Effects Of Single Nucleotide Polymorphisms Of Human UGT2B7 And UGT1A9 On The Selective Glucuronidation Of Some Drugs

UDP-Glucuronosyltransferases (UGTs) are very important drug metabolizing enzymes (DMEs) in human, which catalyze the conjugation of variety of lipophilic substrates with glucuronic acid. The glucuronidation mediated by UGT is the important detoxification mechanism in vivo. UGT1A9 and UGT2B7 are the important members of UGT gene superfamily, which catalyze the glucuronidation of many commonly used drugs in clinic including non-steroidal antiinflammatory drugs (NSAIDs), resveratrol, SN-38, and so on. With the development of modern medical treatment techniques and molecular biological sciences, the functional impact of DMEs genetic polymorphisms on the observed metabolic variability of drug in vivo are widely studied. The functional significance and genotype-phenotype correlation of the DMEs polymorphisms is an ongoing area of research. The different three-dimensional configurations of drugs will lead to the stereoselective metabolisms, such as the stereoselective glucuronidation of chiral drugs. When the biological activity of a chiral molecule is largely associated with one enantiomer, in such cases, any enantioselective metabolism favoring the active antipode would affect the pharmacological and toxicological spectrum of the chiral molecules. To date, however, the effects of DMEs polymorphisms on the selective metabolisms of drugs are not well studied. The aim of this study was to investigate the effects of UGT2B7 and UGT1A9 polymorphisms on the selective glucuronidations of six profens and trans-RES by the recombinant UGTs expressed in Bac-to-Bac system. 1. The expression of recombinant human UGT2B7s and UGT1A9sThe recombinant human metabolizing enzyme is widely used in xenobiotics metabolism studies in vitro. It has been proved as an effective way to detect the substrate spectrum of individual UGT and make clear the sophisticated metabolism mechanism including the stereoselective metabolic mechanism of chiral drugs. The functional significance of drug metabolizing enzymes is a hot topic of research.In this project, we used a Bac-to-Bac baculovirus infected Sf9 insect cell expression system to get stable and high activity recombinant wild types and variants of human UGT2B7 and UGT1A9. The cell homogenates of UGT2B7s and UGT1A9s were subjected to Western blot analysis for identification and determination of relative levels of expressed UGT protein. The catalyzing activity of the recombinant UGTs to 7-HFC was measured and it was proved that the recombinant UGTs were suitable for the further functional study.2. The effect of UGT2B7 genetic polymorphisms on the glucuronidation of six chiral profensProfens all possess one chiral center, but still mostly are available as racemates. Many studies shown that the glucuronidation of profens were stereoselective. The different reactive rates of the two enantiomer may lead to the differential pharmacodynamics and toxicities. To study the effect of UGT2B7 genetic polymorphisms on the glucuronidation of the chiral profens, profens were incubated with the recombinant UGT2B7s expressed in Bac-to-Bac system. Enantiomeric profen glucuronide was biosynthesized by incubating with rat hepatic microsomes and then purified with solid-phase cartridges. The produced glucuronides were quantificated by hydrolysis withβ-glucuronidase and were used as the standards to validate the establised methods. The reversed phase-high performance liquid chromatography (RP-HPLC) assays that had been reported, modified or newly established were employed to simultaneously determine the concentrations of the two diastereoisomeric glucuronides. According to our study, the kinetics of the five profens (except for CPF) glucuronidation in all UGT2B7 allozymes fit the Michaelis-Menten model. The kinetic of CPF exhibited atypical enzymatic profile in UGT2B7s. The four UGT2B7 allozymes exhibited significantly different catalyzing efficiency to the five profens (CPF was not included in), whose order is *1>*71S (except for FePF)>*2>*5. We almost cannot detect the metabolite produced by UGT2B7*5 under the experimental conditions. Although the three variants all exhibited significant changes in the catalytic activities compared with the wild type, there were little differences on the enatioselective glucuronidations were observed.3. The effect of UGT1A9 genetic polymorphisms on the glucuronidation of trans-resveratrol and flurbiprofenResveratrol is a dietary antioxidant polyphenol of plant origin found in grapes, cranberries, peanuts and red wine as the cis-and trans-isomers and is thought to possess a variety of beneficial effects including cardioprotective effects, chemopreventive effects, anti-inflammatory and antioxidant effects. The glucuronidation of RES in human is both regioselective and stereospecific. The major UGT isoforms involved in RES glucuronidation are UGT1A1, UGT1A6 and UGT1A9 yielding the 3-O-glucuronide (R3G) metabolite and the 4'-O-glucuronide (R4'G) metabolite. Although the impact of UGT1A1 and UGT1A6 genetic polymorphisms on the glucuronidation of RES has been studied, the impact of UGT1A9 polymorphisms on the glucuronidation of RES is not clearly established. This study aims at characterizing the effect of UGT1A9 genetic polymorphisms on the glucuronidation trans-RES. The kinetic profiles for the formation of R3G in all the UGT1A9 allozymes exhibited substrate inhibition. It is interesting to note that UGT1A9*2 exhibited a Km similar to *1 but a significantly greater Vmax. UGT1A9*3 and *5 all exhibited a Vmax similar to *1 but a significantly greater Km with *3. The kinetics in all UGT1A9 allozymes with significantly different Km and Ki, values suggests sequential substrate binding to two sites. In the case of R4'G formation, all the UGT1A9s exhibited sigmoidal (autoactivation) profiles. UGT1A9*2 exhibited both significantly greater Km and Vmax compared with*1. UGT1A9*3 and *5 all exhibited a slightly decreased Vmax compared with*1 but a significantly greater Km.UGT1A9 and UGT2B7 present opposite stereoselectivity towards the glucuronidation of FPF. Compared with the UGT1A9*1, the realtive efficiency of the UGT1A9*2 allele to (+)-FPF was almost 1.6-fold higher than that of the UGT1A9*1 protein and similar efficiency to (-)-FPF, resulting in a more significant differences in the stereoselective glucuronidation. For UGT1A9 allozymes, the ratio of (-)-glucuronide to (+)-glucuronide was 0.6,0.3 and 0.4 for *1, *2 and *5, respectively. The two variants all exhibited stronger stereoselectivity to rac-FPF compared with the wild-type.