A Comprehensive Study on the Effect of Human Genetic Polymorphism on the Drug Metabolizing, Properties of CYP

在本研究中，我们专注于细胞色素P450（CYP 450），这是人类药物代谢酶中的一个优势群体，我们的目的是建立一个临床原则，适用于订单治疗，通过综合研究的药物结合和代谢活性的突变体，这些突变体是根据人类基因组单核苷酸多态性（SNP）的信息制备的。我们制备了CYP 1A 2、CYP 2C 9、CYP 2C 19、CYP 2D 6和CYP 3A 4，沿着基于SNP信息的这些CYP的突变体，并测量了这些CYP的药物代谢特性。此外，我们测量了它们的共振拉曼光谱，以阐明受SNP影响的结构因素。此外，我们测量了突变体的药物代谢特性，以研究SNP的影响。我们可以建立一个参数Kd，这是一个指标的药物结合亲和力，是线性相关的Km，代谢活性的措施。此外，Vmax与5-配位血红素的含量呈线性相关。因为Vmax/Km值与药物清除率相关，所以现在清楚的是，Kd和5-配位的静态值揭示了药物清除率。另一方面，我们通过添加底物药物以稳定蛋白质，并在纯化步骤中修饰表面活性剂，成功地制备了CYP 3A 4和CYP 2D 6。因此，我们最终确定，药物代谢的CYP可以使用这些CYP进行综合研究。

In this study, we focused on cytochrome P450 (CYP) which is a dominant group in the human drug metabolizing enzymes, and we aimed at the establishment of a clinical principle which is applicable to order-made treatment, through comprehensive studies on the drug binding and metabolizing activities of mutants which are prepared on the bases of the information on human genomic single-nucleotide polymorphisms (SNPs). We prepared CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, along with mutants of these CYPs based on the SNP information, and measured drug metabolizing properties of these CYPs. In addition, we measured resonance Raman spectra of them in order to clarify structural factors that are affected by SNPs. Furthermore, we measured drug metabolizing properties of the mutants in order to investigate the effect of SNPs. We could establish that a parameter Kd, which is an index of drug binding affinity, is linearly correlated with Km, a measure of metabolizing activity. In addition, Vmax was found to linearly correlate with the content of 5-coordinated heme. Because the value Vmax/Km is correlated with drug clearance, it is now clear that static values of Kd and 5-coordination reveals the drug clearance. On the other hand, we succeeded in the preparation of CYP3A4 and CYP2D6 by adding a substrate drug to stabilize the protein, and modifying surfactants during the purification step. Thus, we finally established that drug metabolism by CYPs can be conprehensively investigated using these CYPs.