Evaluation and optimization of a microcavity plate-based human hepatocyte spheroid model for predicting clearance of slowly metabolized drug candidates.

Abstract

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning® Elplasia® microcavity 96-well microplates enable culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared to microsomes, hepatocyte suspensions, 2D plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted CLint and CLb within 3-fold for 53% (9/17; AAFE=3.9) and 82% (14/17; AAFE=2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE=4.0) and CLb (14/17; AAFE=2.1) without need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 μM itraconazole was within 25% of observed values for 6/8 compounds with 5/8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance, as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. Significance Statement Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance which is improved with the consideration of mass transport within the spheroid. Incubations with 3 µM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.