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Contribution of the N-Glucuronidation Pathway to the Overall in Vitro Metabolic Clearance of Midazolam in Humans

Sanofi-Aventis Recherche, Department of Discovery Metabolism, Pharmacokinetics, and Safety, Montpellier, France (S.K., S.H., R.N., C.A.-F., V.M., F.S., O.F., M.B., F.G., G.F.); Sanofi-Aventis Recherche, Department of Chemical and Analytical Sciences, Toulouse, France (M.R.); and Institut National de la Santé et de la Recherche Médicale U632, Montpellier, France (P.M.)

Midazolam (MDZ) is one of the most commonly used in vivo and in vitro CYP3A4 probe substrates for drug-drug interactions (DDI) studies. The major metabolic pathway of MDZ in humans consists of the CYP3A4-mediated 1'-hydroxylation followed by urinary excretion as 1'-O-glucuronide derivative. In the present study, following incubation of MDZ with human liver microsomes supplemented with UDP-glucuronic acid, two major high-performance liquid chromatography (HPLC) peaks were isolated. HPLC and liquid chromatography/tandem mass spectrometry analyses identified these two metabolites as quaternary direct N-glucuronides of MDZ, thus revealing an additional metabolic pathway for MDZ. ¹H NMR spectrometry studies were performed showing that these two glucuronides were β-N-glucuronides, which could be considered as two different conformers of the same molecule. According to molecular modeling experiments, the two glucuronide derivatives could be involved in atropoisomerism equilibrium. The formation of MDZ N-glucuronide exhibited moderate intersubject variability (at most 4.5-fold difference, n = 10). Among the recombinant human UDP glucuronosyltransferase (UGT) isoforms tested, only isoform UGT1A4 catalyzed the N-glucuronidation of MDZ fitting a Michaelis-Menten model. K_m and V_max values were 29.9 ± 2.4 µM and 659.6 ± 19.0 pmol/min/mg protein, respectively. The N-glucuronide derivative was found in human hepatocytes incubated under control conditions but also in the presence of the well known CYP3A4 inhibitor, ketoconazole. In the context of the in vitro study of CYP3A4-mediated DDI using MDZ and ketoconazole, direct MDZ N-glucuronidation may partly compensate the decrease in MDZ metabolic clearance caused by the addition of the inhibitor, thus potentially leading to underestimation, at least in vitro, of the extent of DDI.