Received for publication November 5, 2007.
Revised January 31, 2008.
Accepted for publication January 31, 2008.

CONTRIBUTION OF THE N-GLUCURONIDATION PATHWAY TO THE OVERALL IN VITRO METABOLIC CLEARANCE OF MIDAZOLAM IN HUMANS

Abstract

Midazolam is one of the most commonly used in vivo and in vitro CYP3A4 probe substrate for drug-drug interactions (DDI) studies. The major metabolic pathway of midazolam in humans consists in the CYP3A4 mediated 1'-hydroxylation followed by urinary excretion as 1'-O-glucuronide derivative. In the present study, following incubation of midazolam with human liver microsomes supplemented with UDPGA, two major HPLC peaks were isolated. HPLC and LC-MS/MS analyses identified these two metabolites as quaternary direct N-glucuronides of midazolam thus revealing an additional metabolic pathway for midazolam. ¹H NMR spectrometry studies were performed demonstrating that these two glucuronides were -N-glucuronides, which could be considered as two different conformers of the same molecule. According to molecular modelling experiments, the two glucuronide derivatives could be involved in atropoisomerism equilibrium. The formation of midazolam N-glucuronide exhibited moderate intersubject variability (at most 4.5-fold difference, n = 10). Among the recombinant human UGTs isoforms tested, only isoform UGT1A4 catalyzed the N-glucuronidation of midazolam fitting a Michaelis-Menten model. K_m and V_max values were of 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 midazolam and ketoconazole, direct midazolam N-glucuronidation may partly compensate the decrease in midazolam metabolic clearance caused by the addition of the inhibitor thus potentially leading to under-estimation, at least in vitro, of the extent of DDI.

Key words: CYP3A, drug interactions, glucuronidation, in vitro-in vivo prediction, isolated hepatocytes

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