Summary. Background: Release of serotonin and activation of serotonin 5HT 2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier-generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT 2A receptor subtype. Objective: To assess whether targeted inhibition of the serotonin 5HT 2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina. Methods: In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT 2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injury + stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin. Results: APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow-time area (index of coronary patency; normalized to baseline coronary flow) averaged 58-59% (P < 0.01) following administration of APD791 vs. 21-28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotoninmediated platelet activation. Conclusion: 5HT 2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model. Keywords: angina, platelets, serotonin, thrombosis.The pathogenesis of platelet-mediated coronary artery thrombosis is complex, with multiple in vivo agonists playing important causal and contributory roles [1,2]. Current clinical therapies target either cyclo-oxygenase 1, the ADP P2Y 12 receptor, or the glycoprotein (GP) IIb/IIIa receptor [3]. However, these therapies, even in combination, are not fully effective in preventing major thrombotic events [3], and are associated with an increase in bleeding [4].Release of serotonin from platelet dense granules and activation of serotonin receptors on platelet surfaces has long been recognized to serve as a potent augmentative stimulus for platelet aggregation [5][6][7][8]. Clinical application of serotonin receptor antagonists for the prevention of thrombotic coronary events has, however, been confounded by their lack of selectivity for the 5HT 2A receptor (i.e. the serotonin receptor subtype expressed on platelets) [9,10]. Accordingly, our aim was to assess the efficacy of APD791 (Arena Pharmaceuticals, Inc., San Diego, CA, USA), a newly developed, potent and highly selective inverse agonist of the 5HT 2A receptor [9], in a well-established preclinical canine model of recurrent thrombosis mimicking unstable angina [11][12][13][14][...

PurposeThis phase 2 study was designed to assess the efficacy, safety and tolerability of immediate-release orally administered ralinepag, a selective, non-prostanoid prostacyclin receptor agonist with a 24-h terminal half-life, compared to placebo in adult patients with symptomatic pulmonary arterial hypertension (PAH).Methods61 PAH patients who were receiving standard care, including mono or dual PAH-targeted background therapy were randomised 2:1 to ralinepag (n=40) or placebo (n=21). The starting dose of ralinepag was 10 μg twice daily. Dosage was then up-titrated as tolerated over the course of the 9-week dose-titration period, to a maximum total daily dose of 600 μg (300 μg twice daily). The primary efficacy end-point was the absolute change in pulmonary vascular resistance (PVR) from baseline to week 22. Additional end-points included percentage change in PVR from baseline, other haemodynamic parameters, 6-min walk distance (6MWD) and safety and tolerability.ResultsRalinepag significantly decreased PVR by 163.9 dyn·s·cm−5 compared to an increase of 0.7 dyn·s·cm−5 with placebo (p=0.02); the least-squares mean change from baseline PVR was −29.8% compared with placebo (p=0.03). 6MWD increased from baseline by 36.2 m with ralinepag and 29.4 m with placebo (p=0.90). Serious adverse events occurred in 10% of ralinepag patients and 29% of placebo patients. Study discontinuations occurred in 13% of ralinepag patients and 10% of placebo patients.SummaryRalinepag reduced PVR compared with placebo in PAH patients on mono (41%) or dual combination (59%) background therapy.

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We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl-)benzamide] a novel 5-hydroxytryptamine 2A (5-HT 2A ) receptor antagonist. APD791 displayed high-affinity binding to membranes (K i ϭ 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC 50 ϭ 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT 2A receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT 2A receptor versus 5-HT 2C and 5-HT 2B receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC 50 ϭ 8.7 and 23.1 nM, respectively).Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC 50 ϭ 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT 2A receptor antagonist with potent activity on platelets and vascular smooth muscle.Serotonin (5-hydroxytryptamine, 5-HT) is a naturally occurring indoleamine found primarily in brain, enterochromaffin tissue, and platelets. 5-HT exerts a multitude of biological effects mediated through interaction with specific cell surface G-protein-coupled receptors (GPCRs). To date, at least 14 different human 5-HT GPCRs are known (Kaumann and Levy, 2006). Among them, 5-HT 2A receptors on vascular smooth muscle cells and platelets play an important role in the regulation of cardiovascular function.The uptake process and storage capacity for 5-HT by platelets are such that minimal amounts of the amine exist in normal plasma. However, upon platelet activation at sites of vessel injury, 5-HT is released from the dense granules in platelets (Ashton et al., 1986). Although 5-HT by itself is only a weak activator of platelet aggregation, it effectively amplifies aggregation induced by other agonists including collagen, ADP, epinephrine, and thrombin (De Clerck and Herman, 1983;De Clerck and Janssen, 1990). Thus, 5-HT This work was supported by Arena Pharmaceuticals, Inc. Article, publication date, and citation information can be found at

The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in the identification of 5c (ralinepag) that has good selectivity in both binding and functional assays with respect to most members of the prostanoid receptor family and a more modest 30- to 50-fold selectivity over the EP3 receptor. In our hands, its potency and efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NS-304) with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile across species. Enterohepatic recirculation most probably contributes to a concentration-time profile after oral administration in the cynomolgus monkey that showed a very low peak-to-trough ratio. Following the identification of an acceptable solid form, 5c was selected for further development for the treatment of PAH.

Serotonin, which is stored in platelets and is released during thrombosis, activates platelets via the 5-HT(2A) receptor. 5-HT(2A) receptor inverse agonists thus represent a potential new class of antithrombotic agents. Our medicinal program began with known compounds that displayed binding affinity for the recombinant 5-HT(2A) receptor, but which had poor activity when tested in human plasma platelet inhibition assays. We herein describe a series of phenyl pyrazole inverse agonists optimized for selectivity, aqueous solubility, antiplatelet activity, low hERG activity, and good pharmacokinetic properties, resulting in the discovery of 10k (APD791). 10k inhibited serotonin-amplified human platelet aggregation with an IC(50) = 8.7 nM and had negligible binding affinity for the closely related 5-HT(2B) and 5-HT(2C) receptors. 10k was orally bioavailable in rats, dogs, and monkeys and had an acceptable safety profile. As a result, 10k was selected further evaluation and advanced into clinical development as a potential treatment for arterial thrombosis.

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