1. Date of download: 6/21/2016 Copyright © The American College of Cardiology. All rights reserved. From: Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects J Am Coll Cardiol. 2005;45(8):1295-1301. doi:10.1016/j.jacc.2005.01.045 Flow chart of study protocols. In the study with multiple daily doses, aspirin (100 mg daily and once at 8 am) was administered for 6 consecutive days and then naproxen (500 mg twice daily, once at 10 am and 10 pm) was co-administered 2 h after aspirin for further 6 days to 4 healthy subjects. After a washout period of at least 14 days, the volunteers reversed the treatment, i.e., low-dose aspirin (100 mg daily at 10 am) was administered 2 h after naproxen (500 mg twice daily, once at 8 am and once at 8 pm) for further 6 days. Blood samples were collected before and up to 26 h after dosing with the first study drug on the 6th, 12th, 27th, and 32nd study day to assess the inhibition of serum thromboxane (TX)B 2 (a capacity index of platelet cyclooxygenase [COX]-1 activity) and lipopolysaccharide-induced prostaglandin E 2 production (a capacity index of monocyte COX-2 activity). Three consecutive urinary samples (time of collection: 0 to 6 h, 6 to 12 h, and 12 to 24 h) were collected before treatment and on days 6, 12, 27, and 32 to evaluate the urinary excretion of 11-dehydro-TXB 2 (a major enzymatic metabolite of TXB 2 that is an index of TXA 2 biosynthesis in vivo). In the study with single dose, aspirin (100 mg) and naproxen (500 mg) were co-administered to 5 healthy subjects, and peripheral blood samples were collected before and up to 14 days after dosing to assess the time-dependent inhibition and recovery of serum TXB 2 production and platelet aggregation ex vivo. Figure Legend:

2. Date of download: 6/21/2016 Copyright © The American College of Cardiology. All rights reserved. From: Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects J Am Coll Cardiol. 2005;45(8):1295-1301. doi:10.1016/j.jacc.2005.01.045 Concentration-response curves for the inhibition of plate let cyclooxygenase (COX)-1 activity by aspirin (A) and naproxen (B). One- milliliter aliquots of washed platelets (1.5 × 10 8 cells) were preincubated with increasing concentrations of aspirin (0.01 to 100 μmol/l) or naproxen (0.01 to 100 μmol/l) for 25 min, and then 0.5 or 10 μmol/l of arachidonic acid (AA) was added for an additional 30 min at 37°C. In panel C, the antagonism of aspirin inhibition of platelet COX-1 by naproxen is shown. Increasing concentrations of naproxen (0.01 to 10 μmol/l) were incubated with washed platelets (1.5 × 10 8 cells/ml) for 5 min before the addition of aspirin (10 or 100 μmol/l) and the incubation continued for additional 20 min at 37°C. After washing twice, platelets were resuspended in 500 μl of Hanks’ balanced salt solution supplemented with 25 mmol/l HEPES, challenged with 10 μmol/l of AA for 30 min at 37°C, and thromboxane (TX)B 2 levels were determined by radioimmunoassay. The data represent the average of inhibition of platelet TXB 2 production from five different donors. IC 50 = concentrations required to inhibit 50% of enzyme activity. Figure Legend:

3. Date of download: 6/21/2016 Copyright © The American College of Cardiology. All rights reserved. From: Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects J Am Coll Cardiol. 2005;45(8):1295-1301. doi:10.1016/j.jacc.2005.01.045 Mean inhibition of platelet cyclooxygenase-1 activity ex vivo, as assessed by the measurement of serum thromboxane (TX)B 2 levels (A), arachidonic acid-induced platelet aggregation ex vivo (B), and TX biosynthesis in vivo, as assessed by the measurement of urinary 11-dehydro-TXB 2 levels (C), in subjects taking low-dose aspirin alone (100 mg daily) for six days (hatched bars) and then readministered with naproxen (500 mg twice daily, with the first dose administered 2 h after aspirin) for further six days (open bars). The solid bars show the effects of the same medications administered in reversed order for further six days after a washout period of 14 days. Values are reported as mean ± SEM, n = 4. All times are hours after the administration of the first study drug. Open bars = aspirin before naproxen (twice daily); solid bars = naproxen (twice daily) before aspirin; hatched bars = aspirin. Figure Legend:

4. Date of download: 6/21/2016 Copyright © The American College of Cardiology. All rights reserved. From: Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects J Am Coll Cardiol. 2005;45(8):1295-1301. doi:10.1016/j.jacc.2005.01.045 Mean inhibition of monocyte cyclooxygenase-2 activity ex vivo, as assessed by measurement of whole-blood lipopolysaccharide (LPS)-induced prostaglandin (PG)E 2 levels in subjects taking low-dose aspirin alone (100 mg daily) for 6 days (hatched bars) and then readministered with naproxen (500 mg twice daily, with the first dose administered 2 h after aspirin) for a further 6 days (open bars). The solid bars show the effect of the same medications, administered in reversed order for further six days after a washout period of 14 days, on monocyte cyclooxygenase-2 activity. The values are reported as mean ± SEM of inhibition (%) of LPS-induced PGE 2 levels caused by the different treatments, n = 4. *p < 0.05, **p < 0.01 versus pre-drug values. All times are hours after the administration of the first study drug. Open bars = aspirin before naproxen (twice daily); solid bars = naproxen (twice daily) before aspirin; hatched bars = aspirin. Figure Legend:

5. Date of download: 6/21/2016 Copyright © The American College of Cardiology. All rights reserved. From: Pharmacodynamic interaction of naproxen with low-dose aspirin in healthy subjects J Am Coll Cardiol. 2005;45(8):1295-1301. doi:10.1016/j.jacc.2005.01.045 Mean inhibition of platelet cyclooxygenase-1 activity ex vivo (solid circles) (as assessed by measurement of serum TXB 2 ) and arachidonic acid-induced platelet aggregation ex vivo (open circles) in subjects taking a single dose of aspirin (100 mg) and naproxen (500 mg). Values are reported as mean ± SEM, n = 5. Serum TXB 2 and platelet aggregation were significantly inhibited versus pre-drug values at 3 h (p < 0.01 and 0.05, respectively), 12 h (p < 0.01), 24 h (p < 0.01), and 48 h (p < 0.01 and 0.05, respectively) after dosing. No significant differences were found at 1, 72, 144, 192, and 336 h after dosing versus pre-drug values. Open circles = platelet aggregation; solid circles = serum TXB 2. Figure Legend: