VASP文献

Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrelJolanta M.Siller-Matula,MD,a Alexander O.Spiel,MD,a Irene ng,MD,b Gerhard Kreiner,MD,bGuenter Christ,MD,b and Bernd Jilma,MD a Vienna,AustriaBackground Clopidogrel is activated by CYP2C19,which also metabolizes proton pump inhibitors(PPI).As proton pump inhibitors are metabolized to varying degrees by CYP2C19,we hypothesized that the reported negative omeprazole-clopidogrel drug interaction may not be a class effect.Methods Responsiveness to clopidogrel was assessed by the vasodilator-stimulated phosphoprotein phosphorylation (VASP)assay and aggregometry(Multiplate Analyzer)in300patients with coronary artery disease(CAD)undergoing percutaneous coronary intervention(PCI).Results The mean platelet reactivity index(PRI,assessed by the VASP assay)was nearly the same in patients with(n=226; PRI=51%)or without PPI treatment(n=74;PRI=49%;P=.724).Likewise,the adenosine diphosphate–induced platelet aggregation did not differ significantly between patients with or without PPI treatment(45vs.41U;P=.619).Similarly,there was no difference in the PRI or the adenosine diphosphate–induced platelet aggregation between patients with pantoprazole(n= 152;PRI=50%;aggregation=47U),esomeprazole(n=74;PRI=54%;aggregation=42U),or without PPI(n=74;PRI=49%; aggregation=41U;P=.382).Conclusion In contrast to the reported negative omeprazole-clopidogrel drug interaction,the intake of pantoprazole or esomeprazole is not associated with impaired response to clopidogrel.(Am Heart J2009;157:148.e1-148.e5.)The antiplatelet effect of clopidogrel is not uniform in all patients and reduced platelet inhibition by clopidogrel is associated with an increased risk of cardiac events.1-4 The variability in the response to clopidogrel has been linked,at least in part,to its cytochrome P450–dependent metabolism steps including CYP2C19and CYP3A4.5-8 Proton pump inhibitors(PPIs),frequently used in patients receiving clopidogrel and aspirin,are also metabolized by CYP2C19and CYP3A4.9It has recently been reported that the PPI omeprazole decreases the antiplatelet effect of clopidogrel possibly due to the inhibition of the CYP2C19enzyme.10,11As all PPIs are metabolized by CYP2C19,12but to a varying degree,we hypothesized that the reported negative omeprazole-clopidogrel drug interaction may not be a class effect.Thus,we compared the responsiveness to clopidogrel with or without concomitant PPI treatment in300patients with coronary artery disease(CAD)undergoing percutaneous coronary intervention(PCI).To quantify the pharmacodynamic effect of clopidogrel with or without PPIs treatment,we performed the vasodilator-stimulated phosphoprotein (V ASP)phosphorylation assay and impedance aggrego-metry.13-15The V ASP assay is specific for clopidogrel and other P2Y12antagonists in the absence of cilostazol.13-18 It has been shown that adjusting the clopidogrel loading dose according to the platelet reactivity index in the V ASP assay may improve the clinical outcome in patients with decreased platelet inhibition by clopidogrel.19,20 MethodsStudy designThe study protocol was approved by the Ethics Committee of the Medical University of Vienna in accordance with the Declaration of Helsinki.Written informed consent was obtained from all study participants before the study entry.Three hundred consecutive patients with coronary artery disease (CAD)undergoing percutaneous coronary intervention(PCI) were enrolled.All patients received a clopidogrel loading dose (600mg)at the start of clopidogrel treatment.Patients had been on clopidogrel(75mg/d)and aspirin(100mg/d)treatment for 3months on average(5days at least)at the time of inclusion. Two different PPIs have been used in the study:pantoprazole and esomeprazole.Blood samples from patients were obtained from the arterial sheath(6F)in the catheterization laboratory immediately after PCI.Part of patients has been analyzed in our previous study.21From the a Department of Clinical Pharmacology,Medical University of Vienna,Vienna, Austria,and b Department of Cardiology,Medical University of Vienna,Vienna,Austria. This study was supported by a grant from the Jubiläumsfond of the Austrian National Bank (Nr.12565).Submitted August24,2008;accepted September25,2008.Reprint requests:Bernd Jilma,MD,Department of Clinical Pharmacology,Medical University of Vienna,Währinger Gürtel18-20,A-1090Vienna,Austria.E-mail:bernd.jilma@meduniwien.ac.at0002-8703/$-see front matter©2009,Mosby,Inc.All rights reserved.doi:10.1016/j.ahj.2008.09.017Analysis of VASP phosphorylation by flow cytometry To determine the V ASP(vasodilator stimulated phospho-protein)phosphorylation state of whole blood,we used a standardized flow cytometric assay(Platelet V ASP;BioCytex, Marseille,France).Blood samples collected in3.8%sodium citrate(BD Vacutainer;Becton Dickinson,Vienna,Austria) were incubated in vitro with adenosine diphosphate and/or prostaglandin E1(PGE1)before fixation.After10minutes, platelets were permeabilized,labeled with a primary mono-clonal antibody against serine239-phosphorylated V ASP(clone 16C2)or its isotype,followed by a secondary fluorescein isothiocyanate–conjugated polyclonal goat-antimouse antibody. All procedures were performed at room temperature.Geo-metric mean fluorescence intensity(GMFI)was determined using a flow cytometer(FACSCalibur System,BD Biosciences, Vienna,Austria).22The platelet population was identified by its forward and side scatter distribution,and10,000platelet events were gated and analyzed for GMFI.Platelet reactivity was expressed as platelet reactivity index(PRI)calculated as PRI%=[(GMFI(PGE1)−GMFI(PGE1+ADP)/GMFI(PGE1)]×100.The ratio is expressed as mean percentage platelet reactivity,which inversely correlates with the clopidogrel effect.The normal value of the PRI measurement using V ASP analysis is69%to100%.21,23The V ASP assay was performed within24hours after blood sampling.AggregometryADP-induced platelet aggregometry is a widely used method to measure the responsiveness to clopidogrel.24,25 Whole blood aggregation was determined using an impe-dance aggregometer(Multiple Platelet Function Analyzer, Dynabyte Medical,Munich,Germany).The system detects the electrical impedance change due to the adhesion and aggregation of platelets on two independent electrode-set surfaces in the test cuvette.26,27A2:1solution of whole blood anticoagulated with hirudin(200U/mL,Dynabyte Medical)and0.9%NaCl was stirred at37°C for3minutes in the test cuvettes,adenosine diphosphate([ADP]6.4μmol/L, Dynabyte Medical)was added,and the increase in electrical impedance was recorded continuously for6minutes.The mean values of the2independent determinations are expressed as the area under the curve of the aggregation tracing.The results measured by the Multiplate Analyzer are reproducible with less than6%variability.26The reference range for the test is29to118U.28Statistical analysisA sample size calculation of our study was based on the observed mean±SD(62±23)of the platelet reactivity index under clopidogrel and omeprazole treatment and the observed mean(50±16)of the platelet reactivity index under clopidogrel alone.11We calculated that we need to include72patients in each group to be able to detect such a 20%relative difference in platelet reactivity index with a power of95%and a2-sidedαvalue of.05.Stepwise multivariable logistic regression analysis was used to estimate possible associations between platelet reactivity index, platelet aggregation,and use of PPI.The logistic model included sex,use of statins(lipophilic versus hydrophilic), angiotensin-converting enzyme(ACE)inhibitors,calcium-channel blockers,diabetes mellitus,arterial hypertension, hypercholesterolemia,previous myocardial infarction,cause for hospitalization,and smoking.Data are reported as mean and95%confidence intervals.Statistical comparisons were performed with the Kruskal-Wallis analysis of variance,theχ2 test,and the Mann-Whitney U test.A2-tailed P value of b.05 was considered significant for the primary end point parameter(platelet reactivity index)between the PPI groups and patients without PPI.All statistical calculations were performed using commercially available statistical software (SPSS Version14.0;SPSS,Chicago,IL).ResultsPatient demographicsPatient demographics are shown in Table I.Most patients had high blood pressure and hyperlipidemia. Almost half of the patients had previous e of β-blockers and statins was high.There were fewer male patients in the pantoprazole(71%)or esomeprazole (80%)groups as compared to patients without any PPI (93%;P=.001;P=.046,respectively).The use of statins was higher in the pantoprazole(82%)or esomeprazole(87%)groups as compared to patients without any PPI(61%;P=.006;P=.001,respectively). The use of ACE inhibitors was higher in the esome-prazole(68%)group as compared to patients without any PPI(47%;P=.018).There were more patients hospitalized for myocardial infarction in the esomepra-zole group(39%)as compared to patients without PPI (20%;P=.002)and fewer patients with stable angina in the esomeprazole group(46%)compared to patients without PPI(66%;P=.008).Comparison of patients with any PPI vs.without PPI The platelet reactivity index(PRI,V ASP assay)was similar in patients with any PPI(n=226,mean51%; 95%CI48-54%)or without any PPI treatment(n=74; mean49%;95%CI43-55%;P=.724).As a consequence, the rate of decreased platelet inhibition by clopidogrel defined by a platelet reactivity index≥69%21was similar in both groups(25%vs20%).Likewise,the ADP-induced platelet aggregation did not differ significantly between patients with any PPI(mean45U;95%CI41-50U)or without any PPI treatment(mean=41U, 95%CI33-48U;P=.619).Comparison of patients with pantoprazole or esomeprazole vs without PPIThere was no difference in the platelet reactivity index or the ADP-induced platelet aggregation between patients with pantoprazole(n=152;PRI=50%;aggregation=47 U),esomeprazole(n=74;PRI=54%;aggregation=42U) or without PPI(n=74;PRI=49%;aggregation=41U; P=.382;Figures1and2).To exclude that minor differences in demographic data may have influenced the platelet reactivity index or the148.e2Siller-Matula et al American Heart JournalJanuary2009ADP-induced platelet aggregation,we performed a multivariable logistic regression analysis.Neither platelet reactivity index nor ADP-induced platelet aggregation were influenced by male sex,intake of statins,ACE inhibitors,or calcium-channel blockers.DiscussionThe intake of pantoprazole or esomeprazole was not associated with a reduced platelet inhibition by clopido-grel as compared to patients without PPI(Figures1and 2).The platelet reactivity index was49%in patients without PPI and,thus,similar to that in a previous study investigating the omeprazole-clopidogrel drug interaction (PRI=50%).11This demonstrates the external validity of the V ASP assay.Previous articles alerted the scientific community that concomitant treatment with omeprazole and clopidogrel may have negative effects.10,11The treating cardiologists, however,were left with the tantalizing question whether they should avoid omeprazole or any other PPI in patients receiving clopidogrel.Our data show that the reported negative effect of omeprazole on platelet function in patients receiving clopidogrel10,11is not seen in patientsTable I.Patient demographicsPatient demographics No PPI(n=74)Pantoprazole(n=152)Esomeprazole(n=74)Age73±1265±1262±13 Gender(male)(n[%])66(93)109(71)†59(80)⁎Cause forhospitalisation(n[%])Silent ischemia7(9)13(9)7(9) Stable angina49(66)87(57)33(45)†Unstable angina3(4)14(9)5(7) Myocardialinfarction15(20)38(25)29(39)†Risk factors/pastmedical history(n[%])Hypertension63(85)132(87)63(85) Smoking37(50)88(58)47(63) Family historyof CAD25(34)44(29)22(30)Diabetes mellitus23(31)53(35)23(31) Hyperlipidemia56(76)119(78)55(75) Prior myocardialinfarction23(31)52(34)24(32) Prior PCI36(49)80(53)38(51) PAOD8(11)20(13)8(11) CVD9(12)14(9)8(11) Laboratory dataWBC(G/L)8±2.57.9±2.87.8±2.9 Platelets(G/L)228±70226±68219±64 CRP(mg/dL) 1.2±2.7 1.3±2.9 1.8±2.6 Hb(g/dL)14±1.412.8±214.5±2.1 Fibrinogen(mg/dL)408±130421±120441±107 Creatinine(mg/dL) 1.3±1.2 1.3±1.2 1.7±1.3 Pre-PCI medications(n[%])ACE inhibitors35(47)73(48)51(68)⁎Calcium-channelblockers13(18)31(20)19(13)Statins45(61)124(82)†65(87)†β-Blockers51(69)122(80)59(80) ASA74(100)152(100)74(100) Clopidogrel74(100)152(100)74(100) PCI dataNumber of stentsper patient1.5±0.7 1.8±0.9 1.8±1.2 Total stent length29±1533±2133±25Data are reported as mean,SD,number of patients(n),or percentages.CVD,Cerebral vascular disease;CRP,C-reactive protein;Hb,hemoglobin;PAOD, peripheral arterial occlusive disease;WBC,white blood cells.⁎P b.05for pantoprazole or esomeprazole versus no PPI.†P b.01for pantoprazole or esomeprazole versus no PPI.Figure1Platelet reactivity index in the VASP phosphorylation assay in patients on clopidogrel with or without PPI:pantoprazole or esomeprazole. Data are presented as mean and95%CI.Figure2Adenosine diphosphate–induced platelet aggregation in patients on clopidogrel with or without PPI:pantoprazole or esomeprazole.Data are presented as mean and95%CI.Siller-Matula et al148.e3American Heart Journal Volume157,Number1treated with pantoprazole or esomeprazole.Therefore, the reported omeprazole-clopidogrel drug interac-tion10,11is probably not a class effect.A possible explanation for the reduced responsiveness to clopido-grel under omeprazole treatment could be the stereo-selective metabolism of omeprazole by CYP2C19,9,29 which might not be apparent for esomeprazole or other PPI.The lack of negative effects of concomitant treatment with pantoprazole or esomeprazole is an important finding because it may have an impact on clinical practice.Our study has97%power for pantoprazole and 88%for esomeprazole to exclude a20%difference of platelet reactivity index as compared to patients without PPI.The PRI was slightly(10%)higher in the esome-prazole group as compared to controls.However,this would require a sample size of762patients to definitely exclude such a drug-drug interaction with the same power(88%).These preliminary findings call for formal clinical trials investigating the effect of different PPIs on the pharmacodynamics and pharmacokinetics of clopido-grel.Moreover,it has to be evaluated if the reported interaction between PPI and clopidogrel has an impact on hard outcome parameters,as significant pharmaco-logical drug-drug interactions do not necessarily have clinical impact.30-32Similar to our study,another trial implicates that the PPI-clopidogrel interaction is not a class effect:con-comitant treatment with lansoprazole did not alter the pharmacokinetics or pharmacodynamics of clopido-grel.7This study also showed that increasing the gastric pH did not influence the platelet inhibitionby clopidogrel.7We selected2methods for evaluating PPI-clopidogrel drug-drug interaction:a very specific V ASP assay and a highly functional ADP-induced aggregometry.There are the following arguments for a combination of the two assays applied.Although the V ASP assay is highly specific for the effect of clopidogrel on the ADP receptor(P2Y12),V ASP is a biochemical marker which may not reflect certain aspects of platelet function: Firstly,activation of the P2Y12receptor by ADP initiates other signalling pathways,which are inde-pendent of V ASP phosphorylation.20Secondly,the antibody16C2used in the V ASP assay recognizes phosphorylation at a serine residue that is also phosphorylated by different cyclic guanosine mono-phosphate-dependent kinases that are not regulated by P2Y12.20Aggregometry is a functional assay based on the stimulation of platelet aggregation with ADP.26We used an impedance aggregometry in whole blood,which eliminates potential disadvantages of the light transmis-sion aggregometry:time-consuming centrifugation steps, variable reproducibility,large required sample volumes, and lengthy processing time.14A good correlation between impedance and light transmission aggregometry has been shown.25Therefore,combination of the V ASP assay and the ADP-induced aggregometry was reasonable.In summary,our study implies that in contrast to the reported negative omeprazole-clopidogrel drug interac-tion,the intake of pantoprazole or esomeprazole is not associated with impaired response to clopidogrel. LimitationsPossible imprecision in study results could have arisen from the nonrandomized study design.Although we adjusted for several variables in the multivariable logistic regression model,it is possible that residual confounding could affect results.Secondly,our study could not compare omeprazole with pantoprazole or esomepra-zole.Therefore,we could not examine whether the data from the previous trial are reproducible. 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