Genetic variability of an uridine-diphosphoglucuronosyltransferase, UGT2B7

grantor: University of TorontoReports of genetically based variations in drug glucuronidation have essentially focused on their relation to Gilbert's Syndrome and Crigler-Najjar type I and II syndromes, which have been linked to abnormalities in the UGT1 gene complex. To date, information about the genetic variability of other UGT isozymes has been limited. Oxazepam is one of a number of C-3 hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R), and active (S), enantiomers. 1. Inhibition studies suggested that the two diastereomeric glucuronidations are catalysed by different UGT isozymes. The inhibition profile of (S)oxazepam glucuronidation suggests that UGT2B7 is the catalysing enzyme. 2. (a) In vivo, the (S) glucuronide was preferentially formed and excreted in nine of eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 $\pm$ 0.79 (mean $\pm$ s.d.) and 3.52 $\pm$ 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p $<$ 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 $\pm$ 3.2 h). In 66 additional subjects, the S/R glucuronide ratio was found to be bimodally distributed, with $\sim$10% of all subjects possessing ratios below an apparent antimode of 2.0. (b) In vitro formation of oxazepam glucuronides by microsomes from four of 37 Caucasian livers were characterized by low S/R glucuronide ratios, and abnormally high K$\rm\sb{m}$ values for the formation of the (S) glucuronide, but not of the (R) glucuronide. 3. (a) First-strand cDNA was synthesized from the mRNA of each liver by reverse-transcription. RFLV analyses and nucleotide sequencing showed that three of the four atypical livers were positive for a point mutation at position 802 (T for C) of the UGT2B7 cDNA coding region. The mutation results in the substitution of histidine for tyrosine at residue 268 of the UGT2B7 enzyme. The three individuals were heterozygous for the mutation. Full-length sequence analysis for the fourth atypical liver, showed no deviations from the wild type sequence of UGT2B7. (b) Expression studies in COS-1 cells showed that the V$\rm\sb{max}$ of glucuronidation of (S)oxazepam was greatly diminished with the variant form of UGT2B7. Glucuronidation efficiencies of estriol and 2-OH-estriol were also significantly reduced, whereas the glucuronidation of ketoprofen, (S)naproxen, and hyodeoxycholic acid were unaffected.Ph.D