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

 目的 体外研究药物对人细胞色素 P450 3A4（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。

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

Objective To study the inhibition degrees of drugs on the human cytochrome P450 3A4 (CYP3A4)(wild type, WT) and its four mutant allelic 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 I118V 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 < I118V. 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.