Bile Acid Transporters and Hepatotoxicity: Novel Mechanisms and In Vitro Models

The clinical relevance of hepatic transport proteins is well-established in drug disposition and safety. Bile acid transporters mediate translocation of bile acids across membranes and maintain bile acid homeostasis. Drug-induced liver injury (DILI) is a major safety concern in drug development and altered bile acid transporter function and/or bile acid homeostasis may cause DILI. Inhibition of the bile salt export pump (BSEP), the primary hepatic bile acid efflux transporter, has been associated with DILI in membrane vesicle studies, but assays that more accurately predict DILI liability are needed. The main objectives of this dissertation were 1) to elucidate DILI mechanisms related to altered bile acid homeostasis, and 2) to develop and characterize in vitro models that retain bile acid transporter regulation for screening compounds to minimize the risk for bile acid-mediated DILI. DILI mechanisms beyond BSEP inhibition were evaluated for hepatotoxic tyrosine kinase inhibitors (TKIs) using sandwich-cultured human hepatocytes. Increased bile acid synthesis via cytochrome P450 7A1 induction and higher taurocholate uptake via increased sodium-taurocholate cotransporting polypeptide (NTCP) were identified as novel mechanisms of TKI-mediated hepatotoxicity. This work highlights that multiple mechanisms of altered bile acid homeostasis may contribute to DILI. Human hepatoma (HuH-7) cells were differentiated over a 2-week culture period using dexamethasone, dimethyl sulfoxide, and overlay to restore normal hepatic function. Differentiated HuH-7 cells expressed key bile acid transporters (e.g., BSEP, NTCP) that were properly localized and functional. This novel in vitro model was sensitive to altered bile acid transport by prototypical hepatotoxic compounds. Unlike membrane vesicles, differentiated HuH-7 cells provide a cellular platform for mechanistic investigation of transporter function and regulation, and for high-throughput screening of compounds for DILI risk. An NTCP-overexpressing Flp-In™ T-REx™-293 cell line was established since NTCP inhibition may be hepatoprotective under cholestatic conditions. Using NTCP-expressing cell lines, everolimus was identified as a novel NTCP inhibitor. This approach can be applied to understand NTCP regulation and conduct efficient inhibitor screening to identify therapeutic compounds. This dissertation research provides a mechanistic and in vitro framework to test compounds for bile acid-dependent DILI risk and transporter inhibition, which is critical for drug development and precision medicine.