| Objectives:1. To detect the genotype of aldehyde dehydrogenase2extracted from human peripheral blood DNA with the DNA microarray method.2. To study the pharmacokinetics of isosorbide dinitrate and its two metabolites in healthy Chinese male volunteers, and then evaluate the relationship between the polymorphisms of aldehyde dehydrogenase and the pharmacokinetics characteristics of them.3. To study the pharmacokinetics of isosorbide5-mononitrate in healthy Chinese male volunteers, and then evaluate the relationship between the polymorphisms of aldehyde dehydrogenase and the pharmacokinetics characteristics of5-ISMN.4. To explore the influence of ALDH2polymorphism on ISDN and5-ISMN, through simulation the acting process between enzyme and substrate by using the molecular docking softwareMethods:1. Among22persons, twenty healthy male subjects with qualified physical examination and laboratory tests were selected. After understanding the content, rights, obligations and risks, all subjects signed the informed consent form.Blank blood samples (3mL) were collected into EDTA-containing tubes, then extracted DNA using the DNA extraction kit. The ALDH2gene-specific primers were amplified by using PCR technology. Then the biotinylated amplification product and the ALDH2genotype alkyl sheet which was fixed on the aldehyde radical piece occurred specific hybridization reaction, and the hybridization signal showed specific color by enzymatic color reaction. The hybridization image was gained by scanning the chip, and to judge the genotype of the samples though the BaiO BE-2.0biochip reading device.2.20healthy male subjects were administered single dose of isosorbide dinitrate spray3.75mg. Blood samples (5mL) were collected into sodium heparin (0.4%)-containing tubes before and2,4,6,8,10,12,15,20,30,45min and1,1.5,2,3,4,6,8,12,24h after administration. Samples were immediately centrifuged at5000rpm for5min and plasma was separated stored at-20℃for analysis. The isosorbide dinitrate and its two metabolites plasma concentrations were determined by GC-ECD method. The main pharmacokinetic parameters (t1/2, Tmax, Cmax, AUC0-t and AUC0-∞) were calculated by DAS software (version2.0.1, by Sun et al, China) and statistically analyzed (Cmax, AUC0-t and AUC0-∞values of the metabolites were corrected by that of the parent).3. These twenty subjects were administered single dose of isosorbide mononitrate spray5mg. Blood samples (5mL) were collected into sodium heparin (0.4%)-containing tubes before and8,15,20,30,45min and1,1.5,2,3,6,8,12,24h after administration. Samples were immediately centrifuged at5000rpm for5min and plasma was separated stored at-20℃for analysis. The isosorbide5-momonitrate plasma concentrations were determined by GC-ECD method. The main pharmacokinetic parameters (t1/2, Tmax, Cmax, AUC0-t and AUC0-∞) were calculated by DAS software (version2.0.1, by Sun et al, China) and statistically analyzed.4. Using GTN as the reference, simulated the acting process between the ALDH2(ALDH2*1/*1, ALDH2*1/*1) and the substrate (GTN, ISDN,5-ISMN).Results:1.20subjects were selected from22subjects and classified into two groups by the ALDH2genotype; that is the homozygous group (n=14) and the heterozygous group (n=6).2. The main pharmacokinetic parameters of isosorbide dinitrate for the homozygous group and the heterozygous group were as follows:t1/2were (0.994±0.309) h and (1.342±0.086) h, Tmax were (0.105±0.018) h and (0.139±0.014) h, Cmax were (33.611±2.065) ng/mL and (42.615±3.003) ng/mL, AUC0-t were (18.999±3.798) ng/mL·h and (31.188±1.427) ng/mL·h, AUC0-∞Were (20.915±4.383) ng/mL-h and (34.576±0.898) ng/mL·h, respectively. The pharmacokinetic parameters of isosorbide5-mononitrate for the homozygous group and the heterozygous group were as follows:t1/2were (4.554±0.858) h and (5.849±0.781) h, Tmax were (0.857±0.128) h and (1.083±0.204) h, Cmax were (63.595±4.941) ng/mL and (83.962±5.512) ng/mL, AUC0-t were (286.433±72.682) ng/mL-h and (482.731±100.705) ng/mL-h, AUC0-∞were (300.173±68.283) ng/mL-h and (519.607±96.383) ng/mL-h, respectively. The pharmacokinetic parameters of isosorbide5-mononitrate for the homozygous group and the heterozygous group were as follows:t1/2were (3.852±0.859) h and (3.940±0.887) h, Tmax were (0.470±0.148) h and (0.667±0.204) h, Cmax were (12.321±1.325) ng/mL and (16.647±2.530) ng/mL, AUC0-t were (36.501±4.564) ng/mL·h and (65.941±13.547) ng/mL·h, AUC0-∞Were (42.407±5.402) ng/mL·h and (75.098±17.972) ng/mL·h, respectively. Compared with the homozygous group, t1/2and Tmax of ISDN prolonged42.16%(p<0.001) and32.38%(p<0.001), Cmax, AUC0-t and AUC0-∞of ISDN increased26.79%(p<0.001),64.16%(p<0.001) and65.32%(p<0.001), respectively; t1/2and Tmax of5-ISMN prolonged28.44%(p<0.05) and26.37%(p<0.05), Cmax, AUC0-t and AUC0-∞of5-ISMN increased4.13%(p>0.05),2.67%(p>0.05) and4.71%(p>0.05), respectively; t1/2and Tmax of2-ISMN prolonged2.28%(p>0.05) and41.91%(p>0.01), Cmax, AUCo-t and AUC0-∞of2-ISMN decreased6.56%(p>0.05),10.05%(p>0.05) and7.12%(p>0.05), respectively.3. The main pharmacokinetic parameters of isosorbide5-mononitrate for the homozygous group and the heterozygous group were as follows:t1/2were (4.981±1.248) h and (5.365±0.824) h, Tmax were (0.821±0.117) h and (0.833±0.119) h, Cmax were (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-∞, were (634.798±83.358) ng/mL-h and (690.937±105.191) ng/mL-h, respectively. Compared with the homozygous group, t1/2and Tmax of5-ISMN prolonged7.72%(p>0.05) and1.45%(p>0.05), respectively, Cmax, AUC0-tand AUCo-∞of5-ISMN increased4.37%(p>0.05),8.37%(p>0.05) and8.84%(p>0.05), respectively.4. The score of the acting process between ALDH2wild enzyme and GTN, ISDN,5-ISMN were6.49,5.45and3.41, repectively; and the score of the acting process between ALDH2mutant enzyme and GTN, ISDN,5-ISMN were3.82,2.42and3.22.Conclusions:The polymorphism of ALDH2had influence on the pharmacokinetics of ISDN. After taken ISDN spray, compared with the homozygous group, t1/2, Tmax, Cmax, AUCo-t and AUC0-∞of ISDN in heterozygous group all increased and the change has statistically significant effect; the corrected pharmacokinetics parameters of2-ISMN between two groups did not show statistically significant effect; t1/2and Tmax of5-ISMN between two groups increased and the increase has statistically significant effect, but other pharmacokinetics parameters of5-ISMN between two groups did not have statistically significant effect.The polymorphism of ALDH2did not had influence on the pharmacokinetics of5-ISMN. After taken ISMN spray, the pharmacokinetics parameters of5-ISMN between two groups did not have statistically significant effect.The binding force between ALDH2wild enzyme and GTN, ISDN were stronger than that between ALDH2mutant enzyme and these two substract; the binding force between ALDH2wild enzyme and5-ISMN was not signantly different from that between ALDH2mutant enzyme and5-ISMN. |
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