title: Characterization of 1'-hydroxymidazolam glucuronidation in human liver microsomes.
abstract: Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhu
authors.forename: David
authors.initials: D
authors.lastname: Bush
authors.forename: George A
authors.initials: GA
authors.lastname: Doss
authors.forename: Stella
authors.initials: S
authors.lastname: Vincent
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Franklin
authors.forename: Shiyao
authors.initials: S
authors.lastname: Xu
mesh_terms: D000818:Animals; D002491:Central Nervous System Agents / Q000378:metabolism; D004008:Diclofenac / Q000494:pharmacology; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008874:Midazolam / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012502:Sapogenins / Q000494:pharmacology; D014535:Uridine Diphosphate Glucuronic Acid / Q000494:pharmacology
chemical_list: D002491:Central Nervous System Agents; D020719:Glucuronides; D011994:Recombinant Proteins; D012502:Sapogenins; D004008:Diclofenac; D014535:Uridine Diphosphate Glucuronic Acid; C007908:hecogenin; C040081:1-hydroxymethylmidazolam; D014453:Glucuronosyltransferase; D008874:Midazolam