ALDH2基因多态性对硝酸异山梨酯和单硝酸异山梨酯药动学影响的研究

发布时间：2018-05-27 14:47

本文选题：ALDH2基因多态性 + 人体药代动力学　；参考：《山东大学》2014年硕士论文

【摘要】：目的：1.采用DNA微阵列芯片法检测从人外周血提取的DNA中线粒体乙醛脱氢酶2(aldehyde dehydrogenase2, ALDH2)基因多态性,并筛选符合临床试验标准的受试者。2.进行中国健康男性志愿者单次喷服硝酸异山梨酯喷雾剂后硝酸异山梨酯(ISDN)及其两代谢物(2-单硝酸异山梨酯和5-单硝酸异山梨酯)药代动力学研究,并评价ALDH2基因多态性对三者药代动力学的影响。3.进行中国健康男性志愿者单次喷服单硝酸异山梨酯喷雾剂后5-单硝酸异山梨酯(5-ISMN)药代动力学研究,并评价ALDH2基因多态性对其药代动力学的影响。4.运用分子对接软件模拟酶与底物的作用过程,以硝酸甘油(GTN)为参考药,从分子水平探讨ALDH2基因多态性对ISDN和5-ISMN药代动力学的影响。方法：1.入组前,采集每位受试者肘静脉血3mL, EDTA抗凝,采用血液DNA提取试剂盒,提取血样DNA; ALDH2基因特异引物经PCR扩增后,将带有生物素标记的扩增产物与固定在醛基基片上的ALDH2基因型检测探针进行特异性杂交反应,并通过酶促显色反应,使特异性杂交信号显色；对芯片进行扫描,得到样品DNA与ALDH2基因位点的野生型和突变型探针杂交形成的杂交图像；经BaiO BE-2.0生物芯片识读仪分析该图像,判断待检样品的基因型。经查体和实验室检查后,筛选出20名健康男性受试者,在了解试验内容、权利、义务和风险的基础上签署知情同意书。 2.筛选出的20名健康男性受试者,按基因型分为野生组和突变杂合组,单次喷服硝酸异山梨酯喷雾剂3.75mg,于给药前和给药后2、4、6、8、10、12、15、20、30、45min和1、1.5、2、3、4、6、8、12、24h,肘静脉取血5mL置于试管中(肝素抗凝),5000rpm、离心5min,取上层血浆,-20℃保存待测。GC-ECD法测定血浆中ISDN、5-ISMN和2-单硝酸异山梨酯(2-ISMN)的浓度,采用DAS2.0.1药代动力学软件分别计算三者的药代动力学参数(t1/2、Tmax、Cmax、 AUC0-t和AUC0-∞),并对两组药代动力学参数进行统计学分析(代谢物Cmax、 AUC0-t和AUC0-t采用校正值比较。参数校正值是代谢物Cmax、AUC0-t、AUC0-∞与母药相应药代动力学参数的比值)。 3.上述受试者,单次喷服单硝酸异山梨酯喷雾剂5mg,于药前和药后8、15、20、30、45min、1、1.5、2、3、6、8、12、24h,肘静脉取血5mL,置于试管中(肝素抗凝),5000rpm、离心5min,取上层血浆,-20℃保存待测。GC-ECD法测定血浆中5-ISMN的浓度,采用DAS2.0.1药代动力学软件计算其药代动力学参数(t1/2、Cmax、AUC0-t和AUC0-∞),并对两组药代动力学参数进行统计学分析。 4.运用SYBYL-X1.1软件,分别模拟ALDH2野生酶、ALDH2突变酶与GTN、ISDN、5-ISMN的作用过程,以GTN为参照药,分别比较两酶与ISDN、5-ISMN的结合力。结果：1.经基因分型,本课题筛选出20名健康男性受试者,其中ALDH2基因野生型(ALDH2*1/*1)14名、突变杂合型(ALDH2*1/*2)6名。 2.20名受试者单次喷服硝酸异山梨酯喷雾剂3.75mg后,野生型和突变杂合型受试者体内ISDN药代动力学参数如下：t1/2分别为(0.994±0.309)h和(1.342±0.086) h, Tmax分别为(0.105±0.018)h和(0.139±0.014)h,Cmax分别为(33.611±2.065)ng/mL和(42.615±3.003)ng/mL, AUC0-t分别为(18.999±3.798)ng/mL·h和(31.188±1.427)ng/mL·h, AUC0-∞分别为(20.915-4.383)ng/mL·h和(34.576±0.898)ng/mL·h。5-ISMN药代动力学参数如下：t1/2分别为(4.554±0.858)h和(5.849±0.781)h,Tmax分别为(0.857±0.128)h和(1.083±0.204)h, Cmax分别为(63.595±4.941)ng/mL和(83.962±5.512)ng/mL, AUC0-t分别为(286.433±72.682)ng/mL·h和(482.731±100.705)ng/mL·h, AUC0-∞分别为(300.173±68.283) ng/mL·h和(519.607±96.383)ng/mL-h;2-ISMN药代动力学参数如下：tl/2分别为(3.852±0.859)h和(3.940±0.887)h,Tmax分别为(0.470±0.148)h和(0.667±0.204)h,Cmax分别为(12.321±1.325)ng/mL和(16.647±2.530)ng/mL, AUC0-t分别为(36.501±4.564)ng/mL·h和(65.941±13.547)ng/mL·h, AUC0-∞分别为(42.407±5.402)ng/mL·h和(75.098±17.972)ng/mL·h。突变杂合组与野生组相比,ISDN的t1/2和Tmax分别延长42.16%(p＜0.001)和32.38%(p＜0.001),Cmax、AUC0-t和AUCo-∞分别增加26.79%(p＜0.01)、64.16%(p＜0.01)和65.32%(p＜0.01)；5-ISMN的t1/2和Tmax分别延长28.44%(p＜0.05)和26.37%(p＜0.05),Cmax、AUC0-t和AUC0-∞的校正值分别增加4.13%(p＞0.05)、2.67%(p＞0.05)和4.71%(p＞0.05)；2-ISMN的t1/2和Tmax分别延长2.28%(p＞0.05)和41.91%(p＞0.05),Cmax、AUC0-t和AUC0-∞的校正值分别增加6.56%(p＞0.05)、10.05%(p＞0.05)和7.12%(p＞0.05)。 3.20名受试者单次喷服单硝酸异山梨酯喷雾剂5mg后,野生型和突变杂合型受试者体内5-ISMN药代动力学参数如下：t1/2分别为(4.981±1.248)h和(5.365±0.824) h, Tmax分别为(0.821±0.117)h和(0.833±0.119)h,Cmax分别为(109.332±5.668)ng/mL和(114.108±4.529) ng/mL, AUC0-t分别为(607.626±68.854) ng/mL·h和(658.459±89.621) ng/mL·h,AUC0-∞分别为(634.798±83.358) ng/mL·h和(690.937±105.191) ng/mL·h。突变杂合组与野生组相比,t1/2和Tmax分别延长了7.72%(p＞0.05)和1.45%(p0.05), Cmax、AUC0-t和AUC0-∞分别增加4.37%(p＞0.05),8.37%(p＞0.05)和8.84%(p＞0.05)。 4. SYBYL-X1.1软件对ALDH2野生酶与GTN、ISDN、5-ISMN作用过程的打分分别为6.49、5.45和3.41,对ALDH2突变酶与GTN、ISDN、5-ISMN作用过程的打分分别为3.82、2.42和3.22。结论：研究结果表明,ALDH2基因多态性影响ISDN的人体药代动力学。喷服硝酸异山梨酯喷雾剂后,与野生组相比,突变杂合组ISDN的t1/2、Tmax, Cmax、AUC0-t和AUC0-∞都增加,且差异具有统计学意义；与野生组相比,突变杂合组5-ISMN的t1/2和Tmax都增加,且差异有统计学意义,而其它药动学校正参数间的差异无统计学意义；但两组2-ISMN校正药代动力学参数间差异无统计学意义。 ALDH2基因多态性不影响5-ISMN的人体药代动力学。喷服单硝酸异山梨酯喷雾剂后,两组间5-ISMN药代动力学参数存在差异,但差异均无统计学意义。 ALDH2野生酶与GTN、ISDN的结合力显著高于ALDH2突变酶与二者的结合力；但两酶与5-ISMN结合力差异不明显。
[Abstract]:Objective: 1. the polymorphisms of mitochondrial acetaldehyde dehydrogenase 2 (aldehyde dehydrogenase2, ALDH2) gene in DNA extracted from human peripheral blood were detected by DNA microarray, and.2. was selected for the single dose of Isosorbide Dinitrate Spray of Chinese healthy male volunteers after single injection of isosorbate nitrate (ISDN) in Chinese healthy male volunteers. Pharmacokinetics of its two metabolites (2- isosorbide mononitrate and 5- isosorbide mononitrate), and evaluation of the effect of ALDH2 gene polymorphism on the pharmacokinetics of the three,.3. pharmacokinetics of 5- mononitrate 5- mononitrate (5-ISMN) after single injection of Isosorbide 5-Mononitrate Spray in Chinese healthy male volunteers was studied and ALDH2 was evaluated for ALDH2 The effect of gene polymorphism on its pharmacokinetics.4. using molecular docking software to simulate the action process of enzyme and substrate, using nitroglycerin (GTN) as the reference drug, to investigate the effect of ALDH2 gene polymorphism on the pharmacokinetics of ISDN and 5-ISMN from the molecular level.
Methods: before 1., the blood samples of each recipient were collected from the elbow vein blood 3mL, EDTA anticoagulant, and blood DNA extraction kit was used to extract the blood sample DNA. The specific primers of ALDH2 gene were amplified by PCR, and the amplified products with biotin markers were specifically hybridized with the ALDH2 genotypic probe fixed on the aldehyde base. The color reaction made the specific hybridization signal color, and the chip was scanned and the hybrid images of the wild type and mutant probe of the DNA and ALDH2 gene loci were hybridized. The image was analyzed by the BaiO BE-2.0 biochip reader to determine the genotype of the samples to be tested.
After physical examination and laboratory examination, 20 healthy male subjects were screened and informed consent was obtained on the basis of knowing the contents, rights, duties and risks of the experiment.
2. selected 20 healthy male subjects were divided into the wild group and the mutant heterozygous group according to the genotype, the single injection of Isosorbide Dinitrate Spray 3.75mg, 2,4,6,8,10,12,15,20,30,45min and 1,1.5,2,3,4,6,8,12,24h before and after administration, and the 5mL in the elbow vein (heparin anticoagulant), 5000rpm, centrifugation 5min, the upper plasma, -20 The concentration of ISDN, 5-ISMN and isosorbide mononitrate (2-ISMN) in plasma was determined by.GC-ECD, and the pharmacokinetic parameters of the three groups were calculated by DAS2.0.1 pharmacokinetics software (t1/2, Tmax, Cmax, AUC0-t and AUC0-) respectively, and the pharmacokinetic parameters of the two groups were statistically analyzed. Comparison of calibration values. Parameter correction is the ratio of metabolites Cmax, AUC0-t, AUC0- infinity to the corresponding pharmacokinetic parameters of the parent drug.
3. the above subjects were given a single dose of Isosorbide 5-Mononitrate Spray 5mg, and 5mL was taken from 8,15,20,30,45min, 1,1.5,2,3,6,8,12,24h, and elbow vein before and after the drug. It was placed in a test tube (heparin anticoagulant), 5000rpm, centrifugation 5min, taking the upper plasma, -20 C and.GC-ECD method to determine the concentration of 5-ISMN in plasma, and the DAS2.0.1 pharmacokinetics software was used. The pharmacokinetic parameters (t1/2, Cmax, AUC0-t and AUC0- infinity) were calculated, and the pharmacokinetic parameters of the two groups were analyzed statistically.
4. SYBYL-X1.1 software was used to simulate the action process of ALDH2 wild enzyme, ALDH2 mutase and GTN, ISDN, 5-ISMN respectively. The binding force of the two enzyme to ISDN and 5-ISMN was compared with GTN as the reference.
Results: 1. according to genotyping, 20 healthy male subjects were screened out in this study. Among them, 14 were wild type (ALDH2*1/*1) of ALDH2 gene, 6 were mutant heterozygous (ALDH2*1/*2).
After a single dose of Isosorbide Dinitrate Spray 3.75mg, the ISDN pharmacokinetic parameters of wild and mutant heterozygous subjects were as follows: t1/2 was (0.994 + 0.309) H and (1.342 + 0.086) h respectively, Tmax was (0.105 + 0.018) H and (0.139 + 0.014) h respectively, Cmax was (33.611 + 2.065) ng/mL and (42.615 + 3.003) ng/mL, AUC0 -t (18.999 + 3.798) ng/mL / h and (31.188 + 1.427) ng/mL. H respectively, AUC0- infinity (20.915-4.383) ng/mL. H and (34.576 + 0.898) ng/mL. H.5-ISMN pharmacokinetic parameters are as follows: t1/2 is (4.554 + 0.858) and (5.849 + 0.781) respectively, respectively (0.857 + 0.128) and (1.083 +) 962 + 5.512) ng/mL, AUC0-t (286.433 + 72.682) ng/mL / h and (482.731 + 100.705) ng/mL. H respectively, AUC0- infinity is (300.173 + 68.283) ng/mL. H and (519.607 + 96.383) ng/mL-h, 2-ISMN pharmacokinetic parameters are as follows: tl/2 is respectively (3.852 + 0.859) and [3.940 +] respectively. Not (12.321 + 1.325) ng/mL and (16.647 + 2.530) ng/mL, AUC0-t (36.501 + 4.564) ng/mL. H and (65.941 + 13.547) ng/mL. H, AUC0- infinity respectively (42.407 + 5.402) ng/mL h and (75.098 + 17.972) ng/mL. H.
Compared with the wild group, the t1/2 and Tmax of ISDN were prolonged by 42.16% (P < 0.001) and 32.38% (P < 0.001), Cmax, AUC0-t and AUCo- infinity were increased by 26.79% (P < 0.01), 64.16% (P < 0.01) and 65.32% (P < 0.01), respectively. Increase 4.13% (P > 0.05), 2.67% (P > 0.05) and 4.71% (P > 0.05); t1/2 and Tmax of 2-ISMN extend 2.28% (P > 0.05) and 41.91% (P > 0.05), Cmax, AUC0-t and AUC0- infinity respectively increase 6.56% (P > 0.05), 10.05% (0.05 > 0.05).
After a single dose of Isosorbide 5-Mononitrate Spray 5mg, the 5-ISMN pharmacokinetics parameters of wild and mutant heterozygous subjects were as follows: t1/2 was (4.981 + 1.248) H and (5.365 + 0.824) h respectively, Tmax was (0.821 + 0.117) H and (0.833 + 0.119) h respectively, Cmax was (109.332 + 5.668) ng/mL and (114.108 + 4.529) ng/mL, AUC0-t were (607.626 + 68.854) ng/mL? H and (658.459 + 89.621) ng/mL? H, AUC0- AUC0- were (634.798 + 83.358) ng/mL? H and (690.937 + 105.191) ng/mL? H., respectively.
Compared with the wild group, the t1/2 and Tmax increased by 7.72% (P > 0.05) and 1.45% (P0.05), Cmax, AUC0-t and AUC0-, respectively 4.37% (P > 0.05), 8.37% (P > 0.05) and 8.84% (P > 0.05), respectively.
4. SYBYL-X1.1 software was divided into 6.49,5.45 and 3.41 for the action of ALDH2 wild enzyme and GTN, ISDN and 5-ISMN, respectively. The action process of ALDH2 mutase and GTN, ISDN and 5-ISMN were 3.82,2.42 and 3.22..
Conclusion: the results show that the polymorphism of ALDH2 gene affects the pharmacokinetics of ISDN in the human body. Compared with the wild group, the t1/2, Tmax, Cmax, AUC0-t and AUC0- infinity of the mutant heterozygous group are all increased, and the difference has statistical significance compared with the wild group. Compared with the wild group, the t1/2 and Tmax of 5-ISMN in the mutant heterozygous group are all increased. Plus, the difference was statistically significant, but there was no statistical difference between the positive parameters of other pharmacokinetic schools, but there was no statistical difference between the two groups of 2-ISMN correction pharmacokinetic parameters.
ALDH2 gene polymorphism did not affect the pharmacokinetics of 5-ISMN in human body. After spraying Isosorbide 5-Mononitrate Spray, there were differences in the pharmacokinetic parameters of 5-ISMN between the two groups, but the difference was not statistically significant.
The binding capacity of ALDH2 wild enzyme to GTN and ISDN was significantly higher than that of ALDH2 mutant enzyme, but the binding force between two enzyme and 5-ISMN was not obvious. Two
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969.1

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