Untargeted Metabolomics Reveal New Endogenous Plasma Biomarkers Associated with CYP3A Inhibition in Humans.

Reliable endogenous biomarkers for assessing CYP3A activity and clinical drug-drug interaction (DDI) risk remain limited. We characterized global metabolomic changes following itraconazole treatment in a fixed-sequence DDI study (11 healthy volunteers) to identify novel CYP3A endogenous biomarkers. Untargeted metabolomic analysis identified 58 plasma metabolites that were significantly modulated (changed by > 1.5-fold or < 0.67-fold, P < 0.05), highlighting the dynamic and systemic impact of CYP3A inhibition on the human metabolome. Bile acids represented the most prominently affected analytes, whereas changes were also observed in endocannabinoids, xanthine metabolism, and food-derived metabolites, revealing both on-target and potential off-target effects of itraconazole. For example, plasma levels of endocannabinoidome metabolites such as linoleoyl ethanolamide and N-linoleoylglycine increased by 2.4- to 6.0-fold (P < 0.05), while bile acids including glycoursodeoxycholate (GUDCA) and glycohyocholate (GHCA) decreased by 2.6- to 12.5-fold (P < 0.01) following itraconazole treatment. Targeted quantification was subsequently performed for the bile acids identified from untargeted analysis and the emerging CYP3A biomarkers, 1β-hydroxy-deoxycholic acid (1βOH-DCA) and its glycine and taurine conjugates. Notably, GHCA, GUDCA and 1βOH-DCA conjugates exhibited pronounced reduction in plasma exposure (~10-fold, P < 0.001) in the itraconazole treatment group. GHCA exhibited lower inter-individual variability than others. In vitro characterization demonstrated CYP3A4-selective hydroxylation of glycochenodeoxycholate to form GHCA, supporting its mechanistic linkage to CYP3A4 activity. Collectively, these findings expand the repertoire of sensitive and mechanistically supported endogenous biomarkers of CYP3A activity. Further evaluation of the newly identified GHCA and emerging bile acid metabolites across diverse CYP3A inhibitors can strengthen their translational utility in DDI risk assessment and drug development.