No abstract

Recent post hoc analyses of several clinical trials with P2Y12 antagonists showed the need for new molecules being fully efficacious as antiplatelet agents and having a reduced propensity to cause major bleeding. We have previously reported the discovery of the 2-phenylpyrimidine-4-carboxamide analogs as P2Y12 antagonists with nanomolar potency in the disease-relevant platelet aggregation assay in human plasma. Herein we present the optimization steps that led to the discovery of clinical candidate ACT-246475 (30d). The key step was the replacement of the carboxylic acid functionality by a phosphonic acid group which delivered the most potent molecules of the program. In addition, low in vivo clearance in rat and dog was achieved for the first time. Since the bioavailability of 30d was low in rat and dog, we developed the bis((isopropoxycarbonyl)oxy)methyl ester prodrug (ACT-281959, 45). Compound 30d showed efficacy in the rat ferric chloride thrombosis model when administered intravenously as parent or orally as its prodrug 45. Moreover, 30d displays a wider therapeutic window as compared to clopidogrel in the rat surgical blood loss model.

The P2Y12 receptor is a validated target for prevention of major adverse cardiovascular events in patients with acute coronary syndrome. The aim of this study was to compare two direct‐acting, reversible P2Y12 antagonists, ACT‐246475 and ticagrelor, in a rat thrombosis model by simultaneous quantification of their antithrombotic efficacy and surgery‐induced blood loss. Blood flow velocity was assessed in the carotid artery after FeCl3‐induced thrombus formation using a Doppler flow probe. At the same time, blood loss after surgical wounding of the spleen was quantified. Continuous infusions of ACT‐246475 and ticagrelor prevented the injury‐induced reduction of blood flow in a dose‐dependent manner. High doses of both antagonists normalized blood flow and completely abolished thrombus formation as confirmed by histology. Intermediate doses restored baseline blood flow to ≥65%. However, ACT‐246475 caused significantly less increase of blood loss than ticagrelor; the difference in blood loss was 2.6‐fold (P < 0.01) at high doses and 2.7‐fold (P < 0.05) at intermediate doses. Potential reasons for this unexpected difference were explored by measuring the effects of ACT‐246475 and ticagrelor on vascular tone. At concentrations needed to achieve maximal antithrombotic efficacy, ticagrelor compared with ACT‐246475 significantly increased carotid blood flow velocity in vivo (P = 0.003), induced vasorelaxation of precontracted rat femoral arteries, and inhibited contraction of femoral artery induced by electrical field stimulation or by phenylephrine. Overall, ACT‐246475 showed a significantly wider therapeutic window than ticagrelor. The absence of vasodilatory effects due to high selectivity of ACT‐246475 for P2Y12 provides potential arguments for the observed safety advantage of ACT‐246475 over ticagrelor.

Selatogrel is a reversible antagonist of the P2Y12 receptor. In rat thrombosis/haemostasis models, selatogrel was associated with lower blood loss than clopidogrel or ticagrelor at equivalent anti-thrombotic effect. Material and methods: We sought to elucidate the mechanism underlying the observed differences in blood loss, using real-time intravital microscopy in mouse. Results: Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to hemostatic seals, were present. The phenotype of these hemostatic seals was dependent on the type of P2Y12 receptor antagonist. In the presence of clopidogrel and ticagrelor, detachment of platelets from the hemostatic seals was increased, indicative of reduced stability. In contrast, in the presence of selatogrel, platelet detachment was not increased. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel reduced laserinduced calcium mobilization in the endothelium, restricted neutrophil adhesion and subsequent fibrin formation and thus reduced fibrin-mediated stabilization of the hemostatic seals. The effects of ticagrelor were also observed in P2Y12 receptor deficient mice, indicating that the effects are off-target and independent of the P2Y12 receptor. In contrast, selatogrel did not interfere with these elements of haemostasis in wild-type or in P2Y12 receptor deficient mice. Conclusion:In the presence of selatogrel the stability of hemostatic seals was unperturbed, translating to an improved blood loss profile. Our data suggest that the mechanism underlying the differences in blood loss profiles of P2Y12 receptor antagonists is by off-target interference with endothelial activation, neutrophil function and thus, fibrin-mediated stabilization of haemostatic seals.

The cyclic undecapeptide cyclosporin A (CsA) has a remarkable spectrum of diverse biological activities, including anti-inflammatory, antifungal, antiparasitic as well as immunosuppressive activities. However, the low water solubility of this drug is a serious problem causing undesirable pharmacological properties such as erratic oral absorption. In order to overcome this problem, the design and synthesis of water-soluble prodrugs of CsA are described. Using the OH-MeBmt-1-group as attachment site, we investigate dipeptide systems exhibiting differential tendencies for intramolecular cyclization [diketopiperazine (DKP) formation] for tailoring the chemoreversible release of the parent CsA. In modulating the chemical and structural features of the dipeptide esters (N-alkylation, side chains, C-terminal Pro), we find conversion rates at physiological conditions ranging from minutes to several days. Together with their thermodynamic stability in the solid state and strongly enhanced solubility in water, these chemoreversible CsA prodrugs represent versatile candidates for therapeutical use.