Stereoselective Conjugation of Oxazepam by Human UDP-Glucuronosyltransferases (UGTs): S-Oxazepam Is Glucuronidated by UGT2B15, While R-Oxazepam Is Glucuronidated by UGT2B7 and UGT1A9

Abstract

ABSTRACT:(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R-and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R-and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Oxazepam is a 1,4-benzodiazepine derivative that is used in clinical practice for its anxiolytic, sedative, and anticonvulsant effects (Greenblatt et al., 1980(Greenblatt et al., , 1981. In humans, this drug is cleared from the body almost exclusively by hepatic glucuronidation, followed by urinary excretion (Abernethy et al., 1983). Oxazepam is formulated as a racemic preparation of S-and R-stereoisomers although the S-enantiomer is thought to be much more active as a benzodiazepine receptor agonist compared with the R-enantiomer (Mohler et al., 1978). Conjugation occurs via the hydroxyl group attached to the asymmetric 3-carbon position yielding diastereomeric glucuronides that are readily separated by routine high pressure liquid chromatography (HPLC 1 ) (Mascher et al., 1984;Patel et al., 1995a).Interindividual variability in the pharmacokinetics and metabolism of (R,S)-oxazepam have been investigated in human volunteers (Patel et al., 1995a). S-Oxazepam glucuronide was found to be formed preferentially over R-oxazepam glucuronide with S/R glucuronide diastereomeric ratios in the plasma and urine of volunteers averaging 3.5 Ϯ 0.6 and 3.9 Ϯ 0.8, respectively. Interestingly, in 2 of 11 subjects (18%), the S/R ratio in the urine was relatively low (Ͻ1.9). Since the plasma clearance of oxazepam in these individuals was also very low (Ͻ0.6 ml/min/kg) compared with other individuals (0.9 -1.4 ml/min/ kg), it was concluded that these differences probably were the result of slower S-oxazepam clearance by glucuronidation in a significant minority of the study population (i.e., a "slow metabolizer" phenotype). Although pharmacodynamic measurements were not made, the relatively slow elimination of oxazepam would be expected to result in prolonged sedation in these individuals.In vitro studies using human liver microsomes (HLMs) showed a similar picture in that S-oxazepam glucuronide was the predominant metabolite (S/R ratios averaging 4.0), and 4 of 37 livers displayed relatively slow oxazepam glucuronidation activities coinciding with low S/R metabolite ratios (Ͻ2.0) (Patel et al., 1995a). Enzyme kinetic analysis showed that the low glucuronidation activity was associated with higher apparent K m values and lower V max values for S-oxazepam glucuronidation in the four atypical livers compared with ...

Pharmacogenomics of human UDP-glucuronosyltransferase enzymes

“…153 As suggested by the authors, these differences could reflect differential stabilities of UGT2B15 proteins under the experimental conditions used for microsome preparation as opposed to enzymatic assays in intact cells. 96,153 Nevertheless, the role of UGT2B15 in the interindividual variability in S-oxazepam glucuronidation remains to be determined in vivo. Of interest, an additional polymorphic site was also reported at codon 86 adjacent to the 85 polymorphism (Figure 3).…”

“…96,97,153,157 A G-T substitution at codon 85 results in an amino-acid change (Asp 85 Tyr) within the putative substrate recognition site of UGT2B15. A number of population genotyping studies were completed to characterize the distribution of UGT2B15*1 and *2 alleles among individuals of distinct ethnic background (Table 5).…”

UGT1A1 polymorphisms are important determinants of dietary carcinogen detoxification in the liver

“…DNA samples and liver microsomes from 48 American subjects were obtained from the International Institute for the Advancement of Medicine (Exton, PA), the National Disease Research Interchange (Philadelphia, PA), and the University of Minnesota Liver Tissue Procurement and Distribution system (Minneapolis, MN), as previously described. 25,26 Population characteristics were previously described. 27 All livers were either intended for transplantation but failed to match the tissue or were normal tissue adjacent to surgical biopsy specimens.…”

UDP-Glucuronosyltransferase (UGT) 2B15 Pharmacogenetics: UGT2B15 D85Y Genotype and Gender Are Major Determinants of Oxazepam Glucuronidation by Human Liver

“…Pharmaceutical substrates of UGT2B15 that have been identified include oxazepam, E-4-hydroxytamoxifen, 5-hydroxyrofecoxib, eugenol, 8-hydroxyquinoline, phenolphthalein, 4Ј-hydroxyphenytoin, and nandrolone (Green et al, 1994;Court et al, 2002;Nishiyama et al, 2002;Kuuranne et al, 2003;Zhang et al, 2003). Oxazepam, a commonly used 1,4-benzodiazepine anxiolytic drug, is of considerable interest because it is cleared primarily by glucuronidation and has been used extensively as an in vivo probe of drug glucuronidation in people (Greenblatt et al, 1980(Greenblatt et al, , 1984Greenblatt, 1981;Sonne, 1993).…”