Effect of UDP-glucuronosyltransferase 1A8 polymorphism on raloxifene glucuronidation.

Raloxifene is an antiestrogen marketed for the treatment of osteoporosis. The major metabolic pathway of raloxifene is glucuronidation at 6- and/or 4'-positions, which is mainly catalyzed by UDP-glucuronosyltransferase 1A8 (UGT1A8) expressed in extrahepatic tissues such as the small intestine and colon. Two non-synonymous allelic variants, termed UGT1A8*2 (518C>G, A173G) and UGT1A8*3 (830G>A, C277Y), have been found in Caucasian, African-American and Asian populations. In this study, the effect of amino acid substitutions in UGT1A8 on raloxifene glucuronidation was studied using recombinant UGT1A8 enzymes of wild-type (UGT1A8.1) and variant UGT1A8 (UGT1A8.2 and UGT1A8.3) expressed in Sf9 cells. Raloxifene 6- and 4'-glucuronidation by UGT1A8.1 exhibited negative allosteric kinetics. The Km and Vmax values of UGT1A8.1 were 15.0 μM and 111 pmol/min/mg protein for 6-glucuronidation, and 9.35 μM and 232 pmol/min/mg protein for 4'-glucuronidation, respectively. The kinetics of raloxifene 6-glucuronidation by UGT1A8.2 was positive allosteric, whereas the kinetics of raloxifene 4'-glucuronidation was negative allosteric. The S50 value of raloxifene 6-glucuronidation was markedly low (1.2%) compared with the Km value of UGT1A8.1, and the Km value for raloxifene 4'-glucuronidation was 29% that of UGT1A8.1. The Vmax value for raloxifene 6-glucuronidation by UGT1A8.2 was comparable to that of UGT1A8.1, whereas the Vmax value for raloxifene 4'-glucuronidation was significantly lower (54%) than that of UGT1A8.1. The activities of raloxifene 6- and 4'-glucuronidation in UGT1A8.3 were markedly lower than those of UGT1A8.1. In mycophenolic acid glucuronidation, the kinetics by wild-type and variant UGT1A8s fitted the Michaelis-Menten model. The Km and Vmax values of UGT1A8.1 were 123 μM and 4820 pmol/min/mg protein, respectively. The Km and Vmax values of UGT1A8.2 were comparable to those of UGT1A8.1. The Km value of UGT1A8.3 was similar to that of UGT1A8.1, whereas the Vmax value was reduced to 2.4% of UGT1A8.1. These findings suggest that A173G and C277Y substitutions of UGT1A8 change the metabolic ability toward raloxifene, and that the polymorphic alleles of UGT1A8 may influence the clinical response and bioavailability of medicines metabolized mainly by UGT1A8.