Biosynthesis of imipramine glucuronide and characterization of imipramine glucuronidation catalyzed by recombinant UGTIA4  

作　　者：Ming-rong QIAN Su ZENG 

机构地区：[1]Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031 China

出　　处：《Acta Pharmacologica Sinica》2006年第5期623-628,共6页中国药理学报（英文版）

基　　金：Project supported by the National Natural Science Foundation of China(No C30100232 and No C30225047).

摘　　要：Aim： To study the profile of imipramine N^＋-glucuronidation using homogenates of recombinant uridine-5＇-dipho sphoglucuronosyltransferase 1 A4 （UGT 1A4） from baculovirus-infected sf9 cells. Methods： Recombinant UGT1A4 was obtained from sf9 cells infected with recombinant baculovirus. Imipramine N^＋-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high pressure liquid chromatography （RP-HPLC） assay method was used to directly measure the concentration of imipramine and its metabolite, imipramine N^＋-glucuronide, with p-nitrophenol as the internal standard. The validated method was used to characterize the activity of recombinant UGT1A4 and carry out kinetic studies on imipramine glucuronidation in vitro. Results： The high concentration of imipramine inhibited glucuronide conjugation, so the formula V=Vmax·SI（Km＋S＋S^2/Ki） was used to calculate the parameters, using MATLAB software. The values of apparent Kin, Ki, and Vmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/min per mg cell homogenate （n=3）, respectively. Conclusion： As a specific substrate of UGT1A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGT1A4 in vitro.Aim： To study the profile of imipramine N^＋-glucuronidation using homogenates of recombinant uridine-5＇-dipho sphoglucuronosyltransferase 1 A4 （UGT 1A4） from baculovirus-infected sf9 cells. Methods： Recombinant UGT1A4 was obtained from sf9 cells infected with recombinant baculovirus. Imipramine N^＋-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high pressure liquid chromatography （RP-HPLC） assay method was used to directly measure the concentration of imipramine and its metabolite, imipramine N^＋-glucuronide, with p-nitrophenol as the internal standard. The validated method was used to characterize the activity of recombinant UGT1A4 and carry out kinetic studies on imipramine glucuronidation in vitro. Results： The high concentration of imipramine inhibited glucuronide conjugation, so the formula V=Vmax·SI（Km＋S＋S^2/Ki） was used to calculate the parameters, using MATLAB software. The values of apparent Kin, Ki, and Vmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/min per mg cell homogenate （n=3）, respectively. Conclusion： As a specific substrate of UGT1A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGT1A4 in vitro.

关 键 词：IMIPRAMINE GLUCURONIDE uridine-5＇- diphosphoglucuronosyltransferase 1A4 BACULOVIRUS solid-phase extraction 

分 类 号：R97[医药卫生—药品]