title: Contribution of the N-glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans.
abstract: 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. (1)H NMR spectrometry studies were performed showing that these two glucuronides were beta-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 microM 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.
authors.affiliation: Department of Discovery Metabolism, Pharmacokinetics, and Safety, Sanofi-Aventis, 371 Rue du Professeur Joseph Blayac, 34184 Montpellier, Cedex 4, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Klieber
authors.forename: Sébastien
authors.initials: S
authors.lastname: Hugla
authors.forename: Robert
authors.initials: R
authors.lastname: Ngo
authors.forename: Catherine
authors.initials: C
authors.lastname: Arabeyre-Fabre
authors.forename: Viviane
authors.initials: V
authors.lastname: Meunier
authors.forename: Freddy
authors.initials: F
authors.lastname: Sadoun
authors.forename: Olivier
authors.initials: O
authors.lastname: Fedeli
authors.forename: Martine
authors.initials: M
authors.lastname: Rival
authors.forename: Martine
authors.initials: M
authors.lastname: Bourrie
authors.forename: François
authors.initials: F
authors.lastname: Guillou
authors.forename: Patrick
authors.initials: P
authors.lastname: Maurel
authors.forename: Gérard
authors.initials: G
authors.lastname: Fabre
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D004347:Drug Interactions; D005260:Female; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007654:Ketoconazole / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008874:Midazolam / Q000378:metabolism*; D008875:Middle Aged
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D020719:Glucuronides; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D014453:Glucuronosyltransferase; D008874:Midazolam; D007654:Ketoconazole