Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport

Abstract

Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na⁺-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [³H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Cl_biliary) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Cl_biliary by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n = 2–4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI.

Introduction

Drug-induced liver injury (DILI), a clinical problem worldwide (reviewed in Lucena et al., 2008, Norris et al., 2008), is a leading reason for withdrawal of approved drugs from clinical use or mandated usage restrictions on drug labels, and frequently contributes to attrition of drug candidates during development (Bakke et al., 1995, Kaplowitz, 2005, Kola and Landis, 2004). Numerous pathophysiological mechanisms underlie hepatotoxicity but remain poorly understood, and many cases of DILI are categorized as idiosyncratic (i.e., rare hepatotoxic reactions of unknown etiology). In addition, DILI is difficult to predict with existing animal models and only approximately half of the drugs that cause human hepatotoxicity can be identified during preclinical testing in rodents, dogs, and monkeys (Olson et al., 2000).

Cholestasis is marked by the accumulation of bile acids and bile constituents within hepatocytes; toxicity ensues due, in part, to the detergent-like effects of bile acids, which cause cellular apoptosis or necrosis and mitochondrial dysfunction (reviewed in Sokol et al., 2006). Many drugs and/or metabolites inhibit transport proteins responsible for the hepatobiliary transport of bile acids (reviewed in Kosters and Karpen, 2008, Pauli-Magnus and Meier, 2006). In addition, genetic and environmental factors that modulate the expression and/or activity of these transport proteins have been linked directly to cholestasis or to an increased susceptibility to its development (reviewed in Kosters and Karpen, 2008, Pauli-Magnus and Meier, 2006, Trauner and Boyer, 2003).

Transport proteins are increasingly recognized as pivotal in determining the pharmacokinetics and the pharmacological or toxicological effects of a number of drugs (Hirano et al., 2006, Szakács et al., 2008). Numerous transport proteins are responsible for the movement of bile acids into and out of hepatocytes (reviewed in Kosters and Karpen, 2008, Trauner and Boyer, 2003, Zollner et al., 2006). The sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp; SLC10A1) and the bile salt export pump (BSEP/Bsep; ABCB11) are the predominant proteins involved in bile acid transport and homeostasis in humans and rats. On the basolateral/sinusoidal membrane of hepatocytes, NTCP/Ntcp mediates the uptake of taurine- and glycine-conjugated (i.e., taurocholate) and unconjugated bile acids from the blood in a sodium-dependent manner. Organic anion-transporting polypeptides (OATPs/Oatps; SLCOs) transport predominantly unconjugated bile acids in a sodium-independent manner. Multidrug resistance-associated proteins (MRP/Mrp) 3 (ABCC3) and 4 (ABCC4), and organic solute transporter (OST) α/β efflux bile acids from hepatocytes back into the blood. On the canalicular/apical membrane, BSEP/Bsep effluxes taurine- and glycine-conjugated bile acids into bile canaliculi, serving as a driving force for bile salt-dependent bile flow and for the concentration of bile acids in bile; MRP2/Mrp2 effluxes glucuronidated and sulfated bile acids.

Several in vitro models exist for assessing compound toxicity due to inhibition of transport proteins. Human and rat sandwich-cultured hepatocytes (SCH) are physiologically relevant in vitro models that maintain many in vivo structural and functional characteristics, including canalicular and basolateral membrane domains, expression and localization of liver-specific proteins, and functional bile excretion into sealed canalicular networks (LeCluyse et al., 1994, LeCluyse et al., 2000, Liu et al., 1998, Liu et al., 1999a, Liu et al., 1999b). These models have been used previously to study the inhibitory effects of drugs (e.g., troglitazone, nefazadone, antiretroviral compounds) on bile acid uptake and biliary excretion (Kemp et al., 2005, Kostrubsky et al., 2006, Marion et al., 2007, McRae et al., 2006).

Because of increased attention to DILI (EMEA, 2008, FDA, 2007), a robust assay utilizing SCH for inhibition of bile acid transport would be beneficial during drug development, allowing early identification of drug candidates that may cause cholestasis in humans. Traditionally, compounds are administered individually in preclinical drug development studies, a time-consuming, labor-intensive, and cost-ineffective practice. Cassette dosing, or the simultaneous administration of multiple compounds, was developed as an alternative screening method. This strategy has been used widely in vitro and in vivo to study the pharmacokinetics and metabolism of drug candidates (reviewed in White and Manitpisitkul, 2001, Zhou et al., 2004). To our knowledge, there are no reports in the literature regarding the use of cassette dosing to identify inhibitors of hepatic bile acid transport. The purpose of the present study was two-fold. First, inhibition of bile acid (taurocholate) transport by hepatotoxic drugs was assessed in rat and human SCH to determine whether it was a common mechanism of hepatotoxicity. Second, the feasibility of using a cassette dosing approach with rat and human SCH to screen for inhibition of taurocholate transport was investigated.