1. PRO-GR: A Prospective, Randomized, Crossover Study of Maintenance High-Dose Clopidogrel vs. Prasugrel in Clopidogrel Resistant Patients With and Without the CYP2C19*2 Loss-of-Function Allele Dimitrios Alexopoulos, MD Patras University Hospital, Patras, Greece

2. Disclosure Statement of Financial Interest I, Dimitrios Alexopoulos DO NOT have a financial interest/arrangement or affiliation with one or more organizations that could be perceived as a real or apparent conflict of interest in the context of the subject of this presentation.

3. I. Xanthopoulou, MD G. Dimitropoulos, MD G. Kasimis, MD A. Panagiotou, MD G. Hahalis MD P. Davlouros, MD Cardiology Department, Patras University Hospital E. Stavrou, PhD A. Athanassiadou, PhD Department of Biology, Faculty of Medicine, University of Patras

4. Price MJ, et al. Eur Heart J 2008 High on treatment platelet reactivity (HTPR), (clopidogrel resistance) and adverse events post- PCI

5. Events and CYP2C19*2 loss of function allele carriage Mega JL et al. N Engl J Med 2009

6. v.Beckerath N et al, EHJ 2008 Doubling the maintenance dose of clopidogrel

7. Wiviott SD et al, Circulation 2007 Prasugrel compared to standard or high dose of Clopidogrel

8. DESIGN Prospective, randomized, crossover, single- center, investigator-initiated study. OBJECTIVE 1. To investigate the antiplatelet effects of prasugrel 10 mg/d versus clopidogrel 150 mg/d in patients with clopidogrel resistance post- PCI. 2. To evaluate the impact of the loss-of-function CYP2C19*2 on platelet reactivity changes.

9. Methods At the time of PCI, clopidogrel naïve patients and those on clopidogrel 75 mg for < 7 days without initial loading received a 600 mg clopidogrel loading dose. At the time of PCI, clopidogrel naïve patients and those on clopidogrel 75 mg for < 7 days without initial loading received a 600 mg clopidogrel loading dose. Patients on clopidogrel 7 days did not receive any additional loading. Patients on clopidogrel 7 days did not receive any additional loading. Use of periprocedural glycoprotein IIb/IIIa inhibitors was allowed, at the operator’s discretion. Use of periprocedural glycoprotein IIb/IIIa inhibitors was allowed, at the operator’s discretion.

10. Methods EXCLUSION CRITERIA a history of bleeding diathesis, a history of bleeding diathesis, chronic oral anticoagulation treatment, chronic oral anticoagulation treatment, known platelet function disorders, known platelet function disorders, PCI or CABG < 3 months, PCI or CABG < 3 months, planned staged PCI in the next 60 days, planned staged PCI in the next 60 days, hemodynamic instability, hemodynamic instability, platelet count <100 000/ μL, platelet count <100 000/ μL, hematocrit <30%, hematocrit <30%, creatinine clearance <25 ml/min, creatinine clearance <25 ml/min, inability to give informed consent, inability to give informed consent,

11. Methods EXCLUSION CRITERIA History of stroke History of stroke Patients 75 years and HTPR were not excluded as they were considered to be at high risk for ischemic events. Patients 75 years and HTPR were not excluded as they were considered to be at high risk for ischemic events.

12. Methods Platelet function determination Peripheral venous blood sampling 24 hours post PCI (48 hrs if IIb/IIIa had be given). Peripheral venous blood sampling 24 hours post PCI (48 hrs if IIb/IIIa had be given). VerifyNowTM (Accumetrics, Inc., San Diego, CA, USA) point-of-care assay. VerifyNowTM (Accumetrics, Inc., San Diego, CA, USA) point-of-care assay. Results are reported as PRU with a value ≥ 235 considered as an indication of HTPR. Results are reported as PRU with a value ≥ 235 considered as an indication of HTPR. Genotyping for CYP2C19*2 (681G>A carriage) Genotyping for CYP2C19*2 (681G>A carriage) Real time PCR (Borlak J, Clin Chem 2002) Real time PCR (Borlak J, Clin Chem 2002)

13. Methods Primary end point: platelet reactivity at the end of the two (precrossover and postcrossover) study periods. Primary end point: platelet reactivity at the end of the two (precrossover and postcrossover) study periods. Secondary end point: HTPR rate. Secondary end point: HTPR rate.

14. Methods Statistical analysis Hierarchical ANCOVA (or mixed-effects) model, with patient indicator as random effect, period and treatment as fixed factors and platelet reactivity at baseline as a covariate. Hierarchical ANCOVA (or mixed-effects) model, with patient indicator as random effect, period and treatment as fixed factors and platelet reactivity at baseline as a covariate. Chi-square Prescott test. Chi-square Prescott test. The study was approved by the ethics committee of the University Hospital of Patras, Greece. The study was approved by the ethics committee of the University Hospital of Patras, Greece. All patients gave written informed consent. All patients gave written informed consent. ClinicalTrials.gov Identifier NCT 01109784 ClinicalTrials.gov Identifier NCT 01109784

15. Study flow chart Patients post PCI with Platelet Reactivity Assessment N=210 PRU≥235 N=71 (33.8%) Clopidogrel 150mg/d N=35 Side effects N=2, low compliance N=1 Complete Day 30 data N=32 Prasugrel 10mg/d N=32 Side effects N=0, lost follow-up N=5 Complete Day 60 data N=27 Prasugrel 10mg/d N=36 Side effects N=0, low compliance N=4 Complete Day 30 data N=32 Clopidogrel 150mg/d N=32 Side effects N=1, lost follow-up N=5 Complete Day 60 data N=26 Randomized N=71

16. Results Safety Pre-crossover period 1 TIMI major bleeding 1 TIMI major bleeding 1 AMI with documented in-stent thrombosis, both allocated to clopidogrel and both excluded from analysis. 1 AMI with documented in-stent thrombosis, both allocated to clopidogrel and both excluded from analysis. 3 patients (allocated to prasugrel) experienced minor bleeding events. 3 patients (allocated to prasugrel) experienced minor bleeding events. Post-crossover period 1 TIMI major bleeding 1 TIMI major bleeding 1 minor bleeding event (both allocated to clopidogrel). 1 minor bleeding event (both allocated to clopidogrel). No deaths or strokes occurred in either treatment group. No deaths or strokes occurred in either treatment group.

17. Results Baseline characteristics of patients with complete Day 30 data Clopidogrel N=32 Prasugrel N=32 P Value Age67.9±10.562.2±10.80.03 Male gender 28(87.5%)29(90.6%)1.0 BMI27.9±4.330.1±3.90.03 Hyperlipidaemia22(68.8%)19(59.4%)0.6 Hypertension22(68.8%)21(65.6%)1.0 Diabetes mellitus 13(40.6%)10(31.2%)0.6 Smoking14(43.8%)16(50.0%)0.8

18. Results Clopidogrel N=32 Prasugrel N=32 Pvalue Proton pump inhibitors 32(100%)30(93.8)0.5 IIb/IIIa inhibitors 6(18.8%)4(12.5)0.7 Platelet Reactivity Units Day 0 292.0±50.4289.9±42.90.9 One CYP2C19*2 allele N=299(31.0%)N=3012(40.0%)0.6 Baseline characteristics of patients with complete Day 30 data

19. Results Prasugrel LS estimates (95%CI) Clopidogrel LS estimates (95%CI) LS mean difference (95%CI) P Day 30 (pre- crossover) N=32 130.2 (103.7- 156.7) N=32 196.9 (170.5- 223.5) 196.9 (170.5- 223.5) -66.8 (-104.3 to -29.3) 0.001 Day 60 (post- crossover) N=27 128.4 (103.2- 153.5) N=26 206.4 (180.8- 232.2) -78.1 (-113.9 to -42.2) <0.001 Combined data (pre and post- crossover) N=59 129.4 (111.1- 147.7) N=58 201.7 (183.2- 220.2) -72.3 (-98.3 to -46.4) <0.001 Platelet reactivity analysis

20. Results Platelet reactivity by treatment sequence

21. ResultsPrasugrelClopidogrelPvalue Day 30 (pre- crossover) N=322(6.3%)N=3210(31.3%)0.022 Day 60 (post- crossover) N=272(7.4%)N=2611(42.3%)0.004 Combined data (pre and post-crossover) N=534(7.5%)N=5319(35.8%)<0.001 HTPR rates

22. Patients individual PR values against the HTPR threshold. HTPR threshold

23. Results Prasugrel LS estimates (95%CI) Clopidogrel LS estimates (95%CI) LS mean difference (95%CI) P Combined data (pre and post- crossover) non-carriersN=37 139.3 (117.1- 161.6) N=35 186.8 (163.8- 209.8) -47.5 (-79.5 to -15.4) 0.004 Combined data (pre and post- crossover) carriers N=20 112.4 (81.5- 143.2) N=20 235.3 (204.7- 265.9) -122.9 (- 166.7 to - 79.2) <0.001 Platelet reactivity in patients with and without the CYP2C19*2 allele

24. Results Platelet reactivity by treatment sequence in non carriers of CYP2C19*2 allele

25. Results Platelet reactivity by treatment sequence in carriers of CYP2C19*2 allele

26. Results Non-carriers Non-carriers 10/34 (29.4%) had HTPR on high clopidogrel. 10/34 (29.4%) had HTPR on high clopidogrel. 3/34 (8.8%) had HTPR on prasugrel (p=0.005). 3/34 (8.8%) had HTPR on prasugrel (p=0.005). Carriers Carriers 9/19 (47.4%) had HTPR on high clopidogrel 9/19 (47.4%) had HTPR on high clopidogrel 1/19 (5.3%) had HTPR on prasugrel (p=.0.007). 1/19 (5.3%) had HTPR on prasugrel (p=.0.007). No period or carry-over effect was found. No period or carry-over effect was found.

27. Conclusions 1. In patients with HTPR post PCI, prasugrel is more effective compared to high clopidogrel in reducing platelet reactivity. 2. This effect is more prominent in patients carrying at least one loss-of- function CYP2C19*2 allele. Dimitrios Alexopoulos PRO-GR: A Prospective, Randomized, Crossover Study of Maintenance High-Dose Clopidogrel vs. Prasugrel in Clopidogrel Resistant Patients With and Without the CYP2C19*2 Loss-of-Function Allele

28. Conclusions 3. In high risk individuals like clopidogrel resistant patients post PCI genotyping seems to be helpful for selection between increasing clopidogrel maintenance dose and prasugrel administration. Dimitrios Alexopoulos PRO-GR: A Prospective, Randomized, Crossover Study of Maintenance High-Dose Clopidogrel vs. Prasugrel in Clopidogrel Resistant Patients With and Without the CYP2C19*2 Loss-of-Function Allele

29. Back up slides

30. G A GGAA GA Real time PCR result picture Real time PCR result explanation (G to A mutation) Sample 1 (purple): homozygous for the normal G allele –GG- Sample 2 (yellow): homozygous for the mutant A allele –AA- Sample 3 (green): heterozygous contains one normal G allele and one mutant A allele –GA-

31. Results Prasugrel LS estimates (95%CI) Clopidogrel LS estimates (95%CI) LS mean difference (95%CI) P *Combined data (pre and post- crossover) N=59 131.5 (113.2- 149.7) N=58 203.7 (185.1- 222.3) -72.2 (-97.9 to -46.5) <0.001 Platelet reactivity analysis Analysis with age and BMI as additional fixed factors