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 S50 values for the formation of O- and N-glucuronides were 43 and 18 μM, respectively, and the corresponding apparent Vmax 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.
Footnotes
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B.Z. and D.B. contributed equally to this work.
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This work was presented in part in N-Glucoronidation of 1′-Hydroxymidazolam in Human Liver Microsomes (B. Zhu, D. Bush, G. A. Doss, S. Vincent, R. B. Franklin, and S. Xu) at the 3rd Pharmaceutical Sciences World Congress, 2007 April 22–25; Amsterdam, The Netherlands.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017962.
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ABBREVIATIONS: UGT, uridine diphosphate glucuronosyltransferase; UDPGA, uridine 5′-diphosphoglucuronic acid; HPLC, high performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NMR, nuclear magnetic resonance.
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↵s⃞ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: Department of Chemistry, The University of Arizona, 1306 E. University Blvd., Tucson, AZ 85721-0041.
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↵2 Current affiliation: Department of Drug Metabolism, Array Biopharma Inc., 3200 Walnut St., Boulder, CO 80301.
- Received August 2, 2007.
- Accepted November 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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