Effects Of The Gene Polymorphisms Of CYP3A4,CYP3A5 And CYP2E1 On The Serum Concentration Of Dl-3-n-butylphthalide And Therapeutic Efficacy In Patients With Acute Ischemic Stroke

ObjectiveResearch for the correlation between the polymorphism of CYP3A4,CYP3A5 and CYP2E1 gene and the serum concentration and efficacy of butylphthalide in patients with acute ischemic stroke.Methods1.HPLC-Orbitrap determination of 3-n-Butylphthalein in Steady State Plasma in Patients with Acute Ischemic StrokeEstablishment of HPLC-ORBITRAP method for quantitative analysis of plasma NBP in patients with acute ischemic stroke in China.NBP was extracted from plasma samples by protein precipitating-methanol;The mobile phase was a mixture of methanol and water（80:20）,The column temperature:25℃,and the flow rate:0.3m L·min^-1.The injection volume:10μL.Run time:5min.MS detection was achieved using a mass spectrometer operated in positive ion electrospray（ESI）modes and orbitrap;The quantitation of NBP in AIS plasma was performed:m/z 191.11→173.10 for NBP and m/z285.08→222.11 for diazepam（Diazepam）.The above methods were used to determine the steady-state plasma NBP concentration in patients with acute ischemic stroke.The plasma drug concentration was calculated using Xcalibur and SPSS software for statistical analysis.2.Effect of CYP3A4*1G CYP3A5*3 CYP2E1*5B genotype on serum NBP concentrations at steady-state in Chinese AIS patientsFrom March 2019 to October 2019,acute ischemic stroke patients meeting the inclusion and exclusion criteria were collected.According to the requirements,the patients were measured the steady state trough concentration by HPLC Orbitrap method.And the genetypes were measured by Hi-single nucleotide polymorphism.SNPs:CYP3A4:*1G、*4、*5、*18、*19、*22;CYP3A5:*3、*4;CYP2E1:*5B、*2.According to the SNP gene polymorphism grouping,the statistical analysis software SPSS17.0 was used to analyze whether subjects with different genotypes had a correlation with the steady-state plasma concentration changes of dl-3-n-butylphthalide.3.Influence of CYP3A4*1G CYP3A5*3 CYP2E1*5B genotype on NBP efficacy in Chinese AIS patientsDetection of CYP3A4,CYP3A5,and CYP2E1 gene polymorphisms in peripheral blood of patients who met the inclusion and exclusion criteria.Group based on their SNP genotyping results.The efficacy measures were T-AOC and the modified Rankin scale（m RS）at day 90.SPSS17.0 was used to analyze the clinical efficacy of different genotypes.Results1.NBP standard curve regression equation:Y=-12261.6+235971X,r=0.9973..The linear range of NBP was 0.025-2.5μg·m L^-1with LLOQ of 5ng·m L^-1.Recovery:97.23%,100.82%,94.32%;RSD:8.68%、3.78%、5.19%.Matrix effect:101.55%,106.99%,105.08%;RSD:6.32%,4.96%,7.60%.The intra-day and inter-day Precision and Accuracy:RE<±15%;The inter-day RSD<10%.6 hours at room temperature,3 times of repeated freezing,thawing at-20℃,-80℃frozen for 1 month and automatic injector placed for 12h:RSD 0.29%-9.29%,RE<±15%.The average steady-state blood concentration of 69 patients was 0.488±0.524μg·m L^-1.2.69 patients were enrolled in this study.The CYP3A4*1G,CYP3A5*3 and CYP2E1*5B allele frequency of 69 patients was 22.46%,24.64%,17.39%.Among all 69Chinese AIS patients,the NBP concentrations were 0.57±0.58μg·m L^-1for CYP3A4*1G CC allele,0.32±0.35μg·m L^-1for TC allele and 0.031μg·m L^-1for TT allele.There was significant difference between CC and TC genotypes（P<0.05）.The NBP concentrations were 0.60±0.58μg·m L^-1for CYP3A5*3 CC allele,0.31±0.33μg·m L^-1for TC allele and0.05±0.02μg·m L^-1for TT allele.There was significant difference between these three genotypes（P<0.05）.The NBP concentrations were 0.56±0.59μg·m L^-1for CYP2E1*5B GG allele,0.39±0.31μg·m L^-1for CG allele and 0.06μg·m L^-1for CC allele.There was significant difference between GG and CG genotypes（P<0.05）.The NBP concentration of the CCCC haplotype in patients was significantly higher than TCTC haplotype（P<0.05）.3.There was significant difference of T-AOC between CYP3A5*3 CC allele and TC allele（P<0.05）,and it was no significant difference between the two genotypes follow-up results（P>0.05）;There was significant difference of T-AOC between CYP2E1*5B GG allele and GC/CC allele（P<0.05）,and it was no significant difference between the three genotypes follow-up results（P>0.05）.Conclusions1.The HPLC-Orbitrap method established in this study can be used to determine the steady-state plasma concentration of butylphthalide in patients with acute ischemic stroke.2.CYP3A4*1G,CYP3A5*3,and CYP2E1*5B gene polymorphisms can affect NBP steady-state plasma concentrations in patients with acute ischemic stroke.CYP3A4*1G and CYP3A5*3 haplotypes are related to NBP plasma concentration,which is significantly higher in CCCC group than in TTTT group.3.CYP3A4*1G,CYP3A5*3 and CTP2E1*5B gene polymorphism is not related to the effect of butylphthalide in patients with acute ischemic stroke;CYP3A5*3 and CYP2E1*5B gene polymorphisms were associated with the T-AOC in serum of patients with acute ischemic stroke（P<0.05）.