Michaud Josée, Leblond Francois A, Naud Judith, Boisvert Caroline, Desbiens Karine, Nicoll-Griffith Deborah A, Pichette Vincent
Service de Néphrologie et Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, Faculté de Médecine, Université de Montréal, 5415 boulevard de l'Assomption, Montréal, Québec, Canada.
J Pharmacol Toxicol Methods. 2007 Mar-Apr;55(2):209-13. doi: 10.1016/j.vascn.2006.07.002. Epub 2006 Aug 4.
Quantification of cytochrome P450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P450 using Supersomes and its application in non-hepatic tissue (e.g., intestine).
Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations.
Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes preparations.
In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P450 in the rat liver and intestine.
细胞色素P450的定量分析是新药研发中的一个主要问题。已有多种检测方法被报道，但对3A亚型或细胞色素P450具有高选择性的方法却很少。苄氧基取代的内酯环氧化酶-2抑制剂3-[(3,4-二氟苄基)氧基]-5,5-二甲基-4-[4-(甲基磺酰基)苯基]呋喃-2(5H)-酮最近已成功用于检测肝脏中细胞色素P450的3A亚型。然而，其对大鼠亚型的选择性以及在其他组织（如肠道）中的适用性仍有待确定。本研究的目的是使用微粒体确定该底物对大鼠细胞色素P450 3A亚型的特异性及其在非肝脏组织（如肠道）中的应用。
通过使用亚型特异性抑制抗体或仅表达一种细胞色素P450亚型的微粒体，确定3-[(3,4-二氟苄基)氧基]-5,5-二甲基-4-[4-(甲基磺酰基)苯基]呋喃-2(5H)-酮对大鼠细胞色素P450 3A亚型的特异性。在大鼠肝脏和肠道微粒体蛋白制剂中检测活性。
抑制抗体实验表明，在肝脏和肠道微粒体中，针对3A亚型的抗体可抑制超过90%的底物代谢。通过测试各种微粒体制剂的代谢活性，进一步确定了该底物对大鼠3A亚型的选择性。
总之，我们的结果证实了3-[(3,4-二氟苄基)氧基]-5,5-二甲基-4-[
