Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human

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Abstract

The large interspecies differences of hepatobiliary transport present a challenge for the allometric prediction of human biliary excretion for drug candidates primarily cleared via hepatobiliary secretion. In the present study, we determined the metabolic stabilities of common fluorescent substrates of hepatobiliary efflux transporters and developed a rapid efflux assay to determine the functional activities of MRP/Mrp, BCRP/Bcrp and P-gp in hepatocytes of four species. The specificities of transporter-mediated dye efflux were confirmed by selective transporter inhibitors. Among tested species, transporter-specific dye efflux kinetics was consistent between freshly isolated and cryopreserved hepatocytes. Hepatocyte elimination half-lives of MRP/Mrp substrates GS-MF and calcein were observed in the rank order of human > monkey > dog > rat. The fourfold higher MRP/Mrp substrate efflux rate of rat hepatocytes compared to human is likely due to the species-specific functional differences of MRP2/Mrp2 expressed on the canalicular membrane. We also observed efficient BCRP-mediated pheophorbide A (PhA) efflux by human and dog hepatocytes, while PhA extrusion in monkey and rat hepatocytes appeared limited. P-gp function measured by DiOC2(3) efflux was minimal in hepatocytes of all origins and no significant species differences were detected. Our results demonstrated marked differences in hepatocyte MRP/Mrp and BCRP/Bcrp activities across species, indicating that they may contribute to the species differences of in vivo hepatobiliary excretion. These results also suggest the potential utility of primary hepatocytes, either fresh or cryopreserved, as an in vitro model to predict interspecies differences in the biliary transport of MRP/Mrp and BCRP/Bcrp substrates.

Introduction

Biliary excretion represents one of the primary elimination routes for xenobiotics and their conjugate metabolites from the body (Arias et al., 1993). Factors interfering with this pathway could have significant impact on drug systemic exposure and are associated with the occurrence of hepatotoxicity (Kostrubsky et al., 2001, Kostrubsky et al., 2003, Stieger et al., 2000). Therefore, a precise knowledge of biliary clearance (CLbile) is critical in predicting the pharmacokinetics and pharmacological/toxic effects of drugs/metabolites primarily excreted into the bile. Species differences in biliary excretion have been long recognized. Mice and rats often exhibit efficient biliary excretion, while monkeys and human are generally considered poor biliary excreters (Mahmood and Sahajwalla, 2002). Consequently, direct extrapolation from preclinical small animal CLbile sometimes leads to significant overestimation of human CLbile. For instance, a 20-fold and 7-fold overestimation of human clearance were observed for susalimod (Pahlman et al., 1999) and napsagatran respectively (Ayrton and Morgan, 2001, Lave et al., 1999). Variations observed in hepatic blood flow and bile flow rates across species could not fully explain such differences (Mahmood, 2005, Mahmood and Sahajwalla, 2002). Therefore, a better understanding of the molecular mechanisms underlying the interspecies difference of biliary elimination would be of great value to improve the accuracy of human CLbile prediction from animal data.

Biliary excretion is an active process. Separate excretory mechanisms for organic anions and cations on the canalicular membrane have been long established. The molecular identities of these hepatic efflux carriers are beginning to be revealed with the cloning of P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs/Mrps) and breast cancer resistance protein (BCRP/Bcrp). The functional role of P-gp in mediating energy-dependent biliary drug elimination was first demonstrated by Kaminoto et al. using rat liver canalicular membrane vesicles (CMV) (Kamimoto et al., 1989). Later studies established P-gp as one of the major carriers responsible for the biliary excretion of amphiphilic cationic drugs (Schinkel et al., 1995, Smit et al., 1993, van Asperen et al., 1999). MRP/Mrp2–6 are also expressed in the liver. MRPs/Mrps commonly accept organic anions as substrates; methotrexate, etoposide, glucuronides and sulfated conjugates are shown transported by either one or multiple MRP/Mrp isoforms (Hirano et al., 2005, Kruh and Belinsky, 2003, Suzuki et al., 2003). Unlike MRP/Mrp3–6 which are expressed on the sinusoidal membrane, MRP2/Mrp2 is uniquely localized on canalicular membrane and facilitates the biliary excretion of a broad range of drugs and conjugation metabolites (Shitara et al., 2005). In addition to P-gp and MRP2/Mrp2, BCRP/Bcrp is also present on the canalicular membrane of hepatocytes and constitutes another biliary excretion route for a diverse variety of organic molecules (Merino et al., 2005). Although information regarding the species difference of hepatic efflux transporters including P-gp and BCRP/Bcrp is currently scarce, marked interspecies difference in MRP2/Mrp2 activity was previously demonstrated in both animal models and isolated CMV (Ishizuka et al., 1999, Shilling et al., 2006). Ishizuka and coworkers reported 40-fold differences in the CLbile of Mrp2 substrate temocaprilat between rat and dog (Ishizuka et al., 1999). Consistently higher uptake activity of Mrp2 probe DNP-SG by rat hepatocyte CMVs was observed compared with dog. It was suggested that the marked difference (10-fold) of hepatic Mrp2 expression between the two species may account for the observed functional difference (Ninomiya et al., 2005). These results highlight Mrp2 as a significant contributor to species differences of in vivo hepatobiliary transport.

Because of the striking species differences in hepatobiliary elimination, an adequate in vitro model which can predict in vivo interspecies biliary excretion difference is needed. A number of in vitro models have been used to investigate biliary excretion mechanisms, including liver derived CMVs and gene transfected cell lines. These systems either lack the complement of cofactors present in the hepatocytes or express transporters at a level not comparable with that of in vivo, therefore their results should not be used to directly compare or predict in vivo biliary transport efficiency. Primary hepatocytes are generally recognized as the closest in vitro surrogate of the liver and are known to express most of the phase I and phase II metabolic enzymes found in liver (Gomez-Lechon et al., 2003, Hengstler et al., 2000). Previous studies demonstrated that the expressions and functions of MRP2/Mrp2, P-gp and BCRP/Bcrp in cultured human and rat hepatocytes (Bi et al., 2006, Hoffmaster et al., 2004, Shitara et al., 2003), are dependent on cell culture conditions (Turncliff et al., 2006). Therefore, cultured hepatocytes may not be an appropriate model to investigate the innate species difference of biliary excretion (Jigorel et al., 2005, Le Bot et al., 1996). In the present study, using hepatocytes from human and three other common preclinical species, we characterize the functions of major canalicular efflux transporters in both fresh and cryopreserved hepatocyte suspensions. The knowledge obtained from the study may provide us the basis to evaluate its potential applications in the prediction of in vivo interspecies biliary excretion differences.

Section snippets

Chemicals and reagents

Pheophorbide A (PhA) was purchased from Frontier Chemical (Salt Lake City, UT). Calcein-acetoxymethyl ester (Calcein-AM), 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)), mitoxantrone, fumitremorgin C (FTC) and antifoam were purchased from Sigma–Aldrich Inc. (St. Louis, MO). 5-Chloromethylfluorescein diacetate (CMFDA), Paclitaxel BODIPY®FL conjugates, Dulbecco's modified Eagle's medium (DMEM) and Hank's balanced salt solution (HBSS) were purchased from Invitrogen (Carlsbad, CA).

Metabolic stability of fluorescent probes

Hepatocytes are known to retain most in vivo enzymatic activities, including both phase I enzymes (cytochrome P450s (CYPs), aldehyde oxidases and monoamine oxidases (MAOs)) and phase II enzymes (UDP-glucuronyltransferases and sulfotransferases). It is possible that some of the enzymes may be reactive with the fluorescent dyes. Thus, we first investigated the metabolic stabilities of commonly used transporter fluorescent substrates. As shown in Fig. 2, the fluorescence intensity of PhA, GS-MF,

Discussion

As a major xenobiotic eliminating organ, the liver possesses an array of important drug metabolizing enzymes and transporters which play key roles in determining xenobiotic disposition and hepatotoxicity. In addition to the well-characterized species differences of hepatic metabolizing enzymes, pronounced interspecies differences in transporter-mediated active biliary excretion were also observed. Although much progress has been made in the molecular identification and characterization of major

Acknowledgements

We would like to thank Drs. Joseph Fleishaker, Jeffrey Stevens, Anup Zutshi and Joseph Ware for their helpful comments and suggestions on our study. We would like to thank Sarah A. South and Kathleen E. Sampson for proofreading the manuscript.

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