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 (Km) and a 3.8- and 2.2-fold higher intrinsic clearance (CLint) 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 CLint values for R-temazepam glucuronidation, with Km values similar to those observed for HLMs and HKMs. For S-temazepam glucuronide formation, UGTs 1A6 and 2B15 exhibited the highest CLint among all UGT1A and UGT2B enzymes, with UGT2B15 exhibiting the lowest Km 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.