Cuttle L, Munns A J, Hogg N A, Scott J R, Hooper W D, Dickinson R G, Gillam E M
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.
Drug Metab Dispos. 2000 Aug;28(8):945-50.
The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C91, 2C92, and 2C93. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C91, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.
抗惊厥药苯妥英（5,5 - 二苯基乙内酰脲）会在5%至10%的患者中引发皮疹，这预示着一种特异反应的开始，该反应可能是由活性药物代谢产物对正常自身蛋白质的共价修饰引起的。苯妥英主要通过细胞色素P450（P450）酶代谢为5 -（对羟基苯基）-5 - 苯基乙内酰脲（HPPH），HPPH可能会进一步代谢为儿茶酚，后者会自发氧化为半醌和醌类物质，这些物质会与蛋白质发生共价修饰。本研究的目的是确定哪些P450催化HPPH代谢为儿茶酚，这被认为是苯妥英生物活化的最终酶促步骤。重组人P450与NADPH - 细胞色素P450还原酶在大肠杆菌中共表达。构建了用于P450 2C19以及P450 2C9的三种主要变体即2C91、2C92和2C93的新型双顺反子表达载体。同时评估了HPPH代谢和共价加合物的形成。P450 2C19是HPPH氧化为儿茶酚代谢产物的最有效催化剂，并且还与最高水平的共价加合物形成相关。P450 3A4、3A5、3A7、2C91和2C9*2也催化HPPH的生物活化，但程度较小。荧光成像分析表明，细菌膜中共价加合物形成的主要靶点是催化性P450形式，这与用人肝微粒体进行的实验结果一致。这些结果表明，P450 2C19以及2C和3A亚家族的其他形式可能既是苯妥英药物 - 蛋白质加合物形成的靶点，也是其催化剂。
