Zhang Y, Guo X, Lin E T, Benet L Z
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, CA, USA.
Pharmacology. 1999 Mar;58(3):147-59. doi: 10.1159/000028277.
4-Dimethylamino-4'-(imidazol-1-yl)chalcone (RL3142) is a newly developed antimalarial cysteine protease inhibitor. Four metabolites (M1-M4) were found in human liver microsomes and their structures were identified by LC/MS/MS. Two primary metabolites, M2 (minor) and M4 (major), were determined to be the N-demethylated product (M2) and the product (M4) resulting from 1,2-hydrogenation of the alpha, beta-unsaturated ketone moiety of the parent compound. A combined approach utilizing selective P450 inhibitors, immunoinhibition with CYP3A and NADPH P450 reductase antibodies, and cDNA expressed human CYP3A4 and NADPH P450 reductase, was used for identification of enzymes responsible for the biotransformation. For formation of M2, both a rabbit CYP3A polyclonal antibody (110 microliter/mg microsomal protein) and ketoconazole (2 micromol/l), a CYP3A inhibitor, showed about 50% inhibitory effects; other specific inhibitors of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 showed no significant effects. For formation of M4, neither CYP3A antibody nor the above mentioned CYP inhibitors exhibited inhibitory effects. Anti-rat NADPH P450 reductase serum (50 microliter/100 microgram microsomal protein) exhibited 70 and 58% inhibitory effects on M2 and M4 formation, respectively. Incubation of RL3142 with cDNA expressed human NADPH P450 reductase yielded formation of M4, but not M2. Carbon monoxide inhibited formation of M2 and M1 (the reduced product of M2), but had no effect on M4 and M3 (the reduced product of M4) formation. Collectively, NADPH P450 reductase solely catalyzed reduction of RL3142 to M4, whereas CYP3A contributed in part to formation of M2.
4-二甲基氨基-4'-(咪唑-1-基)查尔酮(RL3142)是一种新开发的抗疟半胱氨酸蛋白酶抑制剂。在人肝微粒体中发现了四种代谢物(M1-M4)，并通过液相色谱/串联质谱法鉴定了它们的结构。两种主要代谢物，M2(次要)和M4(主要)，被确定为N-去甲基化产物(M2)和母体化合物α,β-不饱和酮部分1,2-氢化产生的产物(M4)。采用选择性P450抑制剂、用CYP3A和NADPH P450还原酶抗体进行免疫抑制以及cDNA表达的人CYP3A4和NADPH P450还原酶的联合方法，用于鉴定负责生物转化的酶。对于M2的形成，兔CYP3A多克隆抗体(110微升/毫克微粒体蛋白)和CYP3A抑制剂酮康唑(2微摩尔/升)均显示出约50%的抑制作用；CYP1A2、CYP2B6、CYP2C9、CYP2C19、CYP2D6和CYP2E1的其他特异性抑制剂均未显示出显著作用。对于M4的形成，CYP3A抗体和上述CYP抑制剂均未表现出抑制作用。抗大鼠NADPH P450还原酶血清(50微升/100微克微粒体蛋白)对M2和M4的形成分别表现出70%和58%的抑制作用。RL3142与cDNA表达的人NADPH P450还原酶孵育产生M4，但不产生M2。一氧化碳抑制M2和M1(M2的还原产物)的形成，但对M4和M3(M4的还原产物)的形成没有影响。总体而言，NADPH P450还原酶单独催化RL3142还原为M4，而CYP3A部分参与M2的形成。
