Prediction of human hepatic clearance with HepaSH monolayers for low-turnover compounds

The pharmacokinetic characterization of drug candidates is an essential step in drug development. To date, primary suspension hepatocytes have been widely used for this purpose; however, their poor stability has limited the application of in vitro systems for compounds with low metabolic turnover rates. Highly functional HepaSH cells, prepared from chimeric mice with humanized livers, maintain a cobblestone-like morphology and cytochrome P450-dependent drug-metabolizing activity for up to 168 h in monolayer culture without medium change using a commercially available long-term hepatocyte culture medium. In this study, we attempted to investigate the utility of long-term culture systems and predict the hepatic clearance of 12 drugs with 9 low and 3 moderate-to-high CL int in humans using multiple HepaSH monolayers. This culture system successfully monitored the depletion of low (such as diazepam and quinidine) and moderate-to-high CL int drugs (midazolam). Two low-clearance drugs, disopyramide and warfarin, showed no depletion over 168 h, indicating limitations in the application of this method for clearance evaluation. Hepatic clearance values obtained from incubation with HepaSH monolayers were predicted for 6 to 8 out of 12 compounds tested with deviations within 3-fold, with an average fold error of 1.14–1.19-fold and an absolute average fold error of 1.52–1.97-fold, roughly correlating with the clinical reference data. In conclusion, a functionally stable culture method for HepaSH monolayers is highly effective for evaluating low-clearance compounds by greatly extending the metabolic reaction time and will be a valuable tool for determining the pharmacokinetic properties of new drug candidates. Significance statement This study demonstrated that combining highly functional HepaSH monolayers with extended drug incubation enables accurate monitoring of low-turnover compound clearance, an outcome that has been difficult to achieve with traditional assays.