Abstract
Midazolam is a widely used index substrate for assessing effects of xenobiotics on CYP3A activity. A previous study involving human hepatocytes showed the primary route of midazolam metabolism, 1'-hydroxylation, shifted to N-glucuronidation in the presence of the CYP3A inhibitor ketoconazole, which may lead to an overprediction of the magnitude of a xenobiotic-midazolam interaction. Because ketoconazole is no longer recommended as a clinical CYP3A inhibitor, indinavir was selected as an alternate CYP3A inhibitor to evaluate the contribution of the N-glucuronidation pathway to midazolam metabolism. The effects of indinavir on midazolam 1'-hydroxylation and N-glucuronidation were first characterized in human-derived in vitro systems. Compared to vehicle, indinavir (10 µM) inhibited midazolam 1’-hydroxylation by rCYP3A4, HLMs, and high-CYP3A activity cryopreserved human hepatocytes by ≥70%; the IC50 obtained with hepatocytes (2.7 µM) was within reported human unbound indinavir Cmax (≤5 µM). Midazolam N-glucuronidation in hepatocytes increased in the presence of indinavir in both a concentration-dependent (1-33 µM) and time-dependent (0-4 h) manner (by up to 2.5-fold), prompting assessment in human volunteers (n=8). As predicted by these in vitro data, indinavir was a strong inhibitor of the 1'-hydroxylation pathway, decreasing the 1'-hydroxymidazolam/midazolam AUC0-12h ratio by 80%. Although not statistically significant, the midazolam N-glucuronide/midazolam AUC0-12h ratio increased by 40%, suggesting a shift to the N-glucuronidation pathway. The amount of midazolam N-glucuronide recovered in urine increased 4-fold but remained <10% of the oral midazolam dose (2.5 mg). A powered clinical study would clarify whether N-glucuronidation should be considered when assessing the magnitude of a xenobiotic-midazolam interaction.
SIGNIFICANCE STATEMENT <I>N</I>-glucuronidation is an understudied primary metabolic pathway of the gold standard index CYP3A substrate midazolam, which if not considered could lead to an overprediction of the magnitude of a xenobiotic-midazolam interaction. Indinavir was characterized as an alternate and selective CYP3A inhibitor to ketoconazole to assess the contribution of the <I>N</I>-glucuronidation pathway to midazolam metabolism in humans. As predicted by established in vitro systems, midazolam <I>N</I>-glucuronide formation increased in eight human subjects in the presence of indinavir, warranting a powered clinical study to asses definitively the contribution of the <I>N</I>-glucuronidation pathway to midazolam metabolism.
- cytochrome P450
- drug-drug interactions
- hepatocytes
- human/clinical
- in vitro-in vivo prediction (IVIVE)
- pharmacokinetics
- The American Society for Pharmacology and Experimental Therapeutics