Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y₁₂antagonist bothin-vitroandex-vivoin human volunteers

Abstract

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E2 (PGE2) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3 µM) used alone or in combination with the P2Y12 antagonist cangrelor (1 µM), both without and with aspirin (100 µM). Platelet aggregation and P-selectin expression were measured in whole blood (n = 10) following stimulation with the thromboxane A2 (TXA2) mimetic U46619 (0.3 or 1 µM) in combination with either the EP3 agonist sulprostone (0.1 µM), or PGE2 (1 µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n = 42) were randomly assigned to receive no background treatment, clopidogrel (300 mg loading dose plus 75 mg daily) or clopidogrel and aspirin (75 mg daily) for 10 days alongside DG-041 (200 mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y12 antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y12 blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.

The EP3 Agonist Sulprostone Enhances Platelet Adhesion But Not Thrombus Formation Under Flow Conditions

“…Our in vitro findings that EP3 receptor stimulation enhanced washed platelet aggregation and adhesion are in line with previous findings, showing an EP3-induced increase of agonist-induced aggregation in human whole blood and PRP [18,21,25] . In addition, we found that adhesion to fibrinogen and collagen under low flow conditions is enhanced based on EP3 receptor activation; however, whole blood thrombus formation on collagen under high flow was not significantly affected by the EP3 receptor agonist sulprostone.…”

“…In addition, the ex vivo experiments showed that DG-041 enhanced the effect of P2Y 12 antagonist clopidogrel [21] . Most remarkably, in contrast to P2Y 12 antagonists, DG-041 does not increase bleeding time in man, rat and mice [21][22][23] . Atherothrombosis is a leading cause of death, and anti-platelet drugs improve the survival rate.…”

Blocking the receptor EP3 to PGE2 as a way to safely prevent atherothrombosis

“…Similarly, a phase I trial did not show any impact on bleeding time in volunteers [22,35] who received high doses of the EP3 blocker DG-041. The maximal tested dose reached 8 times the dose needed to fully inhibit the in vitro potentiating effect of PGE2 on human platelets.…”

Novel Strategies for Assessing Platelet Reactivity

“…The circumstance was a clinical trial in which a new antiplatelet agent, an EP3 receptor antagonist, was administered to volunteers with and without clopidogrel or clopidogrel and aspirin, after which the effectiveness of the drugs as inhibitors of platelet aggregation was assessed [46]. More recently, AGGFix has also been used successfully for assessment of platelet aggregation using the platelet counting approach in blood placed in 96-well plates and also, in parallel, to assess the degree of platelet-leukocyte conjugate formation that occurred, assessed by flow cytometry [47].…”