Characterization of 1'-hydroxymidazolam glucuronidation in human liver microsomes.

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 μM, 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.