Received for publication August 3, 2007.
Revised November 6, 2007.
Accepted for publication November 7, 2007.

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 UDPGA. The structure of the N-glucuronide was confirmed by NMR 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, where 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 S₅₀ values for the formation of O- and N-glucuronides were 43 and 18 µM, respectively, and the corresponding apparent V_max values were 363 and 21 pmol/mg microsomal protein/min. Incubations with recombinant human UGTs indicated that the O-glucuronidation was catalyzed by UGT2B4 and 2B7, while the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation; while 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.

Key words: enzyme kinetics, glucuronidation, liver microsomes, UDP glucuronyltransferases

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