The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug‐induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow‐up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case‐by‐case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.

BackgroundInhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs).MethodsSandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3.ResultsThe ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x).ConclusionsSignificant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.

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ABSTRACT:Drug-induced cholestasis can result from the inhibition of biliary efflux of bile acids in the liver. Drugs may inhibit the hepatic uptake and/or the biliary efflux of bile acids resulting in an increase in serum concentrations. However, it is the intracellular concentration of bile acids that results in hepatotoxicity, and thus serum concentrations may not necessarily be an appropriate indicator of hepatotoxicity. In this study, sandwich-cultured rat hepatocytes were used as an in vitro model to assess the cholestatic potential of drugs using deuterium-labeled sodium taurocholate (d 8 -TCA) as a probe for bile acid transport. Eight drugs were tested as putative inhibitors of d 8 -TCA uptake and efflux. The hepatobiliary disposition of d 8 -TCA in the absence and presence of drugs was measured by using liquid chromatography/tandem mass spectrometry, and the accumulation (hepatocytes and hepatocytes plus bile), biliary excretion index (BEI), and in vitro biliary clearance (Cl biliary ) were reported. Compounds were classified based on inhibition of uptake, efflux, or a combination of both processes. Cyclosporine A and glyburide showed a decrease in total (hepatocytes plus bile) accumulation, an increase in intracellular (hepatocytes only) accumulation, and a decrease in BEI and Cl biliary of d 8 -TCA, suggesting that efflux was primarily affected. Erythromycin estolate, troglitazone, and bosentan resulted in a decrease in accumulation (total and intracellular), BEI, and Cl biliary of d 8 -TCA, suggesting that uptake was primarily affected. Determination of a compound's relative effect on bile acid uptake, efflux, and direct determination of alterations in intracellular amounts of bile acids may provide useful mechanistic information on compounds that cause increases in serum bile acids.