Jang G R, Wrighton S A, Benet L Z
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco 94143, USA.
Biochem Pharmacol. 1996 Sep 13;52(5):753-61. doi: 10.1016/0006-2952(96)00357-7.
Various complementary approaches were used to elucidate the major cytochrome P450 (CYP) enzyme responsible for mifepristone (RU 486) demethylation and hydroxylation in human liver microsomes: chemical and immunoinhibition of specific CYPs; correlation analyses between initial rates of mifepristone metabolism and relative immunodetectable CYP levels and rates of CYP marker substrate metabolism; and evaluation of metabolism by cDNA-expressed CYP3A4. Human liver microsomes catalyzed the demethylation of mifepristone with mean (+/-SD) apparent K(m) and Vmax values of 10.6 +/- 3.8 microM and 4920 +/- 1340 pmol/min/mg protein, respectively; the corresponding values for hydroxylation of the compound were 9.9 +/- 3.5 microM and 610 +/- 260 pmol/min/mg protein. Progesterone and midazolam (CYP3A4 substrates) inhibited metabolite formation by up to 77%. The CYP3A inhibitors gestodene, triacetyloleandomycin, and 17 alpha-ethynylestradiol inhibited mifepristone demethylation and hydroxylation by 70-80%; antibodies to CYP3A4 inhibited these reactions by approximately 82 and 65%, respectively. In a bank of human liver microsomes from 14 donors, rates of mifepristone metabolism correlated significantly with relative immunodetectable CYP3A levels, rates of midazolam 1'-and 4-hydroxylation and rates of erythromycin N-demethylation, marker CYP3A catalytic activities (all r2 > or = 0.85 and P < 0.001). No significant correlations were observed for analyses with relative immunoreactive levels or marker catalytic activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. Recombinant CYP3A4 catalyzed mifepristone demethylation and hydroxylation with apparent K(m) values 7.4 and 4.1 microM, respectively. Collectively, these data clearly support CYP3A4 as the enzyme primarily responsible for mifepristone demethylation and hydroxylation in human liver microsomes.
采用了多种补充方法来阐明在人肝微粒体中负责米非司酮（RU 486）去甲基化和羟基化的主要细胞色素P450（CYP）酶：对特定CYP进行化学和免疫抑制；米非司酮代谢初始速率与相对免疫可检测的CYP水平及CYP标记底物代谢速率之间的相关性分析；以及通过cDNA表达的CYP3A4评估代谢情况。人肝微粒体催化米非司酮的去甲基化，平均（±标准差）表观K(m)和Vmax值分别为10.6±3.8微摩尔和4920±1340皮摩尔/分钟/毫克蛋白；该化合物羟基化的相应值为9.9±3.5微摩尔和610±260皮摩尔/分钟/毫克蛋白。孕酮和咪达唑仑（CYP3A4底物）抑制代谢物形成达77%。CYP3A抑制剂孕二烯酮、三乙酰夹竹桃霉素和17α-乙炔雌二醇抑制米非司酮去甲基化和羟基化达70 - 80%；抗CYP3A4抗体分别抑制这些反应约82%和65%。在来自14名供体的一组人肝微粒体中，米非司酮代谢速率与相对免疫可检测的CYP3A水平、咪达唑仑1'-和4-羟基化速率以及红霉素N-去甲基化速率（标记CYP3A催化活性）显著相关（所有r2≥0.85且P<0.001）。对于CYP1A2、CYP2C9、CYP2C19、CYP2D6或CYP2E1的相对免疫反应水平或标记催化活性分析，未观察到显著相关性。重组CYP3A4催化米非司酮去甲基化和羟基化，表观K(m)值分别为7.4和4.1微摩尔。总体而言，这些数据明确支持CYP3A4是人肝微粒体中负责米非司酮去甲基化和羟基化的主要酶。
