Drug-induced cholestasis assay in primary hepatocytes

Abstract

Drug-induced cholestasis (DIC) is a major cause of clinical failure of drug candidates. Numerous patients worldwide are affected when exposed to marketed drugs exhibiting a DIC signature. Prospective identification of DIC during early compound development remains challenging. Here we describe the optimized in vitro procedure for early assessment and prediction of an increased DIC risk. Our method is based on three principles: Exposure of primary human hepatocyte cultures to test compounds in the absence and presence of a physiologically relevant mixture of endogenous bile salts. Rapid and quantitative assessment of the influence of concomitant bile salt exposure on hepatocyte functionality and integrity after 24 h or 48 h of incubation. Translation of the in vitro result, expressed as a DIC index (DICI) value, into an in vivo safety margin. Using our historical control data, a new (data driven) DICI cut-off value of 0.78 was established for discerning cholestatic and non-cholestatic compounds. Our DIC assay protocol was further improved by now relying on the principle of the no observable adverse effect level (NOAEL) for determining the highest test compound concentration corresponding to a DICI ≥ 0.78. Predicted safety margin values were subsequently calculated for compounds displaying hepatotoxic and/or cholestatic effects in patients, thus enabling evaluation of the performance of our DIC assay. Of note, this assay can be extended to explore the role of drug metabolites in precipitating DIC.

Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes

“…We previously introduced a holistic in vitro screening assay to investigate the cholestatic propensity of a given compound. In this assay, bosentan was not identified as cholestatic at concentrations up to 200 µM (Chatterjee, Richert, et al, 2014;Oorts et al, 2016;Van Brantegem et al, 2020). In order to explain this result, the present study aimed to explore the mechanisms governing the bosentan-induced disturbance in bile salt homeostasis in SCHH.…”

Mouse precision-cut liver slices as an ex vivo model to study drug-induced cholestasis

“…Despite the fact that intracellular BA accumulation is a clear marker for cholestasis, none of these studies considered the composition of BA on DIC development. Some studies do take into account the important role of human BAs in DIC in vitro (Chatterjee et al 2014 ; Hendriks et al 2016 ; Ogimura et al 2017 ; Van Brantegem et al 2020 ; Shinozawa et al 2021 ) and in vivo (Boer et al 2019 ). Most in vitro models, however, do not include the tissue complexity of the PCLS model, which allows for the more faithful tissue response required in multifactorial conditions like cholestasis (Vatakuti et al 2017 ; Starokozhko et al 2017a ).…”

In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach

“…The drug-induced cholestasis (DIC) assay has been developed to distinguish cholestatic compounds from hepatotoxic non-cholestatic and non-hepatotoxic compounds based on their potential to modulate BA disposition in a sandwich-cultured human hepatocytes in vitro model. This assay enables early assessment and prediction of an increased DIC risk [ 103 ] as well as the determination of the drug-induced cholestatic index (DICI). The DICI relies on the ratio of urea produced by the sandwich-cultured human hepatocytes exposed to test compound in the absence or presence of a BA mixture.…”

“…The DICI relies on the ratio of urea produced by the sandwich-cultured human hepatocytes exposed to test compound in the absence or presence of a BA mixture. A DICI cut-off value of 0.78 is used to correlate obtained in vitro results with an in vivo safety margin [ 103 ]. The use of sandwich-cultured human hepatocytes has the advantage of preserving both phenotype and liver-specific functions for prolonged periods of time.…”