1. Preserving Anti-Platelet Activity of Aspirin by Preventing Hydrolysis John C. Somberg*, Vasant V. Ranade*, Imran Qadeer*, Jackie Coleman**, Janos Molnar*** Rush University, Chicago, IL* Accumetrics, San Diego, CA** Academic Pharmaceuticals, Lake Bluff, IL***

2. Purpose of the Study Hydrolysis of aspirin markedly reduces the anti-platelet activity of aspirin (ASA) preventing the development of an effective IV formulation. This study evaluates a new IV formulation that prevents hydrolysis and preserves platelet inhibition.

3. Benefits of ASA ISIS-2: Second International Study of Infarct Survival Reduction in odds of vascular death in days 0–35, by time from pain onset Odds ratio and 95%CI* 0–1 2 3 4 Subtotal for 0–4 5–12 13–24 Subtotal for 5–24 Total for 0–24 The ISIS-2 collaborative group. Lancet 1988; ii: 349–60. * Size of squares indicates how much information available; lines and diamonds indicate 95% CIs 0.51.01.50.51.01.5 Time of randomization (hours from pain onset) SK better Placebo better Aspirin better Placebo better SK; Streptokinase

4. Benefits of ASA Kong et al, AJC 90:622, 2002

5. Feldman and Cryer, AJC 84: 404, 1999

6. Kotake et al. JCPT 31:237, 2006

7. Methods Hydrolysis was determined by HPLC using a Luna column (5µ, 100x46 mm), a constant flow pump (Thermo-Finnigan Spectra System P2000) and a Spectra 200 programmable UV wavelength detector. Platelet function was evaluated ex vivo using the “Verify Now” assay developed by Accumetrics thatemploys a method that is based upon the principle that agonist (arachidonic acid) -induced activated platelets bind to fibrinogen-coated polystyrene beads that agglutinate in whole-blood. Infrared-light transmittance through the chamber increases as the agglutinated platelets and beads fall out of the solution.

9. Rate of Hydrolysis Percent not hydrolyzed Fresh 1 Day 1 month 6 months 40°C 40°C 40 °C New formulation ASA 99.8 99 99 99 Aqueous ASA 97.970 56 11

10. The Effect of ASA Solutions on Platelet Inhibition Percent Inhibition Platelets Fresh 1 month 6 months Aqueous ASA 26 0 0 New ASA Formulation 37 37 39 Vehicle Solution 0 0 0

11. Conclusions  Aqueous solutions of ASA hydrolyzes and this correlates with a diminished ex vivo ASA effect on platelet function  A new formulation of ASA for IV administration shows no hydrolysis at 1 & 6 months of storage at 40°C  This lack of hydrolysis of the new formulation correlates with ex vivo measured platelet inhibition by ASA  Availability of an IV formulation of ASA may be useful in the treatment of acute coronary syndromes