HepaRG™ ability to predict drug-cholestatic liability

Drug-induced liver injury (DILI) accounts for more than 50% of acute liver failure and constitutes therefore a major concern for the pharmaceutical industry in drug pre-and post-marketing scenarios.

Unfortunately, current pre-clinical animal and hepatic in vitro models can only pick-up about 50-60% of clinical DILI cases. Indeed, they have shown clear limitations in drug-cholestasis liability prediction. This can be attributed to interspecies differences in bile acid (BA) synthesis, composition, transport, and regulation but also to the lack of drug-induced cholestasis appropriate biomarkers.

An adverse outcome pathway (AOP) on cholestasis has been recently updated. It depicts the linkage between the most relevant molecular initiating events and the adverse outcomes of bile acid accumulation. 

All the known triggering factors of intrahepatic cholestasis namely transporter expression compromise, cytoskeletal architecture disruption, and bile canaliculi dynamics alteration have been observed in HepaRG cells exposed to cholestatic drugs. 

It has been demonstrated for the first time using the HepaRG model that an in vitro human liver cell line is able to produce and secrete conjugated BA but also to accumulate endogenous BA transiently following exposure to a cholestatic drug. Indeed, HepaRG cells express major BA synthesis enzymes (CYP7A1, CYP8B1, and CYP27A1). In addition, the main hepatobiliary transporters (NTCP, BSEP, MRP2, MDR1, MDR3, and MRP4) are correctly localized to the canalicular and basolateral membrane domains and functionally active in HepaRG cells. Protein activity, as well as mRNA expression of these transporters, are differentially modulated in cholestatic drug-exposed HepaRG cells.

Similar to human hepatocytes, bile canaliculi-like structures of the HepaRG cell line show spontaneous rhythmic motility allowing dynamic evacuation of canalicular content. This motility is strongly altered by cholestatic drugs that induce either bile canaliculi constriction or dilatation. The involvement of Rho Kinase and myosin light chain kinase signaling pathway has been revealed as an underlying mechanism of drug-induced bile canaliculi alteration in HepaRG cells. Disruption of pericanalicular F-actin distribution is another feature of drug-induced cholestasis observed in this cell line.

Two types of cellular response result from the accumulation of BA in HepaRG cells. An adverse response consists mainly of a generation of oxidative stress and endoplasmic reticulum stress associated with a mitochondrial impairment leading to cell death. In parallel, a compensatory response aiming to reestablish BA homeostasis is set up. This adaptive response of decreased bile acid synthesis (CYP7A1 and CYP8B1) and influx (NTCP) and increased expression of basolateral efflux transporters (MRP3 and MRP4) and BA metabolizing enzymes (CYP3A4) seeks to counteract the drug-induced bile acid accumulation.

Altogether, acute and chronic cholestasis studies highlight the HepaRG cell line as a robust and accurate model to flag drug candidates with cholestatic risk according to the most relevant endpoints of cholestasis AOP. 


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Sharanek A, Burban A, Humbert L, Bachour-El Azzi P, Felix-Gomes N, Rainteau D, Guillouzo A. Cellular Accumulation and Toxic Effects of Bile Acids in Cyclosporine A-Treated HepaRG Hepatocytes. Toxicol Sci. 2015 Oct;147(2):573-87. doi: 10.1093/toxsci/kfv155. Epub 2015 Jul 20. PMID: 26198044.

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