Evidence for oxazepam as an in vivo probe of UGT2B15: oxazepam clearance is reduced by UGT2B15 D85Y polymorphism but unaffected by UGT2B17 deletion

Abstract

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• UDP-glucuronosyltransferase (UGT) 2B15 is a major drug glucuronidation enzyme expressed in human liver.• Oxazepam is an isoform-selective probe drug that is being used for in vitro studies of UGT2B15.• The most common UGT2B15 missense polymorphisms (D85Y and K523T) are correlated with variable oxazepam glucuronidation in human liver bank samples.• UGT2B17 is also expressed in liver and has high sequence homology and substrate specificity overlap with UGT2B15. WHAT THIS STUDY ADDS• UGT2B15 D85Y polymorphism is identified as a major determinant of oxazepam disposition, accounting for as much as 34% of interindividual variability in oxazepam apparent oral clearance.• An effect of the UGT2B15 K523T or the UGT2B17 deletion polymorphisms on oxazepam disposition could not be detected.• Provides evidence supporting the use of oxazepam as an isoform-selective in vivo probe for studies of variability in UGT2B15 activity. AIMSAlthough in vitro studies indicate that oxazepam is an isoform-selective substrate probe for UDP-glucuronosyltransferase 2B15, the utility of this drug as an in vivo probe is uncertain. The main aim of this study was to determine whether common missense polymorphisms in the UGT2B15 gene (D85Y and K523T) are associated with altered oxazepam pharmacokinetics and pharmacodynamics. We also determined the possible influence of a common deletion polymorphism in the gene encoding UGT2B17, which shows substantial substrate specificity overlap with UGT2B15. METHODSThirty healthy male subjects were administered 15 mg of oxazepam by mouth followed by plasma oxazepam concentration monitoring for 36 h, and pharmacodynamic testing for 8 h. Genotypes were determined by genomic polymerase chain reaction and commercial 5′-nuclease assays. RESULTSAllele frequencies for D85Y, K523T, UGT2B17del were 47%, 23% and 19%, respectively. Median oxazepam apparent oral clearance was significantly lower in 85YY subjects (1.62 ml min -1 kg -1) compared with 85DD subjects (3.35 ml min -1 kg -1; P = 0.003, Student-Newman-Keuls test), whereas 85DY subjects were intermediate (2.34 ml min -1 kg -1; P = 0.018 vs. 85DD, P = 0.034 vs. 85YY). Regression analysis indicated that UGT2B15 D85Y genotype accounted for 34% of interindividual variability. However, neither UGT2B15 K523T nor UGT2B17del was associated with altered oxazepam disposition. Furthermore, no differences in pharmacodynamic measures, including quantitative electroencephalography, digit-symbol substitution test, self-or observer-rated visual analogue scales, could be demonstrated for any of the polymorphisms evaluated. CONCLUSIONSThese results identify UGT2B15 D85Y as a major determinant of oxazepam clearance, and indicate that oxazepam may be useful as an in vivo probe for glucuronidation by UGT2B15.

Identification and validation of the microRNA response elements in the 3′-untranslated region of the UDP glucuronosyltransferase ( UGT ) 2B7 and 2B15 genes by a functional genomics approach

“…Previous work from our lab identified SNP D85Y (253G>T, rs1902023) in exon 1 of the UGT2B15 gene [13]. Moreover we demonstrated that the high frequency variant allele D85Y (allele frequency 47%) was a major determinant of the observed inter-individual variability ofoxazepam glucuronidation in vivo [14]. However, SNP D85Y could explain only 34% of the observed interindividual variability of hepatic oxazepam glucuronidation in vivo [14].…”

“…Moreover we demonstrated that the high frequency variant allele D85Y (allele frequency 47%) was a major determinant of the observed inter-individual variability ofoxazepam glucuronidation in vivo [14]. However, SNP D85Y could explain only 34% of the observed interindividual variability of hepatic oxazepam glucuronidation in vivo [14]. Consequently, other trans -acting factors could regulate UGT2B7 and UGT2B15 expression and contribute to this variability.…”

“…This was followed by identification and functional validation of the miRNA response elements in the 3’-UTRs by a combination of in silico analysis and luciferase reporter construct assays. Furthermore we determined whether a common SNPin the UGT2B15 3’-UTR (rs3100) that was previously associated with UGT2B15 mRNA allelic imbalance [14] can disrupt miRNA binding. Finally, we measured the transcript amounts of the candidate miRNAs in human liver samples to determine whether they are correlated with hepatic UGT2B7 or UGT2B15 activities.…”

The Glucuronidation of R- and S-Lorazepam: Human Liver Microsomal Kinetics, UDP-Glucuronosyltransferase Enzyme Selectivity, and Inhibition by Drugs

“…UGT1A7, which was observed here to glucuronidate R-lorazepam, also metabolized R-oxazepam. Court et al (2004) and He et al (2009) subsequently showed that Slorazepam, but not R-lorazepam, glucuronidation activity was reduced in HLM from donors expressing UGT2B15*2, and the apparent oral clearance of oxazepam was reduced in subjects homozygous for UGT2B15*2. Overall, the UGT2B15*2 allele was estimated to account for 34% of the interindividual variability in oxazepam clearance (He et al, 2009).…”

“…Court et al (2004) and He et al (2009) subsequently showed that Slorazepam, but not R-lorazepam, glucuronidation activity was reduced in HLM from donors expressing UGT2B15*2, and the apparent oral clearance of oxazepam was reduced in subjects homozygous for UGT2B15*2. Overall, the UGT2B15*2 allele was estimated to account for 34% of the interindividual variability in oxazepam clearance (He et al, 2009). The differential effects of inhibitors on R-and S-oxazepam glucuronidation by HLM further support the involvement of different UGT enzymes in the glucuronidation of the oxazepam enantiomers (Patel et al, 1995b).…”