Identification of reversible OATP1B1 and time-dependent CYP3A4 inhibition as the major risk factors for drug-induced cholestasis (DIC).

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

Novel insights into bile acid detoxification via CYP, UGT and SULT enzymes.

Bile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive approach to identify the key enzymes involved in BA metabolism using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC). We showed that CYP3A4 was a crucial step for the metabolism of several BAs and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study identified CYP3A4, UGT1A3, UGT2B7 and SULT2A1 as the major contributing (metabolic) processes in the BA detoxification network. Inhibition of these enzymes by drug candidates is therefore considered as a critical mechanism in the manifestation of drug-induced cholestasis in humans and should be addressed during the pre-clinical development.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis.

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRß), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFß1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.