TY - JOUR T1 - <strong>Reaction Phenotyping of Low-Turnover Compounds in Long-term Hepatocyte Cultures Through Persistent Selective Inhibition of Cytochromes P450</strong> JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.121.000601 SP - DMD-AR-2021-000601 AU - Sheri R Smith AU - Michael J Lyman AU - Bennett Ma AU - Donald J Tweedie AU - Karsten Menzel Y1 - 2021/01/01 UR - http://dmd.aspetjournals.org/content/early/2021/08/18/dmd.121.000601.abstract N2 - Recognizing the challenges of determining the relative contribution of different drug metabolizing enzymes to the metabolism of slowly metabolized compounds, a cytochrome P450 reaction phenotyping (CRP) method, using co-cultured human hepatocytes (HEPATOPAC®), has been established. In this study, the emphasis on the relative contribution of different cytochrome P450 (CYP) isoforms was assessed by persistently inhibiting CYP isoforms over 7 days with human HEPATOPAC®. CYP isoform selective inhibition was achieved with the chemical inhibitors furafylline (CYP1A2), tienilic acid (CYP2C9), (+)-N-3-benzylnirvanol (CYP2C19), paroxetine (CYP2D6), azamulin (CYP3A), and a combination of 1-aminobenzotriazole and tienilic acid (broad spectrum inhibition of CYPs). We executed this CRP method using HEPATOPAC by optimizing for the choice of CYP inhibitors, their selectivity, and the temporal effect of inhibitor concentrations on maintaining selectivity of inhibition. In general, the established CRP method using potent and selective chemical inhibitors allows to measure the relative contribution of CYPs and to calculate the fraction of metabolism (fm) of low turnover compounds. Several low turnover compounds were used to validate this CRP method by determining their hepatic CLint and fm, with comparison to literature values. We established the foundation of a robust CRP for low turnover compound test system which can be expanded to include inhibition of other drug metabolizing enzymes. This generic CRP assay, using human long-term hepatocyte cultures, will be an essential tool in drug development for new chemical entities in the quantitative assessment of the risk as a victim of drug-drug interactions. Significance Statement An ongoing trend is to develop drug candidates which have limited metabolic clearance. The current studies report a generic approach to conducting reaction phenotyping studies with human HEPATOPAC, focusing on CYP metabolism of low turnover compounds. Potent and selective chemical inhibitors were used to assess the relative contribution of the major human CYPs. Validation was achieved by confirming hepatic CLint and fm for previously reported low turnover compounds. This approach is adaptable for assessment of all drug metabolizing enzymes. ER -