体外研究人细胞色素P450 3A4等位基因多态性对药物代谢和药物相互作用的影响  

Study on the effects of human CYP450 3A4 allelic polymorphism on drug metabolisms and drug-drug interactions in vitro

作　　者：李拴美[1] 刘端[1] 朱娟莉 陈超[1] 

机构地区：[1]西北大学国家微检测系统工程技术研究中心,西安710069 [2]陕西北美基因股份有限公司,西安710069

出　　处：《中国医药生物技术》2009年第3期177-183,共7页Chinese Medicinal Biotechnology

基　　金：国家高技术研究发展计划(863计划)(2006AA020705);陕西省"13115"科技创新工程重大科技专项项目(2007ZDKG-76)

摘　　要：目的体外研究药物对人细胞色素P4503A4(CYP3A4)(野生型,WT)及其4个突变等位基因重组酶CYP3A4*3(M445T)、CYP3A4*4(I118V)、CYP3A4*17(F189S)和CYP3A4*18(L293P)的抑制程度。方法采用荧光高通量法和HPLC法分别测定WT及其4个突变等位基因重组酶催化荧光底物——二甲基荧光素(DBF)脱烷基化和探针底物——硝苯地平氧化反应时的酶动力学参数;且通过测定大扶康、万络、西乐葆、酮康唑、地尔硫卓和维拉帕米的半数抑制浓度(IC50)值,确定6种药物对酶的抑制程度。结果除F189S外,其余4种重组酶均能催化DBF和硝苯地平反应生成相应的产物。各突变等位基因重组酶催化DBF反应的Km值(亲和力)与WT相当,M445T和L293P的内在清除率分别是WT的3.1和3.8倍(P<0.05),I118V是WT的1.3倍(P=0.1010)。各重组酶催化硝苯地平氧化反应的Km值均比WT小,I118V和L293P的内在清除率分别是WT的0.7和2.6倍(P<0.05),M445T与WT相当(P=0.7676)。6种药物对各重组酶的抑制程度强弱均为:酮康唑>地尔硫卓>维拉帕米>大扶康>西乐葆>万络;药物对各重组酶的IC50值大小为:WT<L293P<M445T<I118V。结论等位基因突变导致了酶动力学特征的改变和药物对酶抑制程度的不同,为进一步研究临床联合用药安全性奠定基础。Objective To study the inhibition degrees of drugs on the human cytochrome P450 3A4 （CYP3A4）（wild type, WT） and its four mutant aUelic recombinant enzymes [CYP3A4^＊3（M445T）, CYP3A4^＊4（I118V）, CYP3A4^＊ 17（F189S）, CYP3A4^＊ 18（L293P） ]in vitro. Methods The kinetics parameters of WT and four mutant allelic recombinants were detected in catalyzing fluorescent substrate dibenzylfluorescein （DBF） dealkylation and probe substrate nifedipine oxidization by the fluorescent high throughout and the HPLC. Furthermore, the inhibition degrees of diflucan, vioxx, celebrex, ketoconazole, diltiazem and verapamil to every enzymes were determined by half inhibitory concentration（IC50） values of every enzymes. Results Besides the F189S, other four recombinant enzymes could catalyze DBF and nifedipine to their relevant metabolites. The Km values （substrate affinities） of the mutant allelic recombinants in catalyzing DBF were similar to that of WT. The intrinsic clearance rates of M445T and L293P were 3.1 and 3.8 times higher than that of WT, respectively （P 〈 0.05）. The intrinsic clearance rate of I118V was 1.3 times （P = 0.1010） higher than that of WT. The Km values of the recombinant enzymes in catalyzing nifedipine oxidation were lower than that of WT. The intrinsicclearance rates of Il18V and L293P were 0.7 and 2.6 times higher than that of WT （P 〈 0.05）. The intrinsic clearance rate of M445T was similar to that of WT （P = 0.7676）. The inhibition degrees of 6 drugs showed the following rank order against every recombinant enzymes as measured： ketoconazole 〉 diltiazem 〉 verapamil 〉 diflucan 〉 celebrex 〉 vioxx. The mean IC50 values of every recombinant enzymes by drugs were arranged as WT 〈 L293P 〈 M445T 〈 I 118V. Conclusions The allelic change induced the change of the enzyme kinetic character and inhibition degrees of the drugs on enzymes. This could be used to lay the foundation in studying on the clinical drug combination.

关 键 词：细胞色素P450酶系统 多态性 单核苷酸 半数抑制浓度 药物相互作用 

分 类 号：R96[医药卫生—药理学]