Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation.

Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets. Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the Gαi/o-Gßγ complex in cells with recombinant expression of the P2Y12 receptor. In dose-response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.

The potency of selatogrel, a reversible antagonist of the P2Y12 receptor, is affected by calcium concentration.

Here, we report the in vitro characterization of the P2Y12 receptor antagonist selatogrel (ACT-246475). Binding studies with radiolabeled selatogrel demonstrated that selatogrel is a competitive antagonist of ADP binding to the P2Y12 receptor with a fast onset of action. Consequently, selatogrel was confirmed to be a potent inhibitor of P2Y12-mediated intra-platelet signaling and ADP-induced platelet activation. Characterization of selatogrel in platelet-rich plasma in vitro demonstrated that the mode of anti-coagulation affected the anti-platelet potency. Specifically, in platelet-rich plasma containing physiological calcium concentration (anticoagulated with a direct thrombin inhibitor), selatogrel achieved half-maximal inhibition of ADP-induced platelet aggregation at a 3-fold lower concentration than in conditions with low calcium concentration (anticoagulated with citrate). Furthermore, calcium-dependent reduction in selatogrel potency was observed in whole blood platelet aggregation using the VerifyNow™ system with a 3.7-fold potency loss in low calcium conditions. A comparable potency loss was also observed with the reversible P2Y12 receptor antagonists ticagrelor, cangrelor and elinogrel. Furthermore, receptor-binding experiments using radiolabeled selatogrel confirmed a 3-fold lowering of selatogrel binding affinity to the P2Y12 receptor in low calcium conditions. In conclusion, our data suggest that in low calcium conditions (i.e., citrate-anticoagulated blood), there is a risk of underestimating the potency of reversible P2Y12 receptor antagonists. To avoid overdosing, and a potential increase in bleeding risk, we propose that the ex vivo evaluation of reversible P2Y12 receptor antagonists should be performed with platelet assay systems containing physiological calcium concentration.

The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo.

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

Selatogrel, a reversible P2Y12 receptor antagonist, has reduced off-target interference with haemostatic factors in a mouse thrombosis model.

INTRODUCTION: 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 laser-induced 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.

Insights from In Vitro and Clinical Data to Guide Transition from the Novel P2Y12 Antagonist Selatogrel to Clopidogrel, Prasugrel, and Ticagrelor.

Reduced pharmacodynamic (PD) effects of irreversible oral P2Y12 receptor antagonists have been reported when administered during cangrelor infusion. Therefore, the PD interaction liability of the novel P2Y12 receptor antagonist selatogrel with irreversible (i.e., clopidogrel, prasugrel) and reversible (i.e., ticagrelor) oral P2Y12 receptor antagonists was investigated in vitro and in healthy subjects. In vitro, selatogrel reduced the effects of clopidogrel and prasugrel in a concentration-dependent manner, while additive effects were observed for the combination of selatogrel and ticagrelor. Accordingly, a single-center, randomized, double-blind, two-way crossover study was conducted consisting of six groups. In each group (N = 12), an open-label loading dose of 300 or 600 mg clopidogrel, 60 mg prasugrel, or 180 mg ticagrelor was administered 30 minutes (i.e., at t max of selatogrel) or 12 hours after a single subcutaneous dose of 16 mg selatogrel or placebo. Inhibition of platelet aggregation (IPA) was assessed at various time points up to 48 hours. Reduced IPA was determined when clopidogrel or prasugrel was administered 30 minutes after selatogrel (∼40 and 70% lower IPA, respectively, at 24 hours postdosing). However, when administering prasugrel 12 hours after selatogrel, IPA was not impacted (>90% IPA) and in the case of clopidogrel reduced effects were partially mitigated. Similar IPA was determined for ticagrelor when administered 30 minutes after selatogrel or placebo. In conclusion, reduced IPA was observed for clopidogrel and prasugrel when administered after selatogrel, which can be mitigated by applying an appropriate time interval. No PD interaction with ticagrelor was observed.

The reversible P2Y12 antagonist ACT-246475 causes significantly less blood loss than ticagrelor at equivalent antithrombotic efficacy in rat.

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.

4-((R)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-3-phosphonopropionyl)piperazine-1-carboxylic Acid Butyl Ester (ACT-246475) and Its Prodrug (ACT-281959), a Novel P2Y12 Receptor Antagonist with a Wider Therapeutic Window in the Rat Than Clopidogrel.

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.

Optimization of 2-phenyl-pyrimidine-4-carboxamides towards potent, orally bioavailable and selective P2Y(12) antagonists for inhibition of platelet aggregation.

2-Phenyl-pyrimidine-4-carboxamide analogs were identified as P2Y12 antagonists. Optimization of the carbon-linked or nitrogen-linked substituent at the 6-position of the pyrimidine ring provided compounds with excellent ex vivo potency in the platelet aggregation assay in human plasma. Compound 23u met the objectives for activity, selectivity and ADMET properties.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

Identification of 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (setipiprant/ACT-129968), a potent, selective, and orally bioavailable chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonist.

Herein we describe the discovery of the novel CRTh2 antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 28 (setipiprant/ACT-129968), a clinical development candidate for the treatment of asthma and seasonal allergic rhinitis. A lead optimization program was started based on the discovery of the recently disclosed CRTh2 antagonist 2-(2-benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 5. An already favorable and druglike profile could be assessed for lead compound 5. Therefore, the lead optimization program mainly focused on the improvement in potency and oral bioavailability. Data of newly synthesized analogs were collected from in vitro pharmacological, physicochemical, in vitro ADME, and in vivo pharmacokinetic studies in the rat and the dog. The data were then analyzed using a traffic light selection tool as a visualization device in order to evaluate and prioritize candidates displaying a balanced overall profile. This data-driven process and the excellent results of the PK study in the rat (F = 44%) and the dog (F = 55%) facilitated the identification of 28 as a potent (IC50 = 6 nM), selective, and orally available CRTh2 antagonist.

Evolution of novel tricyclic CRTh2 receptor antagonists from a (E)-2-cyano-3-(1H-indol-3-yl)acrylamide scaffold.

(E)-2-(3-(3-((3-Bromophenyl)amino)-2-cyano-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)acetic acid (1) was discovered in a HTS campaign for CRTh2 receptor antagonists. An SAR around this hit could be established and representatives with interesting activity profiles were obtained. Ring closing tactics to convert this hit series into a novel 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole based CRTh2 receptor antagonist series is presented.

Novel 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids: discovery and hit-to-lead evolution of a selective CRTh2 receptor antagonist chemotype.

Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d]imidazol-1-yl)acetic acid (1), discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist, is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed.

Dose-dependent antithrombotic activity of an orally active tissue factor/factor VIIa inhibitor without concomitant enhancement of bleeding propensity.

The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial thrombosis without prolonging bleeding time. The pharmacodynamic properties of this selective inhibitor are compared to the behaviour of a mixed factor VIIa/factor Xa inhibitor.

Selective and orally bioavailable phenylglycine tissue factor/factor VIIa inhibitors.

We describe the structure-based design and synthesis of highly potent, orally bioavailable tissue factor/factor VIIa inhibitors which interfere with the coagulation cascade by selective inhibition of the extrinsic pathway.

Thrombin binding to GPIbalpha induces integrin alphaIIbbeta3 dependent platelet adhesion to fibrin in ex vivo flowing whole blood.

We have investigated the role of the thrombin/GPIbalpha interaction in the adhesion of platelets to fibrin in a whole blood ex vivo perfusion model at a shear rate of 280 s(-1). Blood was perfused through parallel-plate chambers containing coverslips coated with cells expressing tissue factor, leading to the generation of thrombin and thus, deposition of fibrin onto the exposed cells. Adhesion of platelets to fibrin and thrombus growth were analyzed. Interestingly, when GPIbalpha was removed from the platelet surface by action of mocarhagin, platelet adhesion on fibrin was inhibited. Furthermore, a monoclonal antibody, VM16d, directed against the thrombin binding site on GPIbalpha also inhibited platelet adhesion on fibrin, showing the importance of the thrombin/GPIbalpha interaction.We then looked at the involvement of alphaIIbbeta3 and showed that platelet adhesion and thrombus growth on fibrin were inhibited by the dodecapeptide, whereas lamifiban only inhibited the growth of the platelet thrombus. These results indicated that binding of thrombin to GPIbalpha induced an intracellular signaling leading to the interaction of the platelet integrin alphaIIbbeta3 with the fibrin-dodecapeptide sequence.

Blockade of platelet GPIIB-IIIA (Integrin alphaII(b)beta(3)) in flowing human blood leads to passivation of prothrombotic surfaces.

We examined the impact of platelet activation on platelet adhesion to collagen in flowing human blood. ADP activation of platelets in ex vivo flowing blood resulted in paradoxical inhibition of platelet deposition on collagen. Blockade of fibrinogen binding to platelets by Lamifiban, a competitive antagonist of GPIIb-IIIa (integrin alpha(IIb)beta(3)), reversed this inhibition, leading to a marked increase in integrin alpha(2)beta(1)-dependent platelet adhesion. Analysis of integrin alpha(2)beta(1)-dependent platelet adhesion to collagen indicated that ADP-induced suppression of platelet adhesion is the result of trans-dominant inhibition of integrin alpha(2)beta(1) caused by fibrinogen binding to integrin GPIIb-IIIa. Lamifiban blocked fibrinogen binding, reversing the trans-dominant inhibition of alpha(2)beta(1) dependent adhesion to collagen. The GPIIb-IIIa antagonist resulted in the formation of a non-thrombogenic, passivated surface comprised of an adherent platelet monolayer. This unexpected consequence of blocking fibrinogen binding to GPIIb-IIIa may explain the long-term benefits of short-term GPIIb-IIIa antagonist treatment of Acute Coronary Syndrome patients.

Enhancing the anticoagulant potency of soluble tissue factor mutants by increasing their affinity to factor VIIa.

Complexation of factor VIIa (FVIIa) and tissue factor (TF) initiates the extrinsic pathway of blood coagulation. Inappropriate triggering of this pathway has been linked to thrombotic disorders. We have previously shown that a mutant form ofsoluble tissue factor (sTF, residues 1-219) having Lys165 and Lys166 replaced with alanine residues (hTFAA) gave an antithromboticeffect in a rabbit model of arterial thrombosis. Here we have improved the potency of hTFAA by incorporating amino acid substitutions thatincrease the affinity of sTF for FVIIa. Phage display has been used toselect consensus sequences at two FVIIa-contact regions on sTF, segments 44-50 and 130-140, that individually showed modestly increased (approximately 2-fold) FVIIa-affinity. These consensus sequences have been combined with other previously selected mutations to generate a series of variants (hTFAA-2, hTFAA-3, and hTFAA-4) having 4 to 10-fold increased FVIIa-binding affinity. Surface plasmon resonance measurementsindicate that the increased affinity results mostly from an increase in the association rate constant. All of these variants displayed increased inhibitor potency relative to hTFAA in an assay of Factor X activation catalyzed by the complex of FVIIa with membrane TF. In addition, these affinity improved hTFAA variants are more potent anticoagulants and have increased antithrombotic activity relative to hTFAA in an ex vivo flow chamber model of thrombosis. The potency trend parallels increases in FVIIa-binding affinity; however, the absolute fold increases in potencies are greater than increases in binding affinity, consistent with kinetic studies of the FVIIa-binding event. Through incorporation of 10 amino acid substitutions (hTFAA-3) we have increased the antithrombotic activity of hTFAA by 20-fold.