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. 2020 Sep 25:7:101080.
doi: 10.1016/j.mex.2020.101080. eCollection 2020.

Drug-induced cholestasis assay in primary hepatocytes

Pieter Van Brantegem  1 , Sagnik Chatterjee  2 , Tom De Bruyn  3 , Pieter Annaert  1 , Neel Deferm  1
Affiliations

Drug-induced cholestasis assay in primary hepatocytes

Pieter Van Brantegem et al. MethodsX. .

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.

Keywords: Drug candidate selection; Drug safety evaluation; Drug-induced cholestasis; Drug-induced liver injury; In vitro toxicity; Primary hepatocytes.

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Figures

Image, graphical abstract
Graphical abstract
Fig 1
Fig. 1
Timeline of the experimental part of the DIC assay.
Fig 2
Fig. 2
Urea production of sandwich-cultured human hepatocytes (mean ± SEM, n = 7 batches, measured in triplicate) after 24 h and 48 h of incubation with either 0.2% dimethylsulfoxide (DMSO) or a 50× concentrated bile salt mixture. Statistical significance was evaluated using a Student's t-test (p<0.05).
Fig 3
Fig. 3
ROC curves for different cut-off values of the SM to predict DIC after 24 h and 48 h incubation. The area under the ROC curve was 0.633 (90% CI 0.430–0.836) for 24 h incubation and 0.753 (90% CI 0.553–0.954) for 48 h incubation. The dashed line represents the reference line for classification by random chance.
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