The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B71 (wild type), UGT2B771S (A71S, 211G>T), UGT2B72 (H268Y, 802C>T), UGT2B75 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A91 (wild type), UGT1A92 (C3Y, 8G>A), UGT1A93 (M33T, 98T>C), UGT1A95 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B71, UGT2B72 singles or doubles have lower K values for M3G and M6G, whereas UGT2B75 allozymes perform opposite effects. The double allozymes of UGT1A92 or UGT1A95 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A92-UGT2B71 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CL ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.