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

本研究以人类药物代谢酶中的优势群细胞色素P450 （CYP）为研究对象，通过对基于人类基因组单核苷酸多态性（snp）信息制备的突变体的药物结合和代谢活性进行综合研究，旨在建立一套适用于有序治疗的临床原则。我们根据SNP信息制备了CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4以及这些cypp的突变体，并测定了这些cypp的药物代谢特性。此外，我们测量了它们的共振拉曼光谱，以澄清受snp影响的结构因素。此外，我们还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（药物结合亲和力指标）与Km（代谢活性指标）线性相关。此外，Vmax与5-配位血红素含量呈线性相关。由于Vmax/Km值与药物清除率相关，现在很清楚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来稳定蛋白质，并在纯化步骤中修饰表面活性剂，成功地制备了CYP3A4和CYP2D6。因此，我们最终确定了利用这些CYPs可以全面研究CYPs的药物代谢。

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.