C-DILI ™ Assay: A New Mechanism-Based Human In Vitro Screen for Prediction of Cholestatic Liver Toxicity

Introduction: Drug-induced liver injury (DILI) is a significant cause of drug attrition and market withdrawal, underscoring the importance of the early assessment of hepatotoxicity during drug development. Disruption of bile acid (BA) homeostasis can precipitate liver injury, making the regulation of BA by the liver essential to prevent DILI. Conventional bile salt export pump (BSEP) inhibition assays have poor predictive value, as they do not consider the BA feedback mechanism that mitigates hepatotoxicity. In this study, we present an innovative approach for the preclinical evaluation of the BA-induced hepatotoxic potential of drug candidates. Methods: The C-DILI™ Assay employs two distinct media conditions to differentiate between BA-dependent and BA-independent cytotoxicity by measuring LDH release and ATP depletion in sandwich-cultured human hepatocytes. Seventy-one drugs were evaluated, including 49 unblinded and 22 blinded compounds with varied BSEP inhibition profiles and DILI risk. Results and Discussion: The assay successfully identified 14 drugs with BA-dependent and 7 with BA-independent hepatotoxicity. For instance, troglitazone (Trog) demonstrated BA-dependent cytotoxicity, whereas cyclosporine A exhibited BA-independent cytotoxicity. Notably, antagonism of farnesoid X receptor (FXR) emerged as a common mechanism underlying BA-dependent toxicity, consistent with FXR’s critical role in BA homeostasis. In the blinded assessment, the assay detected nine drugs with BA-dependent cytotoxicity, affirming its utility in elucidating this mechanism of liver toxicity. Of these, six drugs had documented preclinical or clinical hepatotoxicity findings, thus corroborating the strategy’s value in preclinical safety evaluation. This approach provides a comprehensive and clinically relevant framework for the preclinical prediction of BA-dependent liver toxicity, thereby strengthening preclinical DILI safety assessments. Given the varied clinical presentations and mechanisms of DILI, this strategy should be integrated into a multifaceted preclinical DILI assessment paradigm.