Bile acids and bile salts (BA/BS) are substrates of both influx and efflux transporters on hepatocytes. Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/β, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/β is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC50 ≤ 25-50 µM or total Css,plasma/BSEP IC50 ≥ 0.1 may be an indication for further testing to minimize the risk of DILI liability.
Keywords: BSEP; DILI; cholestasis; hepatotoxicity; in vitro ADME; transporters.