Assessment of the in vitro CYPs Activity in Human Liver Organoids: Development and Validation of a Cocktail Method with LC-MS/MS

The metabolic activity of the cytochrome P450 (P450) enzymes is essential for liver detoxification and biotransformation. However, there is currently a lack of methods to simultaneously measure the activities of key P450 enzymes in liver organoids. Here, a sensitive and specific cocktail approach was developed to simultaneously measure the activities of major human P450 enzymes, including CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6, in adult stem cell-derived liver organoids using liquid chromatography-tandem mass spectrometry. The activity of enzymes corresponding to each P450 enzyme substrate was characterized by simultaneously quantifying their derived metabolites in a single 6-minute run employing multiple reaction monitoring in positive electrospray ionization mode. The analytical method has been thoroughly validated, adhering to the rigorous standards outlined by the Food and Drug Administration for bioanalytical method validation. First, liver organoids were successfully established from nontumor tissues of hepatocellular carcinoma patients, exhibiting similar histological characteristics and marker expression to the original tissues. Second, kinetic studies using each characteristic substrate of the 5 major P450 enzymes were conducted to ascertain the optimal concentrations of each P450 enzyme substrate for use in cocktail incubation. No significant differences were found in the concentrations of metabolites produced during incubations, whether they were performed as individual substrates or in a cocktail mixture. Additionally, the cocktail assay was evaluated in the presence of ketoconazole, a selective inhibitor of CYP3A4. These findings demonstrated that the established cocktail approach is both reliable and comprehensive, enabling the simultaneous evaluation of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities in liver organoids. SIGNIFICANCE STATEMENT: This study developed a cocktail method with optimized substrate concentrations to simultaneously measure the activities of 5 major human cytochrome P450 enzymes (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6) in adult stem cell-derived liver organoids. Validated with a selective inhibitor, this approach enables robust phenotypic evaluation of cytochrome P450 enzyme activities, offering critical insights for pharmacokinetic profiling and drug-drug interaction prediction.