Abstract
Pharmacodynamic evaluation of novel antiplatelet agents in clinical trials has traditionally proven problematic due to the inaccessibility of assays to measure the effects on platelet thrombosis in human blood. The present study utilized real-time perfusion chamber technology (RTTP) to compare the antithrombotic activities of PRT128, an orally available, direct-acting, reversible P2Y12 antagonist, to those achieved by chronic dosing of humans with clopidogrel (C, 75 mg/day, ± aspirin (A)). PRT128 was added in vitro to blood collected from healthy volunteers (n=20) at baseline (no treatment) and following A treatment, allowing comparison to C (±A) treatments. Blood samples were monitored using the RTTP assay, where thrombus formation was initiated by a combination of a collagen-coated surface and physiologic shear. 1.25 mM PRT128 provided equivalent inhibition of the thrombotic process achieved by chronic dosing of C (75 mg/d, 14 d)(±A, 325 mg/d, 7 d): higher concentrations (2.5, 5 mM) of PRT128 provided superior inhibition. Although neither P2Y12 antagonist alone affected the initial rate of thrombus formation, both destabilized growing thrombi, to a greater extent in the presence of aspirin, with larger effects achieved by the direct P2Y12 antagonist. Light transmittance platelet aggregation (PA) assays using platelet rich plasma (initiated by ADP (10 mM) or collagen (4 mg/ml)) indicated that the concentration of PRT128 which provided similar inhibition to C (±A) was 0.5–3 mM, while higher concentrations achieved greater inhibition. The RTTP assay was also used to determine the levels of inhibition of thrombosis achieved by C plus A treatment in 5 diabetic patients undergoing stent-placement. Interestingly, 2 of the 5 patients displayed a profile typical of a non-responder (i.e., no observed thrombus destabilization). However, when 2.5 or 5 mM PRT128 was added in vitro to blood from these patients, a clear antithrombotic effect was observed, indicating that the lack of benefit of C plus A therapy in these patients was due to C nonresponsiveness which could be circumvented by the direct-acting antagonist PRT128. Finally, a unique feature of a direct-acting P2Y12 antagonist was revealed using the RTTP assay. Thrombi formed by the perfusion of untreated whole blood for 3 minutes through the perfusion chamber were subsequently exposed to a perfusion of blood treated with PRT128. The second perfusion destabilized thrombi, illustrating the role of ADP acting on P2Y12 in maintaining thrombus stability. In summary, through use of novel perfusion chamber technology, we demonstrate that (1) PRT128, a direct-acting P2Y12 antagonist, can provide superior levels of platelet inhibition than achieved by C (±A), (2) PRT128 can overcome the limitations of C in diabetic patients undergoing PCI who appeared to be C non-responders, (3) direct-acting P2Y12 antagonism can induce destabilization of existing thrombi, and (4) the RTTP assay provides a unique tool for dose selection and evaluation of clinical indications for the further development of PRT128 and other novel antiplatelet agents.
Antiplatelet Activity of Coumarins: In Vitro Assays on COX-1
