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.

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 (IC 50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC 50 0.27 and 0.28 M, respectively) and Fc receptor 1-mediated basophil degranulation (IC 50 0.15 M). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC 50 0.32 M). 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 ϳ67%. 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.

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 collageninduced 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.

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 (Ϫ/Ϫ) 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 (Ϫ/Ϫ) 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.

3414 We used a modified rabbit liver laceration model to demonstrate the effects of PRT064445, a recombinant fXa derivative, to reverse rivaroxaban induced anticoagulation as measured by reduction of blood loss, decrease of unbound fraction of rivaroxaban in plasma and relevant pharmacodynamic markers: anti-fXa activity, prothrombin (PT) and activated thromboplastin (aPTT) times. Recombinant fVIIa (rfVIIa) was tested in the same model for comparison. Anesthetized rabbits were administered vehicle or 1 mg/kg rivaroxaban via IV bolus injection. After 30 minutes to allow the rivaroxaban to biodistribute, vehicle, PRT064445 or rfVIIa was administered as a bolus injection followed by laceration of two liver lobes (5× each lobe: 1-cm long and 3-mm deep incisions with scalpel blade) and blood loss collected on pre-weighed gauze over 15 minutes. Blood loss due to rivaroxaban anticoagulation represented approximately 10% of the animal's total blood volume. Total rivaroxaban concentration in plasma was measured by LC-MS/MS. Free fraction of rivaroxaban (non-protein, non-PRT064445 bound) was assessed using equilibrium dialysis method followed by LC-MS/MS quantitation. Anti-fXa activity was measured using a modified chromogenic LMW heparin kit. PT and aPTT was measured using commercial reagents (HemosIL). Anticoagulation by rivaroxaban (1.65 μM average plasma concentration at 30 min time point) resulted in 2.3-fold and 1.9-fold prolongation of PT and aPTT, respectively, and increased blood loss by 3.2-fold over vehicle. PRT064445 (75 mg/rabbit) administration reduced blood loss due to rivaroxaban anticoagulation by >85% and decreased peak anti-fXa activity by 98%, PT by 74%, aPTT by 66% and the free fraction of rivaroxaban in plasma from 26% to <0.5%. In contrast, rfVIIa (150 μg/kg) had no effect on blood loss but reversed PT by 85% and aPTT by 54%. PRT064445 and rfVIIa alone had no effect on blood loss, but rfVIIa decreased PT by 35%. These data demonstrate that PRT064445 can reduce blood loss due to rivaroxaban induced anticoagulation using a single bolus administration. Reduction of blood loss with PRT06445 correlated to the decrease in the free fraction of rivaroxaban in plasma as well as PD markers anti-fXa activity and PT, while rfVIIa decreased PT but had no effect on blood loss. These and our previous data with reversal of enoxaparin-induced anticoagulation in a rat tail transection model indicate that PRT064445 can be used as a universal antidote for both direct and indirect fXa inhibitors. Disclosures: Hollenbach: Portola Pharmaceuticals: Employment. Lu:Portola Pharmaceuticals: Employment. Tan:Portola Pharmaceuticals: Employment. Lee:Portola Pharmaceuticals: Employment. Athiwat:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.