| Objective: Of all the oral drugs used for anticoagulant therapy of Non-valvular Atrial Fibrillation(NVAF),dabigatran etexilate is the first drug approved by Food and Drug Administration(FDA)in the past sixty years after warfarin.Large-scaled foreign clinical researches proved that darbigatran,as a novel oral anticoagulant,is noninferior to warfarin in anticoagulant effect,but they also suggested that there is great individual difference in plasma concentrations,which is affected by complex factors as well as genetic differences among different populations.In addition,hemorrhagic adverse reactions and antithrombotic effect are closely related with plasma concentrations.At present,clinical therapy lacks antagonists against hemorrhage cause by dabigatran.Although there are foreign reports of relevant studies on the use of dabigatran etexilate for anticoagulant therapy in NVAF patients,clinical evidence-based data about the safety and efficacy of darbigatran during perioperative period of atrial fibrillation ablation in China is far from enough.Therefore,this project aims to establish a rapid and accurate LC-MS/MS method for determination of dabigatran in plasma of NVAF patients,to investigate accuracy of ECA on monitoring of anticoagulant effect for getting a suitable anticoagulation index for clinical reference,and to explore genetic and non-genetic factors influencing plasma concentration of Chinese patients with NVAF.Through the research,anticoagulant therapy for Chinese patients with NVAF can be optimized to lower the risk of stroke and bleeding and to ensure the safety and efficiency of anticoagulant therapy in the perioperative period.Method: This research enrolled 113 NVAF patients undergoing AF ablation as well as taking dabigatran etexilate in the perioperative period for anticoagulation,in our hospital,from January 2016 to December 2017.After continuously taking dabigatran etexilate(110mg or 150mg),all the patients got their plasma concentrations at a steady state,with blood samples separately collected into sodium citrate tubes and EDTA tubes within 10–16 h after the last dose.Meanwhile,the patients' conditions of bleeding and thrombosis were observed and recorded during the treatment.The plasma concentration of dabigatran was determined by LC-MS/MS,using dabigatran-d3 as the internal standard.A single-step precipitation was used for plasma sample preparation by methanol.The separation was performed on an Agilent Eclipse XDB-C18 analytical column using a gradient elution mode with a column temperature of 50?.The mobile phase was a mix of distilled water containing 0.5% formic acid and methanol containing 0.5% formic acid at a flow rate of 0.3 mL/min.Detection and quantification were performed by electrospray ionization(ESI)in the positive ionization mode by multiple reaction monitoring(MRM)of the transitions at m/z 472.3 ?289.1 for dabigatran and m/z 475.3 ?292.3 for dabigatran-d3.At the same time,the ECA method was used to test the anticoagulant effect of dabigatran,compared in correlation with the plasma concentrations measured by the LC-MS/MS method.Single nucleotide polymorphisms(SNP)in ABCB1(rs4148738,rs2235046,rs1128503,rs10276036,rs1202169,rs1202168,rs1202167),CES1(rs8192935,rs4122238)and CES1P2(rs4580160)were detected by SNa Pshot assay and Sanger was applied to gene sequencing of rs4784563 and rs2244613.The data were processed by SPSS 19 statistical software.Quantitative data were analyzed by group t-test,ANOVA or nonparametric analysis.Qualitative data were processed by ?2 test or Fisher accurate test method.The research used Pearson or Spearman test to analyze the correlation.Multiple linear regression analysis and stepwise regression analysis were used to analyze the relationship between the steady-state trough concentration of dabigatran and its influencing factors.Results: 1.The linear range of dabigatran was 5-400 ng/mL(r=0.9999)with lower limit of quantitation of 5 ng/mL by LC-MS/MS.The intra-and inter-day RSDs were all below 10% and accuracy were within ±5% at low,moderate and high concentrations.The extract recoveries were 97.72%-101.22% for dabigatran and the matrix effect was 93.80%-97.40%.Under various storage conditions,the concentrations of dabigatran remained stable.2.The trough concentration of dabigatran in plasma had significant correlation(P<0.0001)with activated partial thromboplastin time(APTT)and international normalized ratio(INR),but correlation coefficients are low(0.608 and 0.440 respectively).There was no statistical difference in the APTT value and the INR value between the bleeding group(37 samples)and the no-bleeding group(74 samples)(P > 0.05).3.There was a significant correlation between the ECA value and the trough concentration of dabigatran in plasma(P<0.0001),and the correlation coefficient was 0.9757.But there was a trend of under-estimating the concentration by ECA value,which is about 0.873 times that of LC-MS/MS.The ECA value was statistically significant(P=0.026)between bleeding group(35 samples)and no-bleeding group(72 samples),and the ECA value in bleeding group was 94.79±67.61ng/mL,which was significantly higher than that in no-bleeding group(66.87±58.42 ng/mL).4.The steady-state trough concentrations of dabigatran of patients who took 110mg(89 samples)or 150mg(24 samples)twice a day were respectively 97.38±61.63 ng/mL and 102.50±67.25ng/mL.CV were 63.29% and 65.61%,respectively.8 patients' concentrations were more than 200 ng/mL,including 2 cases of urinary occult blood 3+,2 cases of urinary occult blood 2+,1 cases of serious gingival bleeding.17 patients' concentrations were under 40 ng/mL,including 1 case of cerebral infarction(33.2 ng/mL).5.The concentration of dabigatran was mainly affected by gender,age and creatinine clearance(Cr Cl).The dose-normalized plasma concentration of dabigatran of female patients was significantly higher than that of male [female vs male: 1.15±0.76(45)vs 0.67±0.37(68),P=0.001],and there was significant difference in bleeding conditions [female vs male: 22(48.9%)vs15(22.06%),P=0.004].With the increase of age,the concentration showed a significant trend of increase,[age < 65 vs 65-75 vs ? 75:0.74±0.43(74)vs 0.99±0.74(32)vs 1.57±0.98(7),P=0.018].Among moderate,mild renal insufficiency patients and normal patients,the concentration had shown a decreasing trend[Cr Cl 30-<50(mL/min)vs 50-<80(mL/min)vs ?80(mL/min): 1.81±0.75(8)vs 1.01±0.67(41)vs 0.66±0.38(60),P=0.001].6.Among the patients with CES1P2 SNP rs4784563,there was a significant difference in the dose-normalized plasma concentration of dabigatran.[AA vs AC+CC: 0.98±0.67(64)vs 0.72±0.48(49),P=0.019].With SNPrs4580160,the concentration of patients was lower in AA+GA genotypes than in GG genotype(P=0.028).For rs10276036 of ABCB1 gene,each minor allele T was associated with the trend of lower bleeding risk [CC vs CT vs TT: 45.65%(21)vs 27.27%(15)vs 8.33%(1),P=0.024].There was a significant relationship between SNP rs1128503 and the bleeding risk(P=0.022),which was the lowest in CC genotype and the highest in TT genotype.Conclusions: The LC-MS/MS method established is accurate,sensitive and specific.This study for the first time in China applied LC-MS/MS to the determination of dabigatran in human plasma for patients with NVAF during atrial fibrillation ablation.The conventional monitoring methods(including TT,INR,APTT)are not suitable for monitoring the anticoagulation effect of darbigatran etexilate.It is creative to use the ECA method in anticoagulant activity monitoring.The ECA method is significantly corelated LC-MS/MS in research results.The LC-MS/MS is more accurate,while the ECA method is more convenient which is expected to be the routine clinical anticoagulant detection and reference index.It is proved in this study that the plasma concentration of dabigatran of Chinese NVAF patients is mainly affected by gender,age and Cr Cl with a large variation,also clinical relevant monitorings should be emphasized.In addition,this study analyzed the related genotype of Chinese patients,discovered the influence of CES1P2 gene and ABCB1 gene polymorphism on the trough concentration and bleeding conditions.According to patients' genotype may be a new idea and direction to optimizing the dose,giving individual therapy and improving the safety and efficacy of perioperative anticoagulant drugs. |
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