Individual Difference in Drug Metabolism and Disposition : Toxicological Significance of Genotypes and Phenotypes of S-mephenytoin 4'-hydroxylase(CYP2C19)

研究表明，S-美苯妥因的4‘-羟基化反应是由细胞色素P42C19介导的。它在一些相关的海因和巴比妥酸盐的代谢中也很重要，在结构上不同的药物如奥美拉唑、普罗瓜尼和西酞普兰的代谢中也是重要的。因此，我们研究了细胞色素P4502C19基因多态性与奥美拉唑代谢的关系，以期为法医学和临床毒理学评估中毒的严重程度和预测结果提供依据。在这项研究中，我们从无血缘关系的健康日本人的血液中制备了DNA样本，并建立了一种基于聚合酶链式反应(PCR)的限制性片段长度多态性快速简便的基因分型方法。使用De Morais等人描述的等位基因特异性引物，通过聚合酶链式反应(PCR)扩增，对CYP2C19进行基因分型。只需稍加修改。用限制性内切酶对扩增产物进行限制性内切酶消化，用聚丙烯酰胺凝胶电泳法进行分析。此外，通过Marinac等人描述的高效液相色谱法测定摄入奥美拉唑后2小时的血清中奥美拉唑和羟基奥美拉唑的浓度，以确定细胞色素P450 2C19的表型。只需稍加修改。因此，所观察到的基因型别分别为：野生型：wt)、野生型(M1)和野生型(M2)。野生型的奥美拉唑羟化指数为-1.15，杂合型为-0.78，同源突变型为1.22。CYP2C19基因分型与表型相关。这些结果表明，药物代谢酶基因分型分析在法医学和临床毒理学中评估中毒的严重程度和预测结果方面将发挥更重要的作用。

The 4'-hydroxylation of S-mephenytoin has been shown to be mediated by CYP2C19. It is also important in the metabolism of a number of relatd hydantoins and barbiturates, as well as in that of structurally dissimilar drugs such as omeprazole, proguanil, and citalopram. As a result, large interphenotypic differences occur in the disposition of these drugs, which may affect their toxicity and efficacy.Therefore, we examined the relationship between genetic polymorphism of CYP2C19 and metabolism of omeprazole in order to assess the severity and to predict the outcome of poisoning for the forensic and clinical toxicology. In this study, we prepared the DNA samples from the blood of unrelated healthy Japanese, and developed a rapid and simple genotyping method using a polymerase chain reaction (PCR) based restriction fragment length polymorphism. Genotyping procedures for the identification of CYP2C19 were performed by PCR amplification with use of the allele-specific primers described by de Morais et al. with minor modifications. PCR products were digested with the restriction enzymes, and were analyzed by polyacrylamide gel electrophoresis. Furthermore, CYP2C19 phenotypes were determined by measuring omeprazole and hydroxyomeprazole concentrations in the serum, collected at 2 hours after omeprazole ingestion, by high performance liquid chromatography described by Marinac et al. with minor modifications. Consequently, the genotypes observed were CYP2C19^*1A (wild type : wt), CYP2C19^*2 (m1), and CYP2C19^*3 (m2). The omeprazole hydroxylation index of wild-type was -1.15, whereas hetero-type was -0.78, and homo-mutated type 1.22. The genotype of CYP2C19 correlated with the phenotype. These results proved that genotyping assays of drug metabolizing enzymes would play more important role in assessing the severity and predicting the outcome of poisoning for forensic and clinical toxicology.