Abstract

To investigate the glucuronidation on the hydroxyl group of carbohydrate-containing drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human liver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). The reaction revealed a marked regioselectivity in rat and humans. Human liver microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on either the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower extent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (V_max/K_m30.0 and 0.06 μl/min/mg, respectively). Phenobarbital, 3-methylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that several UGT isoforms were involved in this process. The biosynthesis of the 2-O-glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UGT1A6 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, suggesting the presence of an endogenous UGT in fibroblasts able to actively glucuronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could reflect the existence of a different enzyme.