Characterization of the UDP-glucuronosyltransferases involved in the glucuronidation of an antithrombotic thioxyloside in rat and humans

To investigate the glucuronidation on the hydroxyl group of carbohydrate-co ntaining drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human l iver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). Th e reaction revealed a marked regioselectivity in rat and humans. Human live r microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on eithe r the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower ext ent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (V-max/K-m 30.0 and 0.06 mu l/min/mg, respectively). Phenobarbital, 3-meth ylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that sever al UGT isoforms were involved ih this process. The biosynthesis of the 2-O- glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UG T1A6 and 2B11. By contrast, the rat liver recombinant UGT1A6 and 2B1 failed to form the 2-O-glucuronide isomers. From all the recombinant UGTs tested, none catalyzed the formation of the 3-O-glucuronide isomer. Interestingly, glucuronidation on the 4-position was found in all the metabolic competent V79 cell lines considered, including the nontransfected V79 cells, suggest ing the presence of an endogenous UGT in fibroblasts able to actively glucu ronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could refl ect the existence of a different enzyme.