Pharmacodynamic Effects of Cangrelor on Platelet P2Y 12 Receptor Mediated Signaling in Prasugrel Treated Patients Fabiana Rollini, Francesco Franchi, Antonio Tello-Montoliu, Ronakkumar Patel, Andrew Darlington, José Luis Ferreiro, Jung Rae Cho, Ana Muniz-Lozano, Bhaloo Desai, Martin M. Zenni, Luis A. Guzman, Theodore A. Bass and Dominick J. Angiolillo. University of Florida College of Medicine-Jacksonville, Jacksonville, FL, USA JACC Cardiovasc Interv Apr;7(4):

Authors and disclosures The present study was funded by a grant from The Medicines Company. The study was designed and undertaken by the investigators who had final responsibility for the decision to publish these results. Dominick J. Angiolillo: Received payment as an individual for: a) Consulting fee or honorarium from Bristol Myers Squibb, Sanofi-Aventis, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Merck, Evolva, Abbott Vascular and PLx Pharma; b) Participation in review activities from Johnson & Johnson, St. Jude, and Sunovion. Institutional payments for grants from Bristol Myers Squibb, Sanofi-Aventis, Glaxo Smith Kline, Otsuka, Eli Lilly, Daiichi Sankyo, The Medicines Company, AstraZeneca, Evolva, Gilead; and has other financial relationships with Esther and King Biomedical Research Grant. José Luis Ferreiro: reports honoraria for lectures from Eli Lilly Co; Daiichi Sankyo, Inc.; Astra Zeneca. Theodore A. Bass: has received research funds (paid to Institution) from Baxter. All other authors have not disclosures to report.

BACKGROUND Despite the more rapid and potent levels of platelet inhibition achieved compared with clopidogrel, pharmacodynamic studies have shown interindividual variability in prasugrel effects (1-5) In particular, delayed antiplatelet effects and high rates of high on platelete reactivity have been shown especially in the early hours after prasugrel administration in patients with STEMI undergoing primary PCI (6,7) The use of orally administered antiplatelet agents may be challenging in patients unable to swallow (e.g. patients sedated, intubated, in shock, or those with nausea or vomiting) These observations support the need for intravenous antiplatelet therapies able to yield more prompt and potent platelet inhibitory effects, which are unlikely to be achieved with oral medications (5) Cangrelor, is a potent intravenous direct-acting and reversible P2Y 12 receptor antagonist which has shown to reduce ischemic complications, including stent thrombosis, in P2Y 12 inhibitor naïve patients undergoing PCI (8,9) 1)Alexopoulos D et al. Curr Pharm Des. 2013;19:5121-6; 2)Bonello L et al. J Am Coll Cardiol. 2011;58:467-73; 3)Alexopoulos D et al. Int J Cardiol. 2012;154:333-4; 4)Ferreiro JL et al. JACC Cardiovasc Interv. 2013;6:182-4; 5)Ferreiro JL et al. Circ Cardiovasc Interv. 2012;5:433-45; 6)Alexopoulos D et al. Circ Cardiovasc Interv. 2012;5: ; 7)Parodi G et al. J Am Coll Cardiol. 2013;61:1601-6; 8)Bhatt DL et al. N Engl J Med. 2013;368: ; 9)Steg PG et al. Lancet. 2013;382:

STUDY AIM The aim of this prospective, randomized, pharmacodynamic investigation was to assess by whole blood vasodilator-stimulated phosphoprotein (VASP) the in vitro effects of cangrelor on platelets from patients with coronary artery disease on maintenance prasugrel 10mg/day therapy treated with two reloading dose (30mg or 60mg) regimens. PRIMARY END-POINT The primary endpoint of the study was the comparison between PRI before and after in vitro incubation with cangrelor at baseline, while patients were on maintenance prasugrel 10mg/day.

Inclusion criteria:  Age between 18 and 74 years old  aspirin (81 mg/day) and prasugrel (10 mg/day) for at least 14 days  stable coronary artery disease Exclusion criteria:  age ≥ 75 years old  active bleeding  prior cerebrovascular event  body weight < 60 kg  clinical instability after the index event  use of oral anticoagulation  platelet count < 100x10 6 /µl  hemoglobin <10 g/dl  creatinine > 2 mg/dl  hepatic enzymes >2.5 times the upper limit of normal  pregnant and lactating females STUDY POPULATION

STUDY DESIGN Patients on aspirin (81 mg/day) and prasugrel (10mg/day) ≥ 14 days post-PCI Prasugrel 30 mg Reload Prasugrel 60 mg Reload Pharmacodynamic testing: 1 hour Pharmacodynamic testing: Baseline Pharmacodynamic testing: 4 hours Pharmacodynamic testing (VASP) with and without in vitro cangrelor 500 nM

PLATELET FUNCTION TEST Whole blood vasodilator-stimulated phosphoprotein (VASP) The VASP assay was used to determine the platelet reactivity index (PRI) according to standard protocols. VASP was performed before and after in vitro incubation with 500 nM cangrelor at each time point. In brief, VASP phosphorylation (VASP-P) was measured by quantitative flow cytometry using commercially available labelled monoclonal antibodies (Biocytex Inc, Marseille, France). A reduced PRI is indicative of greater inhibition of the P2Y 12 signalling pathway.

STATISTICAL ANALYSIS Conformity to the normal distribution was evaluated for continuous variables with the Kolmogorov-Smirnov test Chi-square test or Fisher’s exact test (if expected value in any cell was fewer than 5) were used to compare categorical variables between two groups A repeated measures analysis of variance (ANOVA) model was used to evaluate intra- group comparisons and the overall difference between groups, using Bonferroni approach to correct for multiple comparisons An analysis of covariance (ANCOVA) method with a general linear model, using the baseline value of platelet reactivity as a covariate, was used to evaluate all between- groups comparisons A 2-tailed p value of < 0.05 was considered to indicate a statistically significant difference for all the analyses performed

RESULTS (1) Baseline Characteristics

RESULTS (2) Comparison of platelet reactivity values between 30 mg and 60 mg prasugrel re-loading dose in the absence of cangrelor Baseline1 hour 4 hours Prasugrel 30 mg Prasugrel 60 mg p=0.481 p=0.001 Prasugrel 30 mg Intragroup ANOVA p<0.001 Baseline vs 1hr p<0.001 Baseline vs 4hrs p< hr vs 4hrs p=0.065 Prasugrel 60 mg Intragroup ANOVA p<0.001 Baseline vs 1hr p<0.001 Baseline vs 4hrs p< hr vs 4hrs p=0.001 Platelet Reactivity Index % ANOVA p=0.935

RESULTS (3) Comparison of platelet reactivity values between 30 mg and 60 mg prasugrel re-loading dose in the presence of cangrelor Baseline + cangrelor1 hour4 hours Prasugrel 30 mg + cangrelor Prasugrel 60 mg + cangrelor p=0.604 p=0.334 Platelet Reactivity Index % ANOVA p=0.373 Prasugrel 30 mg + cangrelor Intragroup ANOVA p<0.001 Baseline vs 1h p=0.002 Baseline vs 4 hs p< h vs 4hs p=0.401 Prasugrel 60 mg + cangrelor Intragroup ANOVA p=0.001 Baseline vs 1hr p=0.124 Baseline vs 4 hrs p= hr vs 4 hrs p=0.016

RESULTS (4) p<0.001 p=0.013 p=0.001 ANOVA p=0.001 Platelet Reactivity Index % Comparison of PRI expressed as percentages measured by VASP across time points in the prasugrel 30 mg reload group in presence and absence of cangrelor

RESULTS (5) Comparison of PRI expressed as percentages measured by VASP across time points in the prasugrel 60 mg reload group in presence and absence of cangrelor p<0.001 p=0.002 p=0.325 ANOVA p<0.001 Platelet Reactivity Index %

Baseline + cangrelor1 hour4 hours Δ Platelet Reactivity Index % p>0.999 ANOVA p=0.290 Prasugrel 60 mg + cangrelor Prasugrel 30 mg + cangrelor RESULTS (6) Absolute change or delta (Δ) between PRI values in the absence and presence of cangrelor in each arm (30 mg and 60 mg prasugrel re-load)

CONCLUSIONS In vitro cangrelor is associated with enhanced platelet inhibition when added to platelets of patients on prasugrel maintenance therapy as well as when exposed to a reloading dose Platelet inhibitory effects of in vitro cangrelor are immediate and faster than prasugrel reloading alone Comparable levels of platelet reactivity to that achieved with in vitro cangrelor were observed only 4 hours after a 60 mg prasugrel reloading dose Rollini F. et al. JACC Cardiovasc Interv Apr;7(4):426-34