CYP2C9基因多态性及与Gomisin G作用后对华法林药代动力学的影响

发布时间：2018-09-13 08:25

【摘要】：目的探讨CYP2C9基因多态性及其基因位点CYP2C9*2、CYP2C9*3及CYP2C9*c65与华法林抗凝治疗的维持剂量的相关性：同时预测Gomisin G、华法林与CYP2C9的结合模型,并通过观察戈米辛G对不同基因型CYP2C9酶的作用及其对华法林的药代动力学过程的影响。以期探讨华法林个体用药模式以及戈米辛G应用于临床治疗提供理论和实验依据。方法(1)按照准入标准采集2013年1月-2015年12月年之间于吉林大学第一医院行心脏瓣膜置换术并服用华法林的汉族患者共270例,采集静脉血3-5 mL,血样本经EDTA处理后,采用饱和酚-氯仿方法提取样本的DNA,通过CAPS (cleaved amplification polymorphism sequence -taggde sites)技术也称聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术及常规DNA测序方法对CYP2C9基因的三个候选位点(CYP2C9*2、CYP2C9*3、CYP2C9*c 65)的基因以及等位基因频率进行测定。(1)采用分子对接法对戈米辛G与CYP2C9的作用位点进行预测,CYP2C9的晶体结构从蛋白质数据库(http://www.rcsb.org/pdb)获得,戈米辛G的化学结构由chemdraw软件进行绘制。(2)用高效液相色谱检测戈米辛G在体外对CYP2C9*2、CYP2C9*3、CYP2C9*c65酶的作用、IC50和抑制时间依赖性,建立血浆样品华法林含量测定方法,检测SD大鼠体内戈米辛G对华法林药代动力学的影响。结果(1)所有样品中并未检测到CYP2C9*2 (rs1799853)位点发生突变,仅检测到一种等位基因C,基因型全部为C/C野生型；所有样品中共检测到CYP2C9*3 (rs1057910)位点的两种等位基因,分别为A位点和C位点,共检测出三种基因型,分别为A/A型、A/C型以及C/C型,其中A/A野生型为231例,占85.6%；AC杂合子突变型为25例,占9.26%；C/C纯合子突变型为14例,占5.19%。对等位基因进行分析,结果表明等位基因A频率为94.3%,等位基因C频率为5.7%,CYP2C9*3基因突变与服药维持剂量之间存在相关性(P0.05),A/C型患者服药剂量较A/A型患者降低了18.46%,C/C型患者服药剂量较A/A型降低了76.0%,表明CYP2C9*3的C型位点有突变的可能,患者的华法林服用剂量与未突变者有所不同。CYP2C9*c 65 (rs9332127)位点共检测出G位点和C位点两种等位基因,包括G/G型以及G/C型两种基因型。对G/G野生型进行统计,结果为246例,占91.1%；G/C杂合子突变型为24例,占8.9%；这两种基因突变患者的华法林维持剂量之间不存在明显相关性。(2)分子对接的受体为CYP2C9晶体结构(PDB编号：4GQS),来源于蛋白晶体结构数据库,其中包括与华法林结合的晶体结构,以及相应抑制剂的晶体结构。本研究对CYP2C9代谢戈米辛G的模式进行预测,筛选出戈米辛G作为CYP2C9底物与之结合的晶体结构。底物华法林首先从CYP2C9代谢活性部位被分离开来,然后戈米辛G与CYP2C9的活性位点相结合。如图2-1所示,戈米辛G能够较好地与CYP2C9活性位点相结合,该过程是经由戈米辛G与CYP2C9基因上的Phe476和Gln214以氢键链接而实现的。为了证明戈米辛G是CYP2C9的良好底物,本研究分别对戈米辛G和华法林与CYP2C9结合的活性位点的结构进行分析。如图2-2所示,戈米辛G与CYP2C9结合的活性位点较华法林与CYP2C9结合的活性位点距离更为接近。提示作为CYP2C9的结合底物来说,戈米辛G比华法林更适合。(3)戈米辛G对CYP2C9三种基因型的抑制作用为CYP2C9*3CYP2C9*2 CYP2C9*1,表明戈米辛G对CYP2C9酶的抑制作用存在个体差异。将华法林与戈米辛G联合作用于SD大鼠,研究表明,与对照组相比较,给药组华法林的AUC、Cmax以及CL/F未发生改变,而Tmax以及T1/2则发生延长。表明戈米辛G对华法林的体内药物动力学过程存在一定影响,这可能与戈米辛G能够与华法林竞争CYP2C9酶的活性位点有关,进而抑制了CYP2C9酶对华法林的作用。结论(1)在CYP2C9基因的三个基因位点中,CYP2C9*2和CYP2C9*c 65位点与华法林抗凝治疗剂量无关,无明显相关性；CYP2C9*3位点的多态性与华法林的抗凝治疗剂量大小有相关性；且C型突变患者服用华法林的剂量较少,有统计学意义。(2)戈米辛G是CYP2C9的良好底物,当其与CYP2C9结合后能够抑制华法林与CYP2C9的结合。(3)戈米辛G对CYP2C9酶有抑制作用,且具有个体基因型差异；戈米辛G能够影响华法林的药动学过程,这可能与戈米辛G能够与华法林竞争CYP2C9酶的活性结合位点有关,从而抑制CYP2C9酶对华法林的代谢作用。
[Abstract]:Objective To investigate the association of CYP2C9 gene polymorphisms and CYP2C9*2, CYP2C9*3 and CYP2C9*c65 loci with the maintenance dose of warfarin anticoagulant therapy and to predict the binding model of Gomisin G, warfarin and CYP2C9, and to observe the effect of Gomisin G on different genotypes of CYP2C9 enzymes and its pharmacokinetic process of warfarin. Methods (1) According to the admission criteria, 270 patients of Han nationality who underwent heart valve replacement and warfarin in the First Hospital of Jilin University from January 2013 to December 2015 were collected. The venous blood samples were 3-5 mL. After EDTA treatment, DNA samples were extracted by saturated phenol-chloroform method, and three candidate sites (CYP2C9*2, CYP2C9*3, CYP) of CYP2C9 gene were sequenced by CAPS (cleaved amplification polymorphism sequence-taggde sites), also known as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and conventional DNA sequencing methods. The gene and allele frequencies of 2C9 * C 65 were determined. (1) The interaction sites of Gomicin G and CYP2C9 were predicted by molecular docking. The crystal structure of CYP2C9 was obtained from the protein database (http://www.rcsb.org/pdb), and the chemical structure of Gomicin G was plotted by ChemDraw software. (2) Detection of Gomicin G by high performance liquid chromatography. The effects of CYP2C9*2, CYP2C9*3, CYP2C9*c65 enzymes, IC50 and inhibition time-dependent assays were established to determine the effect of gomicin G on warfarin pharmacokinetics in SD rats. Results (1) Mutations at CYP2C9*2 (rs1799853) were not detected in all the samples, and only one allele was detected. Two alleles at CYP2C9*3 (rs1057910) were detected in all samples, which were A/A, A/C and C/C genotypes, respectively. Among them, 231 were wild type A/A, accounting for 85.6%, 25 were AC heterozygote mutation, accounting for 9.26%; and C/C homozygote mutation was found in 25 samples. Allele A frequency was 94.3%, allele C frequency was 5.7%, CYP2C9*3 gene mutation was correlated with maintenance dose (P 0.05). The dosage of A/C patients was 18.46% lower than that of A/A patients, and the dosage of C/C patients was 76.0% lower than that of A/A patients. CYP2C9*c 65 (rs9332127) locus detected two alleles, including G/G and G/C genotypes. The G/G wild type was counted in 246 cases, accounting for 91.1%; the G/C heterozygote mutation was found in 24 cases. There was no significant correlation between warfarin maintenance doses in 8.9% of the patients with these two mutations. (2) The docked receptor was CYP2C9 crystal structure (PDB number: 4GQS), derived from the protein crystal structure database, including the crystal structure bound to warfarin and the crystal structure of the corresponding inhibitor. The model of metabolism of Gomicin G was predicted, and the crystal structure of Gomicin G was screened out as the substrate of CYP2C9. The substrate warfarin was first isolated from the metabolic active site of CYP2C9, and then combined with the active site of CYP2C9. As shown in Figure 2-1, Gomicin G was able to bind to the active site of CYP2C9, a process in which the substrate warfarin could bind to the active site of CYP2C9. In order to prove that Gomicin G is a good substrate for CYP2C9, the structure of active sites binding to Gomicin G and Warfarin and CYP2C9 was analyzed. As shown in Figure 2-2, the binding sites of Gomicin G and CYP2C9 are more active than warfarin and CYP2C9. It was suggested that Gomicin G was more suitable as the binding substrate of CYP2C9 than warfarin. (3) The inhibitory effect of Gomicin G on CYP2C9 genotypes was CYP2C9 * 3CYP2C9 * 2CYP2C9 * 1, indicating that there were individual differences in the inhibitory effect of Gomicin G on CYP2C9 enzyme. Compared with the control group, the AUC, Cmax and CL/F of warfarin did not change, while Tmax and T1/2 were prolonged in the administration group. This indicated that Gomicin G had some effect on the pharmacokinetic process of warfarin in vivo, which might be related to the fact that Gomicin G could compete with warfarin for the active sites of CYP2C9 enzyme, and thus inhibited the activity of warfarin. Conclusion (1) CYP2C9 * 2 and CYP2C9 * C 65 loci in three CYP2C9 gene loci have no significant correlation with the dose of warfarin anticoagulation therapy, and the polymorphism of CYP2C9 * 3 locus is related to the dose of warfarin anticoagulation therapy, and the dose of warfarin is less in patients with type C mutation. (3) Gomicin G inhibited the binding of warfarin to CYP2C9, and had genotypic differences; Gomicin G could influence the pharmacokinetic process of warfarin, which might compete with warfarin G for CYP2C9 enzyme. The active binding sites are related to the inhibition of CYP2C9 enzyme on warfarin metabolism.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R969

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