SOLUBLE GUANYLATE CYCLASE ACTIVATORS AS COMBINATION ANTI-PLATELET THERAPY WITH P2Y12 INHIBITORS AND PDE INHIBITORS: IN VIVO AND EX VIVO STUDIES

Abstract

Methods Mice were pre-treated with vehicle, prasugrel (0.3 mg/kg), cinaciguat (0.3 mg/kg) + dipyridamole (2.0 mg/kg), or prasugrel + cinaciguat + dipyridamole (triple) and anesthetized. To measure in vivo thrombus formation, the carotid artery was then exposed and thrombosis induced by placement of a piece of filter paper saturated with 10% ferric chloride in contact with the adventitial surface of vessel for 3 minutes. Carotid artery blood flow was monitored by a Doppler probe with the time to form an occlusive thrombus being taken as the time required for blood to stop flowing completely for >1 minutes. Time to occlusion from different treatment groups was compared. To measure ex vivo platelet function, blood was taken from the vena cava of treated mice and aggregation of platelets in whole blood in response to arachidonic acid (AA) 1 mM, PAR-4 amide 30 μM, and collagen 10 μg/ml was then determined using flow cytometry.

Results In mice treated with vehicle, prasugrel and cinaciguat + dipyridamole complete vessel occlusion occurred within 8 minutes. Conversely, triple combination of prasugrel + cinaciguat + dipyridamole blocked thrombus formation (time to occlusion > 24 minutes). In ex vivo platelet function tests, we observed reduced platelet aggregation in mice treated with the triple combination compared to other treatments. Results as mean ± SEM. AA; vehicle 83 ± 9%, prasugrel 67 ± 7%, cinaciguat + dipyridamole 62 ± 9%, triple 27 ± 27%: PAR-4; vehicle 52 ± 11%, prasugrel 27 ± 14%, cinaciguat + dipyridamole 26 ± 16%, triple 11 ± 6%: collagen; vehicle 40 ± 16%, prasugrel 42 ± 10%, cinaciguat + dipyridamole 44 ± 19%, triple 18 ± 11%.

Conclusion Our animal studies suggest that combinations of low doses of cinaciguat, prasugrel and dipyridamole could provide a focused and powerful anti-platelet effect. This could be an effective therapeutic antithrombotic approach with potentially lesser effects at other sGC/PDE sites, particularly the vascular smooth muscle, reducing the incidence of headache and hypotension.