Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrel

Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrel Jolanta M. Siller-Matula, MD, a Alexander O. Spiel, MD, a Irene M. Lang, MD, b Gerhard Kreiner, MD, b Guenter Christ, MD, b and Bernd Jilma, MD a Vienna, Austria Background 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 omeprazoleclopidogrel 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) in 300 patients 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 (45 vs. 41 U; 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 = 47 U), esomeprazole (n = 74; PRI = 54%; aggregation = 42 U), or without PPI (n = 74; PRI = 49%; aggregation = 41 U; 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 J 2009;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 CYP2C19 and CYP3A4. 5-8 Proton pump inhibitors (PPIs), frequently used in patients receiving clopidogrel and aspirin, are also metabolized by CYP2C19 and CYP3A4. 9 It has recently been reported that the PPI omeprazole decreases the antiplatelet effect of clopidogrel possibly due to the inhibition of the CYP2C19 enzyme. 10,11 As all PPIs are metabolized by CYP2C19, 12 but 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 in 300 patients with coronary From 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 August 24, 2008; accepted September 25, 2008. Reprint requests: Bernd Jilma, MD, Department of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: bernd.jilma@meduniwien.ac.at 0002-8703/$ - see front matter 2009, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2008.09.017 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 (VASP) phosphorylation assay and impedance aggregometry. 13-15 The VASP assay is specific for clopidogrel and other P2Y12 antagonists 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 VASP assay may improve the clinical outcome in patients with decreased platelet inhibition by clopidogrel. 19,20 Methods Study design The 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 (600 mg) at the start of clopidogrel treatment. Patients had been on clopidogrel (75 mg/d) and aspirin (100 mg/d) treatment for 3 months on average (5 days 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. 21

148.e2 Siller-Matula et al American Heart Journal January 2009 Analysis of VASP phosphorylation by flow cytometry To determine the VASP (vasodilator stimulated phosphoprotein) phosphorylation state of whole blood, we used a standardized flow cytometric assay (Platelet VASP; BioCytex, Marseille, France). Blood samples collected in 3.8% sodium citrate (BD Vacutainer; Becton Dickinson, Vienna, Austria) were incubated in vitro with adenosine diphosphate and/or prostaglandin E1 (PGE1) before fixation. After 10 minutes, platelets were permeabilized, labeled with a primary monoclonal antibody against serine 239-phosphorylated VASP (clone 16C2) or its isotype, followed by a secondary fluorescein isothiocyanate conjugated polyclonal goat-antimouse antibody. All procedures were performed at room temperature. Geometric mean fluorescence intensity (GMFI) was determined using a flow cytometer (FACSCalibur System, BD Biosciences, Vienna, Austria). 22 The platelet population was identified by its forward and side scatter distribution, and 10,000 platelet 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 VASP analysis is 69% to 100%. 21,23 The VASP assay was performed within 24 hours after blood sampling. Aggregometry ADP-induced platelet aggregometry is a widely used method to measure the responsiveness to clopidogrel. 24,25 Whole blood aggregation was determined using an impedance 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. 26,27 A 2:1 solution of whole blood anticoagulated with hirudin (200 U/mL, Dynabyte Medical) and 0.9% NaCl was stirred at 37 C for 3 minutes in the test cuvettes, adenosine diphosphate ([ADP] 6.4 μmol/l, Dynabyte Medical) was added, and the increase in electrical impedance was recorded continuously for 6 minutes. The mean values of the 2 independent determinations are expressed as the area under the curve of the aggregation tracing. The results measured by the Multiplate Analyzer are reproducible with less than 6% variability. 26 The reference range for the test is 29 to 118 U. 28 Statistical analysis A 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. 11 We calculated that we need to include 72 patients in each group to be able to detect such a 20% relative difference in platelet reactivity index with a power of 95% and a 2-sided α value 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, calciumchannel blockers, diabetes mellitus, arterial hypertension, hypercholesterolemia, previous myocardial infarction, cause for hospitalization, and smoking. Data are reported as mean and 95% confidence intervals. Statistical comparisons were performed with the Kruskal-Wallis analysis of variance, the χ 2 test, and the Mann-Whitney U test. A 2-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 Version 14.0; SPSS, Chicago, IL). Results Patient demographics Patient demographics are shown in Table I. Most patients had high blood pressure and hyperlipidemia. Almost half of the patients had previous PCI. Use 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 esomeprazole (68%) group as compared to patients without any PPI (47%; P =.018). There were more patients hospitalized for myocardial infarction in the esomeprazole 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, VASP assay) was similar in patients with any PPI (n = 226, mean 51%; 95% CI 48-54%) or without any PPI treatment (n = 74; mean 49%; 95% CI 43-55%; P =.724). As a consequence, the rate of decreased platelet inhibition by clopidogrel defined by a platelet reactivity index 69% 21 was similar in both groups (25% vs 20%). Likewise, the ADPinduced platelet aggregation did not differ significantly between patients with any PPI (mean 45 U; 95% CI 41-50 U) or without any PPI treatment (mean = 41 U, 95%CI 33-48 U; P =.619). Comparison of patients with pantoprazole or esomeprazole vs without PPI There 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 = 42 U) or without PPI (n = 74; PRI = 49%; aggregation = 41 U; P =.382; Figures 1 and 2). To exclude that minor differences in demographic data may have influenced the platelet reactivity index or the

American Heart Journal Volume 157, Number 1 Siller-Matula et al 148.e3 Table I. Patient demographics Figure 1 Patient demographics No PPI (n = 74) Pantoprazole (n = 152) Esomeprazole (n = 74) Age 73 ± 12 65 ± 12 62 ± 13 Gender (male) 66 (93) 109 (71) 59 (80) Cause for hospitalisation Silent ischemia 7 (9) 13 (9) 7 (9) Stable angina 49 (66) 87 (57) 33 (45) Unstable angina 3 (4) 14 (9) 5 (7) Myocardial 15 (20) 38 (25) 29 (39) infarction Risk factors/past medical history Hypertension 63 (85) 132 (87) 63 (85) Smoking 37 (50) 88 (58) 47 (63) Family history 25 (34) 44 (29) 22 (30) of CAD Diabetes mellitus 23 (31) 53 (35) 23 (31) Hyperlipidemia 56 (76) 119 (78) 55 (75) Prior myocardial 23 (31) 52 (34) 24 (32) infarction Prior PCI 36 (49) 80 (53) 38 (51) PAOD 8 (11) 20 (13) 8 (11) CVD 9 (12) 14 (9) 8 (11) Laboratory data WBC (G/L) 8 ± 2.5 7.9 ± 2.8 7.8 ± 2.9 Platelets (G/L) 228 ± 70 226 ± 68 219 ± 64 CRP (mg/dl) 1.2 ± 2.7 1.3 ± 2.9 1.8 ± 2.6 Hb (g/dl) 14 ± 1.4 12.8 ± 2 14.5 ± 2.1 Fibrinogen (mg/dl) 408 ± 130 421 ± 120 441 ± 107 Creatinine (mg/dl) 1.3 ± 1.2 1.3 ± 1.2 1.7 ± 1.3 Pre-PCI medications ACE inhibitors 35 (47) 73 (48) 51 (68) Calcium-channel 13 (18) 31 (20) 19 (13) blockers Statins 45 (61) 124 (82) 65 (87) β-blockers 51 (69) 122 (80) 59 (80) ASA 74 (100) 152 (100) 74 (100) Clopidogrel 74 (100) 152 (100) 74 (100) PCI data Number of stents 1.5 ± 0.7 1.8 ± 0.9 1.8 ± 1.2 per patient Total stent length 29 ± 15 33 ± 21 33 ± 25 Data 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.05 for pantoprazole or esomeprazole versus no PPI. P b.01 for pantoprazole or esomeprazole versus no PPI. ADP-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. Platelet reactivity index in the VASP phosphorylation assay in patients on clopidogrel with or without PPI: pantoprazole or esomeprazole. Data are presented as mean and 95% CI. Figure 2 Adenosine diphosphate induced platelet aggregation in patients on clopidogrel with or without PPI: pantoprazole or esomeprazole. Data are presented as mean and 95% CI. Discussion The intake of pantoprazole or esomeprazole was not associated with a reduced platelet inhibition by clopidogrel as compared to patients without PPI (Figures 1 and 2). The platelet reactivity index was 49% in patients without PPI and, thus, similar to that in a previous study investigating the omeprazole-clopidogrel drug interaction (PRI = 50%). 11 This demonstrates the external validity of the VASP assay. Previous articles alerted the scientific community that concomitant treatment with omeprazole and clopidogrel may have negative effects. 10,11 The 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 clopidogrel 10,11 is not seen in patients

148.e4 Siller-Matula et al American Heart Journal January 2009 treated with pantoprazole or esomeprazole. Therefore, the reported omeprazole-clopidogrel drug interaction 10,11 is probably not a class effect. A possible explanation for the reduced responsiveness to clopidogrel under omeprazole treatment could be the stereoselective 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 has 97% power for pantoprazole and 88% for esomeprazole to exclude a 20% difference of platelet reactivity index as compared to patients without PPI. The PRI was slightly (10%) higher in the esomeprazole group as compared to controls. However, this would require a sample size of 762 patients 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 clopidogrel. Moreover, it has to be evaluated if the reported interaction between PPI and clopidogrel has an impact on hard outcome parameters, as significant pharmacological drug-drug interactions do not necessarily have clinical impact. 30-32 Similar to our study, another trial implicates that the PPI-clopidogrel interaction is not a class effect: concomitant treatment with lansoprazole did not alter the pharmacokinetics or pharmacodynamics of clopidogrel. 7 This study also showed that increasing the gastric ph did not influence the platelet inhibition by clopidogrel. 7 We selected 2 methods for evaluating PPI-clopidogrel drug-drug interaction: a very specific VASP assay and a highly functional ADP-induced aggregometry. There are the following arguments for a combination of the two assays applied. Although the VASP assay is highly specific for the effect of clopidogrel on the ADP receptor (P2Y12), VASP is a biochemical marker which may not reflect certain aspects of platelet function: Firstly, activation of the P2Y12 receptor by ADP initiates other signalling pathways, which are independent of VASP phosphorylation. 20 Secondly, the antibody 16C2 used in the VASP assay recognizes phosphorylation at a serine residue that is also phosphorylated by different cyclic guanosine monophosphate-dependent kinases that are not regulated by P2Y12. 20 Aggregometry is a functional assay based on the stimulation of platelet aggregation with ADP. 26 We used an impedance aggregometry in whole blood, which eliminates potential disadvantages of the light transmission aggregometry: time-consuming centrifugation steps, variable reproducibility, large required sample volumes, and lengthy processing time. 14 A good correlation between impedance and light transmission aggregometry has been shown. 25 Therefore, combination of the VASP assay and the ADPinduced aggregometry was reasonable. In summary, our study implies that in contrast to the reported negative omeprazole-clopidogrel drug interaction, the intake of pantoprazole or esomeprazole is not associated with impaired response to clopidogrel. Limitations Possible 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 esomeprazole. Therefore, we could not examine whether the data from the previous trial are reproducible. References 1. Snoep JD, Hovens MM, Eikenboom JC, et al. Clopidogrel nonresponsiveness in patients undergoing percutaneous coronary intervention with stenting: a systematic review and meta-analysis. Am Heart J 2007;154:221-31. 2. Siller-Matula J, Schror K, Wojta J, et al. Thienopyridines in cardiovascular disease: focus on clopidogrel resistance. Thromb Haemost 2007;97:385-93. 3. Angiolillo DJ, Guzman LA, Bass TA. Current antiplatelet therapies: benefits and limitations. Am Heart J 2008;156:S3-9. 4. Gladding P, Webster M, Ormiston J, et al. Antiplatelet drug nonresponsiveness. Am Heart J 2008;155:591-9. 5. Savi P, Pereillo JM, Uzabiaga MF, et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000;84:891-6. 6. Pereillo JM, Maftouh M, Andrieu A, et al. Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metab Dispos 2002;30:1288-95. 7. Small DS, Farid NA, Payne CD, et al. Effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel. J Clin Pharmacol 2008;48: 475-84. 8. Lotfi A, Schweiger MJ, Giugliano GR, et al. High-dose atorvastatin does not negatively influence clinical outcomes among clopidogrel treated acute coronary syndrome patients a Pravastatin or Atorvastatin Evaluation and Infection Therapy Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22) analysis. Am Heart J 2008; 155:954-8. 9. Li XQ, Andersson TB, Ahlstrom M, et al. Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities. Drug Metab Dispos 2004;32: 821-7. 10. Gilard M, Arnaud B, Cornily JC, et al. Influence of omeprazole on the antiplatelet action of clopidogrel associated with aspirin: the randomized, double-blind OCLA (Omeprazole CLopidogrel Aspirin) study. J Am Coll Cardiol 2008;51:256-60. 11. Gilard M, Arnaud B, Le Gal G, et al. Influence of omeprazol on the antiplatelet action of clopidogrel associated to aspirin. J Thromb Haemost 2006;4:2508-9.

American Heart Journal Volume 157, Number 1 Siller-Matula et al 148.e5 12. Ishizaki T, Horai Y. Review article: cytochrome P450 and the metabolism of proton pump inhibitors emphasis on rabeprazole. Aliment Pharmacol Ther 1999;13(Suppl 3): 27-36. 13. Cairns JA, Eikelboom J. Clopidogrel resistance: more grist for the mill. J Am Coll Cardiol 2008;51:1935-7. 14. Gurbel PA, Becker RC, Mann KG, et al. Platelet function monitoring in patients with coronary artery disease. J Am Coll Cardiol 2007;50: 1822-34. 15. Manolopoulos P, Glenn JR, Fox SC, et al. Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: a new finding that may influence the design of antithrombotic agents. Platelets 2008;19:134-45. 16. Sudo T, Ito H, Kimura Y. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 2003;14:381-90. 17. Judge HM, Buckland RJ, Sugidachi A, et al. The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses. Platelets 2008;19:125-33. 18. Bonello L, Paganelli F, Arpin-Bornet M, et al. Vasodilatorstimulated phosphoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events. J Thromb Haemost 2007;5:1630-6. 19. Bonello L, Camoin-Jau L, Arques S, et al. Adjusted clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index decrease rate of major adverse cardiovascular events in patients with clopidogrel resistance: a multicenter randomized prospective study. J Am Coll Cardiol 2008; 51:1404-11. 20. Kleiman NS. Will measuring vasodilator-stimulated phosphoprotein phosphorylation help us optimize the loading dose of clopidogrel? J Am Coll Cardiol 2008;51:1412-4. 21. Siller-Matula J, Lang I, Christ G, et al. Calcium channel blockers reduce the antiplatelet effect of clopidogrel. J Am Coll Cardiol 2008; 52:1557-63. 22. Siller-Matula JM, Panzer S, Jilma B. Reproducibility and standardised reporting of the vasodilator-stimulated phosphoprotein phosphorylation assay. Platelets 2008;19:551-4. 23. Morel O, Faure A, Ohlmann P, et al. Impaired platelet responsiveness to clopidogrel identified by flow cytometric vasodilator-stimulated phosphoprotein (VASP) phosphorylation in patients with subacute stent thrombosis. Thromb Haemost 2007;98:896-9. 24. Seyfert UT, Haubelt H, Vogt A, et al. Variables influencing Multiplate (TM) whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals. Platelets 2007;18: 199-206. 25. Sibbing D, Braun S, Jawansky S, et al. Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment. Thromb Haemost 2008;99:121-6. 26. Toth O, Calatzis A, Penz S, et al. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood. Thromb Haemost 2006;96:781-8. 27. Penz SM, Reininger AJ, Toth O, et al. Glycoprotein Ibalpha inhibition and ADP receptor antagonists, but not aspirin, reduce platelet thrombus formation in flowing blood exposed to atherosclerotic plaques. Thromb Haemost 2007;97:435-43. 28. Mueller T, Dieplinger B, Poelz W, et al. Utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate(R) analyzer-comparison with two flow cytometric methods. Thromb Res 2007;121:249-58. 29. Abelo A, Andersson TB, Antonsson M, Naudot AK, Skanberg I, Weidolf L. Stereoselective metabolism of omeprazole by human cytochrome P450 enzymes. Drug Metab Dispos 2000;28:966-72. 30. Lau WC, Waskell LA, Watkins PB, et al. Atorvastatin reduces the ability of clopidogrel to inhibit platelet aggregation: a new drug-drug interaction. Circulation 2003;107:32-7. 31. Saw J, Brennan DM, Steinhubl SR, et al. Lack of evidence of a clopidogrel-statin interaction in the CHARISMA trial. J Am Coll Cardiol 2007;50:291-5. 32. Angiolillo DJ, Alfonso F. Clopidogrel-statin interaction: myth or reality? J Am Coll Cardiol 2007;50:296-8.