Lasker J M, Wester M R, Aramsombatdee E, Raucy J L
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Arch Biochem Biophys. 1998 May 1;353(1):16-28. doi: 10.1006/abbi.1998.0615.
Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, including S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidize S-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolized S-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0-6.4 vs 2.4-4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km = 650 microM, Vmax = 3.71 min-1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km = 178-407 microM, Vmax = 2.95-7.08 min-1). Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0. 001), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liver samples from 13-17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88. 5 +/- 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 +/- 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-Km omeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.
具有涉及CYP2C酶的药物代谢多态性的个体，对包括S-美芬妥英、奥美拉唑、华法林、甲苯磺丁脲和非甾体抗炎药在内的重要治疗药物表现出氧化不足。虽然已证明在酵母或大肠杆菌中表达的重组CYP2C19和CYP2C9蛋白能够氧化这些药物，但从人肝脏中分离出的相应天然P450的氧化能力却不清楚。为此，我们使用传统色谱技术从人肝脏样本中纯化了CYP2C19、CYP2C9和CYP2C8，并检测了它们氧化S-美芬妥英、奥美拉唑和甲苯磺丁脲的能力。重组后，CYP2C19代谢S-美芬妥英和奥美拉唑的速率分别比完整肝微粒体高11倍和8倍，而CYP2C9和CYP2C8对这些底物均未表现出明显的代谢活性。CYP2C19也被证明是甲苯磺丁脲代谢的有效催化剂，其周转速率与CYP2C9制剂相似（形成的羟基甲苯磺丁脲为2.0 - 6.4 vs 2.4 - 4.3 nmol/分钟/nmol P450）。CYP2C19介导的甲苯磺丁脲羟基化的动力学参数（Km = 650 microM，Vmax = 3.71分钟-1）与CYP2C9催化反应的参数（Km = 178 - 407 microM，Vmax = 2.95 - 7.08分钟-1）有些相似。多克隆CYP2C19抗体显著降低了人肝微粒体对S-美芬妥英4'-羟基化（98%抑制）和奥美拉唑5-羟基化（85%抑制）。CYP2C19抗体也强烈抑制（>90%）微粒体甲苯磺丁脲羟基化，这与用CYP2C9抗体观察到的抑制（>85%）相似。此外，在来自13 - 17个不同受试者的肝脏样本中，免疫反应性CYP2C19含量、S-美芬妥英4'-羟化酶活性（r = 0.912；P < 0.001）和奥美拉唑5-羟化酶活性（r = 0.906；P < 0.001）之间发现了极好的相关性。同样观察到微粒体甲苯磺丁脲羟基化与CYP2C9含量之间存在显著关系（r = 0.664；P < 0.02），但与CYP2C19含量无关（r = 0.393；P = 0.184）。最后，免疫定量显示，在这些人肝脏样本中，CYP2C9（88.5 +/- 36 nmol/mg）的表达比CYP2C19（17.8 +/- 14 nmol/mg）高5倍，比CYP2C8（11.5 +/- 12 nmol/mg）高近8倍。我们的结果与用重组CYP2C酶获得的结果一样，表明CYP2C19是人肝脏中S-美芬妥英4'-羟基化和低Km奥美拉唑5-羟基化的主要决定因素。尽管CYP2C19具有甲苯磺丁脲羟化酶活性，但其在肝脏中的低表达水平（相对于CYP2C9）可能使其在肝脏甲苯磺丁脲代谢及其任何多态性中不发挥重要作用。
