微粒体环氧化物水解酶基因（EPHX1）介导的复方丹参方对华法林药动学及药效学影响

In clinical, Compound Danshen Formula often combined with Warfarin based on the anticoagulant and antiplatelet aggregation function. However, warfarin with narrow therapeutic window and higher intra-individual variation was easy to have the drug-drug interactions. Our previous research had already evaluated the pharmacokinetics and pharmacodynamics effects of Compound Danshen Dropping Pill (CDDP) on warfarin by population pharmacokinetic method. The results revealed that CDDP may have interaction with warfarin only in patients of EPHX1 A/A. This project will conduct a further study base on the previous research, which will be more comprehensive and precise to investigate the interaction of the two: Firstly, the transfected EPHX1 cells which could code mEH enzyme will be employed. Warfarin and CDDP will be cultured with substrate in cell, measure the content of substrate and its metabolite, obtain the relationship between Warfarin, CDDP and mEH enzyme. Then illustrate the effect of CDDP on the metabolism of warfarin in cell level. Secondly, the transgenic rats will be used. These rats will be administrated with single or multiple dose of warfarin and CDDP, detect plasma and tissue concentration of warfarin and its metabolite, monitor the four indicators of coagulation and activity of mEH enzyme, clarify the effect of CDDP on pharmacokinetics and pharmacodynamics of Warfarin. This project associated with in vitro and in vivo experiments will ensure safe medication in clinical, promote the rational combination of Warfarin and CDDP, then bring into full play to advantages of the combination of Chinese and Western medicine.

复方丹参方与华法林均具有抗凝、抗血小板聚集作用，临床上常联合使用。但华法林的治疗窗较窄、个体内变异大，为易发生药物相互作用的高风险药物。课题组前期采用群体药动学的方法评价了复方丹参滴丸（CDDP）对华法林药动学及药效学的影响，结果显示仅EPHX1 A/A基因的患者可能存在相互作用。本项目拟在此研究基础上，全面精准化的明确EPHX1介导的二者相互作用：首先采用编码mEH酶的特异EPHX1细胞，将华法林、CDDP与酶底物在细胞上共同培养，测定底物及其代谢物的含量，得出华法林、CDDP与mEH酶的关系，进而明确细胞水平上CDDP对华法林代谢的影响；其次采用EPHX1转基因大鼠，经过单、多次给药后，测定血浆及组织中华法林及其代谢物的浓度，监测凝血四项指标及mEH酶活性，阐明动物体内CDDP对华法林药动学及药效学的影响。体内外关联，以保障临床用药安全，促进二者的合理联合使用，充分发挥中西医结合优势。

复方丹参方与华法林均具有抗凝、抗血小板聚集作用，临床上常联合使用。但华法林的治疗窗较窄、个体内变异大，为易发生药物相互作用的高风险药物。课题组前期采用群体药动学的方法评价了复方丹参滴丸（CDDP）对华法林药动学及药效学的影响，结果显示仅EPHX1 A/A基因的患者可能存在相互作用。. 本项目拟在此研究基础上，全面精准化的明确EPHX1介导的二者相互作用：首先，招募EPHX1 A/A健康人，服用CDDP与华法林钠片后，监测华法林的血药浓度及凝血指标的变化，结果显示联合用药后华法林体内浓度均无显著性差异，而联合给药后可使受试者的PT、TT延长，INR增高，推测CDDP可能并不影响华法林在体内的血药浓度，却可以增强华法林的抗凝效果，因此CDDP与华法林在EPHX1 A/A人体内可能存在药效学的相互作用；其次，以人肝癌细胞（HepG2）构建过表达EPHX1-HepG2-G/A/Blank细胞系，将华法林与CDDP在细胞上进行孵育，结果显示在EPHX1-HepG2-A细胞系上，加用CDDP之后，mEH的表达量显著下降，差异具有统计学意义，而在EPHX1-HepG2-G细胞系上mEH的表达量并没有显著的统计学意义，确证了CDDP与华法林在特异转染的EPHX1细胞内的相互作用；最后，通过western blot测定华法林药效作用位点维生素K环氧化物还原酶（VKOR）及4种凝血因子（FII、FVII、FIX、FX）的含量，结果显示在EPHX1-HepG2-G细胞系上，加用CDDP之后，VKOR及4种凝血因子的表达量并没有显著差异，而在EPHX1-HepG2-A细胞系上，加用CDDP之后，VKOR的表达量显著下降，4种凝血因子的表达量显著升高，由此推测活化的凝血因子的表达量可能降低导致凝血功能降低，出血风险增加。. 依据体内-体外实验结果，最终验证了EPHX1 A/A介导的CDDP对华法林药效学的影响，涉及的机制可能是影响了华法林凝血作用的VKOR酶及相关的凝血因子。本研究全面明确了复方丹参方与华法林相互作用关系并对其机制进行了初探，为评价中西药相互作用提供了研究方法。