Watanabe Yuichiro, Nakajima Miki, Yokoi Tsuyoshi
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan.
Drug Metab Dispos. 2002 Dec;30(12):1462-9. doi: 10.1124/dmd.30.12.1462.
Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.
对曲格列酮在人肝脏和肠道微粒体以及重组尿苷二磷酸葡萄糖醛酸基转移酶（UGTs）中的葡萄糖醛酸化作用进行了全面表征。杆状病毒感染昆虫细胞中的所有重组UGT同工型（UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT1A7、UGT1A8、UGT1A9、UGT1A10、UGT2B7和UGT2B15）均表现出曲格列酮葡萄糖醛酸基转移酶活性。特别是在胃、肠道和结肠等肝外组织中表达的UGT1A8和UGT1A10显示出高催化活性，其次是UGT1A1和UGT1A9。当底物浓度超过200μM时，重组UGT1A10和UGT1A1中曲格列酮葡萄糖醛酸化的动力学表现出非典型的底物抑制模式。在曲格列酮浓度为6至200μM时，根据米氏方程，重组UGT1A10中的K(m)值为11.1±5.8μM，V(max)值为33.6±3.7 pmol/min/mg蛋白。在重组UGT1A1中，K(m)值为58.3±29.2μM，V(max)值为12.3±2.5 pmol/min/mg蛋白。人肝脏和空肠微粒体中曲格列酮葡萄糖醛酸化的动力学也表现出非典型模式。人肝脏微粒体中曲格列酮葡萄糖醛酸化的K(m)值为13.5±2.0μM，V(max)值为34.8±1.2 pmol/min/mg，人空肠微粒体中的K(m)值为8.1±0.3μM，V(max)为700.9±4.3 pmol/min/mg蛋白。当根据体外动力学参数、微粒体蛋白含量和组织重量估算内在清除率时，人肠道中曲格列酮葡萄糖醛酸化比人肝脏中的高3倍。13名个体的肝脏微粒体中曲格列酮葡萄糖醛酸基转移酶活性的个体差异最大为2.2倍。曲格列酮葡萄糖醛酸基转移酶活性与主要由UGT1A1催化的β-雌二醇3-葡萄糖醛酸基转移酶活性显著相关（r = 0.579，p < 0.05）。胆红素（IC(50)=1.9μM）对人肝脏微粒体混合液中的曲格列酮葡萄糖醛酸基转移酶活性有强烈抑制作用，胆红素是UGT1A1的典型底物。这些结果表明，人肝脏中曲格列酮葡萄糖醛酸化主要由UGT1A1催化。5名个体肠道的S-9样品中曲格列酮葡萄糖醛酸基转移酶活性的个体差异为8.2倍。大黄素（IC(50)=15.6μM）对人空肠微粒体中的曲格列酮葡萄糖醛酸基转移酶活性有强烈抑制作用，大黄素是UGT1A8和UGT1A10的典型底物，而胆红素（IC(50)=154.0μM）的抑制作用较弱。因此，提示人肠道中曲格列酮葡萄糖醛酸化可能主要由UGT1A8和UGT1A10催化。
