SLCO1B1与CYP2C9之间的相互作用机理及在个体化用药中的应用

It is very important to comprehensively consider genetic polymorphism in drug metabolism enzymes and drug transporters in personlized medicine. Our previous work indicates that there may be some relations between SLCO1B1 and CYP2C9. However, study of interaction between SLCO1B1 and CYP2C9 has not been available in literature. Our project is to invetigate transport of series of CYP2C9 substrates via OATP1B1 by using transgenic OATP1B1 cells, explore the interaction between CYP2C9 substrates and OATP1B1 and quantitative structure activity relationship. A double transfected OATP1B1/CYP2C9 cell line will be established to describe the relationship between transport ability of CYP2C9 substrates via OATP1B1 and intrinsic clearance rate of intracellular drug metabolism via CYP2C9. Clinical studies in volunteers will be conducted to investigate the influence of SLCO1B1 gene polymorphism on CYP2C9 phenotype and single-nucleotide polymorphism of CYP2C9 on OATP1B1 phenotye. Clinical liver samples will be used to study the relations among gene polymorphism, mRNA and protein level of CYP2C9 and SLCO1B1, and the role of nuclear receptor-related factors in interplay of CYP2C9 and SLCO1B1. The molecular mechanism of interation between CYP2C9 and SLCO1B1 will be revealed and thus provide the scientific evidence for personalized medicine and rational drug use.

综合考虑代谢酶与转运体基因多态性对个体化用药十分重要。我们前期工作发现SLCO1B1和CYP2C9存在某种联系，而两者的相互作用机理尚不清楚。本项目应用OATP1B1转基因细胞研究一系列CYP2C9底物经OATP1B1转运的性质，探究CYP2C9底物与OATP1B1相互作用方式和构效关系。建立OATP1B1-CYP2C9双转染细胞模型，探索CYP2C9底物经OATP1B1的转运与经细胞内CYP2C9代谢的内在清除率之间的关系。开展人体试验，揭示SLCO1B1基因多态性对CYP2C9活性影响以及CYP2C9基因多态性对OATP1B1活性影响。利用临床肝脏样本，研究CYP2C9、OATP1B1在基因多态性、mRNA和蛋白水平的相关性，以及相关核受体因子在CYP2C9和OATP1B1相互关系中的作用。揭示SLCO1B1与CYP2C9相互作用的分子机理，为指导个体化合理用药提供科学依据。

SLCO1B1和CYP2C9可能存在某种联系，而两者相互作用的研究国内外未见报道。本项目利用OATP1B1转基因细胞研究系列CYP2C9底物经OATP1B1转运，探究CYP2C9底物与OATP1B1相互作用方式和定量关系。通过RT-PCR、Western blotting和瑞舒伐他汀在单克隆细胞中的积聚试验，筛选出功能稳定、表达量高的人OATP1B1*1a/*15细胞株。证实HEK293-OATP1B1*1a细胞对托拉塞米的摄取呈现出良好的时间依赖性，细胞内积聚符合米氏方程特征。运用稳定表达OATP1B1 的HEK293细胞，通过OATP1B1经典底物瑞舒伐他汀的摄取实验，对33种中药化学成分中人OATP1B1潜在抑制剂进行了筛选。首次发现苦玄参苷等17种对瑞舒伐他汀的摄取均有抑制作用，而白藜芦醇等8种能显著增加细胞中瑞舒伐他汀的摄取。建立OATP1B1-CYP2C9双转染细胞模型，探索CYP2C9底物经OATP1B1的转运与经细胞内CYP2C9代谢的内在清除率之间的关系。通过人CYP2C9表达质粒的构建、细胞转染、专属底物验证单克隆细胞 CYP2C9 功能，证实与不转染CYP2C9的细胞相比，单克隆细胞有显著功能。对托拉塞米在HEK293-OATP1B1*1a-CYP2C9单克隆细胞中的摄取与代谢进行考察。以HEK293-MOCK-CYP2C9和HEK293-OATP1B1*1a-CYP2C9细胞模型考察33种中药化学成分对托拉塞米摄取和代谢的作用，研究OATP1B1和CYP2C9在肝脏药物处置中的共同作用。证实HEK293-OATP1B1-CYP2C9细胞中环孢素作用后托拉塞米代谢下降是由于摄取被显著抑制所致。发现一些中药单体能同时抑制OATP1B1和CYP2C9从而导致托拉塞米的摄取与代谢量均下降；一些中药单体是OATP1B1的底物和CYP2C9的抑制剂，通过OATP1B1摄取进入细胞从而抑制CYP2C9代谢托拉塞米。证实熊去氧胆酸、鹅去氧胆酸和牛黄胆酸钠抑制托拉塞米代谢和OATP1B1摄取，牛黄胆酸钠通过OATP1B1摄取进入细胞从而抑制CYP2C9对托拉塞米代谢。CYP2C9*3对OATP1B1底物匹伐他汀PK有重要影响，但SLCO1B1多态性对CYP2C9底物氟伐他汀PK无显著影响。循证发现转运体与药物代谢酶之间相互作用在评价DDI临床数据时应予以强调。