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.
Highlights
Drug-induced cholestasis (DIC) is a major cause of clinical failure of drug candidates
We describe a non-destructive assay for in vitro hepatocyte-based prediction of drug-induced cholestasis
The in vitro assay is based on quantifying changes in the ability of primary hepatocytes to convert toxic ammonia to non-toxic urea when co-exposed to a test compound and bile salts
Summary
Predicted safety margin values were subsequently calculated for compounds displaying hepatotoxic and/or cholestatic effects in patients, enabling evaluation of the performance of our DIC assay. By determining the ratio of urea formation after incubation with test compound in absence and in presence of the bile salt mixture, the DIC index (DICI) value can be calculated. Based on previously published protocols, a DICI value lower than 0.80 was used to flag a test compound at that specific concentration for an increased in vitro DIC risk [6,7,8]. The 90% confidence interval (CI) of the DICI values for the control conditions (i.e. incubation with 0.2% dimethylsulfoxide (DMSO) or the bile salt mixture alone) throughout all our previously performed human hepatocyte-based experiments, Fig. 1. We reassessed our previously generated data and verified the SM for prediction of DIC in vivo using our assay after 48 h incubation with receiver operating characteristic (ROC) analysis
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