Olga Gorchakova a, Nicolas von Beckerath b, *, Meinrad Gawaz a, Adrienne Mocz b, Alexander Joost b, Albert Schömig a,b, Adnan Kastrati b.

European Heart Journal (24) 25, 1898 192 Clinical research Antiplatelet effects of a mg loading dose of clopidogrel are not attenuated in patients receiving atorvastatin or simvastatin for at least 4 weeks prior to coronary artery stenting Olga Gorchakova a, Nicolas von Beckerath b, *, Meinrad Gawaz a, Adrienne Mocz b, Alexander Joost b, Albert Schömig a,b, Adnan Kastrati b a Medizinische Klinik rechts der Isar, Technische Universität München, Munich, Germany b Department of Cardiology, Deutsches Herzzentrum, Technische Universität München, Lazarettstrasse 36, 636 Munich, Germany Received 2 October 23; revised 11 October 23; accepted 13 October 23 KEYWORDS Clopidogrel; Statins; Platelets; Pharmacology Aims To test prospectively whether the antiplatelet effect of a mg loading dose of clopidogrel is attenuated in patients receiving atorvastatin and simvastatin for at least 4 weeks prior to coronary artery stenting. Methods and results Blood samples were obtained at least 2 h after receiving 1 mg aspirin and mg clopidogrel and prior to coronary stenting from 9 patients without statin therapy and 9 patients with statin (atorvastatin and simvastatin) therapy for at least 4 weeks. Maximal and residual platelet aggregation was evaluated with optical aggregometry in response to ADP (5 and 2 lmol/l). Surface expression of IIb/IIIa (CD61) and P-selectin (CD62) was assessed with whole blood flow-cytometry at baseline and following stimulation (5 and 2 lmol/l ADP). Inhibition of ADP-induced platelet aggregation was not impaired in the presence of concomitant statin therapy. Moreover, patients with and without statin therapy did not differ in respect to all flow-cytometric parameters obtained. Conclusion The antiplatelet effect of a high, mg loading dose of clopidogrel is not diminished in patients receiving atorvastatin and simvastatin for at least 4 weeks prior to coronary stenting. c 24 Published by Elsevier Ltd on behalf of The European Society of Cardiology. Introduction * Corresponding author. Tel.: +49 89 1218 11; fax: +49 89 1218 4593. E-mail address: beckerath@dhm.mhn.de (N. von Beckerath). Dual antiplatelet therapy with clopidogrel and aspirin is now the standard regimen to prevent thrombosis following coronary stent insertion. Clopidogrel is a prodrug that is extensively metabolised in the liver. The active shortlived metabolite is a thiol derivate that is formed by oxidation to 2-oxo-clopidogrel and subsequent hydrolysis. 1 Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, were consistently effective in randomised trials for primary and secondary prevention of cardiovascular events. Since statins also reduce the incidence of adverse events following coronary stenting, 2 clopidogrel and statins are often given together in this clinical setting. In a recent report on a large consecutive series of patients 195-668X/$ - see front matter c 24 Published by Elsevier Ltd on behalf of The European Society of Cardiology. doi:1.116/j.ehj.23.1.39

Antiplatelet effects of a mg loading dose of clopidogrel are not attenuated in patients 1899 undergoing coronary stenting from 1995 to 1999 roughly % of the patients received statins. 2 Lau and coworkers reported a concerning attenuation of the antiaggregatory effect of clopidogrel due to concomitant maintenance therapy with atorvastatin. 3 Patients in that study received a 3 mg loading dose clopidogrel before stenting. ADP-induced platelet aggregation was assessed before administration of the loading dose and 24 h later with a point-of-care test. The authors also provided evidence that the observed drug drug interaction was related to the cytochrome P45 3A4 (CYP3A4) pathway. 3 Simvastatin, another CYP3A4-metabolised statin, is believed to exert the same drug drug interaction with clopidogrel as atorvastatin. 4 Administration of a high, mg loading dose clopidogrel before coronary stenting exerts the maximal antiaggregatory effect of clopidogrel within 2 h, 5 has the potential to overcome individual differences in response to clopidogrel 6,7 and has been shown to be clinically useful in patients undergoing percutaneous coronary interventions. 8 Recently, no attenuation of the antiaggregatory effect of mg has been described after administration of single doses of the CYP3A4-metabolised statins, atorvastatin and simvastatin. 9 Since CYP3A4-mediated drug drug interactions may occur only at steady state, 1 it is not known whether this high dose of clopidogrel is capable of overcoming the interfering action of long-term therapy with CYP3A4-metabolised statins. This prospective study was planned to test whether the antiplatelet effect of the mg loading dose of clopidogrel is attenuated in patients receiving atorvastatin or simvastatin for at least 4 weeks prior to elective coronary stenting. Methods Study population Eligible for this prospective study were patients scheduled for elective coronary stenting for symptomatic coronary artery disease. Patients with a history of myocardial infarction (MI) within 14 days and stroke within 3 months as well as patients with haemodynamic instability, malignancies, active bleeding and bleeding diathesis, oral anticoagulation therapy with a coumarin derivate, recent treatment (less than 14 days) with a GP IIb/IIIa antagonist or a platelet count <1 1 9 /l were not included. This also applied to patients that presented with chest pain at rest and STsegment changes in >2 ECG leads and/or troponin-t level >.3 ng/ml. Additional exclusion criteria were concomitant medication with known CYP3A4 inducers or inhibitors 11 except calcium channel blockers 12 as well as an impaired renal function resulting in a serum creatinine >2 mg/dl and any liver disease resulting in a bilirubin >2 mg/dl. The first study group comprised 9 consecutive patients who were not on statin therapy. The second study group consisted of 9 consecutive patients treated with atorvastatin or simvastatin for at least the previous 4 weeks. Enrolment was done before knowing the results of platelet function evaluation. The study complies with the Declaration of Helsinki. Approval from the institutional ethics committee and written informed consent from all subjects participating in this study had been obtained. Patients received 1 mg aspirin and a mg loading dose of clopidogrel orally at least 2 h before the intervention. Aggregometry Citrated blood samples were obtained from the arterial sheath immediately before the intervention and then processed within min by operators unaware of the patients medication. Platelet aggregation was evaluated by optical aggregometry in platelet-rich plasma (PRP) using a Chrono-log lumi-aggregometer (Probe & go Labordiagnostica, Endingen, Germany) with a constant stirring rate of 1 rpm at 37 C as previously described. 13 The final platelet count was adjusted to 3 1 9 /l with autologous platelet-poor plasma. Platelet-rich plasma (% light transmission) and platelet-poor plasma (1% light transmission) served as references. After baseline adjustment ADP (5 or 2 lmol/l) was added and aggregation recorded for 5 min. The analysed parameters were maximal aggregation (%) and aggregation after 5 min = residual aggregation (%). Flow-cytometry The evaluation of surface expression of platelets receptors was performed in whole blood. 14 The following monoclonal antibodies were used: anti-cd61 PE (anti-gp IIb/IIIa labelled with phycoerythrine; Serotec, Düsseldorf, Germany) and anti-cd62 FITC (anti-p-selectin labelled with fluorescein isothiocyanate; Beckman Coulter, Krefeld, Germany). In brief, citrated blood (1 ll) was diluted with 49 ll phosphate buffered saline (PBS) and gently mixed. Both antibodies (5 ll each) and ADP or PBS (5 ll) were added to 35 ll diluted citrated blood and incubated at room temperature for 2 min. The samples were fixed with 3 ll of 1% buffered paraformaldehyde and then analysed on a Becton Dickenson FACScan flow-cytometer. Mean intensity of immunofluorescence was used as an index of antibody binding and receptor surface expression. Statistical analysis The primary endpoint of the study was maximal ADP (5 lmol/l)- induced aggregation immediately before the intervention and at least 2 h after ingestion of the mg loading dose clopidogrel and 1 mg aspirin. The number of patients included in the study was based on the assumption that concomitant therapy with atorvastatin or simvastatin results in a 25% increase (from ± 2% to 5 ± 2% [mean ± standard deviation]) of maximal ADP (5 lmol/l)-induced aggregation. Choosing a power of 9% and a two-sided a value of.5 we calculated that a sample size of at least 86 was required in each group (nquery advisor, version 5.). Continuous variables are expressed as mean ± S.D. The unpaired t-test or Wilcoxon rank-sum test were used for 2-group comparisons. One-way analysis of variance (ANOVA) was used for multiple group comparisons. Categorical variables were compared using v 2 test. Statistical analyses were performed using S-Plus software (Mathsoft Inc., Seattle, WA). Statistical significance was accepted for P values <.5. Results Table 1 shows the baseline clinical characteristics and concomitant cardiovascular medication of the patients without statin therapy and those with atorvastatin or simvastatin therapy. Treatment with aspirin and the high loading dose clopidogrel resulted in a pronounced inhibition of ADPinduced platelet aggregation. Maximal ADP-induced

19 O. Gorchakova et al. Table 1 Clinical characteristics of patients according to statin treatment No statins (n = 9) Statins (n = 9) P Age (years) 65.5 ± 9.1 64.4 ± 9.7.42 Female (%) 22 (24.4) 18 (2).35 Active smokers (%) 22 (24.4) 12 (13.3).6 Arterial hypertension (%) 64 (71.1) 51 (59.3).1 Hypercholesterolemia (%) 53 (58.9) (88.9) <.1 Diabetes mellitus (%) 16 (17.8) 21 (23.3).36 Previous myocardial infarction (%) 19 (21.1) 31 (34.4).5 Previous CABG (%) 7 (7.8) 13 (14.4).15 Platelet count, 1 3 /ll 226 54 23 ± 61.65 Time from clopidogrel loading (h) a 4.8 (3. 6.9) 4.7 (2.6 7.3).49 Concomitant medication ACE inhibitors (%) 43 (47.8) 51 (56.7).23 ß-blockers (%) 62 (68.9) 74 (82.2).4 Calcium channel blockers (%) 6 (6.7) 9 (1.).42 CABG denotes coronary artery bypass graft surgery and ACE angiotensin-converting enzyme. a Time from clopidogrel loading to blood draw is expressed as median (interquartile range) and comparison is made by the use of Wilcoxon rank-sum test. platelet aggregation was 41.8 ± 19.8% and 5.7 ± 21.3% for 5 and 2 lmol/l, respectively (n = 1). Table 2 shows all obtained aggregometric and flow-cytometric parameters according to the presence or absence of concomitant statin therapy. Inhibition of ADP-induced platelet aggregation was not impaired in the presence of concomitant statin therapy (see also Fig. 1a). Patients with and without statin therapy did not differ in respect to all flow-cytometric parameters obtained (Table 2). Maximal aggregation in response to 5 lmol/l ADP was similar in patients without statin therapy and those concomitantly treated with atorvastatin or simvastatin (Fig. 1b). The antiaggregatory effect clopidogrel was not diminished even in the presence of high doses of atorvastatin and simvastatin (Fig. 1c). We also assessed the potential influence of calcium channel blockers, another group of CYP3A4 substrates, on the antiplatelet effect of clopidogrel. ADP (5 lmol/l)-induced aggregation was similar in patients with and without calcium channel blocker therapy (42. ± 17.2% vs. 41.8 ± 2.1%; P =.96). Discussion Recently, Lau et al. reported an inhibition of the antiaggregatory effect of clopidogrel by concomitant therapy with atorvastatin, a CYP3A4-metabolised statin. 3 In the first part of their study 44 patients (16 without, 9 with pravastatin and 19 with atorvastatin therapy) were studied that received a 3 mg loading dose of clopidogrel before coronary stenting. In addition, all patients received 325 mg aspirin 15 and adjunctive therapy with eptifibatide. Platelet aggregation was measured before clopidogrel (and eptifibatide) administration and 24 h later (after cessation of the eptifibatide effect) as well as 6 to 8 days later. The effect of concomitant atorvastatin therapy was dose-dependent. Treatment with mg atorvastatin almost completely inhibited the antiplatelet effect of clopidogrel. Moreover, the attenuation of the antiaggregatory effect of clopidogrel persisted until day 6 to 8. In the second and third part of the study in which altogether 27 volunteers were involved and in a recent in Table 2 Effect of the mg clopidogrel loading dose on ADP-induced platelet aggregation and receptor surface expression in the absence and presence of statin therapy No statins (n = 9) Statins (n = 9) P Aggregation (%) Maximal aggregation (5 lmol/l ADP) 43.5 ± 2.4.1 ± 19.2.25 Residual aggregation (5 lmol/l ADP) 31. ± 24.3 24. ± 23..5 Maximal aggregation (2 lmol/l ADP) 52. ± 2.8 49.3 ± 21.8. Residual aggregation (2 lmol/l ADP) 43.1 ± 27. 37.2 ± 27.8.15 Surface expression of receptors (mean intensity of immunofluorescence) CD61 baseline 567. ± 86.1 552.9 ± 12.7.33 CD61 (5 lmol/l ADP) 9.8 ± 82.2 5.1 ± 84.1.71 CD 61 (2 lmol/l ADP) 612. ± 82.1 7.2 ± 83..7 CD62 baseline 147.2 ± 22.2 152.7 ± 25.7.13 CD62 (5 lmol/l ADP) 225.1 ± 45.8 227.2 ± 45.4.75 CD 62 (2 lmol/l ADP) 229.9 ± 49.5 232.6 ± 44.2.71 Data are expressed as mean ± S.D.

Antiplatelet effects of a mg loading dose of clopidogrel are not attenuated in patients 191 (a) (b) (c) 1 2 1 2 1 2 1,5 2, 2,5 3, 3,5 4, 4,5 123456789 No statins No statins n=14 No statins1 mg n=38 2 mg P=.25 P=.42 n=58 Atorvastatin P=.45 n=6 mg n=4 1 mg Statins n=32 Simvastatin Atorvastatin Simvastatin n=17 2 mg n=11 mg Fig. 1 Mean ± standard error of the mean (S.E.M.) of maximal aggregation in response to ADP (5 lmol/l) in patients with and without statin therapy (a), in patients with atorvastatin and simvastatin therapy and without statin therapy (b) and according to atorvastatin and simvastatin dosages (c). vitro study evidence was provided that CYP3A4 is the major cytochrome metabolising clopidogrel. 3,4 In our study, we did not observe an attenuation of the antiplatelet effect of clopidogrel in the presence of atorvastatin and simvastatin therapy as demonstrated by all aggregometric and flow-cytometric parameters obtained. In addition to maximal aggregation, we also evaluated residual aggregation. Residual aggregation is particularly helpful in assessing the secondary phase of ADP-induced platelet aggregation that is predominantly mediated by ADP-binding to the P2Y12 receptor. 16 We also did not find an influence of concomitant calcium channel blocker therapy on platelet inhibition achieved by the mg loading dose of clopidogrel. These data, though, have to be interpreted with caution since only 15 out of the 1 patients received these drugs. It is important to note the most important difference between the study of Lau et al. 3 and our study: the different loading dose of clopidogrel. The high, mg loading dose of clopidogrel may overcome differences in drug-response and drug drug interactions observed with the 3 mg loading dose used in the study of Lau et al. 3 The findings of the present study conducted in clinical settings common to patients undergoing coronary artery stenting complement the data provided recently by Müller and colleagues who did not find any reduction of the antiplatelet effects of mg clopidogrel after single 2 mg doses of atorvastatin or simvastatin. 9 Other recent studies have evaluated patients on longterm clopidogrel therapy and did not find a significant interaction between statins and clopidogrel. In the CRE- DO (clopidogrel for the reduction of events during observation) study, the 1-year adverse event rate in the clopidogrel group was 7.6% for those treated with CYP3A4-metabolised statins and 5.4% for those treated with other statins (P =.51). 17 In the MITRA PLUS (maximal individual therapy of acute myocardial infarction PLUS) registry, the long term mortality was 3.2% in the group receiving atorvastatin and 2.7% in the group that received other statins (p =.49). 18 Two limitations of the present study deserve consideration: the lack of randomisation between patients with and without statin therapy and the assessment of platelet function at a single time point prior to the intervention. Although this time point is probably most useful in the clinical setting of percutaneous coronary intervention, baseline measurements before loading might have provided additional information. Conclusion The antiplatelet effect of a high, mg loading dose of clopidogrel is not diminished in patients receiving atorvastatin and simvastatin for at least 4 weeks prior to coronary stenting. Acknowledgement The invaluable and skilful contribution of M. Eichinger is highly appreciated. References 1. Savi P, Pereillo JM, Uzabiaga MF et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2;84:891 6. 2. Schömig A, Mehilli J, Holle H et al. Statin treatment following coronary artery stenting and one-year survival. J Am Coll Cardiol 22;:854 61. 3. Lau WC, Waskell LA, Watkins PB et al. Atorvastatin reduces the ability of clopidogrel to inhibit platelet aggregation: a new drug drug interaction. Circulation 23;17:32 7. 4. Clarke TA, Waskell LA. The metabolism of clopidogrel is catalyzed by human cytochrome p45 3A and is inhibited by atorvastatin. Drug Metab Dispos 23;31:53 9.

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