Andexanet alfa effectively reverses edoxaban anticoagulation effects and associated bleeding in a rabbit acute hemorrhage model.

INTRODUCTION: Increasing use of factor Xa (FXa) inhibitors necessitates effective reversal agents to manage bleeding. Andexanet alfa, a novel modified recombinant human FXa, rapidly reverses the anticoagulation effects of direct and indirect FXa inhibitors. OBJECTIVE: To evaluate the ability of andexanet to reverse anticoagulation in vitro and reduce bleeding in rabbits administered edoxaban. MATERIALS AND METHODS: In vitro studies characterized the interaction of andexanet with edoxaban and its ability to reverse edoxaban-mediated anti-FXa activity. In a rabbit model of surgically induced, acute hemorrhage, animals received edoxaban vehicle+andexanet vehicle (control), edoxaban (1 mg/kg)+andexanet vehicle, edoxaban+andexanet (75 mg, 5-minute infusion, 20 minutes after edoxaban), or edoxaban vehicle+andexanet prior to injury. RESULTS: Andexanet bound edoxaban with high affinity similar to FXa. Andexanet rapidly and dose-dependently reversed the effects of edoxaban on FXa activity and coagulation pharmacodynamic parameters in vitro. In edoxaban-anticoagulated rabbits, andexanet reduced anti-FXa activity by 82% (from 548±87 to 100±41 ng/ml; P<0.0001), mean unbound edoxaban plasma concentration by ~80% (from 100±10 to 21±6 ng/ml; P<0.0001), and blood loss by 80% vs. vehicle (adjusted for control, 2.6 vs. 12.9 g; P = 0.003). The reduction in blood loss correlated with the decrease in anti-FXa activity (r = 0.6993, P<0.0001) and unbound edoxaban (r = 0.5951, P = 0.0035). CONCLUSION: These data demonstrate that andexanet rapidly reversed the anticoagulant effects of edoxaban, suggesting it could be clinically valuable for the management of acute and surgery-related bleeding. Correlation of blood loss with anti-FXa activity supports the use of anti-FXa activity as a biomarker for assessing anticoagulation reversal in clinical trials.

The novel kinase inhibitor PRT062070 (Cerdulatinib) demonstrates efficacy in models of autoimmunity and B-cell cancer.

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate-mediated signaling or activation in B and T cells nor T-cell antigen receptor-mediated signaling in T cells, providing evidence for selectivity of action. Potent antitumor activity was observed in a subset of B-cell lymphoma cell lines. After oral dosing, PRT062070 suppressed inflammation and autoantibody generation in a rat collagen-induced arthritis model and blocked B-cell activation and splenomegaly in a mouse model of chronic B-cell antigen receptor stimulation. PRT062070 is currently under evaluation in a phase I dose escalation study in patients with B-cell leukemia and lymphoma (NCT01994382), with proof-of-concept studies in humans planned to assess therapeutic potential in autoimmune and malignant diseases.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa.

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

The selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and function in vitro and in vivo and augments the activity of fludarabine in chronic lymphocytic leukemia.

B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.

Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 µM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 µM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 µM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Thienopyridines, but not elinogrel, result in off-target effects at the vessel wall that contribute to bleeding.

Clinical studies with clopidogrel or prasugrel show that although increased inhibition of P2Y(12) and platelet function improves efficacy, bleeding is also increased. Other preclinical and clinical studies have suggested a greater therapeutic index (TI) with reversible inhibitors and disproportionate effects of thienopyridines on bleeding at high doses. We used multiple in vivo (FeCl(3)-induced arterial thrombosis in mesenteric arteries, blood loss after tail transsection, and platelet deposition and wound closure time in a micropuncture model in mesenteric veins) and ex vivo (light transmittance aggregometry, prothrombin time, and activated partial thromboplastin time) mouse models to 1) compare the TI of clopidogrel, prasugrel, and elinogrel, a reversible, competitive antagonist, with that in P2Y(12)(-/-) mice and 2) determine whether the bleeding consequences of the thienopyridines are attributed only to the inhibition of P2Y(12). Data indicated greater (elinogrel) and decreased (thienopyridines) TI compared with that in P2Y(12)(-/-) mice. The impaired TI associated with the thienopyridines was not attributed to non-P2Y(12) activities on platelet function or coagulation but was related to a direct effect at the vessel wall (inhibition of vascular tone). Further analysis showed that the prasugrel off-target effect was dose- and time-dependent and of a reversible nature. In conclusion, the TI of thienopyridines in the mouse may be decreased by P2Y(12)-independent off-target effects at the vessel wall, whereas that of elinogrel may be enhanced by the reversible, competitive nature of the antiplatelet agent.

PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model.

Heparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.

Platelet aggregation induces platelet aggregate stability via SLAM family receptor signaling.

Platelet aggregation is a dynamic entity, capable of directing its own growth and stability via the activation of signaling cascades that lead to the expression and secretion of various secondary agonists. Here we show that the signaling pathways triggered during platelet aggregation include an intrinsic pro-thrombotic activity mediated by 2 homophilic adhesion molecules, CD84 and CD150 (SLAM [signaling lymphocyte activation molecule]), which are tyrosine phosphorylated in a platelet aggregation-dependent fashion. The 2 CD84/SLAM adapter proteins, SAP (SLAM-associated protein) and EAT-2 (EWS-activated transcript-2), were found in platelets; only SAP, however, was found to immunoprecipitate with tyrosine-phosphorylated SLAM. The immobilized extracellular domain of CD84 promoted microaggregate formation, while SAP-deficient platelets demonstrated defective spreading on immobilized CD84, demonstrating a functional role in platelets for SLAM family interactions. Finally, analysis of SLAM-deficient mice revealed an overall defect in platelet aggregation in vitro and a delayed arterial thrombotic process in vivo. The data indicate that signaling of the adhesion molecules in the SLAM family, activated by proximity during aggregation, further stabilize platelet-platelet interactions in thrombosis.

P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.