Membrane vesicle ABC transporter assays for drug safety assessment.

The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance-associated protein 2, 3, or 4 (MRP2-4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug-induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter. This window is used to determine the degree to which each test compound inhibits transporter activity. This assay battery is helpful for prioritizing and rank-ordering compounds within a chemical series with respect to each other and in the context of known inhibitors of transporter activity and/or liver injury. This model can be used to influence the drug development process at an early stage and provide rapid feedback regarding the selection of compounds for advancement to in vivo safety evaluations. A detailed protocol for the high-throughput assessment of ABC transporter function is provided, including specific recommendations for curve-fitting to help ensure consistent results.

Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development.

The bile salt export pump (BSEP) is an efflux transporter, driving the elimination of endobiotic and xenobiotic substrates from hepatocytes into the bile. More specifically, it is responsible for the elimination of monovalent, conjugated bile salts, with little or no assistance from other apical transporters. Disruption of BSEP activity through genetic disorders is known to manifest in clinical liver injury such as progressive familial intrahepatic cholestasis type 2. Drug-induced disruption of BSEP is hypothesized to play a role in the development of liver injury for several marketed or withdrawn therapeutics. Unfortunately, preclinical animal models have been poor predictors of the liver injury associated with BSEP interference observed for humans, possibly because of interspecies differences in bile acid composition, differences in hepatobiliary transporter modulation or constitutive expression, as well as other mechanisms. Thus, a BSEP-mediated liver liability may go undetected until the later stages of drug development, such as during clinical trials or even postlicensing. In the absence of a relevant preclinical test system for BSEP-mediated liver injury, the toxicological relevance of available in vitro models to human health rely on the use of benchmark compounds with known clinical outcomes, such as marketed or withdrawn drugs. In this study, membrane vesicles harvested from BSEP-transfected insect cells were used to assess the activity of more than 200 benchmark compounds to thoroughly investigate the relationship between interference with BSEP function and liver injury. The data suggest a relatively strong association between the pharmacological interference with BSEP function and human hepatotoxicity. Although the most accurate translation of risk would incorporate pharmacological potency, pharmacokinetics, clearance mechanisms, tissue distribution, physicochemical properties, indication, and other drug attributes, the additional understanding of a compound's potency for BSEP interference should help to limit or avoid BSEP-related liver liabilities in humans that are not often detected by standard preclinical animal models.