Comparative analysis of sex-based, vendor-based, and species differences in cytochrome P450 metabolism.

Hepatic clearance is crucial as it directly impacts drug exposure, efficacy, and safety. Cytochrome P450 (CYP450) enzymes play a pivotal role in drug metabolism and exhibit differences based on sex, species, and commercial liver microsome vendors. These variables can directly influence translational accuracy when preclinical data are applied to human drug development. In this study, we evaluated metabolic stability of isozyme-selective compounds across human, rat, and mouse liver microsomes, incorporating both male and female microsomes and multiple vendors. Our analysis revealed three layers of variability: (1) sex-specific differences consistent with prior clinical observations, where certain substrates displayed markedly faster clearance in one sex; (2) interspecies divergence, such as male-predominant isoforms in rodents without direct human orthologs; and (3) vendor-related discrepancies, where the same species-sex pool yielded divergent stability outcomes depending on microsome source. Together, these findings illustrate the combined effects of sex, species, and vendor source that contribute to variability in CYP450-mediated metabolism. By systematically comparing these factors, our work underscores the importance of considering these variables during early preclinical studies. Accounting for these sources of variability may improve the translational reliability of in vitro assays, reduce costly late-stage failures, and better support the development of safe and effective therapeutics.