CYP2C9基因多態(tài)性及與Gomisin G作用后對華法林藥代動力學的影響
發(fā)布時間：2018-09-13 08:25
【摘要】：目的探討CYP2C9基因多態(tài)性及其基因位點CYP2C9*2、CYP2C9*3及CYP2C9*c65與華法林抗凝治療的維持劑量的相關(guān)性：同時預(yù)測Gomisin G、華法林與CYP2C9的結(jié)合模型,并通過觀察戈米辛G對不同基因型CYP2C9酶的作用及其對華法林的藥代動力學過程的影響。以期探討華法林個體用藥模式以及戈米辛G應(yīng)用于臨床治療提供理論和實驗依據(jù)。方法(1)按照準入標準采集2013年1月-2015年12月年之間于吉林大學第一醫(yī)院行心臟瓣膜置換術(shù)并服用華法林的漢族患者共270例,采集靜脈血3-5 mL,血樣本經(jīng)EDTA處理后,采用飽和酚-氯仿方法提取樣本的DNA,通過CAPS (cleaved amplification polymorphism sequence -taggde sites)技術(shù)也稱聚合酶鏈式反應(yīng)-限制性片段長度多態(tài)性(PCR-RFLP)技術(shù)及常規(guī)DNA測序方法對CYP2C9基因的三個候選位點(CYP2C9*2、CYP2C9*3、CYP2C9*c 65)的基因以及等位基因頻率進行測定。(1)采用分子對接法對戈米辛G與CYP2C9的作用位點進行預(yù)測,CYP2C9的晶體結(jié)構(gòu)從蛋白質(zhì)數(shù)據(jù)庫(http://www.rcsb.org/pdb)獲得,戈米辛G的化學結(jié)構(gòu)由chemdraw軟件進行繪制。(2)用高效液相色譜檢測戈米辛G在體外對CYP2C9*2、CYP2C9*3、CYP2C9*c65酶的作用、IC50和抑制時間依賴性,建立血漿樣品華法林含量測定方法,檢測SD大鼠體內(nèi)戈米辛G對華法林藥代動力學的影響。結(jié)果(1)所有樣品中并未檢測到CYP2C9*2 (rs1799853)位點發(fā)生突變,僅檢測到一種等位基因C,基因型全部為C/C野生型；所有樣品中共檢測到CYP2C9*3 (rs1057910)位點的兩種等位基因,分別為A位點和C位點,共檢測出三種基因型,分別為A/A型、A/C型以及C/C型,其中A/A野生型為231例,占85.6%；AC雜合子突變型為25例,占9.26%；C/C純合子突變型為14例,占5.19%。對等位基因進行分析,結(jié)果表明等位基因A頻率為94.3%,等位基因C頻率為5.7%,CYP2C9*3基因突變與服藥維持劑量之間存在相關(guān)性(P0.05),A/C型患者服藥劑量較A/A型患者降低了18.46%,C/C型患者服藥劑量較A/A型降低了76.0%,表明CYP2C9*3的C型位點有突變的可能,患者的華法林服用劑量與未突變者有所不同。CYP2C9*c 65 (rs9332127)位點共檢測出G位點和C位點兩種等位基因,包括G/G型以及G/C型兩種基因型。對G/G野生型進行統(tǒng)計,結(jié)果為246例,占91.1%；G/C雜合子突變型為24例,占8.9%；這兩種基因突變患者的華法林維持劑量之間不存在明顯相關(guān)性。(2)分子對接的受體為CYP2C9晶體結(jié)構(gòu)(PDB編號：4GQS),來源于蛋白晶體結(jié)構(gòu)數(shù)據(jù)庫,其中包括與華法林結(jié)合的晶體結(jié)構(gòu),以及相應(yīng)抑制劑的晶體結(jié)構(gòu)。本研究對CYP2C9代謝戈米辛G的模式進行預(yù)測,篩選出戈米辛G作為CYP2C9底物與之結(jié)合的晶體結(jié)構(gòu)。底物華法林首先從CYP2C9代謝活性部位被分離開來,然后戈米辛G與CYP2C9的活性位點相結(jié)合。如圖2-1所示,戈米辛G能夠較好地與CYP2C9活性位點相結(jié)合,該過程是經(jīng)由戈米辛G與CYP2C9基因上的Phe476和Gln214以氫鍵鏈接而實現(xiàn)的。為了證明戈米辛G是CYP2C9的良好底物,本研究分別對戈米辛G和華法林與CYP2C9結(jié)合的活性位點的結(jié)構(gòu)進行分析。如圖2-2所示,戈米辛G與CYP2C9結(jié)合的活性位點較華法林與CYP2C9結(jié)合的活性位點距離更為接近。提示作為CYP2C9的結(jié)合底物來說,戈米辛G比華法林更適合。(3)戈米辛G對CYP2C9三種基因型的抑制作用為CYP2C9*3CYP2C9*2 CYP2C9*1,表明戈米辛G對CYP2C9酶的抑制作用存在個體差異。將華法林與戈米辛G聯(lián)合作用于SD大鼠,研究表明,與對照組相比較,給藥組華法林的AUC、Cmax以及CL/F未發(fā)生改變,而Tmax以及T1/2則發(fā)生延長。表明戈米辛G對華法林的體內(nèi)藥物動力學過程存在一定影響,這可能與戈米辛G能夠與華法林競爭CYP2C9酶的活性位點有關(guān),進而抑制了CYP2C9酶對華法林的作用。結(jié)論(1)在CYP2C9基因的三個基因位點中,CYP2C9*2和CYP2C9*c 65位點與華法林抗凝治療劑量無關(guān),無明顯相關(guān)性；CYP2C9*3位點的多態(tài)性與華法林的抗凝治療劑量大小有相關(guān)性；且C型突變患者服用華法林的劑量較少,有統(tǒng)計學意義。(2)戈米辛G是CYP2C9的良好底物,當其與CYP2C9結(jié)合后能夠抑制華法林與CYP2C9的結(jié)合。(3)戈米辛G對CYP2C9酶有抑制作用,且具有個體基因型差異；戈米辛G能夠影響華法林的藥動學過程,這可能與戈米辛G能夠與華法林競爭CYP2C9酶的活性結(jié)合位點有關(guān),從而抑制CYP2C9酶對華法林的代謝作用。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R969
本文編號：2240600
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R969
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