Characterization of 1′-Hydroxymidazolam Glucuronidation in Human Liver Microsomes

Abstract

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 Nglucuronide 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 M, 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.

Metabolism of 1′- and 4-Hydroxymidazolam by Glucuronide Conjugation Is Largely Mediated by UDP-Glucuronosyltransferases 1A4, 2B4, and 2B7

“…Because our inhibition and recombinant experiments do not support a significant role for UGT1A4 in 1Ј-hydroxymidazolam Oglucuronidation, the observed correlation may be derived from the involvement of UGT2B7 in lamotrigine glucuronidation (Rowland et al, 2006). These results are compatible with those from a previous report by Zhu et al (2008).…”

“…2). These results are consistent with those from previous reports (Zhu et al, 2008). When 4-hydroxymidazolam was incubated with the HLMs, two glucuronide conjugates (m/z 518) were detected.…”

“…When 4-hydroxymidazolam was incubated with the HLMs, two glucuronide conjugates (m/z 518) were detected. On the basis of the structures of the 1Ј-hydroxymidazolam O-and N-glucuronides, which were reported by Zhu et al (2008), it is reasonable to assume that glucuronic acid was conjugated to 4-hydroxymidazolam at the same N and OH positions. These two metabolites were designated as 4-hydroxymidazolam glucuronide I and II because we could not distinguish 4-hydroxymidazolam O-glucuronide from the N-glucuronide.…”

“…Glucuronide conjugates of 1Ј-hydroxymidazolam account for 60 to 70% of the midazolam dose (Heizmann and Ziegler, 1981). Zhu et al (2008) recently identified an N-linked glucuronide conjugate of 1Ј-hydroxymidazolam by incubation with human liver microsomes (HLMs). Although the O-and N-glucuronidation of 1Ј-hydroxymidazolam is catalyzed by UGT2B4/2B7 and 1A4, respectively (Zhu et al, 2008), the specific hepatic UGT isoforms involved in 4-hydroxymidazolam glucuronidation have not been identified.…”

Hepatic UDP-Glucuronosyltransferase Is Responsible for Eslicarbazepine Glucuronidation

“…UGT2B7 and UGT2B4 have been reported to be involved in O-glucuronidation of 1Ј-hydroxymidazolam, a metabolite of midazolam, whereas 1Ј-hydroxymidazolam N-glucuronidation is catalyzed by UGT1A4 (Zhu et al, 2008). O-glucuronidation of several coumarins, anthraquinones, and flavonoids was observed with UGT2B17 (Turgeon et al, 2003).…”