Pharmacodynamic effects of EV-077: results of an in vitro pilot investigation in healthy volunteers

Abstract

Antiplatelet agents are important for the treatment of patients with atherosclerotic disease manifestations. Antiplatelet agents act by inhibiting platelet activation and aggregation processes, which are mediated by several activating factors including thromboxane A2 (TxA2), adenosine diphosphate (ADP), and thrombin. In particular, TxA2 induced platelet activation is a pivotal pharmacological target as supported by the undisputable benefit of aspirin in secondary prevention of ischemic events [1, 2]. TxA2 is a metabolite of arachidonic acid (AA) deriving from the cyclooxygenase (COX)-1 enzyme, which transforms AA into prostaglandin H2 (PGH2), which is ultimately metabolized into TxA2 by thromboxane synthase (TS) [3]. Low dose of aspirin mainly inhibits COX-1 and subsequently TxA2 production. However, up to one-third of aspirin treated patients aspirin show persistent TxA2 production, which in turn exposes these subjects to an increased risk of atherothrombotic events [4]. These findings underscore the need to identify new TxA2 modulators. TxA2 receptor (TP receptor) antagonists, TS inhibitors, and dual TS inhibitors and TP receptor antagonists have been recently developed [3, 5]. Such antagonists and inhibitors have also beneficial effects on TP receptor mediated vascular inflammation and oxidative stress, thus on the progression and development of atherosclerotic lesions [6]. EV-077 is a novel dual TS inhibitor and TP receptor antagonist in early phases of clinical investigation. Limited data is currently available on the pharmacodynamic effects of this agent on human platelets [7]. In this pilot investigation we describe the pharmacodynamics effects on human platelets of escalating in vitro concentrations of EV-077. Ten healthy volunteers aged 35.1 ± 3.6 years old were recruited for this study. None of the subjects were taking any type of medication, including over the counter and herbal supplements. Venous blood was collected by antecubital puncture, in hirudin (25 lg/mL) and sodium citrate (0.150 M) containing tubes. Plasma citrate concentration was adjusted according to the haematocrit and the resulting free plasma citrate were adjusted to a final plasma concentration of 20 mmol/L as previously described [8]. Whole-blood hirudin-tubes were incubated with escalating concentrations of EV-077 (vehicle, 1, 10, 30, 100, and 300 nM) for 10 min at 37 8C. EV-077 was provided by Evolva SA (Reinach, Switzerland). Platelet-rich plasma (PRP) was obtained by centrifugation of whole-blood citrated tubes at 1179 g for 10 min. After measuring the platelet count, PRP was adjusted to 250,000/lL ± 10 %. This PRP was incubated with vehicle or 100 nM of EV-077 for 10 min at 37 C. Platelet aggregation was determined by impedance aggregometry using Multiplate Analyzer (MEA) (Dynabyte Medical, Munich, Germany) for whole-blood samples stimulated with AA (1 mM) (Dynabite Medical, Munich, Germany) and the TxA2 analog U-46619 (7 lM) (Calbiochem, Merck KGaA, Darmstadt, Germany), as well as with an agonist indirectly related to the TxA2 pathway, collagen (3 lg/mL) (Dynabite Medical, Munich, Germany). Lighttransmission platelet aggregometry (LTA) was performed using PRP by the turbidimetric method in a two-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp., A. Tello-Montoliu F. Rollini B. Desai G. Pasqualino R. Patel D. J. Angiolillo (&) University of Florida College of Medicine-Jacksonville, 655 West 8th Street, Jacksonville, FL 32209, USA e-mail: dominick.angiolillo@jax.ufl.edu