1. Aspirinn doses in diabetics - AngiolilloSlide 1 Davide Capodanno, MD, Aasita Patel, MD, Kodlipet Dharmashankar, MD, José Luis Ferreiro, MD, Masafumi Ueno, MD, Murali Kodali, MD, Salvatore D. Tomasello, MD, Piera Capranzano, MD, Naveen Seecheran, MD, Andrew Darlington, MD, Antonio Tello-Montoliu, MD, PhD, Bhaloo Desai, PhD, Theodore A. Bass, MD, Dominick J. Angiolillo, MD, PhD University of Florida, College of Medicine, Jacksonville, USA Pharmacodynamic Effects of Different Aspirin Dosing Regimens in Type 2 Diabetes Mellitus Patients with Coronary Artery Disease Circ Card Interv 2011 Ahead of print

2. Aspirinn doses in diabetics - AngiolilloSlide 2  The reduced life-span and increased turnover rates of platelets have been suggested to have a contributing role in the differential pharmacodynamic response profiles to antiplatelet therapy in type 2 diabetes mellitus (T2DM) patients.  Aspirin has only a 20-minute half-life and therefore the accelerated thrombopoiesis which characterizes T2DM patients does not allow newly generated platelets entering the circulation to be sufficiently exposed to aspirin. Background Winocour, et al. Eur J Clin Invest. 1994;24 (Suppl 1):34-7 Di Minno G, et al Blood. 1983;61:1081-5 Patrono C. N Engl J Med. 1994;330:1287-94

3. Aspirinn doses in diabetics - AngiolilloSlide 3  It may be hypothesized that an increase in the frequency, rather than the dose, of aspirin administration may be a more effective strategy to inhibit platelet reactivity in diabetic patients as this may enable COX-1 blockade of newly generated platelets  Therefore, the aim of the present pilot investigation was to evaluate how increasing the frequency of aspirin administration, remaining within the daily recommended therapeutic doses, affects antiplatelet responsiveness in T2DM patients with coronary artery disease (CAD) Rationale and objective

4. Aspirinn doses in diabetics - AngiolilloSlide 4 Schematic of circadian release of platelets into bloodstream from bone marrow and impact of a single daily dose of aspirin on newly generated platelets in type 2 diabetes mellitus Platelets from patients with type 2 diabetes mellitus (T2DM) have a reduced life-span and increased turnover rates, leading to enhanced bone marrow megakaryocyte generation and release of new and hyper-reactive platelets into the bloodstream. Aspirin has only a 20-minute half-life and therefore the accelerated thrombopoiesis which characterizes T2DM patients does not allow newly generated platelets entering the circulation to be sufficiently exposed to aspirin if given once daily. This may lead to a considerable proportion of circulating platelets with uninhibited cyclooxigenase-1 (COX-1) activity that continue to generate high levels of serum thromboxane and therefore promote activation of circulating platelets (acetylated and non-acetylated) via thromboxane receptors (TP) on the platelet surface. A twice daily administration of aspirin may allow newly generated platelets released into the bloodstream to be COX-1 inhibited, thus achieving more optimal blockade of platelet activation processes in T2DM.

5. Aspirinn doses in diabetics - AngiolilloSlide 5 Visit 1 81 mg od Visit 2 81 mg bid Visit 3 162 mg od Visit 4 162 mg bid Visit 5 325 mg od run-in phase Screening phase Patients modified their aspirin regimen on a weekly basis according to the following scheme Pharmacodynamic assessments included:  light transmittance aggregometry (LTA) following arachidonic acid, collagen and adenosine diphosphate (ADP) stimuli  VerifyNow-Aspirin assay;  serum thromboxane B 2 (TXB 2 ) levels

6. Aspirinn doses in diabetics - AngiolilloSlide 6 T2DM patients screened N = 82 Eligible to participate to the study N = 48 Agreed to participate to the study N = 36 Completed all five treatment regimens and entered the final analysis N = 20 Excluded * N = 34 active bleeding or bleeding diathesis concomitant use of other antithrombotic drugs recent treatment (30 days) with a glycoprotein IIb/IIIa antagonist platelet count 100*10 6 /l acute coronary or cerebrovascular event within 3 months; serum creatinine > 2 mg/dL; baseline ALT > 2.5 times the upper limit of normal HbA1C > 10% Patient population * Key exclusion criteria

7. Aspirinn doses in diabetics - AngiolilloSlide 7 (n = 20) Age (years±SD)59±7 Male, n (%)10 (50) BMI (Kg/m 2 ±SD)33±9 Risk factors, n (%) Smoking6 (30) Hypertension19 (95) Dyslipidemia17 (85) Insulin-treated8 (40) Medical history, n (%) Prior MI1 (5) Prior stroke0 (0) Prior CABG2 (10) Multivessel CAD6 (30) Baseline clinical characteristics

8. Aspirinn doses in diabetics - AngiolilloSlide 8 (n = 20) Beta-blockers11 (55) ACE inhibitors18 (90) Ca 2+ antagonists11 (55) Lipid-lowering agents CYP 3A4 pathway metabolized8 (40) Non-CYP 3A4 pathway metabolized0 (0) Proton pump inhibitors5 (25) Medical therapy Laboratory data (n = 20) Platelet count (1,000/mm 3 ±SD)241±66 Hematocrit (%±SD)42±4 HbA1C (%±SD)7.1±1.3 Creatinine (g/dl±SD)1.0±0.3

9. Aspirinn doses in diabetics - AngiolilloSlide 9 Assay 81 mg od 162 md od 325 mg od 81 mg od vs. 162 mg od P value 81 mg od vs. 325 mg od P value 162 mg od vs. 325 mg od P value 81 mg od vs. 162 mg od vs 325 mg od P for trend Arachidonic acid (1 mmol/L), % 2±0.92±0.7 1.000 Collagen (2 µg/mL), % 44±2339±1435±15 0.2850.0830.3740.157 ADP (5 µmol/L), % 49±1354±1054±11 0.1110.8510.6120.192 ADP (20 µmol/L), % 66±771±1168±7 0.0330.4590.1450.109 VN-ASA, ARU 455±51432±62431±58 0.0870.1260.9220.121 Serum TXB 2, pg/ml 107±14341±7922±210.0080.0300.3280.008 Dose comparison in once daily administration Data are expressed as means±SD. ADP indicates adenosine diphosphate; VN-ASA indicates VerifyNow-Aspirin assay; TXB 2 indicates thromboxane B 2

10. Aspirinn doses in diabetics - AngiolilloSlide 10 Assay81 mg bid162 mg bidP value Arachidonic acid (1 mmol/L), % 2±0.52±1.4 0.106 Collagen (2 µg/mL), % 32±1433±14 0.895 ADP (5 µmol/L), % 54±1350±15 0.360 ADP (20 µmol/L), % 69±1167±14 0.476 VN-ASA, ARU 420±41423±52 0.777 Serum TXB 2, pg/ml34±5019±210.165 Dose comparison in twice daily administration Data are expressed as means±SD. ADP indicates adenosine diphosphate; VN-ASA indicates VerifyNow-Aspirin assay; TXB 2 indicates thromboxane B 2

11. Aspirinn doses in diabetics - AngiolilloSlide 11 Assay 162 mg od 81 mg bid P 325 mg od 162 mg bid P Arachidonic acid (1 mmol/L), % 2±0.72±0.5 0.772 2±0.72±1.4 0.094 Collagen (2 µg/mL), % 39±1432±14 0.060 35±1533±14 0.490 ADP (5 µmol/L), % 54±1054±13 0.857 54±1150±15 0.273 ADP (20 µmol/L), % 71±1169±11 0.343 68±767±14 0.751 VN-ASA, ARU 432±62420±41 0.345 431±58423±52 0.551 Serum TXB 2, pg/ml 41±7934±500.71622±2119±210.579 Comparison of single versus staggered daily administration of the same aspirin dose Data are expressed as means±SD. ADP indicates adenosine diphosphate; VN-ASA indicates VerifyNow-Aspirin assay; TXB 2 indicates thromboxane B 2

12. Aspirinn doses in diabetics - AngiolilloSlide 12 Comparison of different aspirin regimens by collagen induced light transmission aggregometry (A) and VerifyNow-ASA (B) A B

13. Aspirinn doses in diabetics - AngiolilloSlide 13 p = 0.003 Changes in thromboxane B 2 levels across the study phases Thromboxane B 2 levels are expressed as pg/ml. Error bars indicate standard deviations of the mean. Error bars indicate standard deviations; od indicates once daily administration; bid indicates twice daily administration.

14. Aspirinn doses in diabetics - AngiolilloSlide 14 Conclusions Aspirin dosing regimens are associated with different pharmacodynamic effects in platelets from T2DM patients and stable CAD. In particular, a twice daily low-dose aspirin administration is associated with greater platelet inhibition than a once daily administration as assessed by aspirin sensitive assays, and a dose- dependent effect is observed on serum TXB2 levels. The clinical implications of a modified aspirin regimen tailored to T2DM patients warrants further investigation.

15. Aspirinn doses in diabetics - AngiolilloSlide 15 Circ Card Interv 2011 Ahead of print