-6, M3II-9 and M4I-10, respectively, at a 1:1000 dilution (Alexis Biochemicals, San Diego, CA). CYP3A1/2 protein expression was determined with a polyclonal antibody against rat CYP3A1/2 at a 1:1500 dilution 121 (Xenotech, Kansas City, KS). Protein loading was normalized to β-actin expression using a β-actin antibody at a dilution of 1:5000 (C4; Chemicon, San Fransisco, CA). Immunoreactive protein bands were detected by chemilumiscence using a Bio-Rad VersaDoc imaging system and densitometry analysis was achieved using the Quantity One software package v.4.1 (Bio-Rad Laboratories, Hercules, CA). Isolated Perfused Liver Study. Recirculating isolated perfused liver (IPL) studies were performed with male Wistar wild-type and TR- rat livers as described previously (Brouwer and Thurman, 1996). Briefly, livers were perfused in situ with oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) after cannulation of the bile duct and portal vein. Livers were removed from the body cavity and placed into a humidified perfusion chamber at 37 ˚C. Perfusion was continued with oxygenated buffer containing 20% (v/v) heparinized male rat blood at a flow rate of 20 ml/min. Livers were allowed to equilibrate for 10-15 min prior to bolus administration of 24 nmol of trabectedin (preliminary recovery studies in the perfusion system without the liver indicated that the actual dose delivered to the IPL was decreased by 50% due to nonspecific binding/degradation in blood-containing perfusate). Liver viability was assessed by monitoring portal pressure (<15 cm of H2O), observing gross morphology, and measuring bile flow in perfused livers from control and TR- rats (>0.8 and >0.3 µl/min/g liver, respectively). Taurocholate (0.5 µmol/min, in saline) was infused to maintain bile flow. Bile was collected in toto every 10 min, and the volume was determined gravimetrically (specific gravity 1.0). Perfusate samples (0.5 ml) were immediately centrifuged following collection to obtain plasma for analysis. 122 Analytical Methods. Bile and perfusate samples were analyzed by liquid chromatography with detection by tandem mass spectrometry (Applied Biosystems API 4000 triple quadrupole with TurboIonSpray interface, MDS Sciex, Concord, ON, Canada). Trabectedin and cimetidine (internal standard) were eluted from an Aquasil C18 column (dp=5µm, 2.1 x 50 mm, Thermo-Electron, Waltham, MA) using a mobile phase gradient (A: water with 0.1% formic acid, B: methanol with 0.1% formic acid); 0-2.5 min hold at 95% B, a linear gradient to 10% B from 2.5-3 min, 3-4 min hold at 10% B; flow rate was 0.75 mL/min (Shimadzu solvent delivery system, Columbia, MD). Trabectedin and cimetidine were detected in positive ion mode using multiple reaction monitoring; trabectedin: 744.2 495.2 m/z, cimetidine: 250.9 156.9 m/z. Trabectedin was quantified with calibration curves prepared in the appropriate matrix using analyte:internal standard (trabectedin:cimetidine) peak area ratios. The lower limit of detection was 1 nM for all analytes and standard curves ranged from 1 to 1000 nM. Statistical Analysis. Statistical significance was assessed using two-way analysis of variance with Bonferroni’s post hoc test, except where the groups being compared had unequal variances, in which case log-transformed data were assessed. SCRH data were presented as the mean ± S.E.M. when at least three sets of independent experiments were performed in triplicate. In all cases, p < 0.05 was considered to be statistically significant. 123 RESULTS DEX effect on trabectedin-mediated cytotoxicity in SCRH from wild-type rats Trabectedin-mediated cytotoxicity was defined as LDH release, relative to a Triton X-100 treated control, from hepatocytes into the culture medium. Following 2 hr of trabectedin exposure, LDH release was not increased relative to untreated SCRH (data not shown). However, cytotoxicity was observed after 24 hr of continuous trabectedin exposure to SCRH (Figure 4.1). From day 2 to day 4, additional DEX (1 µM) was added to the culture medium containing 0.1 µM DEX to examine the effect of DEX on trabectedin-mediated cytotoxicity; SCRH were incubated with trabectedin for 24 hr starting on day 3. This pretreatment of SCRH with additional DEX (1 µM) decreased toxicity of trabectedin compared to vehiclepretreated SCRH (Figure 4.1). Trabectedin-mediated cytotoxicity in SCRH from TR- rats Primary hepatocytes from Mrp2-deficient TR- rats were utilized to examine the involvement of Mrp2 in trabectedin-mediated cytotoxicity at 0.1 µM DEX. Unexpectedly, cytotoxicity caused by exposure to 50 nM and 100 nM trabectedin in TR- SCRH was reduced compared to that in wild-type SCRH (Figure 4.2). BSO effect on trabectedin-mediated cytotoxicity in wild-type and TR- SCRH Elevated levels of glutathione in TR- compared to wild-type rat hepatocytes has been proposed as one mechanism that can protect the hepatocyte from druginduced hepatotoxicity (Lu et al., 1996). To determine the effect of GSH depletion on trabectedin cytotoxicity, BSO (500 µM), an inhibitor of γ-glutamylcysteine synthetase which is the rate-liming step in GSH synthesis, was incubated for 24 hr prior to 124 trabectedin exposure in SCRH from wild-type and TR- rats (Griffith and Meister, 1979). Interestingly, toxicity in wild-type SCRH pretreated with BSO was reduced compared to vehicle-pretreated wild-type SCRH (Figure 4.3). Trabectedin-mediated cytotoxicity in BSO-pretreated TR- SCRH was not different from vehicle-pretreated TR- SCRH (data not shown). Alteration of Mrp2, 3, 4 and CYP3A1/2 protein expression by DEX and BSO When SCRH were cultured with additional DEX (1 µM), the fold increase in expression of CYP3A1/2 and Mrp2 was 5.0 ± 2.5 and 2.5 ± 0.6, respectively, compared to vehicle, whereas the fold increase in protein expression of Mrp3 and Mrp4 was 1.2 ± 0.7 and 1.4 ± 0.4, respectively (Figure 4.4). Interestingly, in BSOpretreated SCRH, the fold increase in Mrp4 expression was 3.1 ± 0.7 whereas the fold increase in Mrp2, Mrp3 and CYP3A1/2 protein expression was 1.5 ± 0.7, 1.9 ± 0.4 and 1.2 ± 0.9, respectively. Hepatobiliary disposition of trabectedin in IPLs IPLs from wild-type and TR- rats were utilized to examine the involvement of Mrp2 in trabectedin hepatobiliary disposition. Trabectedin concentrations in perfusate were not different between livers from wild-type and TR- rats; trabectedin was rapidly extracted by the liver and concentrations were negligible after 20 min (Figure 4.5.a). Biliary recovery of trabectedin represented less than 3% of the dose in wild-type rat livers, consistent with extensive metabolism of trabectedin in rats as reported previously (Figure 4.5.b) (Allen et al., 2002). Biliary recovery of trabectedin in IPLs from TR- rats was decreased by ~75% compared to wild-type IPLs. 125 DISCUSSION SCRH were selected to investigate the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity because they maintain metabolic enzyme activity and develop functional bile canalicular networks with proper localization of hepatic transport proteins (Liu et al., 1998; LeCluyse et al., 1999; Liu et al., 1999a; Hoffmaster et al., 2004a). Previous experiments in freshly isolated rat hepatocytes cultured in a conventional configuration were unable to replicate the hepatoprotective effect of DEX against trabectedin-mediated cytotoxicity when rats were pretreated orally with 20 mg/kg/day of DEX 24 hr prior to hepatocyte isolation (Donald et al., 2004). When freshly isolated hepatocytes are cultured on a single collagen substratum (conventional configuration), hepatocytes do not form bile canalicular networks, do not re-establish cell polarity, and gradually lose expression of CYP enzymes (LeCluyse et al., 1996; George et al., 1997). The loss of CYP expression over time in culture of human hepatocytes varies for each isoform (George et al., 1997); mRNA levels of some P450s such as CYP1A2 and CYP2C9 did not change over time in culture whereas CYP2E1 and CYP3A4 expression declined gradually over time in culture. Therefore, induction of CYP3A protein by DEX prior to hepatocyte isolation may be lost during culture in a conventional configuration. In contrast, incubation of SCRH with additional DEX (1 µM) increased CYP3A1/2 protein levels compared to vehicle-treated SCRH (Figure 4.4) and may provide hepato-protection from trabectedin-mediated cytotoxicity. Vectorial transport studies in polarized cell monolayers examining cellular accumulation of [14C] trabectedin in the absence and presence of P-gp inhibitors 126 indicated that trabectedin is a substrate for P-gp (Beumer et al., 2007a). However, P-gp was only able to confer resistance to trabectedin when expressed at very high levels, suggesting that the intrinsic clearance of P-gp for trabectedin is low. Even though trabectedin seems to be more toxic than its metabolites based on previous cytotoxicity studies (Brandon et al., 2005), limited information is available on the interaction of other hepatic transport proteins with trabectedin. The present studies are the first to demonstrate the involvement of Mrp2 in the biliary excretion of trabectedin in whole rat livers. Although the biliary excretion of unchanged trabectedin was low as a percentage of the total dose, the biliary excretion of trabectedin in IPLs from TR- rats was decreased ~75% compared to wild-type IPLs (Figure 4.5.b). As shown in Figure 4.4, induction of Mrp2 protein expression by additional DEX (1µM) in SCRH may provide hepato-protection from trabectedin-mediated cytotoxicity by increasing the biliary excretion of parent compound and/or derived metabolites. Interestingly, TR- SCRH were more resistant to trabectedin-mediated cytotoxicity relative to wild-type controls, despite the increased potential for trabectedin accumulation in TR- hepatocytes due to impaired biliary excretion. This observation could be explained by modulation of the expression/activity of hepatic basolateral efflux transport proteins and metabolic enzymes in TR- rats. Mrp2- deficient rats are well-known to exhibit higher Mrp3 expression (Hirohashi et al., 1998; Ogawa et al., 2000; Johnson et al., 2006). Mrp3 mediates hepatic basolateral efflux of substrates from hepatocytes to the blood, and is thought to compensate for impaired biliary excretion of organic anions during Mrp2 deficiency (Hirohashi et al., 127 2000; Xiong et al., 2000). In addition, the expression and activity of several phase I and II metabolic enzymes that may be involved in trabectedin detoxification are modulated in TR- rats. For example, the total P450 content and activity of CYP1A1/2 and CYP2B1/2 were significantly higher in TR- compared to wild-type rats, although testosterone 6β-hydroxylation mediated by CYP3A1/2 was lower in TR- compared to wild-type rats (Newton et al., 2005; Silva et al., 2005). TR- rats exhibit up-regulated UGT1a (Johnson et al., 2006). Since Mrp2 plays an important role in GSH biliary excretion, impaired function of Mrp2 in TR- rats has been attributed to higher hepatocellular concentrations of GSH relative to wild-type rats (Lu et al., 1996). The important role of GSH in detoxification of the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine, was demonstrated recently in TR- rats (Silva et al., 2005); TR- rats were more resistant to acetaminophen hepatotoxicity compared to wild-type rats, but this protection against acetaminophen hepatotoxicity in TR- rats was completely reversed by BSO treatment. Previous studies in cultured rat hepatocytes have documented that 200 µM BSO reduced GSH levels by 50% after 5 hr (Sinbandhit-Tricot et al., 2003). Interestingly, in the present study, pretreatment of SCRH with BSO (500 µM) for 48 hr decreased trabectedin–mediated cytotoxicity relative to vehicle-pretreated SCRH. A novel finding in the current study was induction of Mrp4 in SCRH associated with BSO treatment. MRP4 is a basolateral protein involved in the transport of xenobiotics and endogenous substrates such as cyclic nucleotides (cAMP and cGMP) and bile acids (van Aubel et al., 2002; Reid et al., 2003b; Rius et al., 2003; Tian et al., 2006). MRP4 mediates the efflux of GSH across the 128 basolateral membrane of hepatocytes into blood by co-transport with monoanionic bile acids (Rius et al., 2003). BSO may play a role as a GSH-depleting agent not only by inhibiting synthesis, but also by enhancing hepatic efflux of GSH out of the hepatocyte by increasing the expression of Mrp4. Whether upregulation of Mrp4 by BSO is a direct effect, or an indirect response associated with oxidative stress due to GSH depletion, requires further investigation. MRP4 mRNA and protein expression were significantly up-regulated in liver samples from patients with cholestasis, indicating a potential role of MRP4 in protecting the liver by decreasing the intracellular accumulation of detergent-like bile acids (Gradhand et al., 2007). Hence, the protective role of BSO in trabectedin-mediated cytotoxicity may be due to increased efflux of trabectedin or toxic metabolites by Mrp4, although the affinity of trabectedin for Mrp4 is unknown. In conclusion, the hepato-protective effect of DEX on trabectedin toxicity may be due to increased metabolism by CYP3A1/2 as well as enhanced biliary excretion by Mrp2. Although the interaction of trabectedin and/or derived metabolites with basolateral transport proteins such as Mrp3 and 4 remains to be elucidated, the unexpected attenuation of trabectedin toxicity in SCRH from TR- rats, and in SCRH pretreated with BSO, may be associated with increased basolateral excretion of trabectedin /metabolites by Mrp3 and/or Mrp4. 129 REFERENCES Beumer, J. H., Buckle, T., Ouwehand, M., Franke, N. E., Lopez-Lazaro, L., Schellens, J. H., Beijnen, J. H., van Tellingen, O.,2007a. Trabectedin (ET-743, Yondelis) is a substrate for P-glycoprotein, but only high expression of Pglycoprotein confers the multidrug resistance phenotype. Invest New Drugs. 25, 1-7. Beumer, J. H., Rademaker-Lakhai, J. M., Rosing, H., Hillebrand, M. J., Bosch, T. M., Lopez-Lazaro, L., Schellens, J. H., Beijnen, J. H.,2007b. Metabolism of trabectedin (ET-743, Yondelis) in patients with advanced cancer. Cancer Chemother Pharmacol. 59, 825-837. Beumer, J. H., Rademaker-Lakhai, J. M., Rosing, H., Lopez-Lazaro, L., Beijnen, J. H., Schellens, J. H.,2005. Trabectedin (Yondelis, formerly ET-743), a mass balance study in patients with advanced cancer. Invest New Drugs. 23, 429436. Blay, J. Y., Le Cesne, A., Verweij, J., Scurr, M., Seynaeve, C., Bonvalot, S., Hogendoorn, P., Jimeno, J., Evrard, V., van Glabbeke, M., Judson, I.,2004. A phase II study of ET-743/trabectedin ('Yondelis') for patients with advanced gastrointestinal stromal tumours. Eur J Cancer. 40, 1327-1331. Brandon, E. F., Meijerman, I., Klijn, J. S., den Arend, D., Sparidans, R. W., Lazaro, L. L., Beijnen, J. H., Schellens, J. H.,2005. In-vitro cytotoxicity of ET-743 (Trabectedin, Yondelis), a marine anti-cancer drug, in the Hep G2 cell line: influence of cytochrome P450 and phase II inhibition, and cytochrome P450 induction. Anticancer Drugs. 16, 935-943. Brandon, E. F., Sparidans, R. W., Guijt, K. J., Lowenthal, S., Meijerman, I., Beijnen, J. H., Schellens, J. H.,2006. In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis, Trabectidin), a novel marine anti-cancer drug. Invest New Drugs. 24, 3-14. Brouwer, K. L., Thurman, R. G.,1996. Isolated perfused liver. Pharm Biotechnol. 8, 161-192. Cui, Y., Konig, J., Buchholz, J. K., Spring, H., Leier, I., Keppler, D.,1999. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol. 55, 929-937. Delaloge, S., Yovine, A., Taamma, A., Riofrio, M., Brain, E., Raymond, E., Cottu, P., Goldwasser, F., Jimeno, J., Misset, J. L., Marty, M., Cvitkovic, E.,2001. Ecteinascidin-743: a marine-derived compound in advanced, pretreated sarcoma patients--preliminary evidence of activity. J Clin Oncol. 19, 12481255. 130 Donald, S., Verschoyle, R. D., Greaves, P., Gant, T. W., Colombo, T., Zaffaroni, M., Frapolli, R., Zucchetti, M., D'Incalci, M., Meco, D., Riccardi, R., Lopez-Lazaro, L., Jimeno, J., Gescher, A. J.,2003. Complete protection by high-dose dexamethasone against the hepatotoxicity of the novel antitumor drug yondelis (ET-743) in the rat. Cancer Res. 63, 5902-5908. Donald, S., Verschoyle, R. D., Greaves, P., Orr, S., Jimeno, J., Gescher, A. J.,2004. Comparison of four modulators of drug metabolism as protectants against the hepatotoxicity of the novel antitumor drug yondelis (ET-743) in the female rat and in hepatocytes in vitro. Cancer Chemother Pharmacol. 53, 305-312. Fattinger, K., Funk, C., Pantze, M., Weber, C., Reichen, J., Stieger, B., Meier, P. J.,2001. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther. 69, 223-231. FDA,2007. Drug induced liver toxicity. Funk, C., Pantze, M., Jehle, L., Ponelle, C., Scheuermann, G., Lazendic, M., Gasser, R.,2001. Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology. 167, 83-98. George, J., Goodwin, B., Liddle, C., Tapner, M., Farrell, G. C.,1997. Time-dependent expression of cytochrome P450 genes in primary cultures of welldifferentiated human hepatocytes. J Lab Clin Med. 129, 638-648. Gradhand, U., Lang, T., Schaeffeler, E., Glaeser, H., Tegude, H., Klein, K., Fritz, P., Jedlitschky, G., Kroemer, H. K., Bachmakov, I., Anwald, B., Kerb, R., Zanger, U. M., Eichelbaum, M., Schwab, M., Fromm, M. F.,2007. Variability in human hepatic MRP4 expression: influence of cholestasis and genotype. Pharmacogenomics J. Griffith, O. W., Meister, A.,1979. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem. 254, 7558-7560. Grosso, F., Dileo, P., Sanfilippo, R., Stacchiotti, S., Bertulli, R., Piovesan, C., Jimeno, J., D'Incalci, M., Gescher, A., Casali, P. G.,2006. Steroid premedication markedly reduces liver and bone marrow toxicity of trabectedin in advanced sarcoma. Eur J Cancer. 42, 1484-1490. Hirohashi, T., Suzuki, H., Chu, X. Y., Tamai, I., Tsuji, A., Sugiyama, Y.,2000. Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2). J Pharmacol Exp Ther. 292, 265-270. 131 Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., Sugiyama, Y.,1998. Hepatic expression of multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats. Mol Pharmacol. 53, 10681075. Hirohashi, T., Suzuki, H., Sugiyama, Y.,1999. Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J Biol Chem. 274, 15181-15185. Hoffmaster, K. A., Turncliff, R. Z., LeCluyse, E. L., Kim, R. B., Meier, P. J., Brouwer, K. L.,2004. P-glycoprotein expression, localization, and function in sandwichcultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm Res. 21, 1294-1302. Izbicka, E., Lawrence, R., Raymond, E., Eckhardt, G., Faircloth, G., Jimeno, J., Clark, G., Von Hoff, D. D.,1998. In vitro antitumor activity of the novel marine agent, ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients. Ann Oncol. 9, 981-987. Johnson, B. M., Zhang, P., Schuetz, J. D., Brouwer, K. L.,2006. Characterization of transport protein expression in multidrug resistance-associated protein (Mrp) 2-deficient rats. Drug Metab Dispos. 34, 556-562. Kaplowitz, N.,2005. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov. 4, 489499. Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A. M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M., Edwards, P. A.,2002. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem. 277, 2908-2915. Kawabe, T., Chen, Z. S., Wada, M., Uchiumi, T., Ono, M., Akiyama, S., Kuwano, M.,1999. Enhanced transport of anticancer agents and leukotriene C4 by the human canalicular multispecific organic anion transporter (cMOAT/MRP2). FEBS Lett. 456, 327-331. Kiuchi, Y., Suzuki, H., Hirohashi, T., Tyson, C. A., Sugiyama, Y.,1998. cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3). FEBS Lett. 433, 149-152. Konig, J., Nies, A. T., Cui, Y., Leier, I., Keppler, D.,1999. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta. 1461, 377-394. Laverdiere, C., Kolb, E. A., Supko, J. G., Gorlick, R., Meyers, P. A., Maki, R. G., Wexler, L., Demetri, G. D., Healey, J. H., Huvos, A. G., Goorin, A. M., 132 Bagatell, R., Ruiz-Casado, A., Guzman, C., Jimeno, J., Harmon, D.,2003. Phase II study of ecteinascidin 743 in heavily pretreated patients with recurrent osteosarcoma. Cancer. 98, 832-840. LeCluyse, E., Bullock, P., Madan, A., Carroll, K., Parkinson, A.,1999. Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes. Drug Metab Dispos. 27, 909-915. LeCluyse, E. L., Bullock, P. L., Parkinson, A., Hochman, J. H.,1996. Cultured rat hepatocytes. Pharm Biotechnol. 8, 121-159. Leslie, E. M., Watkins, P. B., Kim, R. B., Brouwer, K. L.,2007. Differential inhibition of rat and human Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1)by bosentan: a mechanism for species differences in hepatotoxicity. J Pharmacol Exp Ther. 321, 1170-1178. Liu, X., Brouwer, K. L., Gan, L. S., Brouwer, K. R., Stieger, B., Meier, P. J., Audus, K. L., LeCluyse, E. L.,1998. Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration. Pharm Res. 15, 1533-1539. Liu, X., Chism, J. P., LeCluyse, E. L., Brouwer, K. R., Brouwer, K. L.,1999. Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab Dispos. 27, 637-644. Lu, S. C., Cai, J., Kuhlenkamp, J., Sun, W. M., Takikawa, H., Takenaka, O., Horie, T., Yi, J., Kaplowitz, N.,1996. Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats. Hepatology. 24, 253-258. Luttringer, O., Theil, F. P., Lave, T., Wernli-Kuratli, K., Guentert, T. W., de Saizieu, A.,2002. Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem Pharmacol. 64, 1637-1650. Newton, D. J., Wang, R. W., Evans, D. C.,2005. Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats. Life Sci. 77, 1106-1115. Nies, A. T., Keppler, D.,2007. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 453, 643-659. Ogawa, K., Suzuki, H., Hirohashi, T., Ishikawa, T., Meier, P. J., Hirose, K., Akizawa, T., Yoshioka, M., Sugiyama, Y.,2000. Characterization of inducible nature of MRP3 in rat liver. Am J Physiol Gastrointest Liver Physiol. 278, G438-446. Paulusma, C. C., Kool, M., Bosma, P. J., Scheffer, G. L., ter Borg, F., Scheper, R. J., Tytgat, G. N., Borst, P., Baas, F., Oude Elferink, R. P.,1997. A mutation in the 133 human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology. 25, 1539-1542. Reid, G., Wielinga, P., Zelcer, N., van der Heijden, I., Kuil, A., de Haas, M., Wijnholds, J., Borst, P.,2003. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A. 100, 9244-9249. Reid, J. M., Kuffel, M. J., Ruben, S. L., Morales, J. J., Rinehart, K. L., Squillace, D. P., Ames, M. M.,2002. Rat and human liver cytochrome P-450 isoform metabolism of ecteinascidin 743 does not predict gender-dependent toxicity in humans. Clin Cancer Res. 8, 2952-2962. Rius, M., Nies, A. T., Hummel-Eisenbeiss, J., Jedlitschky, G., Keppler, D.,2003. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology. 38, 374-384. Ryan, D. P., Supko, J. G., Eder, J. P., Seiden, M. V., Demetri, G., Lynch, T. J., Fischman, A. J., Davis, J., Jimeno, J., Clark, J. W.,2001. Phase I and pharmacokinetic study of ecteinascidin 743 administered as a 72-hour continuous intravenous infusion in patients with solid malignancies. Clin Cancer Res. 7, 231-242. Sasaki, M., Suzuki, H., Ito, K., Abe, T., Sugiyama, Y.,2002. Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2). J Biol Chem. 277, 6497-6503. Silva, V. M., Thibodeau, M. S., Chen, C., Manautou, J. E.,2005. Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity. Biochem Pharmacol. 70, 1832-1839. Sinbandhit-Tricot, S., Cillard, J., Chevanne, M., Morel, I., Cillard, P., Sergent, O.,2003. Glutathione depletion increases nitric oxide-induced oxidative stress in primary rat hepatocyte cultures: involvement of low-molecular-weight iron. Free Radic Biol Med. 34, 1283-1294. Taamma, A., Misset, J. L., Riofrio, M., Guzman, C., Brain, E., Lopez Lazaro, L., Rosing, H., Jimeno, J. M., Cvitkovic, E.,2001. Phase I and pharmacokinetic study of ecteinascidin-743, a new marine compound, administered as a 24hour continuous infusion in patients with solid tumors. J Clin Oncol. 19, 12561265. Tian, Q., Zhang, J., Chan, S. Y., Tan, T. M., Duan, W., Huang, M., Zhu, Y. Z., Chan, E., Yu, Q., Nie, Y. Q., Ho, P. C., Li, Q., Ng, K. Y., Yang, H. Y., Wei, H., Bian, J. S., Zhou, S. F.,2006. Topotecan is a substrate for multidrug resistance associated protein 4. Curr Drug Metab. 7, 105-118. 134 Turncliff, R. Z., Meier, P. J., Brouwer, K. L.,2004. Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes. Drug Metab Dispos. 32, 834-839. van Aubel, R. A., Smeets, P. H., Peters, J. G., Bindels, R. J., Russel, F. G.,2002. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol. 13, 595-603. Westley, I. S., Brogan, L. R., Morris, R. G., Evans, A. M., Sallustio, B. C.,2006. Role of Mrp2 in the hepatic disposition of mycophenolic acid and its glucuronide metabolites: effect of cyclosporine. Drug Metab Dispos. 34, 261-266. Xiong, H., Turner, K. C., Ward, E. S., Jansen, P. L., Brouwer, K. L.,2000. Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats. J Pharmacol Exp Ther. 295, 512-518. Yovine, A., Riofrio, M., Blay, J. Y., Brain, E., Alexandre, J., Kahatt, C., Taamma, A., Jimeno, J., Martin, C., Salhi, Y., Cvitkovic, E., Misset, J. L.,2004. Phase II study of ecteinascidin-743 in advanced pretreated soft tissue sarcoma patients. J Clin Oncol. 22, 890-899. Zamek-Gliszczynski, M. J., Hoffmaster, K. A., Humphreys, J. E., Tian, X., Nezasa, K., Brouwer, K. L.,2006. Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat. J Pharmacol Exp Ther. 319, 459-467. Zelek, L., Yovine, A., Brain, E., Turpin, F., Taamma, A., Riofrio, M., Spielmann, M., Jimeno, J., Misset, J. L.,2006. A phase II study of Yondelis (trabectedin, ET743) as a 24-h continuous intravenous infusion in pretreated advanced breast cancer. Br J Cancer. 94, 1610-1614. 135 Cytotoxicity (% of control) 35 30 25 20 * 15 10 * 5 * * 0 1 10 50 100 1000 Trabectedin (nM) Figure 4.1. Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated from wild-type Wistar rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1-1000 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (0.1 µM DEX; solid bars) or additional DEX (1 µM; white bars); *, p < 0.05. 136 Cytotoxicity (% of control) 35 30 25 20 15 10 * * 50 100 5 0 1 10 Trabectedin (nM) Figure 4.2. Trabectedin-mediated cytotoxicity in SCRH from wild-type and TR- rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1 - 100 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from wild-type (solid bars) or TR- (white bars) SCRH; *, p < 0.05. 137 Cytotoxicity (% of control) 35 30 25 20 * 15 10 5 * * 0 50 100 1000 Trabectedin (nM) Figure 4.3. Effect of BSO on trabectedin-mediated cytotoxicity. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (50, 100 and 1000 nM) exposure. BSO (500 µM) was added to the culture medium 24 h prior to trabectedin exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (solid bars) or BSO-pretreated (white bars) SCRH; *, p < 0.05. 138 Mrp2 Mrp3 Actin CYP3A1/2 Mrp4 Actin Figure 4.4. Representative Western blot from whole cell lysates for Mrp2, 3, 4 and CYP3A1/2 in SCRH pretreated with vehicle (0.1 µM DEX), additional DEX (1 µM) or BSO (500 µM) (25 µg protein/well; n=3). 139 Trabectedin in Perfusate (nM) A 60 50 40 30 20 10 0 0 5 10 20 30 40 50 60 70 80 90 B Trabectedin Biliary Excretion (% of dose) Time (min) 4 3 2 1 0 0 5 10 20 30 40 50 60 70 80 90 Time (min) Figure 4.5. Trabectedin perfusate concentrations (A) and cumulative biliary excretion (B) in isolated perfused livers from wild-type (solid circle) and TR- (white triangle) rats. Data are presented as mean ± S.D., n= 3/group. 140 CHAPTER 5 Interactions Between Sulindac, Sulindac Sulfone, Sulindac Sulfide and Hepatic Transport Proteins This Chapter will be submitted to the Journal of Pharmacology and Experimental Therapeutics ABSTRACT Sulindac, a nonsteroidal anti-inflammatory drug (NSAID) used clinically to relieve symptoms of rheumatoid arthritis and osteoarthritis, is associated with a higher incidence of hepatotoxicity compared to other NSAIDS. The role of hepatic transport proteins in sulindac-mediated hepatotoxicity remains to be evaluated. Accordingly, model substrates for hepatic transport proteins [[3H]taurocholate (TC), [3H]estradiol 17-β-glucuronide (E217G), nitrofurantoin (NF)] were utilized in rat and human sandwich-cultured hepatocytes (SCH) and suspended hepatocytes to determine whether sulindac and/or sulindac metabolites (S-sulfone and S-sulfide) interfere with hepatic transport protein activity. In rat SCH, compared to control, Ssulfone and S-sulfide (100 µM) decreased TC biliary excretion index (BEI) by 42% and 55%, respectively, but had no effect on E217G BEI; in contrast, sulindac/metabolites (100 µM) decreased both TC and E217G in vitro biliary clearance (Clbiliary) by 38-83% and 81-97%, respectively, consistent with impaired uptake of TC and E217G and impaired excretion of TC. In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na+-dependent TC initial uptake (IC50 of 25.6±10.7 and 12.7±5.3 µM, respectively) and Na+-independent E217G initial uptake (IC50 of 12.6±5.1 and 6.5±1.2 µM, respectively). In rat SCH, compared to control, sulindac, S-sulfone and S-sulfide (100 µM) decreased NF BEI and Clbiliary by 30-57%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na+dependent TC initial uptake (IC50 of 42.2 and 3.1 µM, respectively). In conclusion, inhibition of multiple hepatic transport proteins (Na+/taurocholate cotransporting protein, organic anion transporting polypeptides, bile salt export pump, breast cancer 142 resistance protein) by sulindac/metabolites may play a role in clinically significant drug interactions and/or liver injury. 143 INTRODUCTION Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) indicated for the relief of signs and symptoms of arthritic conditions, including osteoarthritis and rheumatoid arthritis. Sulindac is reduced by the aldehyde oxidase system to sulindac sulfide (S-sulfide) (Figure 5.1), which has analgesic and anti-inflammatory properties. S-sulfide is converted by flavin-containing monooxygenase 3 back to sulindac and to sulindac sulfone (S-sulfone), which has been recognized as a promising antiproliferative agent in colon cancer (Tatsumi et al., 1983; Davies and Watson, 1997; Hamman et al., 2000). Among NSAIDs, sulindac has been associated most consistently with hepatotoxicity (Aithal and Day, 2007), particularly with cholestatic hepatitis, and to a lesser extent with hepatocellular damage (Tarazi et al., 1993). Despite the high incidence of liver injury, the mechanism(s) underlying sulindacmediated hepatotoxicity remain(s) to be elucidated. Inhibition of hepatic transport proteins has been proposed as one mechanism of drug-induced liver injury (DILI). Inhibition of the canalicular bile salt export pump in rats (Bsep; ABCB11) by hepatotoxic drugs such as troglitazone and bosentan may lead to elevated hepatic concentrations of detergent-like bile acids, which can disrupt cellular function (Fattinger et al., 2001; Funk et al., 2001a). Several hepatic transport proteins other than Bsep also are involved in bile acid transport, acting in concert to maintain homeostasis of bile acids. In humans, conjugated and unconjugated bile acids are taken up primarily by Na+-dependent taurocholate cotransporting polypeptide (NTCP; SLC10A1) with high affinity, as well as by Na+independent organic anion transporting polypeptides (OATPs; SLCOs). Once taken 144 up by the hepatocytes, bile acids are excreted into the canalicular lumen primarily by the bile salt export pump (BSEP). Alternatively, bile acids can be excreted into sinusoidal blood by basolateral hepatic transport proteins such as the multidrug resistance-associated protein (MRP; ABCC) 3, MRP4 and the organic solute transporter (OST) α/β (Kosters and Karpen, 2008). The involvement of hepatic transport proteins in DILI and some drug interactions has been established (Smith et al., 2005b; Pauli-Magnus and Meier, 2006). Furthermore, interactions between sulindac and hepatic transport proteins have been demonstrated. Sulindac inhibited organic anion transporter (OAT) 1mediated [14C]para-aminohippurate uptake (IC50 ~36 µM) and OAT3-mediated [3H]estrone sulfate uptake (IC50 ~3 µM) in stably transfected cells; sulindac also was the most potent inhibitor among tested NSAIDs of organic cation transporter (OCT) 1 and 2 in stably transfected cells (Khamdang et al., 2002). Sulindac also inhibited [3H]methotrexate transport in MRP2- and MRP4-overexpressing membrane vesicles, with IC50 values of ~38 µM and ~2 µM, respectively (El-Sheikh et al., 2006); MRP2 functions to excrete compounds from the hepatocyte into bile. Finally, S-sulfide was shown to be a potent inhibitor of MRP4-mediated leukotriene B4 and C4 transport in MRP4-overexpressing membrane vesicles (Rius et al., 2008). Sulindac is metabolized extensively by the liver in rats as well as in humans, and plasma concentrations of sulindac metabolites are sustained due to extensive enterohepatic recycling (Dujovne et al., 1983). In rats, S-sulfone is the major metabolite in the plasma. After intravenous administration of sulindac (10 mg/kg) in rats, the plasma area under the concentration-time curve (AUC) for S-sulfone was 145 ~2.5-fold higher than that of sulindac, whereas the AUC for S-sulfide was only ~40% that of sulindac (Duggan et al., 1978); Clbiliary of sulindac and S-sulfone were comparable, whereas S-sulfide Clbiliary was only 40% of that of sulindac. In humans, after oral administration of sulindac, peak plasma total concentrations (Cmax) were achieved after 1 h for sulindac (10-34 µM), and after 2-4 h for S-sulfone (4-19 µM) and S-sulfide (3-33 µM) (Davies and Watson, 1997); the apparent Clbiliary of sulindac and S-sulfone, measured by an occludable T-tube that was placed in the common bile duct of patients following elective gallbladder surgery, was ~25-fold and ~18-fold greater, respectively, than that of S-sulfide (Dobrinska et al., 1983). Several groups have reported a proportional relationship between S-sulfide concentrations and the incidence of hepatotoxicity, emphasizing the potential for metabolites, in addition to the parent drug to cause hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986). The aim of the present study was to examine the interactions between hepatic transport proteins and sulindac, S-sulfone and S-sulfide. First, interactions between sulindac/metabolites and the model substrates [3H]taurocholate (TC; substrate for Ntcp/Oatp/Bsep), [3H]estradiol 17-β-glucuronide (E217G; substrate for Oatp/Mrp2) and nitrofurantoin (NF; substrate for breast cancer resistance protein (Bcrp, a canalicular efflux transport protein) were examined in rat sandwich-cultured hepatocytes (SCH). Second, the inhibitory potency (IC50) of sulindac metabolites on Na+-dependent TC initial uptake and Na+-independent E217G initial uptake was examined in freshly-isolated rat hepatocytes. Third, the impact of sulindac/metabolites on the hepatobiliary disposition of TC was examined in human SCH, and suspended human hepatocytes were used to determine the IC50 of 146 sulindac metabolites on Na+-dependent TC initial uptake. Results suggested that inhibition of multiple transport proteins by sulindac/metabolites could play a role in sulindac-mediated liver injury. 147 MATERIALS AND METHODS Chemicals and Reagents. [3H]TC, [3H]E217G and [14C]inulin were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Dulbecco’s modified Eagle’s medium (DMEM) and MEM non-essential amino acids were purchased from Invitrogen (Carlsbad, CA). BioCoatTM culture plates, MatrigelTM extracellular matrix, and insulin/transferrin/selenium culture supplement (ITSTM) were purchased from BD Biosciences Discovery Labware (Bedford, MA). NF, TC, E217G, sulindac, S-sulfone, S-sulfide, penicillin-streptomycin solution, dexamethasone, Hanks’ Balanced Salt Solutions (HBSS), HBSS modified (HBSS without Ca2+ and Mg+) and Triton X-100 were purchased from Sigma-Aldrich, Inc (St. Louis, MO). All other chemicals and reagents were of analytical grade and were readily available from commercial sources. Culture of Primary Rat and Human Hepatocytes in a Sandwich Configuration. Primary rat hepatocytes were cultured as described previously (Marion et al., 2007). Briefly, hepatocytes were seeded at a density of 1.75×106 cells/well onto 6-well BioCoatTM plates. Approximately 24 hours later, hepatocytes were overlaid with 2 mL of 0.25 mg/mL BD MatrigelTM basement membrane matrix in DMEM containing 1% (v/v) ITS+TM, 0.1 µM dexamethasone, 2 mM L-glutamine, 1% (v/v) MEM nonessential amino acids, 100 units penicillin G sodium/mL and 100 µg streptomycin sulfate/mL. CellzDirect (Durham, NC) kindly donated human hepatocytes cultured in 6-well plates, seeded at a density of 1.5×106 cells/well, and overlaid with Matrigel™. Hepatocytes were cultured for 4 days (rat) or 7-8 days (human) to allow extensive 148 formation of canalicular networks between cells before experimentation; medium was changed daily. Transport Studies using Rat and Human Sandwich-Cultured Hepatocytes (SCH). The method to determine substrate accumulation in SCH has been described previously (Liu et al., 1999e). Briefly, SCH were washed twice with 2 ml of warm HBSS containing Ca2+ (standard; cells+bile) or Ca2+-free (cells), followed by incubation in the same buffer for 10 min at 37˚C. Subsequently, cells were incubated at 37˚C for 10 min with 1.5 mL standard HBSS containing NF (5 µM), [3H]TC (1 µM; 100 nCi/mL), or [3H]E217G (1 µM; 100 nCi/mL) in the presence of sulindac or its metabolites (0-100 µM). After 10 min, cells were washed 3 times with ice-cold standard HBSS. Cells were lysed either with 1 mL of methanol/water (70/30; v/v) (NF studies) or 0.5% (v/v) Triton X-100 in phosphate-buffered saline (TC and E217G studies), and plates were shaken for at least 20 min. Samples of cells+bile and cells were quantified by HPLC-MS/MS for NF, and by liquid scintillation spectroscopy (Packard Tricarb, Packard Corp., Meriden, CT) for [3H]TC or [3H]E217G, and transport function was normalized to the protein content of the hepatocytes using a BCA protein assay (Pierce, Rockford, IL). The biliary excretion index (BEI; %) and in vitro biliary clearance (Clbiliary; mL/min/kg) were calculated using B-CLEAR® technology (Qualyst, Inc., Raleigh, NC) based on the following equations: BEI(%) = Accumulation cells +bile − Accumulation cells × 100 Accumulation cells +bile In Vitro Biliary Clearance (mL/min/kg ) = Accumulation cells +bile − Accumulation cells AUC medium 149 where AUCmedium represents the area under the substrate concentration-time curve, which was determined by multiplying the initial substrate concentration in the incubation medium by the incubation time (10 min). Clbiliary values were converted to milliliter per minute per kilogram based on previously reported values for liver weight (200 mg/g liver weight for rats; 160 mg/g liver weight for humans) and protein content in liver tissue (40 g/kg body weight for rats; 25.7 g/kg body weight for humans) (Davies and Morris, 1993; Wilson et al., 2003). HPLC/MS/MS Analysis of NF. NF accumulation in cells+bile and cells were quantified by HPLC/MS/MS as described previously (Yue et al., 2008). Briefly, samples obtained from rat SCH were deproteinated with 1 mL of methanol/water (70/30, v/v), and sonicated for 20 sec. After the removal of protein by centrifugation at 4°C (12000 g) for 3 min, the supernatant (20 µL) was mixed with 100 µL methanol and water (3.8:1) containing internal standard (ethyl warfarin, 10 mM). A Shimadzu solvent delivery system (Columbia, MD) and a Leap HTC Pal thermostatted autosampler (Carrboro, NC) connected to an Applied Biosystems API 4000 triple quadruple mass spectrometer with a Turbo Spray ion source (Applied Biosystems, Foster City, CA) were used for analysis. Tuning, operation, integration and data analysis were performed in negative ionization mode using multiple reaction monitoring (Analyst software v.1.4.1, Applied Biosystems). NF and internal standard were eluted from an Aquasil C18 column (50 × 2.1 mm, dp = 5 µm; Thermo Electron Corporation, San Jose, CA) with 20 µL injection volume at a flow rate of 0.75 mL/min. Initial gradient conditions (100% 10 mM ammonium acetate aqueous solution) were held for 0.75 min. From 0.75 to 1.39 min, the mobile phase composition increased 150 linearly to 40% methanol, and the eluent was directed to the mass spectrometer. At 3.3 min, the methanol composition was increased to 90%. The flow was held at 90% methanol until 4 min. At 4 min, the column was equilibrated with 100% 10 mM ammonium acetate aqueous solution. The total run time, including equilibration, was 5 min per injection. Eight-point calibration curves (2-1000 nM) were constructed based on peak area ratios of analyte to internal standard (ethyl warfarin) using the following transitions: 236.8 151.8 (NF) and 320.8 160.9 (ethyl warfarin). All points on the curves back-calculated to within ± 15% of the nominal value. Transport Studies using Suspended Primary Rat and Human Hepatocytes. [3H]TC and [3H]E217G initial uptake was determined in freshly-isolated hepatocytes as described previously (Leslie et al., 2007). Briefly, hepatocytes were washed twice with ice-cold standard HBSS modified with 10 mM Tris and 5 mM glucose (Na+containing condition) or Na+-free choline buffer (10 mM Tris, 5 mM glucose, 5.4 mM KCl, 1.8 mM CaCl2, 0.9 mM MgSO4, 10 mM HEPES, and 137 mM choline). Hepatocytes were suspended at 1×106 cells/mL in the same buffer, placed on ice, and used immediately in experiments. Hepatocytes (2 mL) in 16 × 100 mm test tubes were incubated at 37˚C in an orbital shaking water bath for 3 min. Vehicle control (0.1 % DMSO), S-sulfone or S-sulfide (0.5–100 µM) was added, followed by [3H]TC (1 µM; 60 nCi/mL) or [3H]E217G (1 µM; 60 nCi/mL). At 30 sec (TC) or 90 sec (E217G), samples (200 µL) were placed into 0.4-mL polyethylene tubes on top of a layer of silicone oil:mineral oil (82:18, v/v; 100 µL) and a bottom layer of 3 M KOH (50 µL), and immediately centrifuged. Radioactivity in the cell pellet and supernatant was quantified by liquid scintillation counting. The adherent fluid volume was 151 determined by the incubation of cells with [14C]inulin. At the end of each experiment, an aliquot (10 µl) of suspended cells was used to determine protein concentrations by the BCA protein assay. Determination of Inhibitory Potency of Sulindac Metabolites on Na+-dependent [3H]TC Uptake or Na+-independent [3H]E217G Uptake. The data obtained from substrate transport studies ([3H]TC and [3H]E217G) in suspended hepatocytes were plotted against the inhibitor concentration (S-sulfone and S-sulfide). Based on standard model selection criteria including residual distribution, Akaike’s information criterion and visual inspection, the sigmoidal inhibitory Emax model was selected to estimate the concentration for 50% inhibition of uptake (IC50) using WinNonlin 5.0.1 (Pharsight, Mountain View, CA). E = Emax × (1− Cr Cr + IC50r ) where E is the rate of substrate uptake, Emax is maximum rate of substrate uptake, C is the inhibitor concentration, and r is the curve shape factor. Statistical Analysis. Data are expressed as mean and the associated S.E.M. Statistical differences were determined using one-way analysis of variance with Bonferroni’s post hoc test using SigmaStat 2.03 (Aspire Software International Ashburn, VA). Differences were considered significant at p < 0.05. 152 RESULTS Effect of Sulindac and Metabolites on TC Hepatobiliary Disposition in Rat SCH. [3H]TC was used as a model substrate to examine the impact of sulindac and metabolites on hepatic uptake and biliary excretion of bile acids in rat SCH (Figure 5.2A-C). The accumulation of TC (1 µM, 100 nCi/mL, 10 min) in cells+bile in the presence of 100 µM S-sulfide was significantly decreased compared to vehicle control. TC BEI in the presence of 1 and 10 µM sulindac, S-sulfone and S-sulfide was not statistically different from those values in vehicle control. TC BEI was decreased significantly by 100 µM S-sulfone (42% decrease) and S-sulfide (55% decrease), but TC BEI in the presence of 100 µM sulindac was not different from those values in vehicle control. Sulindac (10 µM; 53% decrease), S-sulfone (100 µM; 76% decrease) and S-sulfide (100 µM; 84% decrease) significantly inhibited TC Clbiliary. Effect of Sulindac and Metabolites on E217G Hepatobiliary Disposition in Rat SCH. [3H]E217G was used as a model substrate to examine the impact of sulindac and metabolites on Oatp-mediated uptake and Mrp2-mediated biliary excretion in rat SCH (Figure 5.3A-C). Accumulation of E217G (1 µM, 100 nCi/mL, 10 min) in cells+bile and in cells was decreased significantly in the presence of sulindac (100 µM), S-sulfone (10 and 100 µM) and S-sulfide (10 and 100 µM). E217G BEI was not affected by sulindac, S-sulfone or S-sulfide (1-100 µM), whereas E217G Clbiliary was decreased significantly by S-sulfone (1, 10 and 100 µM) and S-sulfide (100 µM), but not by sulindac. 153 Effect of Sulindac and Metabolites on NF Hepatobiliary Disposition in Rat SCH. NF, a specific Bcrp substrate in rat SCH (Yue, 2008), was used as a model substrate to examine the impact of sulindac and metabolites on Bcrp-mediated biliary excretion in rat SCH (Figure 5.4). NF accumulation (5 µM, 10 min) in cells+bile was not significantly different in the presence of 100 µM sulindac, Ssulfone or S-sulfide, but cellular accumulation was increased significantly in the presence of S-sulfide. NF BEI was decreased by 30%, 39% and 57% in the presence of 100 µM sulindac, S-sulfone and S-sulfide, respectively, compared to vehicle control. Clbiliary of NF was decreased, with more potent inhibition by S-sulfide (50% decrease), followed by sulindac (33% decrease) and S-sulfone (33% decrease). Influence of S-sulfone and S-sulfide on Na+-dependent Initial Uptake of TC and Na+-independent Initial Uptake of E217G in Suspended Rat Hepatocytes. To further examine the mechanisms of inhibition of hepatic transport proteins by sulindac metabolites, the inhibitory potency of S-sulfone and S-sulfide on the initial uptake of TC and E217G in the absence or presence of Na+ was determined in suspended rat hepatocytes. Initial uptake was determined at 30 sec for TC (1 µM, 60 nCi/mL) based on preliminary studies (data not shown). E217G initial uptake (1 µM, 60 nCi/mL, 90 sec) was determined under Na+-free conditions to exclude Na+dependent E217G uptake, based on previously published data demonstrating that E217G uptake was partially dependent on sodium (Brock and Vore, 1984). The IC50 values for inhibition of Na+-dependent TC initial uptake by S-sulfone and S-sulfide were 25.6±10.7 µM (Figure 5.5A) and 12.7±5.3 µM (Figure 5.5B), respectively. The 154 IC50 values for inhibition of Na+-independent E217G initial uptake by S-sulfone and S-sulfide were 12.6±5.1 µM (Figure 5.6A) and 6.5±1.2 µM (Figure 5.6B), respectively. Effect of Sulindac and Metabolites on TC Hepatobiliary Disposition in Human SCH. In human SCH, [3H]TC accumulation in cells+bile and cells (1 µM, 100 nCi/mL, 10 min) in the presence of 10 and 100 µM sulindac and S-sulfone was comparable to vehicle control, but S-sulfide at 10 and 100 µM inhibited TC accumulation by 52% and 87%, respectively (Figure 5.7). TC BEI was not decreased by sulindac, Ssulfone or S-sulfide, but the Clbiliary of TC was decreased by 50% and 88% with 10 and 100 µM S-sulfide, respectively. Influence of S-sulfone and S-sulfide on Na+-dependent TC Initial Uptake in Suspended Human Hepatocytes. The initial uptake of [3H]TC (1 µM, 60 nCi/mL) in suspended human hepatocytes was determined at 30 sec based on previously published data demonstrating that TC uptake was linear for at least 2 min in freshly-isolated human hepatocytes (Shitara et al., 2003). The IC50 for inhibition of Na+-dependent TC initial uptake by S-sulfone and S-sulfide were 42.2 µM (Figure 5.8A) and 3.1 µM (Figure 5.8B), respectively. 155 DISCUSSION Among the NSAIDs, sulindac has been associated with the highest incidence of DILI, which may involve interference with hepatic transport protein function. As such, the present study was designed to examine interactions between sulindac/metabolites and hepatic transport proteins using TC, E217G and NF as model substrates for Ntcp-/Bsep-, Oatp-/Mrp2- and Bcrp-mediated transport, respectively, in primary rat and human hepatocytes. BEI and Clbiliary from SCH were used as indices of hepatobiliary disposition of each model substrate; BEI is a measure of net canalicular efflux, whereas Clbiliary encompasses both uptake and efflux. Both the BEI and Clbiliary of TC in rat SCH were decreased significantly by Ssulfone and S-sulfide (100 µM) (Figure 5.2A-C). Moreover, both metabolites inhibited Na+-dependent TC initial uptake in suspended rat hepatocytes (IC50 of 26 and 13 µM, respectively). Collectively, these data indicate that S-sulfone and S-sulfide inhibit Ntcp and Bsep. The balance between bile acid uptake and excretion in hepatocytes may be influence the occurrence of DILI. Indeed, the preferential inhibition of rat Ntcp rather than human NTCP by bosentan has been proposed as a plausible mechanism for protection of the rat against bosentan-mediated hepatotoxicity by preventing the further uptake of bile acids from blood when hepatocyte bile acid concentrations are increased due to bosentan-mediated Bsep/BSEP inhibition (Leslie et al., 2007). In rats, E217G is taken up by multiple Oatps, whereas biliary excretion of E217G is governed by Mrp2. The decreased hepatocellular accumulation of E217G 156 in the presence of S-sulfone and S-sulfide in rat SCH, combined with the inhibition of E217G Clbiliary but not BEI, suggested that sulindac metabolites inhibited Oatpmediated uptake but not Mrp2-mediated biliary excretion of E217G (Figure 5.3A-C). Subsequent investigations in suspended rat hepatocytes revealed that S-sulfone and S-sulfide were potent inhibitors (IC50 of 13 and 6.5 µM, respectively) of E217G initial uptake, consistent with inhibition of Oatp 1a1, 1a3, 1a4, 1a5 and 1b2. NF was chosen as a model substrate for Bcrp-mediated transport based on recent data demonstrating that NF is a specific Bcrp substrate in rat SCH (Yue et al., 2008). Several clinical studies have reported that genetic polymorphisms in BCRP (ABCG2) can influence drug disposition. For example, the ABCG2 C421A polymorphism, which is associated with decreased protein expression and function in vitro (Imai et al., 2002), increased plasma concentrations of several BCRP substrates, including rosuvastatin, diflomotecan, and sulfasalazine (Morisaki et al., 2005; Zhang et al., 2006a; Yamasaki et al., 2008). In the present study, sulindac/metabolites significantly decreased NF BEI and Clbiliary in rat SCH, consistent with inhibition of Bcrp (Figure 5.4). NF has been associated with hepatotoxicity; although cholestatic and mixed patterns of NF-mediated hepatotoxicity have been reported, hypersensitivity or immunologic injury were most common (Thiim and Friedman, 2003). NF-mediated oxidative stress via depletion of GSH or NADPH has been postulated as one mechanism for NF-mediated hepatotoxicity (Thiim and Friedman, 2003). Increased accumulation of NF in hepatocytes may occur in patients who have impaired BCRP activity (due to genetic variations or inhibitors), thus increasing the likelihood of NF-mediated hepatotoxicity. 157 Investigations are underway to determine whether sulindac and metabolites alter human BCRP-mediated transport. Due to the limited availability of human liver tissue combined with prominent roles of bile acid toxicity, only TC was tested further in human SCH to examine the effect of sulindac/metabolites on the hepatobiliary disposition of TC. In human SCH, TC accumulation in cells+bile, and the TC Clbiliary, were markedly decreased by Ssulfide without affecting TC BEI. These findings may reflect, at least in part, Ssulfide-mediated inhibition of NTCP. The IC50 (3.1 µM) of Na+-dependent TC initial uptake by S-sulfide determined in human hepatocyte suspensions, which do not contain serum, was near the unbound plasma concentration range of S-sulfide (~0.14-1.5 µM) based on reported Cmax values of 3-33 µM and an unbound fraction (fu) of 4.6% after oral administration of a therapeutic dose of sulindac (Davies and Watson, 1997). However, the IC50 (42.2 µM) of Na+-dependent TC initial uptake by S-sulfone was ~100-fold greater than the unbound plasma concentration range (~0.1-0.4 µM) of S-sulfone (Cmax, 4-19 µM; fu, 2.1%). Although the IC50 for inhibition of Na+-dependent TC initial uptake by S-sulfide and S-sulfone was higher than unbound plasma concentrations, it is probable that initial inhibitor concentrations are higher than unbound plasma concentrations in the portal vein when sulindac is administered orally, since hepatic blood flow consists of ~85% the portal venous blood, which contains drug absorbed directly from the gastrointestinal tract (Ito et al., 1998). In addition, it is plausible that the extensive enterohepatic recycling of sulindac and metabolites may result in prolonged inhibition of hepatic transport 158 proteins due to much higher concentrations in the portal circulation (Dujovne et al., 1983). The FDA has recommended an in vivo evaluation of drug interactions when the estimated [I]/Ki ratio is greater than 0.1, where [I] represents the mean steadystate Cmax value for total drug (bound plus unbound) following administration of the highest proposed clinical dose (Huang et al., 2008). In theory, the IC50 values determined in the present study would be identical to Ki values, regardless of the mechanism of inhibition, based on previous data demonstrating the relationships between the Ki and IC50 values (Cheng and Prusoff, 1973); that is, the Ki value is equal to the IC50 value under conditions of either noncompetitive or uncompetitive inhibition, whereas under competitive inhibition conditions, the IC50 value will be identical to Ki(1+S/Km), where S is the substrate concentration and Km is an approximate measure of the affinity of the substrate for the enzyme. Because the TC concentration (1 µM) used for the IC50 determination in the present study was lower than the previously reported Km for the uptake of TC in rat (18 µM) and human hepatocytes (4.8 µM), the Ki values would be equal to or less than the IC50 value (Kouzuki et al., 1998; Shitara et al., 2003). With this assumption, the [I]/Ki ratio determined using data from human hepatocytes ranged from 0.9-10.6 for S-sulfide, and from 0.1-0.4 for S-sulfone, based on Cmax values of 3-33 µM and 4-19 µM, respectively. These findings suggest that S-sulfide and S-sulfone may interact with NTCP-mediated transport of drugs and/or bile acids in humans (Davies and Watson, 1997). 159 In conclusion, the present studies demonstrated that hepatic transport proteins (Ntcp/NTCP, Oatps, Bsep, Bcrp) are inhibited by sulindac and metabolites. Considering the important role of Ntcp/NTCP, Oatp and Bsep in hepatic uptake and excretion of bile acids in the liver, impaired hepatic transport of bile acids by sulindac/metabolites may disturb bile acid homeostasis, resulting in sulindacmediated liver injury. Based on the present study demonstrating inhibition of multiple transport proteins by sulindac and metabolites, the possibility of drug-xenobiotic interactions should be considered when sulindac is coadministered with other drugs/xenobiotics, warranting careful monitoring for liver injury. 160 REFERENCES Aithal GP and Day CP (2007) Nonsteroidal anti-inflammatory drug-induced hepatotoxicity. Clin Liver Dis 11:563-575, vi-vii. Brock WJ and Vore M (1984) Characterization of uptake of steroid glucuronides into isolated male and female rat hepatocytes. J Pharmacol Exp Ther 229:175181. Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099-3108. Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10:1093-1095. Davies NM and Watson MS (1997) Clinical pharmacokinetics of sulindac. A dynamic old drug. Clin Pharmacokinet 32:437-459. Dobrinska MR, Furst DE, Spiegel T, Vincek WC, Tompkins R, Duggan DE, Davies RO and Paulus HE (1983) Biliary secretion of sulindac and metabolites in man. Biopharm Drug Dispos 4:347-358. Duggan DE, Hooke KF, Noll RM, Hucker HB and Van Arman CG (1978) Comparative disposition of sulindac and metabolites in five species. Biochem Pharmacol 27:2311-2320. Dujovne CA, Pitterman A, Vincek WC, Dobrinska MR, Davies RO and Duggan DE (1983) Enterohepatic circulation of sulindac and metabolites. Clin Pharmacol Ther 33:172-177. El-Sheikh AA, van den Heuvel JJ, Koenderink JB and Russel FG (2006) Interaction of Non-Steroidal Anti-Inflammatory Drugs with MRP2/ABCC2- and MRP4/ABCC4-Mediated Methotrexate Transport. J Pharmacol Exp Ther. Fattinger K, Funk C, Pantze M, Weber C, Reichen J, Stieger B and Meier PJ (2001) The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther 69:223-231. Funk C, Pantze M, Jehle L, Ponelle C, Scheuermann G, Lazendic M and Gasser R (2001) Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167:83-98. 161 Hamman MA, Haehner-Daniels BD, Wrighton SA, Rettie AE and Hall SD (2000) Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes. Biochem Pharmacol 60:7-17. Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, Qiu W, Rahman A, Sobel S, Stifano T, Thummel K, Wei XX, Yasuda S, Zheng JH, Zhao H and Lesko LJ (2008) New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 48:662-670. Imai Y, Nakane M, Kage K, Tsukahara S, Ishikawa E, Tsuruo T, Miki Y and Sugimoto Y (2002) C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance. Mol Cancer Ther 1:611-616. Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H and Sugiyama Y (1998) Prediction of pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. Pharmacol Rev 50:387-412. Khamdang S, Takeda M, Noshiro R, Narikawa S, Enomoto A, Anzai N, Piyachaturawat P and Endou H (2002) Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal antiinflammatory drugs. J Pharmacol Exp Ther 303:534-539. Kosters A and Karpen SJ (2008) Bile acid transporters in health and disease. Xenobiotica 38:1043-1071. Kouzuki H, Suzuki H, Ito K, Ohashi R and Sugiyama Y (1998) Contribution of sodium taurocholate co-transporting polypeptide to the uptake of its possible substrates into rat hepatocytes. J Pharmacol Exp Ther 286:1043-1050. Laffi G, La Villa G, Pinzani M, Ciabattoni G, Patrignani P, Mannelli M, Cominelli F and Gentilini P (1986) Altered renal and platelet arachidonic acid metabolism in cirrhosis. Gastroenterology 90:274-282. Leslie EM, Watkins PB, Kim RB and Brouwer KL (2007) Differential inhibition of rat and human Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1)by bosentan: a mechanism for species differences in hepatotoxicity. J Pharmacol Exp Ther 321:1170-1178. Liu X, LeCluyse EL, Brouwer KR, Lightfoot RM, Lee JI and Brouwer KL (1999) Use of Ca2+ modulation to evaluate biliary excretion in sandwich-cultured rat hepatocytes. J Pharmacol Exp Ther 289:1592-1599. 162 Marion TL, Leslie EM and Brouwer KL (2007) Use of sandwich-cultured hepatocytes to evaluate impaired bile acid transport as a mechanism of drug-induced hepatotoxicity. Mol Pharm 4:911-918. Morisaki K, Robey RW, Ozvegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B and Bates SE (2005) Single nucleotide polymorphisms modify the transporter activity of ABCG2. Cancer Chemother Pharmacol 56:161-172. Pauli-Magnus C and Meier PJ (2006) Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44:778-787. Rius M, Hummel-Eisenbeiss J and Keppler D (2008) ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4). J Pharmacol Exp Ther 324:86-94. Shitara Y, Li AP, Kato Y, Lu C, Ito K, Itoh T and Sugiyama Y (2003) Function of uptake transporters for taurocholate and estradiol 17beta-D-glucuronide in cryopreserved human hepatocytes. Drug Metab Pharmacokinet 18:33-41. Smith NF, Figg WD and Sparreboom A (2005) Role of the liver-specific transporters OATP1B1 and OATP1B3 in governing drug elimination. Expert Opin Drug Metab Toxicol 1:429-445. Tarazi EM, Harter JG, Zimmerman HJ, Ishak KG and Eaton RA (1993) Sulindacassociated hepatic injury: analysis of 91 cases reported to the Food and Drug Administration. Gastroenterology 104:569-574. Tatsumi K, Kitamura S and Yamada H (1983) Sulfoxide reductase activity of liver aldehyde oxidase. Biochim Biophys Acta 747:86-92. Thiim M and Friedman LS (2003) Hepatotoxicity of antibiotics and antifungals. Clin Liver Dis 7:381-399, vi-vii. Wilson ZE, Rostami-Hodjegan A, Burn JL, Tooley A, Boyle J, Ellis SW and Tucker GT (2003) Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver. Br J Clin Pharmacol 56:433-440. Yamasaki Y, Ieiri I, Kusuhara H, Sasaki T, Kimura M, Tabuchi H, Ando Y, Irie S, Ware J, Nakai Y, Higuchi S and Sugiyama Y (2008) Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans. Clin Pharmacol Ther 84:95-103. Yue W, Abe K and Brouwer KL (2008) Knocking Down Breast Cancer Resistance Protein (Bcrp) by Adenoviral Vector-Mediated RNA Interference (RNAi) in Sandwich-Cultured Rat Hepatocytes: A Novel Tool To Assess the Contribution of Bcrp to Drug Biliary Excretion. Mol Pharm. 163 Yue W, Abe K, Brouwer KLR (2008) Knocking Down Breast Cancer Resistance Protein (Bcrp) by Adenoviral Vector-Mediated RNA Interference (RNAi) in Sandwich-Cultured Rat Hepatocytes: A Novel Tool to Assess the Contribution of Bcrp to Drug Biliary Excretion. Molecular Pharmaceutics In review. Zhang W, Yu BN, He YJ, Fan L, Li Q, Liu ZQ, Wang A, Liu YL, Tan ZR, Fen J, Huang YF and Zhou HH (2006) Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin Chim Acta 373:99-103. 164 Sulindac H3CS H CH3 F CH2COOH Sulindac sulfide Sulindac sulfone Figure 5.1. Chemical Structures of Sulindac, Sulindac Sulfone (S-sulfone) and Sulindac Sulfide (S-sulfide). 165 60 TC Accumulation (pmol/mg protein) A 50 40 30 20 10 0 CTL 1 10 100 Sulindac (µM) BEI (%) 78 ± 5 79 ± 4 61 ± 7 in vitro Clbiliary (ml/min/kg) 32 ± 5 26 ± 1a 15 ± 1 * 69 ± 13 20 ± 3a 100 TC Accumulation (pmol/mg protein) B 80 60 40 20 0 CTL 1 10 100 S-sulfone (µM) * BEI (%) 82 ± 2 84 ± 2a 76 ± 3a 47 ± 12 in vitro Clbiliary (ml/min/kg) 45 ± 10 31 ± 7b 20 ± 4b 11 ± 5 100 TC Accumulation (pmol/mg protein) C * * 80 60 40 20 0 CTL 1 10 100 S-sulfide (µM) * BEI (%) 83 ± 2 79 ± 2a 79 ± 2a 37 ± 17 in vitro Clbiliary (ml/min/kg) 49 ± 8 32 ± 6b 31± 9b 7.5 ± 5.0 * Figure 5.2. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in Rat SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of increasing concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH. BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. 166 A * E217G Accumulation (pmol/mg protein) 180 * 160 140 120 100 80 60 40 20 0 CTL 1 10 100 Sulindac (µM) BEI (%) 18 ± 3 15 ± 6 8±3 12 ± 6 in vitro Clbiliary (ml/min/kg) 21 ± 7 19 ± 9 7.0 ± 2.0 4.1 ± 2.2 B * E217G Accumulation (pmol/mg protein) 160 * 140 120 100 80 60 40 20 0 CTL 1 10 100 S-Sulfone (µM) BEI (%) 18 ± 3 10 ± 4 in vitro Clbiliary (ml/min/kg) 20 ± 3 10 ± 2 * 160 13 ± 12 * * C E217G Accumulation (pmol/mg protein) 10 ± 3 * 6.4 ± 0.8 3.4 ± 2.0 * 140 120 100 80 60 40 20 0 CTL 1 10 100 S-Sulfone (µM) BEI (%) 18 ± 3 10 ± 4 in vitro Clbiliary (ml/min/kg) 20 ± 3 10 ± 2 * 10 ± 3 13 ± 12 * * 6.4 ± 0.8 3.4 ± 2.0 Figure 5.3. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of E217G in Rat SCH. Accumulation of [3H]E217G (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of increasing concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH. BEI and in vitro Clbiliary of E217G were calculated as described in Materials and Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. 167 * NF Accumulation (pmol/mg protein) 80 60 40 20 0 CTL Sulindac S-sulfone S-sulfide BEI (%) 64 ± 1 45 ± 1* 40 ± 1* 27 ± 1* in vitro Clbiliary (ml/min/kg) 5.9 ± 0.7 4.3 ± 0.1* 3.6 ± 0.1* 2.9 ± 0.2* Figure 5.4. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of NF in Rat SCH. Accumulation of NF (5 µM, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of 100 µM sulindac, S-sulfone or Ssulfide in rat SCH. BEI and in vitro Clbiliary of NF were calculated as described in the Materials and Methods. Data are presented as the mean ± S.E.M (n=3 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. 168 IC50 = 25.6 ± 10.7 µM 100 80 60 40 20 + Na -dependent TC uptake (% control) A 0 0.1 1 10 100 1000 S-sulfone (µM) 120 IC50 = 12.7 ± 5.3 µM 100 80 60 40 20 + Na -dependent TC uptake (% control) B 0 0.1 1 10 100 1000 S-sulfide (µM) Figure 5.5. Inhibition of Na+-dependent TC Initial Uptake in Suspended Rat Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake (1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free buffer from [3H]TC uptake in Na+-containing buffer in the presence of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and error bars represent the mean ± S.E.M. of n=3 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.4±0.1 and 0.8±0.2 for Ssulfone and S-sulfide, respectively. 169 E217G uptake (% control) A 120 IC50 = 12.6 ± 5.1 µM 100 80 60 40 20 0 0.1 1 10 100 1000 S-Sulfone (µM) E217G uptake (% control) B 100 IC50 = 6.5 ± 1.2 µM 80 60 40 20 0 0.1 1 10 100 1000 S-Sulfide (µM) Figure 5.6. Inhibition of Na+-independent E217G Initial Uptake in Suspended Rat Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-independent [3H]E217G initial uptake (1 µM, 60 nCi/ml, 90 sec) was determined in Na+-free buffer in the presence of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and error bars represent the mean ± S.E.M. of n=3 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.8±0.2 and 1±0.2 for S-sulfone and S-sulfide, respectively. 170 TC Accumulation (pmol/mg protein) 350 300 250 200 150 100 50 0 CTL 10 100 10 100 10 100 Sulindac (µ µM) S-sulfone (µ µM) S-sulfide (µ µM) BEI (%) 59 57 44 54 52 60 49 in vitro Clbiliary (ml/min/kg) 27 22 18 19 21 13 3.3 Figure 5.7. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in Human SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of 10 and 100 µM sulindac, S-sulfone or S-sulfide in human SCH. BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods. Bars and error bars denote mean ± one-half of the range from n=2 livers in duplicate. 171 IC50 = 42.2 µM 100 80 60 40 20 + Na -dependent TC uptake (% control) A 0 0.1 1 10 100 1000 IC50 = 3.1 µM 100 80 60 40 20 + B Na -dependent TC uptake (% control) S-sulfone (µM) 0 0.01 0.1 1 10 100 1000 S-sulfide (µM) Figure 5.8. Inhibition of Na+-dependent TC Initial Uptake in Suspended Human Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake (1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free buffer from those values in Na+-containing buffer in the presence of increasing concentrations of S-sulfone (1-300 µM) and S-sulfide (0.1-100 µM). Open and closed circles denote individual data from n=2 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.6 and 0.9 for S-sulfone and S-sulfide, respectively. 172 CHAPTER 6 Summary and Future Directions The objective of this dissertation research was to examine the role of hepatic transport proteins in drug disposition and drug-induced liver injury. Although a number of studies have focused on elucidating the transport mechanisms responsible for hepatic uptake and biliary excretion of drugs, the effect of one or more transport protein(s) on overall drug disposition and drug-induced liver injury has not been fully characterized. To address these questions, a series of experiments was designed using a multi-experimental approach. Sandwich-cultured hepatocytes (SCH) have been used instead of primary hepatocytes cultured in monolayer to examine the hepatobiliary disposition of substrates because SCH exhibit polarized architecture, form extensive canalicular networks over time in culture, and maintain function of metabolic enzymes and transport proteins. The maintenance of tight junction integrity in the presence of Ca2+ enables substrate accumulation in the canalicular spaces. Biliary clearance data estimated in rat and human SCH have been correlated with in vivo biliary clearance for several therapeutic compounds (Liu et al., 1999a; Ghibellini et al., 2007b; Abe et al., 2008). However, the use of SCH is limited when determining the contribution of a specific transport protein to overall drug hepatobiliary disposition because specific inhibitors or probe substrates for individual transport proteins have not been identified. Alternatively, membrane vesicles overexpressing a single transport protein can be used to determine substrate specificity or affinity for transport proteins. Isolated liver perfusion is an established technique. This approach enables careful and comprehensive assessment of hepatic disposition of drugs and metabolites by examining the rate of appearance of substrate in bile and in the fluid perfusing the 174 liver vasculature. In mice, this technique presents a number of technical challenges due to the small size of the liver. In recent years, our laboratory has modified successfully the technique in rats for use in mice (Nezasa et al., 2006). The underlying hypothesis of this doctoral dissertation research was that multiple transport proteins are involved in drug disposition and drug-induced liver injury. This hypothesis was examined by a series of experiments described below: 1. Elucidate the mechanism of differences in the hepatobiliary disposition of acetaminophen and metabolites in male and female mice (Chapter 2). In situ mouse liver perfusion using male and female wild-type and Bcrp-knockout mice was performed to test the hypothesis that the biliary excretion of phase II conjugates was influenced by higher Bcrp expression in male mouse livers than female mouse livers. Acetaminophen and phase II metabolites (i.e, acetaminophen sulfate and glucuronide) were chosen based on previous findings demonstrating that Bcrp plays an important role in the biliary excretion of these conjugates in mice. Comprehensive pharmacokinetic models were developed to describe the hepatobiliary disposition of acetaminophen and metabolites in mouse livers. 2. Assess the utility of an integrated in vitro approach [SCH and modeling/simulation] to predict alterations in drug hepatobiliary disposition (Chapter 3). These experiments tested the hypothesis that rat and human SCH can be used to mimic the in vivo disposition of troglitazone (TGZ) and 175 generated metabolites in rats and humans. Additionally, pharmacokinetic modeling and Monte Carlo simulation approaches coupled with data from SCH were utilized to examine the consequences of altered biliary excretion, which might be due to drug interactions or disease states, on hepatic exposure in hypothetical “populations” of hepatocytes. 3. Examine the potential role of hepatic transport proteins in drug-induced liver injury (Chapters 4 and 5). Trabectedin (a promising anticancer agent) and sulindac (a non-steroidal anti-inflammatory drug) were chosen based on previously published findings regarding the incidence of liver injury mediated by these drugs in humans. 1) Dexamethasone (DEX) decreased liver injury mediated by trabectedin in rats and humans, but this was not mimicked in rat hepatocytes cultured on a fibronectin matrix. The hypothesis underlying this aim (Chapter 4) was that Mrp2 and CYP3A1/2 played a role in the hepato-protective effect of DEX against trabectedin-mediated hepatotoxicity. Trabectedin-mediated cytotoxicity was examined in SCH. The role of Mrp2 in biliary excretion of trabectedin was evaluated in isolated perfused livers from wild-type and Mrp2-deficient TR- rats. 2) The hypothesis underlying this aim (Chapter 5) was that the interaction of sulindac as well as its metabolites with hepatic transport proteins altered the hepatobiliary disposition of bile acids and other endogenous or exogenous compounds, and these interactions played a role in sulindac-mediated liver 176 injury. Probe substrates including nitrofurantoin (NF), taurocholate (TC), and estradiol-17β-glucuronide (E217G) were used to examine the interaction between sulindac/metabolites and Bcrp-, Ntcp/Oatp/Bsep-, Oatp/Mrp2mediated transport, respectively, in rat or human SCH, and in suspended hepatocytes. Mechanism of differences in the hepatobiliary disposition of phase II conjugates in male and female mice (Chapter 2). Sex-related variations in pharmacokinetics have been considered as a potential determinant for the clinical effectiveness of therapeutic agents by affecting ADME (absorption, disposition, metabolism and elimination). Emerging evidence suggests that sex-related variations in the expression and activity of drug transport proteins may contribute to differences in therapeutic response or toxicity. BCRP is highly expressed in the liver and mediates the excretion of a wide variety of drugs and environmental toxicants into bile, as well as specific metabolites (e.g., sulfate and glucuronide conjugates) formed in the liver. Interestingly, it has been reported that BCRP protein expression in liver is sex-dependent in mice and humans (Merino et al., 2005b). Furthermore, several anticancer drugs that are known BCRP substrates (e.g., topotecan, methotrexate, and doxorubicin) exhibit sex-dependent disposition in humans. Only a limited number of studies have explored relationships between BCRP and sex-related treatment efficacy; the specific role of BCRP in this therapeutically-important phenomenon has not been elucidated. 177 In situ mouse liver perfusion was applied to four different groups of mice; male and female wild-type and Bcrp-deficient mice (C57BL/6) were used to compare the perfusate disposition and biliary excretion of acetaminophen sulfate (AS) and acetaminophen glucuronide (AG) in the presence of differential BCRP expression (i.e, higher in males than in females). Male and female Bcrp-deficient mice, obtained through collaboration with Eli Lilly and Co., provided a unique opportunity to ascertain whether mechanisms other than Bcrp expression modulate hepatic disposition of acetaminophen metabolites between male and female mice. AG and AS biliary excretion in female wild-type mice was 3- and 2.5-fold lower, respectively, compared to male wild-type mice, reflecting male-predominant Bcrp expression. In Bcrp-deficient mice, sex-dependent differences in AG biliary excretion were not observed, indicating that Bcrp was likely responsible for the sex difference in wildtype mice. Unexpectedly, excretion of AG and AS into the perfusate was also sexdependent; basolateral excretion of AG was higher in male wild-type mouse livers while AS basolateral excretion was higher in female wild-type mouse livers. Pharmacokinetic modeling was used to describe the disposition of acetaminophen, AG and AS in perfused mouse livers. The AG biliary excretion rate constant (Kbile,AG) was ~7-fold higher in male wild-type mouse livers, but was similar between male and female Bcrp-deficient mouse livers. Likewise, the rate constant for AS biliary excretion (Kbile,AS) in male livers was ~2.5-fold higher than in female wild-type mouse livers. Sex-dependent differences in metabolite formation rate constants were observed for AG (~2-fold higher in males) and AS (~2-4-fold higher in females). The basolateral excretion rate constant of AS (Kperf,AS), but not AG 178 (Kperf,AG), differed based on sex; the AS basolateral excretion rate constant was ~2fold higher in females, potentially due to higher Mrp4 expression in female livers. Overall, these results demonstrated that sex-dependent differences in the hepatic disposition of AG and AS were due, in part, to sex-related BCRP expression. Other factors relating to formation of conjugates as well as conjugate transport from the liver into blood likely played a role in disposition of AG and AS. Sex-related differences in BCRP expression may cause clinically significant alterations in the disposition of drugs that are BCRP substrates. These exciting results provide a new framework for investigations directed towards elaborating the mechanisms underlying sex-related differences in drug disposition and drug interactions resulting in altered drug response and/or toxicity associated with therapeutic agents and environmental chemicals. An integrated in vitro approach [SCH and modeling/simulation] to predict alterations in hepatobiliary drug disposition (Chapter 3). Troglitazone (Rezulin; TGZ), an anti-diabetic and anti-inflammatory drug, was withdrawn from the market in 2000 due to severe hepatotoxicity. Inhibition of BSEP by TGZ and TGZ-sulfate (TS) has been hypothesized to be responsible for the cholestatic effect; BSEP was inhibited more potently by TS than by TGZ, demonstrating the importance of metabolite-mediated hepatotoxicity in addition to that of the parent drug. Unfortunately, preclinical toxicological studies failed to predict this hepatotoxicity (Smith, 2003). The aim of this study was to evaluate the capability of an in vitro model, rat and human SCH, to mimic the in vivo metabolism and hepatobiliary disposition of 179 TGZ in rats and humans. Consistent with the in vivo metabolism of TGZ, TGZ was extensively metabolized to TS and to a lesser extent to TG and TGZ-quinone (TQ) in rat and human SCH. Moreover, TS was the predominant metabolite in medium and hepatocytes in both rat and human SCH. A secondary objective of this specific aim was to examine the impact of impaired biliary excretion on the hepatobiliary disposition of TS and TG using data from SCH coupled with pharmacokinetic modeling and Monte Carlo simulations. Parameters governing the hepatobiliary disposition of TGZ and its metabolites in SCH were estimated by compartmental pharmacokinetic modeling. Subsequently, Monte Carlo simulations were performed by utilizing parameter estimates and variance associated with nonlinear least-squares regression and uncontrolled error (e.g., experimental or analytical error; 15%) to examine the impact of altered biliary excretion on accumulation of TS and TG in hepatocytes and medium. In a hypothetical “population” of hepatocytes, cellular accumulation of substrate [representative of liver] was highly sensitive to altered biliary transport, but changes in biliary excretion exhibited only a modest effect on substrate in medium [representative of blood]. Data from Monte Carlo simulations at each time point were utilized to estimate the number of observations necessary to detect a statistically significant difference in TS or TG accumulation in hepatocytes and medium when the rate constant for biliary excretion of TS or TG was modulated [2-fold increase or decrease from experimentally-determined parameter estimates]. The number of observations required to detect a statistically significant difference for TS and TG was less in hepatocytes than in medium at all time points, supporting the hypothesis 180 that cellular accumulation of these substrates is more sensitive to changes in altered biliary excretion than accumulation in medium. In clinical studies, plasma concentrations are typically used to examine the effect of altered hepatic transport function (e.g., drug interactions in biliary excretion, polymorphisms of canalicular transport proteins). However, Monte Carlo simulations suggest that plasma concentrations may not always reflect changes in hepatic exposure when biliary excretion pathways are modulated. Previous findings have demonstrated that BCRP plays an important role in TS biliary excretion in humans (Enokizono et al., 2007). Simulations suggest that TS concentrations in the liver would be higher in subjects with less functional BCRP (due to drug-drug interactions, disease state alterations, and/or polymorphisms), resulting in more potent BSEP inhibition, thereby increasing the susceptibility of some patients to TS-mediated cholestatic effects. The potential role of hepatic transport proteins in drug-induced liver injury. Drug-induced liver injury has been the most common reason for the withdrawal of approved drugs from the market. Although drug-induced liver injury has been attributed to many factors including covalent adduct formation, immunotoxicity, and impaired bioenergetics, accumulating evidence suggests that inhibition of hepatic transport proteins by a drug and/or derived metabolite may mediate liver injury. Many studies have focused on interactions with BSEP because inhibition of BSEP results in accumulation of detergent-like bile acids in the liver which may lead to hepatic injury. However, when drugs or metabolites are intrinsically hepatotoxic, impaired function of one or more transport proteins other 181 than BSEP which are responsible for hepatobiliary disposition of a drug or its metabolites can also contribute to drug-induced liver injury. 1) Trabectedin (Chapter 4) Liver injury mediated by trabectedin (ET-743), a promising anticancer agent, has been the most common side effect during phase I/II clinical trials (Beumer et al., 2005b). Interestingly, pretreatment with DEX in rats and humans markedly decreased ET-743-mediated hepatotoxicity, which was attributed to DEX-mediated CYP3A4 induction. However, the protection by DEX against trabectedin-mediated hepatotoxicty could not be mimicked in primary rat hepatocytes cultured on a fibronectin matrix. The objective of this specific aim was to examine whether SCH could predict the hepatoprotective effect of DEX against trabectedin-mediated hepatotoxicity, and to further examine the involvement of Mrp2 in ET-743 biliary excretion using isolated perfused rat livers (IPL). Pretreatment with DEX in wild-type rat SCH significantly decreased trabectedin-mediated cytotoxicity compared to vehicle control. In addition to inducing CYP3A1/2, DEX also induces Mrp2. To investigate the involvement of Mrp2, trabectedin-mediated cytotoxicity was compared between wild-type and Mrp2deficient TR- rats. Surprisingly, trabectedin-mediated cytotoxicity was less extensive in TR- than wild-type rat SCH. One possible explanation for this unexpected finding was that TR- rat hepatocytes were protected from trabectedin-mediated hepatotoxicity because of a higher level of glutathione in these hepatocytes due to impaired Mrp2-mediated excretion. To examine this hypothesis, trabectedinmediated cytotoxicity was examined in wild-type rat SCH pretreated with buthionine 182 sulfoximine (BSO), a glutathione depleting agent. Trabectedin-mediated cytotoxicity was significantly decreased in BSO-pretreated compared to vehicle-pretreated SCH. Consistent with previous findings (Turncliff et al., 2004), immunoblot analysis demonstrated that Mrp2 and CYP3A1/2 protein expression in rat SCH was increased by DEX pretreatment. Interestingly, BSO pretreatment markedly increased expression of Mrp4 and to a lesser extent Mrp2 and Mrp3 compared to vehicle control. To examine the involvement of Mrp2 in trabectedin hepatobiliary disposition, IPL studies were performed in wild-type and TR- rats. Trabectedin biliary excretion in IPLs from TR- rats was decreased by ~75% compared to wild-type IPLs, suggesting that Mrp2 plays a significant role in trabectedin biliary excretion. Collectively, the hepatoprotection that DEX provides in trabectedin-mediated hepatotoxicity may be due to the combined effect of increased metabolism by CYP3A1/2 and enhanced biliary excretion by Mrp2. Increased expression of basolateral transport proteins such as Mrp3 and/or Mrp4 due to BSO-pretreatment in TR- rat SCH may be one mechanism underlying the unexpected attenuation of trabectedin-mediated hepatotoxicity. 2) Sulindac, sulindac sulfone and sulindac sulfide (Chapter 5) Sulindac is converted to two major metabolites; sulindac sulfide (S-sulfide) has anti-inflammatory activity and sulindac sulfone (S-sulfone) has been recognized recently as a chemopreventive agent in colon cancer. After oral administration of sulindac, plasma concentrations of sulindac and its metabolites were comparable in humans (Davies and Watson, 1997). Among other NSAIDS, sulindac was associated most consistently with hepatotoxicity (Aithal and Day, 2007), but the 183 mechanism(s) of sulindac-mediated hepatotoxicity are not fully characterized. It has been shown that S-sulfide concentrations in plasma correlated in a linear manner with the incidence of hepatotoxicity, suggesting that S-sulfide may be involved in sulindac-mediated hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986). The potential role of transport proteins in drug-induced liver injury has been hypothesized recently and some studies have demonstrated the interaction between sulindac and transport proteins (e.g., MRP2, MRP4, OAT1, OAT3, and OCT3) (Khamdang et al., 2002; El-Sheikh et al., 2006). However, only limited information about the interaction between sulindac metabolites and hepatic transport proteins is available (e.g., Ssulfide-mediated MRP4 inhibition) (Rius et al., 2008). Therefore, the aim of this study was to determine the interaction of hepatic transport proteins with sulindac as well as its metabolites using nitrofurantoin (NF), taurocholate (TC) and estradiol-17βglucuronide (E217G) as probe substrates for Bcrp-, Ntcp/Oatp/Bsep- and Oatp/Mrp2-mediated transport. In rat SCH, the in vitro Clbiliary of NF was decreased significantly by 100 µM sulindac (33% decrease), S-sulfone (33% decrease) and S-sulfide (42% decrease) without affecting NF accumulation in the hepatocyte and bile compartments, suggesting that Bcrp-mediated transport was inhibited by sulindac and its metabolites. Sulindac (10 µM; 53% decrease), S-sulfone (100 µM; 76% decrease) and S-sulfide (100 µM; 84% decrease) significantly inhibited in vitro Clbiliary of TC; the biliary excretion index (BEI) of TC was inhibited significantly by 100 µM S-sulfone (43% decrease) and S-sulfide (55% decrease) but not by sulindac. The BEI of E217G was not decreased in the presence of sulindac, S-sulfone and S-sulfide, but 184 in vitro Clbiliary of E217G was significantly decreased by S-sulfone and S-sulfide, suggesting more potent inhibition in the hepatic uptake process than biliary excretion. Suspended hepatocytes were selected as the model system to further characterize the inhibitory kinetics of sulindac metabolites on TC and E217G uptake, because it is easier to determine initial uptake rates using this system compared to SCH. In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na+-dependent TC initial uptake (IC50 of 25.6±10.7 µM and 12.7±5.3 µM, respectively), and Na+independent E217G initial uptake (IC50 of 12.6±5.1 µM and 6.5±1.2 µM, respectively). In addition, S-sulfide markedly decreased TC accumulation in the hepatocytes and bile compartments as well as in vitro TC Clbiliary, but did not affect TC BEI in human SCH. The IC50 of S-sulfide (3.1 µM) on Na+-dependent TC uptake determined in suspended human hepatocytes was ~14-fold lower than for S-sulfone (42.2 µM). Collectively, studies conducted to address this specific aim revealed that hepatic transport proteins (Ntcp/NTCP, Oatp, Bsep or Bcrp) are inhibited by sulindac and its metabolites. Considering the important role of Ntcp/NTCP, Oatp and Bsep in hepatic uptake and excretion of bile acids in the liver, impaired hepatic transport of bile acids by sulindac and its metabolites may disturb bile acid homeostasis, resulting in sulindac-mediated hepatotoxicity. Although the contribution of BCRP to bile acid transport is minor, inhibition of BCRP-mediated transport by sulindac and its metabolites may result in increased hepatic accumulation of potentially toxic metabolites such as S-sulfide or other co-administered drugs. Several studies with sulindac alone or combined with other drugs (e.g., lovastatin, celecoxib, doxetaxel, carboplatin, and vinorelbine) have been conducted recently to examine the 185 therapeutic effect of sulindac as an antiproliferative agent for colon cancer (Jones et al., 2005; Hoang et al., 2006; Xiao and Yang, 2008). Based on the present study demonstrating inhibition of multiple transport proteins by sulindac and its metabolites, the possibility of drug-drug interactions should be examined carefully when sulindac is coadministered with other drugs, and careful monitoring for hepatotoxicity would be warranted. Future directions This dissertation research has prompted a number of proposed future studies regarding the impact of sex-dependent transport protein expression on overall drug pharmacokinetics, the utility of pharmacokinetic modeling and simulation coupled with SCH to examine the impact of transport modulation on drug disposition, and assessing multiple types of interactions between compounds and transport proteins. These topics are detailed below. Sex differences in drug transport Discrepancies in drug metabolism between sexes are currently thought to play a role in determining sex-differences in pharmacokinetic parameters. For example, significant sex-differences in some key CYP450 enzymes have been demonstrated. A recent study analyzed 38 data sets containing the clearance values of 10 xenobiotics which were CYP3A [but not P-glycoprotein (P-gp)] substrates in healthy young male and female subjects; the overall mean of weightednormalized clearance values was significantly different between males and females 186 (Greenblatt and von Moltke, 2008). Although some animal and human data suggest sex differences in expression of transport proteins, evidence for sex-dependent pharmacokinetic differences in relation to transport proteins in humans are less apparent. For example, despite higher P-gp expression in livers of men compared to women, phenotyping with fexofenadine did not demonstrate sex differences in clearance (Schuetz et al., 1995; Kim et al., 2001). There are several potential reasons to explain the less apparent transportermediated pharmacokinetic differences in male and female subjects. First, the overlapping substrate specificity among hepatic transport proteins may be a confounding factor in assessing sex-dependent differences in drug transport. For example, multiple transport mechanisms such as BSEP and MRP4 are involved in the hepatobiliary disposition of fexofenadine which was used in P-gp phenotyping (Matsushima et al., 2008). Although higher P-gp expression in male subjects results in decreased biliary excretion of fexofenadine in the liver, BSEP-mediated biliary excretion and/or MRP3-mediated basolateral excretion of fexofenadine could completely offset the impact of differences in P-gp expression. Second, tissuespecific sex-dependent expression of transport proteins can be another confounding factor. Sex-dependent hepatic Bcrp/BCRP expression in mice and humans was not observed in rats (Merino et al., 2005b), but Bcrp expression in the kidney was higher in male rats (Tanaka et al., 2005). Although this finding may suggest apparent species differences in sex-dependent expression of transport proteins, it also suggests that differential mechanisms may be involved in the tissue-specific regulation of transport proteins. Therefore, sex-dependent expression of transport 187 proteins in one organ may not change the overall pharmacokinetics due to equal expression of the same transport protein in other organs between males and females. Overall, it is still challenging to examine the possible influence of sex on transporter-mediated drug disposition due to the apparent complexity of transport mechanisms as mentioned above. It is plausible that transporter-dependent sex differences in drug disposition between males and females may influence therapeutic outcome and/or toxicological response if a drug is targeting one specific organ for its therapeutic efficacy (e.g., statins). Data regarding sex-dependent differences in transport proteins in clearance organs (e.g., liver, kidney) can be incorporated in physiologically-based pharmacokinetic modeling to predict the impact of sex-dependent transport functions on the overall drug disposition. Pharmacokinetic modeling and simulation coupled with data from SCH The utility of SCH as an in vitro model to examine phase I and II metabolism and hepatic transport mechanisms has been established (Chapter 3, (Liu et al., 1999c; Hoffmaster et al., 2005; Turncliff et al., 2006; Ghibellini et al., 2007a). Pharmacokinetic modeling has been employed to describe the hepatobiliary disposition of model substrates, and subsequent simulation studies utilizing parameter estimates from pharmacokinetic modeling have been performed to further examine the impact of altered function of one or more hepatic transport proteins on overall hepatobiliary disposition (Chandra et al., 2005; Hoffmaster et al., 2005; Turncliff et al., 2006; Ghibellini et al., 2008). However, these simulations did not 188 incorporate uncertainty associated with parameter estimates or experimental/analytical error, thereby resulting in a less reliable prediction of the true phenomenon. A Monte Carlo simulation is a stochastic method using random numbers and probability to analyze the behavior of a complex system that involves uncertainty and to determine how random variation or error influences the sensitivity, performance, or reliability of a model (Rubinstein, 2008). Recently, Monte Carlo simulations have been applied to the design and execution of clinical trials, and to evaluating methods to predict drug-drug interactions (Kato et al., 2003; Michelson et al., 2006). Monte Carlo simulations using data from rat SCH were employed to predict the impact of altered biliary excretion on the hepatobiliary disposition of TS and TG in a hypothetical “population” of hepatocytes, and to examine the impact of a first-order leakage process of TS and TG from the bile canaliculi of SCH into the incubation medium on the accumulation of TS and TG in hepatocytes and medium. Interestingly, pharmacokinetic modeling revealed the presence of a first-order leakage process for TS and TG from bile canaliculi to the incubation medium in SCH after 20 min of incubation. The mechanism(s) responsible for this leakage process in SCH have not been characterized. In future studies, it would be beneficial to examine the hepatobiliary disposition of drugs which undergo enterohepatic recycling as the parent drug, or without deconjugation, [e.g., rosuvastatin, TS (Kawai et al., 2000; Nezasa et al., 2002)] because it may reflect a fundamental physiological process of bile excretion in vivo and subsequent reuptake into the liver after intestinal absorption. On the other hand, when the time-course data with more than 189 a 20-min incubation time in SCH is used to predict in vivo drug biliary clearance, the leakage process in SCH may result in underestimation of biliary clearance. As shown in Chapters 4 and 5, the SCH system is a useful in vitro model to examine the potential role of transport proteins in the hepatobiliary disposition of a drug that is associated with liver injury due to its intrinsically toxic properties, or its interaction with hepatic transport proteins. However, elucidating the role of hepatic transport proteins in the hepatobiliary disposition of hepatotoxic drugs does not answer the important question of how impaired hepatic transport mechanisms result in hepatotoxicity. Although increased accumulation of detergent-like bile acids in the liver due to drug/metabolite-mediated BSEP inhibition has been proposed as one mechanism for liver injury associated with some hepatotoxic drugs, the role of other transport proteins in drug-induced hepatotoxicity remains to be determined. In order to answer these questions, SCH can be used to measure the activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) along with the accumulation of drugs/metabolites in medium, hepatocytes, and bile at predetermined time points. Pharmacokinetic modeling can be constructed to explain the flux of drug/metabolites in SCH. Changes in the activity of ALT and ALP due to drug exposure can be linked to pharmacokinetic parameters as toxicodynamic responses when developing an integrated pharmacokinetic/toxicodynamic model. This approach may provide a mechanistic understanding of the role of hepatic transport proteins in drug-mediated hepatotoxicity. Several clinical studies have demonstrated that genetic polymorphisms in BCRP (ABCG2) can influence drug disposition. For example, the ABCG2 C421A 190 polymorphism, which is associated with decreased protein expression and function in vitro (Imai et al., 2002), increased plasma concentrations of several BCRP substrates such as rosuvastatin, diflomotecan, and sulfasalazine (Morisaki et al., 2005; Zhang et al., 2006a; Yamasaki et al., 2008). However, the ABCG2 C421A polymorphism had no effect on the plasma pharmacokinetics of NF, which was tested as a potential clinical probe substrate to assess BCRP activity in humans (Adkison et al., 2008). Hepatobiliary disposition of the BCRP substrates, whose pharmacokinetics were examined in subjects, can be assessed in human SCH. Pharmacokinetic modeling/simulation of the data from human SCH can be utilized to test whether the impact of hypothetical changes in the biliary excretion pathway on hepatobiliary disposition of BCRP substrates is consistent with previously reported in vivo findings in humans. SCH with partial and/or complete BCRP knockdown using RNAi, which has been recently optimized for Bcrp knockdown in rat SCH (Yue, 2008), can be used to confirm the reliability of predicting the consequences of altered biliary excretion. Interaction between compounds and hepatic transport proteins The results of this dissertation research demonstrate that compounds interact with transport proteins in multiple ways. For example, BSO increased protein expression of Mrp4 in rat SCH (Chapter 4). BSO inhibits γ-glutamylcysteine synthetase, the rate-limiting step in GSH synthesis (Griffith and Meister, 1979), and has been used as a glutathione (GSH)-depleting agent. Considering that GSH is excreted with monoanionic bile acids by Mrp4 (Rius et al., 2003), increased Mrp4 191 expression by BSO may augment GSH depletion in hepatocytes. Interestingly, Aleksunes et al. demonstrated that the transcription factor NFE2-related factor 2 (Nrf2) was involved in the upregulation of Mrp3 and Mrp4 as one mechanism of neutralizing oxidative stress during acetaminophen hepatotoxicity (Aleksunes et al., 2008). Therefore, BSO-mediated upregulation of Mrp4 may be mediated by an indirect response associated with oxidative stress due to GSH depletion. Pharmacokinetic modeling of the hepatobiliary disposition of acetaminophen and metabolites in mouse livers (Chapter 2) required an intervening compartment between the sites of conjugate formation and excretion into bile to describe the prolonged biliary excretion of AG and AS during the washout phase. These data suggest the presence of a discrete compartment with cytosolic localization and functional Bcrp activity that explains the unidirectional flux of AG and AS prior to biliary excretion. This hypothesis may be supported by a recent study demonstrating subcellular localization of BCRP in folate-deprived MCF cells as a process of cellular adaptation to folate deprivation. (Ifergan et al., 2005). Additionally, the presence of functional P-gp in the Golgi and the mitochondrial membrane has been reported in doxorubicin-resistant K562 leukemia cells (Munteanu et al., 2006). Intracellular sequestration of substrates mediated by transport proteins, which are functionally expressed in cytosolic organelles, may affect overall hepatobiliary disposition of substrates, and further studies are warranted. 192 193 Summary This dissertation work furthered our understanding of the role of hepatic transport proteins in drug disposition and drug-induced liver injury using several model compounds. First, the hepatobiliary disposition of AS and AG was influenced by sex-dependent Bcrp expression in mice. Clinically-significant sex-dependent disposition of drugs/metabolites that are BCRP substrates is plausible due to malepredominant BCRP expression in humans. Second, the similarity of TGZ metabolism and disposition in rat and human SCH with those in rats and humans in vivo further established the utility of the SCH model as an in vitro tool to predict in vivo disposition in rats and humans. In addition, pharmacokinetic modeling and simulation coupled with data obtained from the SCH model predicted likely alterations in drug/metabolite hepatobiliary disposition in humans. Third, the potential involvement of transport proteins other than BSEP in drug-induced liver injury was proposed for trabectedin (Mrps) and sulindac/metabolites (Ntcp, Oatp or Bcrp). 194 REFERENCES Abe K, Bridges AS, Yue W and Brouwer KL (2008) In vitro biliary clearance of angiotensin II receptor blockers and 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors in sandwich-cultured rat hepatocytes: comparison with in vivo biliary clearance. J Pharmacol Exp Ther 326:983-990. Adkison KK, Vaidya SS, Lee DY, Koo SH, Li L, Mehta AA, Gross AS, Polli JW, Lou Y and Lee EJ (2008) The ABCG2 C421A polymorphism does not affect oral nitrofurantoin pharmacokinetics in healthy Chinese male subjects. Br J Clin Pharmacol 66:233-239. Aithal GP and Day CP (2007) Nonsteroidal anti-inflammatory drug-induced hepatotoxicity. Clin Liver Dis 11:563-575, vi-vii. Aleksunes LM, Slitt AL, Maher JM, Augustine LM, Goedken MJ, Chan JY, Cherrington NJ, Klaassen CD and Manautou JE (2008) Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2. Toxicol Appl Pharmacol 226:74-83. Beumer JH, Schellens JH and Beijnen JH (2005) Hepatotoxicity and metabolism of trabectedin: a literature review. Pharmacol Res 51:391-398. Chandra P, Johnson BM, Zhang P, Pollack GM and Brouwer KL (2005) Modulation of hepatic canalicular or basolateral transport proteins alters hepatobiliary disposition of a model organic anion in the isolated perfused rat liver. Drug Metab Dispos 33:1238-1243. Davies NM and Watson MS (1997) Clinical pharmacokinetics of sulindac. A dynamic old drug. Clin Pharmacokinet 32:437-459. Duggan DE, Hooke KF, Noll RM, Hucker HB and Van Arman CG (1978) Comparative disposition of sulindac and metabolites in five species. Biochem Pharmacol 27:2311-2320. El-Sheikh AA, van den Heuvel JJ, Koenderink JB and Russel FG (2006) Interaction of Non-Steroidal Anti-Inflammatory Drugs with MRP2/ABCC2- and MRP4/ABCC4-Mediated Methotrexate Transport. J Pharmacol Exp Ther. Enokizono J, Kusuhara H and Sugiyama Y (2007) Involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion and intestinal efflux of troglitazone sulfate, the major metabolite of troglitazone with a cholestatic effect. Drug Metab Dispos 35:209-214. Ghibellini G, Bridges AS, Generaux CN and Brouwer KL (2007a) In vitro and in vivo determination of piperacillin metabolism in humans. Drug Metab Dispos 35:345-349. 195 Ghibellini G, Leslie EM, Pollack GM and Brouwer KL (2008) Use of tc-99m mebrofenin as a clinical probe to assess altered hepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies. Pharm Res 25:1851-1860. Ghibellini G, Vasist LS, Leslie EM, Heizer WD, Kowalsky RJ, Calvo BF and Brouwer KL (2007b) In vitro-in vivo correlation of hepatobiliary drug clearance in humans. Clin Pharmacol Ther 81:406-413. Greenblatt DJ and von Moltke LL (2008) Gender has a small but statistically significant effect on clearance of CYP3A substrate drugs. J Clin Pharmacol 48:1350-1355. Griffith OW and Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 254:7558-7560. Hoang T, Kim K, Merchant J, Traynor AM, McGovern J, Oettel KR, Sanchez FA, Ahuja HG, Hensing TA, Larson M and Schiller JH (2006) Phase I/II study of gemcitabine and exisulind as second-line therapy in patients with advanced non-small cell lung cancer. J Thorac Oncol 1:218-225. Hoffmaster KA, Zamek-Gliszczynski MJ, Pollack GM and Brouwer KL (2005) Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5]enkephalin. Drug Metab Dispos 33:287-293. Ifergan I, Jansen G and Assaraf YG (2005) Cytoplasmic confinement of breast cancer resistance protein (BCRP/ABCG2) as a novel mechanism of adaptation to short-term folate deprivation. Mol Pharmacol 67:1349-1359. Imai Y, Nakane M, Kage K, Tsukahara S, Ishikawa E, Tsuruo T, Miki Y and Sugimoto Y (2002) C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance. Mol Cancer Ther 1:611-616. Jones SF, Kuhn JG, Greco FA, Raefsky EL, Hainsworth JD, Dickson NR, Thompson DS, Willcutt NT, White MB and Burris HA, 3rd (2005) A phase I/II study of exisulind in combination with docetaxel/carboplatin in patients with metastatic non-small-cell lung cancer. Clin Lung Cancer 6:361-366. Kato M, Tachibana T, Ito K and Sugiyama Y (2003) Evaluation of methods for predicting drug-drug interactions by Monte Carlo simulation. Drug Metab Pharmacokinet 18:121-127. Kawai K, Hirota T, Muramatsu S, Tsuruta F, Ikeda T, Kobashi K and Nakamura KI (2000) Intestinal absorption and excretion of troglitazone sulphate, a major biliary metabolite of troglitazone. Xenobiotica 30:707-715. 196 Khamdang S, Takeda M, Noshiro R, Narikawa S, Enomoto A, Anzai N, Piyachaturawat P and Endou H (2002) Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal antiinflammatory drugs. J Pharmacol Exp Ther 303:534-539. Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI, Taylor A, Xie HG, McKinsey J, Zhou S, Lan LB, Schuetz JD, Schuetz EG and Wilkinson GR (2001) Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther 70:189-199. Laffi G, La Villa G, Pinzani M, Ciabattoni G, Patrignani P, Mannelli M, Cominelli F and Gentilini P (1986) Altered renal and platelet arachidonic acid metabolism in cirrhosis. Gastroenterology 90:274-282. Liu X, Chism JP, LeCluyse EL, Brouwer KR and Brouwer KL (1999a) Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab Dispos 27:637-644. Liu X, LeCluyse EL, Brouwer KR, Gan LS, Lemasters JJ, Stieger B, Meier PJ and Brouwer KL (1999b) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 277:G12-21. Matsushima S, Maeda K, Hayashi H, Debori Y, Schinkel AH, Schuetz JD, Kusuhara H and Sugiyama Y (2008) Involvement of multiple efflux transporters in hepatic disposition of fexofenadine. Mol Pharmacol 73:1474-1483. Merino G, van Herwaarden AE, Wagenaar E, Jonker JW and Schinkel AH (2005) Sex-dependent expression and activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP/ABCG2) in liver. Mol Pharmacol 67:1765-1771. Michelson S, Sehgal A and Friedrich C (2006) In silico prediction of clinical efficacy. Curr Opin Biotechnol 17:666-670. Morisaki K, Robey RW, Ozvegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B and Bates SE (2005) Single nucleotide polymorphisms modify the transporter activity of ABCG2. Cancer Chemother Pharmacol 56:161-172. Munteanu E, Verdier M, Grandjean-Forestier F, Stenger C, Jayat-Vignoles C, Huet S, Robert J and Ratinaud MH (2006) Mitochondrial localization and activity of Pglycoprotein in doxorubicin-resistant K562 cells. Biochem Pharmacol 71:1162-1174. Nezasa K, Takao A, Kimura K, Takaichi M, Inazawa K and Koike M (2002) Pharmacokinetics and disposition of rosuvastatin, a new 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor, in rat. Xenobiotica 32:715-727. 197 Nezasa K, Tian X, Zamek-Gliszczynski MJ, Patel NJ, Raub TJ and Brouwer KL (2006) Altered hepatobiliary disposition of 5 (and 6)-carboxy-2',7'dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice. Drug Metab Dispos 34:718-723. Rius M, Hummel-Eisenbeiss J and Keppler D (2008) ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4). J Pharmacol Exp Ther 324:86-94. Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G and Keppler D (2003) Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38:374-384. Rubinstein RY (2008) Simulation and the Monte Carlo method. John Wiley & Sons, Hoboken, N.J. Schuetz EG, Furuya KN and Schuetz JD (1995) Interindividual variation in expression of P-glycoprotein in normal human liver and secondary hepatic neoplasms. J Pharmacol Exp Ther 275:1011-1018. Smith MT (2003) Mechanisms of troglitazone hepatotoxicity. Chem Res Toxicol 16:679-687. Tanaka Y, Slitt AL, Leazer TM, Maher JM and Klaassen CD (2005) Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice. Biochem Biophys Res Commun 326:181-187. Turncliff RZ, Hoffmaster KA, Kalvass JC, Pollack GM and Brouwer KL (2006) Hepatobiliary disposition of a drug/metabolite pair: Comprehensive pharmacokinetic modeling in sandwich-cultured rat hepatocytes. J Pharmacol Exp Ther 318:881-889. Turncliff RZ, Meier PJ and Brouwer KL (2004) Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes. Drug Metab Dispos 32:834-839. Xiao H and Yang CS (2008) Combination regimen with statins and NSAIDs: a promising strategy for cancer chemoprevention. Int J Cancer 123:983-990. Yamasaki Y, Ieiri I, Kusuhara H, Sasaki T, Kimura M, Tabuchi H, Ando Y, Irie S, Ware J, Nakai Y, Higuchi S and Sugiyama Y (2008) Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans. Clin Pharmacol Ther 84:95-103. Yue W, Abe K, Brouwer KLR (2008) Knocking Down Breast Cancer Resistance Protein (Bcrp) by Adenoviral Vector-Mediated RNA Interference (RNAi) in Sandwich-Cultured Rat Hepatocytes: A Novel Tool to Assess the Contribution of Bcrp to Drug Biliary Excretion. Molecular Pharmaceutics In review. 198 Zhang W, Yu BN, He YJ, Fan L, Li Q, Liu ZQ, Wang A, Liu YL, Tan ZR, Fen J, Huang YF and Zhou HH (2006) Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin Chim Acta 373:99-103. 199 APPENDIX 1 GF120918 (Elacridar) Inhibits Multidrug ResistanceAssociated Protein 3 (MRP3)- and MRP4-Mediated Transport. This Chapter will be submitted to Drug Metabolism and Disposition as Short Communication. ABSTRACT GF120918 (GW918) has been used extensively as a potent and selective inhibitor of P-glycoprotein-(P-gp; MDR1) and breast cancer resistance protein(BCRP; ABCG2) mediated transport. Interestingly, pharmacokinetic modeling of data generated in our laboratory supports a potential interaction between GW918 and hepatic basolateral efflux proteins. The inhibition, by GW918, of both biliary and basolateral excretion routes may cause unexpected hepatic accumulation of drugs. The aim of the present study was to examine the interaction between GW918 and hepatic basolateral efflux transport proteins, specifically MRP3 and MRP4. Effects of GW918 (P-gp/BCRP inhibitor) and MK571 (MRP inhibitor; positive control) on MRP3- and MRP4-mediated transport were examined in inside-out HEK293 membrane vesicles overexpressing MRP3 or MRP4 using two model substrates (Estradiol-17-β-glucuronide, E217G and methotrexate, MTX). In the presence of physiologic concentration of reduced glutathione (GSH, 3 mM), MK571 (18 µM) significantly inhibited MRP3- and MRP4-mediated E217G and MTX transport. Interestingly, GW918 (18 µM) significantly inhibited MRP3mediated E217G transport, and MRP4-mediated E217G (30%) and MTX (60%) transport when GSH was coadministered. In conclusion, GW918, which previously was designated as a specific canalicular transport inhibitor, also inhibits MRP3and MRP4-mediated hepatic basolateral transport. 201 INTRODUCTION GW918 (GF120918 ; elacridar; N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide) has been used extensively as a potent and selective inhibitor for Pglycoprotein (P-gp; MDR1) and breast cancer resistance protein (BCRP; ABCG2) in vitro as well as in vivo. For example, the systemic exposure of topotean was increased 10-fold in mice in the presence of GW918 (Jonker et al., 2000); the apparent oral bioavailability of topotecan combined with GW918 was improved from 40% to 97.1% in patients (Kruijtzer et al., 2002). Recently, a phase I clinical study was conducted to determine an optimal oral dosing schedule for coadministration of topotecan and GW918 (Kuppens et al., 2007). Although GW918 has been designated as a canalicular transport inhibitor, pharmacokinetic modeling of in vitro/in vivo data generated in our laboratory indicated that GW918 inhibits a hepatic basolateral efflux process. However, the exact mechanism of an interaction between GW918 and hepatic basolateral export proteins remains to be elucidated. Dual inhibition of biliary and basolateral excretion by GW918 may cause unexpected hepatic accumulation of drugs, thereby impacting drug efficacy and/or toxicity. A previous study suggested GW918-mediated inhibition of basolateral transport of [D-Pen2, D-Pen5]-enkephalin (DPDPE), an opioid peptide, in isolated perfused rat liver experiments (Hoffmaster et al., 2004b). In this study, GW918 decreased steady-state perfusate concentrations of DPDPE due to increased perfusate clearance (Hoffmaster et al., 2004b). 202 These unexpected findings, particularly in Mrp2-deficient TR- rat livers where DPDPE biliary clearance was decreased, could be explained by the increased liver/perfusate partitioning and inhibition of hepatic basolateral efflux of DPDPE in the presence of GW918. Furthermore, Tian et al. also observed a significant decrease in the rate constant representing basolateral efflux of spiramycin by GW918 in isolated perfused mouse livers (Tian et al., 2007). An interaction between GW918 and MRP4 was proposed by Lee et al. who examined the role of Bcrp in brain disposition of mitoxantrone and dehydroepiandrosterone sulfate (DHEAS) in mice (Lee et al., 2005b). The brain uptake of [3H]DHEAS and [3H]mitoxantrone in Bcrp-deficient mice was comparable with that in wild-type and P-gp-deficient mice, indicating a minor role of Bcrp and P-gp in the active efflux transport of these substrates at the blood brain barrier (BBB). Interestingly, the inhibitory effect of GW918 on the brain disposition of [3H]DHEAS and [3H]mitoxantrone was still observed in P-gpdeficient mice, suggesting a possible contribution of one or more GW918-sensitive efflux transporters that are distinct from Bcrp and/or P-gp at the BBB. Based on the abundant expression of MRP4 mRNA at the BBB, it was proposed that MRP4 plays a role in efflux of DHEAS and mitoxantrone at the BBB, and that GW918 may affect the brain uptake of these substrates by inhibiting MRP4. This study was aimed to identify interactions between GW918 and hepatic basolateral transport proteins MRP3 and MRP4. Estradiol 17-β-glucuronide (E217G) and methotrexate (MTX) were selected as model substrates for MRP3 and MRP4 since the kinetics of both compounds have been characterized previously in MRP3- and MRP4-overexpressing membrane vesicles; E217G is 203 transported with moderate affinity and low capacity for MRP3 [Km = 25.6 µM; Vmax = 75.6 pmol/mg protein/min] and for MRP4 [Km = 30.3 µM; Vmax = 102 pmol/mg protein/min] (Zeng et al., 2000; Chen et al., 2001); MTX is transported with low affinity and high capacity for MRP3 [Km = 776 µM; Vmax = 288 pmol/mg protein/min] and for MRP4 [Km = 220 µM; Vmax = 240 pmol/mg protein/min] (Zeng et al., 2000; Chen et al., 2002) . 204 MATERIALS AND METHODS Chemicals. Estradiol-17-β-glucuronide (E217G), rifampicin, AMP, ATP, pyruvic acid and gentamicin sulfate were purchased from Sigma Chemical Co (St. Louis, MO, USA); creatine phosphate and creatine kinase were purchased from Roche Diagnostics (Indianapolis, IN, USA), and MK571 was purchased from Cayman Chemical Co (Ann Arbor, MI, USA). [3H]E217G (46.9 Ci/mmol; purity>97%) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). [3H]Methotrexate, disodium salt (MTX; 24 mCi/mmol; purity>99%) was purchased from Moravek Biochemicals (Brea, CA). All other chemicals and reagents were of analytical grade and readily available from commercial sources. Transient Transfection of HEK293 Cells with MRP3 or MRP4. The pcDNA3.1(+)/MRP3 was kindly provided by Dr. Susan Cole, Queen’s University, Ontario, Canada. Recombinant pcDNA3.1/Hygro plasmids containing human MRP4 cDNA were kindly provided by Dr. Dietrich Keppler, German Cancer Research Center, Heidelberg, Germany. HEK293 cells were cultured in T-150 flasks with DMEM (high glucose with phenol red containing 10% FBS). After cellcounting with trypan blue in 1:10 dilutions, 4.6 × 106 cells in serum-free DMEM were seeded in T-150 flasks. After 24-h of cell seeding, HEK293 cells were transiently transfected using FuGENE 6 kit (Roche Molecular Biochemicals, Laval, PQ, Canada) per the manufacturer’s instructions. Briefly, 46.77 µl of Fugene reagent was diluted with 1553 µl of serum free DMEM without antibiotics and incubated for 5 min at room temperature per transfection. DNA was added to the mixture of serum free medium and Fugene reagent and incubated for 45 min at 205 room temperature. After incubation, the total mixture was added directly to the T150 flask. Incubation was continued until cells were confluent (~60-72 hr). Membrane Vesicle Preparation. MRP3- or MRP4-overexpressing membrane vesicles were prepared as described previously (Leslie et al., 2001). Transfected HEK293 cells were homogenized in buffer (250 mM sucrose/50 mM Tris, 0.25 mM CaCl2, and EDTA-free protease inhibitor, pH 7.4). Cells were disrupted by N2 cavitation (200 PSI, 5 min). The exploded cell suspension was mixed with 0.5 M EDTA and centrifuged at 1900 rpm for 10 min at 4˚C to remove nuclei. The supernatant was layered onto 10-12 ml of 35% (w/w) sucrose/1 mM EDTA/50 mM Tris, pH 7.4 in high speed centrifuge tubes. After centrifugation at 100,000g at 4˚C for 1 hr using SW28 rotor (Beckman), the interface was placed in a 25 mM sucrose/50 mM Tris, pH 7.4 buffer solution and centrifuged at 100,000g at 4˚C for 30 min. The pellets were resuspended with 250 mM sucrose/50 mM Tris, pH 7.4 and centrifuged at 100,000g at 4˚C for 30 min. The membranes were washed with buffer (250 mM sucrose, 50 mM Tris, pH 7.4) and resuspended by passing them through a 27-gauge needle (10-20 times) to make the suspension homogeneous and aid in vesicle formation. The membranes were aliquoted and stored at – 80˚C until use. Western Blot Analysis. Overexpression of MRP3 and MRP4 in membrane vesicles was confirmed by Western blot analysis. After determination of protein concentration using the BCA protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL), samples were diluted to a final protein concentration of 2 µg/10 µl with 4X NuPAGE sample buffer, dithiothreitol (DTT; 10% final sample volume) and 206 water. Samples were loaded onto a NuPAGE 4-12% 10-well SDS polyacrylamide gel and separated by electrophoresis in a NOVEX Minicell II blot with NuPAGE running buffer at 180 V until the marker reached the bottom of the gel (~2 hr). Proteins in the SDS polyacrylamide gel were transferred onto a PVDF membrane at 30V for 90 min with NuPAGE transfer buffer. Following transfer, the membrane was incubated in 5% nonfat dry milk solution in Tris-buffered saline (TBS) with 0.1% (v:v) Tween-20 for 30-60 min at room temperature. After blocking, the membrane was incubated with anti-MRP3 antibody (M3II-9; Alexis Biochemicals) and anti-MRP4 antibody (M4I-10; Alexis Biochemicals) for 2 hr at room temperature or over-night at 4˚C and rinsed three times at 10 min intervals. Subsequently, the membrane was incubated with 15 mL horseradish peroxidaselinked secondary antibodies, and rinsed three times. MRP3 and MRP4 in membranes were treated with SuperSignal West Dura (Pierce Biotechnology) and detected with a VersaDoc 1000 molecular imager (Bio-Rad Laboratories, Hercules, CA). Membrane Vesicle Transport Studies. Uptake of [3H]E217G and [3H]MTX into membrane vesicles was performed by a rapid filtration method as described previously (Leslie et al., 2001). Membrane vesicles (5 µg of protein per time point) were pre-warmed for 1 min at 37˚C and added to Tris/sucrose (TS) buffer, supplemented with an ATP-regeneration mixture (4 mM ATP or AMP, 10 mM MgCl2, 10 mM creatine phosphate and 100 µg/ml creatine kinase) in a final volume of 40 µl. The reaction mixture was incubated with [3H]E217G or [3H]MTX at 37˚C in the presence of 10 mM DTT or 3 mM GSH solubilized in 10 mM DTT. 207 Initial uptake for E217G and MTX was determined at 1.5 min and 5 min, respectively, based on previously published studies (Zeng et al., 2000; Chen et al., 2001; Chen et al., 2002). At designated times, 800 µl of ice-cold TS buffer was added to the sample mixture. Diluted samples were filtered through Type A/E glass fiber filters (Pall Life Sciences, East Hills, NY) by a rapid filtration method and washed twice with TS buffer. Radioactivity associated with the filters was measured by liquid scintillation counting. substituted with AMP. In control experiments, ATP was Net ATP-dependent transport was calculated by subtracting uptake measured in the presence of AMP from uptake measured in the presence of ATP. Measurements were corrected for the amount of ligand bound to the filters (usually <2% of total radioactivity). To evaluate the inhibitory effects of GW918 or MK571 (18 µM) on [3H]E217G and [3H]MTX uptake mediated by MRP3 and MRP4, GW918 or MK571 was added into the reaction mixture, and then the transport assay was performed as described previously. MK571 was utilized as a positive control to ensure functional inhibition of MRP3- and MRP4mediated uptake. GW918 and MK571 were dissolved in dimethyl sulfoxide (DMSO) and diluted with incubation medium. The final DMSO content was less than 0.1 % in the reaction mixture. In all transport assays, the same concentration of DMSO was used as a control. Data Analysis. All data are expressed as mean ± SE unless otherwise indicated. The effect of GW918 and MK571 on MRP3- and MRP4-mediated uptake was assessed using a one-way analysis of variance followed by Bonferroni’s post hoc test. Differences were considered to be statistically significant when p < 0.05 208 RESULTS Western blot analysis confirmed overexpression of MRP3 and MRP4 in membrane vesicles (Figure A.1). To examine functional activity of MRP3- or MRP4-overexpressing membrane vesicles, [3H]E217G was used as a model substrate at initial concentrations of 5 µM. At the 0.5-min time point, ATP- dependent [3H]E217G uptake was markedly higher in MRP3- and MRP4membrane vesicles with 56.7 ± 15.3 pmol/mg protein and 29.8 ± 6.1 pmol/mg protein, respectively, compared to 7.4 ± 4.5 pmol/mg protein in control (CTL) membrane vesicles prepared from nontransfected HEK293 cells (Figure A.2). ATP-dependent MRP3-mediated [3H]E217G uptake rate in the absence of GSH was ~26% stimulated by GW918, compared to CTL (Figure A.3.A). In MRP4overexpressing membrane vesicles, uptake of [3H]E217G in the absence of GSH was ~30% decreased in the presence of GW918, compared to in the absence of GW918 (Figure A.3.B). MK571 (18 µM), a known inhibitor of MRPs, was used as a positive control to ensure functional inhibition of MRP-mediated [3H]E217G uptake. MK571 inhibited ~47% of MRP3-mediated [3H]E217G uptake while transport of MRP4-mediated [3H]E217G uptake was completely inhibited in the presence of MK571 in the absence of GSH (Figure A.3.A and A.3.B). MTX was employed as a substrate with low affinity and high capacity for MRP3 and MRP4 to examine the interaction between GW918 and basolateral transport proteins. In MRP3-overexpressing membrane vesicles, ATP-dependent MRP3-mediated [3H]MTX uptake in the absence of GSH was not affected by GW918 (Figure A.3.C). Transport of MRP4-mediated [3H]MTX uptake in the 209 absence of GSH was ~15% decreased by GW918 (Figure A.3.D). The degree of inhibition of [3H]MTX uptake by MK571 was comparable between values obtained in MRP3-overexpressing membrane vesicles (75% inhibition) and those in MRP4overexpressing membrane vesicles (82% inhibition). The effect of GSH on MRP3- and MRP4-mediated [3H]E217G and [3H]MTX uptake was examined in the presence of GW918 or MK571 because previous reports indicated that GSH is necessary for MRP4-mediated transport of some substrates (Rius et al., 2003; Rius et al., 2008). DTT (10 mM) was added to the reaction mixture as a vehicle control since GSH was solubilized in DTT. ATPdependent MRP3- and MRP4-mediated [3H]E217G and MRP3-mediated [3H]MTX uptake was comparable in the absence or presence of GSH, but MRP4-mediated [3H]MTX uptake was significantly decreased In the presence of GSH, compared to DTT control (Figure A. 4.A-D). ATP-dependent MRP3-mediated [3H]E217G uptake in the presence of GSH was not affected by GW918 (Figure A.4.A). In MRP4-overexpressing membrane vesicles, transport of [3H]E217G in the presence of GSH was ~30% decreased by GW918, compared to without GW918 (Figure A.4.B). MRP3mediated [3H]E217G uptake and MRP4-mediated [3H]E217G uptake was inhibited ~50% and ~100% when MK571 and GSH were included in the incubation medium (Figure A.4.A and A.4.B). In MRP3-overexpressing membrane vesicles, ATP-dependent [3H]MTX uptake in the presence of GSH was significantly decreased (15%) by GW918, compared to without GW918 (Figure A.4.C). Transport of MRP4-mediated 210 [3H]MTX uptake in the presence of GSH was ~60% decreased by GW918 (Figure A.4.D). The degree of inhibition of [3H]MTX uptake by MK571 in the presence of GSH was comparable (~90% inhibition) between values obtained in MRP3overexpressing membrane vesicles and membrane vesicles (Figure A.4.C and A.4.C). 211 those in MRP4-overexpressing DISCUSSION The present study demonstrated that GW918 inhibited MRP3-mediated [3H]MTX transport (~15% inhibition) and MRP4-mediated transport of [3H]E217G (~30% inhibition) and [3H]MTX (~60% inhibition) when GSH was present. In contrast, GW918 did not inhibit MRP3-mediated [3H]E217G transport. Compoundspecific GW918 inhibition of MRP3-mediated transport may be due to different affinities of the substrate for MRP3; E217G represents a substrate with moderate affinity and MTX a substrate with low affinity. GW918-mediated inhibition of MRP4 transport may cause organ-specific differences in drug disposition, depending on the localization of MRP4 throughout the body. Considering the sinusoidal localization of MRP4 in the liver, GW918mediated inhibition of MRP4 transport coupled with inhibition of canalicular transport proteins including P-gp and BCRP, may increase hepatic accumulation of drug, thus causing unexpected hepatic toxicity or compromised therapeutic efficacy. In contrast to the liver, MRP4 along with P-gp and BCRP are expressed on the apical domain in the kidney and brain. Therefore, inhibition of MRP4 transport by GW918 may result in more potent inhibition of drug efflux in the kidney and brain than expected. While the bile salt export pump (BSEP) is the major canalicular bile salt transport proteins in hepatocytes, MRP4, but not MRP3, plays an important role in hepatic basolateral efflux of bile acids in the presence of GSH (Zeng et al., 2000; Byrne et al., 2002; Rius et al., 2003). Interestingly, affinities of bile acids for MRP4 in the presence of GSH were comparable to those obtained for BSEP, 212 emphasizing the importance of MRP4 as an alternate route of bile acid excretion from hepatocytes into blood (Byrne et al., 2002; Rius et al., 2003). Up-regulation of MRP4 under cholestatic conditions has been observed in mice and rats, as well as in BSEP-deficient patients suffering from progressive familial intrahepatic cholestasis 2 (PFIC2) (Schuetz et al., 2001; Denk et al., 2004; Keitel et al., 2005). Therefore, up-regulation of MRP4 under cholestatic conditions has been recognized as a protective mechanism against hepatotoxicity by preventing accumulation of bile acids in hepatocytes through MRP4-mediated basolateral efflux of bile acids. Considering the hepatoprotective role of MRP4, GW918mediated inhibition of MRP4 transport may predispose the hepatocyte to toxicity when canalicular excretion of bile acids also is impaired due to disease states or drug interactions. In conclusion, the present study demonstrated that GW918 significantly inhibited MRP4-mediated transport in the presence of GSH in inside-out membrane vesicles. GW918 also impaired MRP3-mediated transport, but the extent of inhibition was less than for MRP4. Although GW918 has been used extensively as a P-gp and BCRP inhibitor, the potential inhibition of other hepatic basolateral transport proteins such as MRP4 and MRP3 may confound data interpretation when this inhibitor is employed. 213 REFERENCES Byrne JA, Strautnieks SS, Mieli-Vergani G, Higgins CF, Linton KJ and Thompson RJ (2002) The human bile salt export pump: characterization of substrate specificity and identification of inhibitors. Gastroenterology 123:1649-1658. Chen ZS, Lee K and Kruh GD (2001) Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6mercaptopurine and 6-thioguanine. J Biol Chem 276:33747-33754. Chen ZS, Lee K, Walther S, Raftogianis RB, Kuwano M, Zeng H and Kruh GD (2002) Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system. Cancer Res 62:3144-3150. Denk GU, Soroka CJ, Takeyama Y, Chen WS, Schuetz JD and Boyer JL (2004) Multidrug resistance-associated protein 4 is up-regulated in liver but downregulated in kidney in obstructive cholestasis in the rat. J Hepatol 40:585-591. Hoffmaster KA, Zamek-Gliszczynski MJ, Pollack GM and Brouwer KL (2004) Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems. J Pharmacol Exp Ther 311:1203-1210. Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH and Schinkel AH (2000) Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan. J Natl Cancer Inst 92:16511656. Keitel V, Burdelski M, Warskulat U, Kuhlkamp T, Keppler D, Haussinger D and Kubitz R (2005) Expression and localization of hepatobiliary transport proteins in progressive familial intrahepatic cholestasis. Hepatology 41:1160-1172. Kruijtzer CM, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC, Paul EM and Schellens JH (2002) Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 20:2943-2950. Kuppens IE, Witteveen EO, Jewell RC, Radema SA, Paul EM, Mangum SG, Beijnen JH, Voest EE and Schellens JH (2007) A phase I, randomized, open-label, parallel-cohort, dose-finding study of elacridar (GF120918) and oral topotecan in cancer patients. Clin Cancer Res 13:3276-3285. Lee YJ, Kusuhara H, Jonker JW, Schinkel AH and Sugiyama Y (2005) Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: a minor role of breast cancer resistance protein. J Pharmacol Exp Ther 312:44-52. 214 Leslie EM, Ito K, Upadhyaya P, Hecht SS, Deeley RG and Cole SP (2001) Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfurcontaining analog. J Biol Chem 276:27846-27854. Rius M, Hummel-Eisenbeiss J and Keppler D (2008) ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4). J Pharmacol Exp Ther 324:86-94. Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G and Keppler D (2003) Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38:374-384. Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C, Lamba J, Kim RB, Ramachandran V, Komoroski BJ, Venkataramanan R, Cai H, Sinal CJ, Gonzalez FJ and Schuetz JD (2001) Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J Biol Chem 276:39411-39418. Tian X, Li J, Zamek-Gliszczynski MJ, Bridges AS, Zhang P, Patel NJ, Raub TJ, Pollack GM and Brouwer KL (2007) Roles of P-glycoprotein, Bcrp, and Mrp2 in biliary excretion of spiramycin in mice. Antimicrob Agents Chemother 51:3230-3234. Zeng H, Liu G, Rea PA and Kruh GD (2000) Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res 60:4779-4784. 215 A. CTL vesicle MRP3 vesicle B. MRP3 CTL vesicle MRP4 vesicle MRP4 Figure A.1. Representative western blot of MRP3 (A) and MRP4 (B) in MRP3and MRP4- overexpressing membrane vesicle preparations. Membrane vesicles were prepared from HEK293 cells transiently transfected with MRP3 or MRP4 cDNA. Equal amounts of protein (7 µg/lane) were used for immunoblot (n=3). 216 ATP dependent E217G uptake (pmol/mg protein/0.5 min) 80 60 40 20 0 CTL MRP3 MRP4 Figure A.2. Functional activity of MRP3- or MRP4-overexpressing membrane vesicles. Membrane vesicles were incubated with 5 µM of [3H]E217G for 0.5 min at 37˚C. Net ATP-dependent transport was calculated by subtracting uptake measured in the presence of AMP from uptake measured in the presence of ATP (mean ± SD from a single experiment in triplicate). 217 80 60 40 20 0 CTL GW918 MK571 * 180 D 160 140 120 100 80 60 40 20 0 CTL GW918 MRP4-mediated E217G uptake rate (pmol/mg protein/1.5 min) MRP3-mediated E217G uptake rate (pmol/mg protein/1.5 min) B 100 35 30 25 20 15 10 5 0 CTL MK571 100 80 60 40 20 0 CTL MK571 GW918 * MRP4-mediated MTX uptake rate (pmol/mg protein/5 min) C MRP3-mediated MTX uptake rate (pmol/mg protien/5 min) A GW918 MK571 Figure A.3. Inhibitory effect of GW918 and MK571 on MRP3- and MRP4mediated [3H]E217G and [3H]MTX uptake in the absence of GSH. Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± S.E; n=3 in triplicate). Membrane vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). Net ATP-dependent uptake was calculated by subtracting values in the presence of 4 mM AMP from those in the presence of 4 mM ATP. 218 60 40 20 0 CTL CTL MRP3-mediated MTX uptake rate (pmol/mg protein/5 min) GW918 15 10 5 0 CTL D 200 150 100 50 0 GW918 CTL GW918 DTT 250 DTT 20 MK571 * CTL * 25 GSH 300 CTL MRP4-mediated E217G uptake rate (pmol/mg protein/1.5 min) 80 DTT C B * 100 MK571 MRP4-mediated MTX uptake rate (pmol/mg protein/5 min) MRP3-mediated E217G uptake rate (pmol/mg protein/1.5 min) A * 250 200 150 100 50 0 CTL DTT GSH MK571 GSH CTL GW918 MK571 GSH Figure A.4. Inhibitory effect of GW918 and MK571 on MRP3- and MRP4mediated [3H]E217G and [3H]MTX uptake in the presence of GSH (3 mM). Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). Membrane vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). GSH (3 mM), which is solubilized in 10 mM DTT (vehicle control), was added into the reaction mixture. Net ATP-dependent uptake was calculated by subtracting values in the presence of 4 mM AMP from those in the presence of 4 mM ATP. 219 APPENDIX 2 Data Summary Figure 2.2. APAP concentrations (nM) in outflow perfusate from male wild-type (●) and Bcrp-deficient (○), and female wild-type (▼) and Bcrp-deficient (∆) mouse liver perfusions. Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40 min followed by an additional 30-min infusion with drug-free buffer (n = 3-4/group). Wild-type mice Time (min) 0 10 20 30 35 40 40.3 42 46 50 60 70 Bcrp-deficient mice Male Female Male Female 0 334 ± 48 372 ± 19 343 ± 18 356 ± 23 332 ± 28 338 ± 88 229 ± 87 57 ± 41 0 0 0 0 297 ± 32 332 ± 116 379 ± 63 358 ± 41 357 ± 49 345 ± 34 206 ± 20 22 ± 26 0 0 0 0 324 ± 20 333 ± 29 349 ± 29 336 ± 39 355 ± 14 321 ± 49 153 ± 10 22 ± 25 0 0 0 0 354 ± 53 412 ± 8 406 ± 11 376 ± 39 384 ± 32 346 ± 91 188 ± 40 37 ± 33 0 0 0 221 Figure 2.3. Cumulative biliary excretion and basolateral excretion of AG (A and B, respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; p < 0.05). AG (nmol/g liver) Cumulative Biliary Excretion Cumulative Basolateral Excretion Wild-type mice Male Female 1.02 ± 0.40 19.06 ± 2.09 222 0.42 ± 0.19 12.47 ± 1.58 Bcrp-deficient mice Male Female 0.32 ± 0.02 18.42 ± 1.92 0.22 ± 0.08 12.35 ± 2.19 Figure 2.4. Cumulative biliary excretion and basolateral excretion of AS (A and B, respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; N.D.:not detected; p < 0.05). AS (nmol/g liver) Cumulative Biliary Excretion Cumulative Basolateral Excretion Wild-type mice Male Female 0.14 ± 0.05 1.02 ± 0.31 223 0.07 ± 0.03 3.37 ± 0.73 Bcrp-deficient mice Male Female 0 1.09 ± 0.50 0 2.91 ± 0.96 Figure. 2.6. Representative outflow perfusate rate (pmol/min/g liver) of AG (●) and AS (■), and biliary excretion rate (pmol/min/g liver) of AG (○) and AS (□) in male and female wild-type (solid symbols) and Bcrp-deficient (open symbols) mouse livers (N/A, not available; N/D, not detectable). Male Wild-type Mouse Liver Outflow Perfusate Rate (pmol/min/g liver) Observed Time (min) 0 5 15 25 37.5 44 48 55 65 Biliary Excretion Rate (pmol/min/g liver) Predicted Observed AG AS AG AS 0 296.87 599.62 579.43 619.80 566.82 315.37 145.49 62.91 0 6.40 44.05 35.71 50.54 19.16 0.00 0.00 0.00 0 218.09 545.92 644.76 676.33 516.10 326.50 137.90 39.74 0 26.50 40.24 40.67 40.68 19.03 5.35 0.43 0.01 Time (min) 0 5 15 25 35 45 55 65 AG AS AG AS 0 0 2.02 8.44 16.21 28.44 25.28 14.40 0 0 2.71 3.52 3.02 5.54 1.95 1.51 0 0.40 3.99 9.23 14.91 20.26 22.39 22.83 0 0.38 2.04 3.13 3.73 3.68 2.20 1.21 Female Wild-type Mouse Liver Outflow Perfusate Rate (pmol/min/g liver) Observed Predicted Time (min) 0 5 15 25 37.5 44 48 55 65 AG AS AG AS 0 220.34 464.22 460.86 501.22 259.86 116.06 30.36 21.78 0 68.75 111.48 119.32 125.00 28.52 0 0 0 0 222.76 443.40 476.05 480.90 307.03 148.33 37.94 5.30 0 102.62 123.24 123.33 123.33 33.37 4.44 0.09 0 224 Time (min) 0 5 15 25 35 45 55 65 Predicted Biliary Excretion Rate (pmol/min/g liver) Observed Predicted AG AS AG AS 0 0 1.47 3.92 6.61 10.13 11.79 6.05 0 0 1.57 1.84 1.69 1.66 1.27 0 0 0.24 1.99 4.21 6.48 8.51 9.02 9.03 0 0.31 1.31 1.89 2.21 2.07 1.17 0.64 Male Bcrp-deficient Mouse Liver Outflow Perfusate Rate (pmol/min/g liver) Observed Time (min) 0 5 15 25 37.5 44 48 55 65 Predicted AG AS AG AS 0 294.34 516.36 486.93 451.61 295.18 65.85 37.09 0 0 1.44 19.87 20.31 16.67 13.69 0 0 0 0 279.21 483.85 499.94 501.13 278.11 106.70 17.32 1.24 0 11.45 19.27 19.80 19.83 10.65 3.87 0.56 0.03 Biliary Excretion Rate (pmol/min/g liver) Observed Time (min) 0 5 15 25 35 45 55 65 Predicted AG AS AG AS 0 0.00 1.57 3.83 7.34 6.84 8.90 3.86 N/D N/D N/D N/D N/D N/D N/D N/D 0 0.36 2.55 4.84 6.85 8.22 7.52 6.53 N/A N/A N/A N/A N/A N/A N/A N/A Female Bcrp-deficient Mouse Liver Biliary Excretion Rate Outflow Perfusate Rate (pmol/min/g liver) (pmol/min/g liver) Observed Predicted Observed Predicted Time (min) 0 5 15 25 37.5 44 48 55 65 AG AS AG AS 0 16.31 146.33 428.06 488.61 374.24 338.91 181.65 49.53 0 41.36 80.18 126.21 91.74 26.83 0.00 0.00 0.00 0 89.67 275.19 374.86 435.64 386.06 303.41 193.53 101.36 0 67.67 85.18 85.31 85.31 27.01 4.42 0.13 0.00 225 Time (min) 0 5 15 25 35 45 55 65 AG AS AG AS 0 0 1.13 2.98 4.31 7.00 8.67 3.97 N/D N/D N/D N/D N/D N/D N/D N/D 0 0.10 1.06 2.62 4.40 6.10 6.86 6.90 N/A N/A N/A N/A N/A N/A N/A N/A Figure 3.2. Accumulation of TGZ, TS, and TG in rat SCH. Rat SCH were treated for 10 min with 5 µM TGZ, TS, or TG and accumulation (pmol/mg protein) was measured in cells + bile (solid bars) and in cells (open bars). Values represent the mean of triplicate determinations from n=1 experiment (TGZ) or mean ± SEM of triplicate determinations in n=3 experiments (TS and TG). TGZ TS TG Accumulation (pmol/mg protein) Cells + Bile Cells 1577 1560.2 2274.8 ± 547.9 1939.3 ± 461.5 907.8 ± 247.0 605.4 ± 171.2 226 Figure 3.3. Mass vs. time profiles of TGZ (●), TS (○), TG (▼) and TQ (∆) in the medium initially containing 10 µM TGZ in day 4 rat SCH (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. Accumulation in Medium (pmol/well) Observed Predicted Time (min) TGZ TS TG TQ TGZ TS TG TQ 10 20 30 60 90 120 14141±1394 12956±513 12165±552 9533±748 7035±461 5294±431 72±10 446±88 922±85 2782±302 4489±397 5670±404 29±2 178±14 361±24 579±117 881±172 1034±221 150±11 204±35 190±13 151±19 128±19 98±21 13773 12646 11612 8989 6958 5386 80 324 977 2849 4488 5794 45 139 278 622 887 1092 115 166 184 170 135 105 227 Figure 3.4. Mass vs. time profiles of TGZ (●), TS (○) and TG (▼) in the hepatocytes in day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. Accumulation in Hepatocytes (pmol/well) Observed Predicted Time (min) TGZ TS TG TGZ TS TG 10 20 30 60 90 120 226 ± 49 296 ± 34 235 ± 14 206 ± 58 110 ± 38 45 ± 17 819 ± 179 1072 ± 264 1187 ± 333 1378 ± 276 1296 ± 280 1182 ± 243 59 ± 5 76 ± 7 64 ± 15 46 ± 6 37 ± 11 24 ± 3 233 217 199 154 119 92 582 1013 1229 1360 1285 1197 53 61 58 47 38 31 228 Figure 3.5. Mass vs. time profiles of TS (○) and TG (▼) in the bile canaliculi of day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model derived from the scheme depicted in Figure 3.1 to the data. Accumulation in Bile (pmol/well) Observed Predicted Time (min) TS TG TS TG 10 20 30 60 90 120 138 ± 70 234 ± 22 202 ± 40 174 ± 68 191 ± 51 112 ± 18 28 ± 2 50 ± 2 49 ± 10 36 ± 4 26 ± 13 20 ± 5 60 247 172 182 174 162 17 53 44 33 26 22 229 Figure 4.1. Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated from wild-type (WT) Wistar rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1 - 1000 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (0.1 µM DEX; solid bars) or additional DEX (1 µM ; white bars); *, p < 0.05. Trabectedin Concentration (nM) Vehicle-treated WT SCRH (0.1 µM DEX) DEX-treated WT SCRH (1 µM DEX) 1 10 50 100 1000 0.1 ± 0.1 1.3 ± 0.8 18.9 ± 4.6 21.5 ± 4.1 25.7 ± 3.2 0.1± 0.1 0.1 ± 0.3 5.1 ± 1.1 6.9 ± 0.8 11.6 ± 2.3 230 Figure 4.2. Trabectedin-mediated cytotoxicity in SCRH from WT and TR- rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1 - 100 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from wild-type (solid bars) or TR- (white bars) SCRH; *, p < 0.05. - Trabectedin Concentration (nM) WT SCRH (0.1 µM DEX) TR SCRH (0.1 µM DEX) 1 10 50 100 0.4 ± 0.2 0.9 ± 0.4 23.0 ± 1.7 28.3 ± 0.7 0.2 ± 0.2 0.6 ± 0.6 7.5 ± 3.5 10.2 ± 0.2 231 Figure 4.3. Effect of BSO on trabectedin-mediated cytotoxicity. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (50, 100, and 1000 nM) exposure. BSO (500 µM) was added to the culture medium 24 h prior to trabectedin exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (solid bars) or BSO-pretreated (white bars) SCRH; *, p < 0.05. Trabectedin Concentration (nM) Vehicle-treated WT SCRH BSO-treated WT SCRH (500 µM BSO) 50 100 1000 18.9 ± 4.6 21.5 ± 4.1 25.7 ± 3.2 3.8 ± 1.9 7.8 ± 2.1 13.4 ± 2.0 232 Figure 4.5. Trabectedin perfusate concentrations (Figure 4.5.a) and cumulative biliary excretion (Figure 4.5.b) in isolated perfused livers from WT (solid circle) and TR- (white triangle) rats. Data are presented as mean ± S.D., n= 3/group. Perfusate Concentrations (nM) Time WT Rat Livers TR- Rat Livers (min) 5 35.1 ± 1.2 38.2 ± 11.5 10 7.3 ± 6.4 13.9 ± 3.2 20 0.5 ± 0.8 2.2 ± 2.0 30 0 0.2 ± 0.3 40 0 0 50 0 0 60 0 0 70 0 0 80 0 0 90 0 0 Cumulative Biliary Excretion (% of dose) Time WT Rat Livers TR- Rat Livers (min) 5 0.08 ± 0.09 0 10 0.84 ± 0.54 0.08 ± 0.10 20 1.79 ± 0.84 0.37 ± 0.29 30 2.13 ± 0.96 0.49 ± 0.37 40 2.26 ± 1.00 0.53 ± 0.39 50 2.31 ± 1.01 0.54 ± 0.41 60 2.34 ± 1.02 0.56 ± 0.41 70 2.36 ± 1.01 0.56 ± 0.42 80 2.38 ± 1.00 0.56 ± 0.42 90 2.39 ± 0.99 0.57 ± 0.42 233 Figure 5.2. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in Rat SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of increasing concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH. BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. TC accumulation (pmol/mg protein) in rat SCH Cells+bile Cells Concentration Mean SEM Mean SEM (µM) 0 50.6 4.0 10.6 1.7 1 41.6 3.5 9.0 2.3 Sulindac 10 31.0 6.5 12.6 4.8 100 35.5 4.1 11.0 3.2 0 67.2 13.6 11.2 1.4 1 46.2 16.9 7.6 1.4 S-Sulfone 10 33.5 7.1 8.2 2.2 100 22.9 8.8 9.6 2.7 0 75.2 9.1 11.1 1.5 1 50.4 8.3 10.8 2.1 S-Sulfide 10 48.2 13.1 9.4 2.3 100 17.6 7.7 8.1 1.8 234 Figure 5.3. Effect of sulindac and metabolites on hepatobiliary disposition of E217G in rat SCH. Accumulation of [3H]E217G (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of increasing concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH. BEI and in vitro Clbiliary of E217G were calculated as described in Materials and Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. E217G accumulation (pmol/mg protein) in rat SCH Cells+bile Cells Concentration Mean SEM Mean SEM (µM) 0 143.2 24.0 116.4 14.9 1 146.9 15.4 123.2 3.9 Sulindac 10 110.2 9.9 101.4 12.4 100 42.2 0.5 37.1 2.2 0 141.1 10.0 116.0 6.1 1 120.0 6.7 107.9 7.8 S-Sulfone 10 80.9 3.5 78.8 5.5 100 30.9 3.2 26.7 2.9 0 141.1 10.0 116.0 6.1 1 113.2 8.2 104.2 5.8 S-Sulfide 10 71.9 8.6 59.3 4.9 100 8.7 1.9 7.7 1.8 235 Figure 5.4. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of NF in Rat SCH. Accumulation of NF (5 µM, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of 100 µM sulindac, S-sulfone or Ssulfide in rat SCH. BEI and in vitro Clbiliary of NF were calculated as described in the Materials and Methods. Data are presented as the mean ± S.E.M (n=3 livers in triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc test. NF accumulation (pmol/mg protein) in rat SCH Cells+bile Cells Concentration Mean SEM Mean SEM (µM) CTL 58.4 4.9 20.9 1.1 Sulindac 100 61.4 0.3 34.0 0.0 S-Sulfone 100 59.7 1.4 36.5 0.6 S-Sulfide 100 68.9 8.6 50.7 7.1 236 Figure 5.5. Inhibition of Na+-dependent TC initial uptake in suspended rat hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake (1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free buffer from [3H]TC uptake in Na+-containing buffer in the presence of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and error bars represent the means ± S.E.M. of n=3 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.4±0.1 and 0.8±0.2 for Ssulfone and S-sulfide, respectively. S-Sulfone Observed Predicted Concentration (µM) Mean SEM 0.5 1 5 10 30 50 75 100 72.4 71.4 68.7 53.0 51.3 39.1 47.6 36.3 21.2 10.6 17.1 8.8 19.3 0.7 0.0 7.1 82.8 78.5 65.8 59.3 48.4 43.3 39.4 36.7 237 S-Sulfide Observed Predicted Mean SEM 89.5 84.6 69.9 54.2 43.0 21.1 13.3 12.0 22.1 19.5 22.2 6.7 15.4 7.4 0.0 6.5 93.0 88.4 67.8 54.8 33.5 25.0 19.5 16.1 Figure 5.6. Inhibition of Na+-independent E217G unitial uptake in suspended rat hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-independent [3H]E217G initial uptake (1 µM, 60 nCi/ml, 90 sec) was determined in Na+-free buffer in the presence of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and error bars represent the means ± S.E.M. of n=3 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.8±0.2 and 1±0.2 for S-sulfone and S-sulfide, respectively. S-Sulfone Observed Predicted Concentration (µM) Mean SEM 0.5 1 5 10 30 50 75 100 103.3 86.7 66.4 50.3 34.0 23.2 16.3 20.2 0.0 14.9 20.3 14.7 6.2 5.6 0.0 7.4 93.0 88.4 67.8 54.7 33.4 25.0 19.4 16.1 238 S-Sulfide Observed Predicted Mean SEM 93.1 84.5 56.7 56.8 16.0 12.4 5.5 8.1 0.0 5.6 13.3 6.7 5.5 2.6 0.0 2.2 93.0 86.9 56.5 39.3 17.6 11.3 7.8 5.9 Figure 5.7. Effect of sulindac and metabolites on hepatobiliary disposition of TC in human SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid bars) and cells (white bars) was measured in the presence of 10 and 100 µM sulindac, S-sulfone or S-sulfide in human SCH. BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods. Bars and error bars denote means ± one-half of the range from n=2 livers in duplicate. TC accumulation (pmol/mg protein) in human SCH Cells+bile Cells Concentration Mean Range/2 Mean Range/2 (µM) CTL Sulindac S-Sulfone S-Sulfide 10 100 10 100 10 100 255.1 61.2 108.0 49.5 209.5 226.8 190.6 215.9 119.9 33.5 22.3 33.4 21.0 24.7 13.8 19.7 90.9 127.8 88.7 103.7 48.4 15.4 20.2 17.3 20.9 8.3 13.4 7.1 239 Figure 5.8. Inhibition of Na+-dependent TC initial uptake in suspended human hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake (1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free buffer from those values in Na+-containing buffer in the presence of increasing concentrations of S-sulfone (1-300 µM) and S-sulfide (0.1-100 µM). Open and closed circles denote individual data from n=2 livers in duplicate. The dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data using WinNonlin; the curve shape factor (r) was estimated as 0.6 and 0.9 for S-sulfone and S-sulfide, respectively. S-Sulfone Concentration (µM) 0.1 0.5 1 5 10 30 50 100 300 Observed (n=1) 95.8 91.2 69.9 55.3 55.3 32.8 Observed (n=2) 90.8 87.7 72.9 74.8 47.8 36.1 19.7 S-Sulfide Predicted 90.7 76.6 71.9 55.2 47.1 37.3 23.2 240 Observed (n=1) 55.6 37.2 16.6 0.0 0.0 0.0 Observed (n=2) Predicted 95.1 96.1 87.6 59.1 37.6 11.1 6.2 0.0 74.2 38.8 24.9 10.6 6.8 3.7 Figure A.2. Functional activity of MRP3- or MRP4-overexpressing membrane vesicles. Membrane vesicles were incubated with 5 µM of [3H]E217G for 0.5 min at 37˚C. Net ATP-dependent transport was calculated by subtracting uptake measured in the presence of AMP from uptake measured in the presence of ATP (mean ± SD from a single experiment in triplicate). CTL MRP3 MRP4 E217G transport (pmol/mg protein/0.5 min) Mean SD 7.4 4.5 56.7 15.3 29.8 6.1 241 Figure A.3. Inhibitory Effect of GW918 and MK571 on MRP3- and MRP4- mediated [3H]E217G and [3H]MTX uptake in the absence of GSH. Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± S.E; n=3 in triplicate). Membrane vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). Net ATP-dependent uptake was calculated by subtracting values in the presence of 4 mM AMP from those in the presence of 4 mM ATP. CTL GW918 MK571 CTL GW918 MK571 E217G transport (pmol/mg protein/1.5 min) MRP3 MRP4 Mean SEM Mean SEM 64.7 7.1 22.4 6.4 81.4 9.3 15.9 4.8 34.5 2.0 0.5 0.5 MTX transport (pmol/mg protein/5 min) MRP3 MRP4 Mean SD Mean SD 128.1 36.6 78.6 15.6 128.7 35.1 67.9 7.5 32.2 25.1 14.0 15.4 242 Figure A.4. Inhibitory Effect of GW918 and MK571 on MRP3- and MRP4- mediated [3H]E217G and [3H]MTX uptake in the presence of GSH (3 mM). Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). Membrane vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single experiment in triplicate). GSH (3 mM), which is solubilized in 10 mM DTT (vehicle control), was added into the reaction mixture. Net ATP-dependent uptake was calculated by subtracting values in the presence of 4 mM AMP from those in the presence of 4 mM ATP. E217G transport (pmol/mg protein/1.5 min) MRP3 MRP4 Mean SD Mean SD CTL (DTT) 71.4 6.9 20.8 2.2 CTL 69.4 9.5 20.7 2.8 GW918 74.4 1.6 14.7 3.2 MK571 32.9 3.3 0.0 0.4 MTX transport (pmol/mg protein/5 min) MRP3 MRP4 Mean SD Mean SD CTL (DTT) 234.3 7.6 221.2 10.4 CTL 237.4 15.7 184.4 4.2 GW918 199.3 7.2 79.1 8.9 MK571 18.4 2.2 26.5 2.0 243