P19-20 A Multi-Substrate Approach for Simultaneous Assessment of CYP Enzymatic Activities in various In vitro Hepatic Models

The cytochrome P450 (CYP) system plays a pivotal role in both xenobiotic and endogenous compound metabolism, making it essential for predicting drug toxicity and pharmacokinetics. Characterizing both basal activity and compound-induced modulation of CYPs across species is crucial in early toxicological evaluation. Assessing CYP activity via intrinsic clearance offers a more physiologically relevant understanding of metabolism compared to traditional Vmax measurements. We report the development of a novel substrate cocktail, Xenomix™, which enables simultaneous measurement of the activity of 9 CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5) in diverse in vitro hepatic systems. Two mixes were developed: one with 10 to 14 substrates, and a corresponding calibration mix of their metabolites for LC-MS/MS. Unlike substrate depletion methods, CYP activities were quantified via metabolite formation to provide a more specific and accurate measurement. The method was applied to human and animal liver microsomes and to hepatocytes in suspension and in monolayer. Initial screening used human and animal liver microsomes. Five human liver microsomal batches (20 donors each) showed consistent CYP activity profiles across batches. The cocktail was similarly applied to 14 batches of primary human hepatocytes (10–20 donors each) and hepatocytes from multiple animal species, all yielding reproducible results. High inter-batch consistency was also observed with 2D-differentiated HepaRG™ cells and HepaSH™ hepatocytes. Potential drug–drug interactions (DDIs) within the cocktail were evaluated by comparing single substrate and full cocktail incubations. Some DDIs were noted, particularly involving phenacetin and mephenytoin, prompting the optimization of a new cocktail version comprising 14 substrates to monitor 12 CYP isoforms (adding CYP1A1, CYP2J2, and distinguishing between CYP3A4 and CYP3A5). Validation in human microsomes confirmed that clearance rates in cocktail incubations matched those seen in single substrate tests, indicating no metabolic interference. Further validation in induction studies using 2D-differentiated HepaRG™ cells involved a 48-hour exposure to omeprazole, phenobarbital, and rifampicin, known inducers of CYP1A1/2, CYP2B6, and CYP3A4/5, respectively. The fold induction of CYP activities determined by Xenomix™ was compared to standard Vmax assays. The intrinsic clearance method not only captured CYP induction profiles similar to reference assays but also demonstrated superior sensitivity across a broad CYP panel. Additionally, expected CYP3A4 induction levels were obtained after exposure to weak, moderate, and strong inducers, confirming quantitative reliability. Overall, this multi-substrate approach offers a powerful, scalable, and sensitive tool for evaluating CYP enzymatic activity and induction with broad applications in drug metabolism, toxicology, and regulatory science.