Identification of human hepatic UDP-glucuronosyltransferases involved in the glucuronidation of temazepam.

Temazepam is a commonly used 1,4-benzodiazepine that exists as a racemic mixture of R and S enantiomers that are metabolized primarily by glucuronidation via the UDP-glucuronosyltransferases (UGT) enzymes. The goal of the present study was to identify the individual UGTs responsible for catalyzing temazepam enantiomer glucuronidation. There was a 3.7- and 4.5-fold lower substrate concentration at half-maximal velocity (K) and a 3.8- and 2.2-fold higher intrinsic clearance (CL) observed for S-temazepam versus R-temazepam glucuronide formation in human liver microsomes (HLMs) and human kidney microsomes (HKMs), respectively, suggesting a higher clearance of S-temazepam in vivo. Reaction phenotyping and kinetic analysis suggested that UGTs 1A3, 1A9, 2B4, 2B7, and 2B17 exhibited glucuronidation activity against both R- and S-temazepam, whereas UGTs 1A6 and 2B15 specifically glucuronidated S-temazepam. UGTs 1A9 and 2B7 exhibited the highest activity for the R-temazepam enantiomer, whereas UGTs 1A6 and 2B15 demonstrated the highest S-temazepam glucuronidation activity, further suggesting UGT stereospecificity against temazepam enantiomers. UGTs 2B7 and 1A9 displayed the highest CL values for R-temazepam glucuronidation, with K values similar to those observed for HLMs and HKMs. For S-temazepam glucuronide formation, UGTs 1A6 and 2B15 exhibited the highest CL among all UGT1A and UGT2B enzymes, with UGT2B15 exhibiting the lowest K among all UGTs tested for any temazepam enantiomer. In both HLMs and HKMs, fluconazole (an inhibitor of UGT2B7) significantly inhibited R-temazepam glucuronidation, whereas serotonin (a specific substrate of UGT1A6) significantly inhibited S-temazepam glucuronidation. These data suggest that several enzymes including UGTs 2B7, 2B15, 1A6 and 1A9 may be important in temazepam glucuronidation. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensively characterize the major UDP-glucuronosyltransferases (UGT) enzymes involved in the metabolism of temazepam. Multiple hepatic UGTs were shown to glucuronidate temazepam enantiomers, with R-temazepam metabolized primarily by UGT2B7, whereas UGT1A6 and UGT2B15 were involved in S-temazepam glucuronide formation. This study implicates specific UGT enzymes in the stereospecific glucuronidation of temazepam.