Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care.

To cite this article: Wong PC, Crain EJ, Watson CA, Xin B. Favorable therapeutic index of the direct factor Xa inhibitors, apixaban and rivaroxaban, compared with the thrombin inhibitor dabigatran in rabbits. J Thromb Haemost 2009; 7: 1313-20.Summary. Background: Apixaban is an oral, direct factor Xa (FXa) inhibitor in late-stage clinical development. This study assessed effects of the direct FXa inhibitors, apixaban and rivaroxaban, vs. the direct thrombin inhibitor, dabigatran, on venous thrombosis (VT), bleeding time (BT) and clotting times in rabbits. Methods: We induced the formation of non-occlusive thrombus in VT models by placing threads in the vena cava, and induced bleeding by the incision of cuticles in anesthetized rabbits. Apixaban, rivaroxaban and dabigatran were infused IV to achieve a stable plasma level. Clotting times, including the activated partial thromboplastin time (aPTT), prothrombin time (PT), modified PT (mPT) and thrombin time (TT), were measured. Results: Apixaban, rivaroxaban and dabigatran exhibited dose-related efficacy in preventing VT with EC 50 of 65, 33 and 194 nM, respectively. At doses for 80% reduction of control thrombus, apixaban, rivaroxaban and dabigatran prolonged BT by 1.13 ± 0.02-, 1.9 ± 0.1-* and 4.4 ± 0.4-fold*, respectively (*P < 0.05, vs. apixaban). In the treatment model, these inhibitors equally prevented growth of a preformed thrombus. Antithrombotic doses of apixaban and rivaroxaban prolonged aPTT and PT by <3-fold with no effect on TT. Dabigatran was ‡50-fold more potent in prolonging TT than aPTT and PT. Of the clotting assays studied, apixaban, rivaroxaban and dabigatran responded the best to mPT. Conclusion: Comparable antithrombotic efficacy was observed between apixaban, rivaroxaban and dabigatran in the prevention and treatment of VT in rabbits. Apixaban and rivaroxaban exhibited lower BT compared with dabigatran at equivalent antithrombotic doses. The clinical significance of these findings remains to be determined.

Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was identified as a potent and selective tool compound for proof of concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis models and did not prolong bleeding times. This demonstrates proof of concept for the FXIa mechanism in animal models with a reversible, small molecule inhibitor.

BMS-262084 is a 4-carboxy-2-azetidinone-containing irreversible inhibitor of FXIa, which is selective over other coagulation proteases. We evaluated the in vitro and in vivo properties of BMS-262084 in rabbits. Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrolytic-mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. BMS-262084 was infused IV from 1 h before thrombus induction or cuticle transection to the end of the experiment. In vitro, BMS-262084 prolonged activated partial thromboplastin time (aPTT) with EC(2x) (concentration required to double aPTT) of 10.6 μM in rabbit plasma, and did not prolong prothrombin time (PT), thrombin time (TT) and HepTest. In vivo, BMS-262084 produced dose-dependent antithrombotic effects in rabbits with antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT of 0.4, 0.7 and 1.5 mg/kg/h IV, respectively. BMS-262084 increased ex vivo aPTT dose-dependently without changes in PT and TT. The antithrombotic effect of BMS-262084 was significantly correlated with its ex vivo aPTT, supporting the use of ex vivo aPTT as a pharmacodynamic biomarker. BMS-262084 did not alter ex vivo rabbit platelet aggregation to ADP and collagen. BT (fold-increase) determined at 3 and 10 mg/kg/h of BMS-262084 were 1.17 ± 0.04 and 1.52 ± 0.07*, respectively (*P < 0.05 vs. control). This study demonstrated that BMS-262084 prevented experimental thrombosis at doses with low BT effects in rabbits, and suggests that a small molecule FXIa inhibitor may represent a promising antithrombotic therapy.

phenyl]-N-[3-fluoro-2Ј-(methylsulfonyl)[1,1Ј-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide] is a synthetic, competitive, and selective inhibitor of coagulation factor Xa (fXa) (K i : 0.15 nM in humans, 0.3 nM in rabbit). The objective of this study was to compare effects of DPC423, enoxaparin (low-molecular-weight heparin), and argatroban (thrombin inhibitor) on arterial thrombosis and hemostasis in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding, respectively. Compounds were infused i.v. continuously from 60 min before artery injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. Antithrombotic ED 50 values were 0.4 mg/kg/h for enoxaparin (n ϭ 6), 0.13 mg/kg/h for argatroban (n ϭ 6), and 0.6 mg/kg/h for DPC423 (n ϭ 12). DPC423 at the maximum antithrombotic dose increased activated partial thromboplastin time and prothrombin time (n ϭ 6) by 1.8 Ϯ 0.07-and 1.8 Ϯ 0.13-fold, respectively, without changes in thrombin time and ex vivo thrombin activity. The antithrombotic effect of DPC423 was significantly correlated with its ex vivo anti-fXa activity (r ϭ 0.86). DPC423 at 1, 3, and 10 mg/kg p.o. increased carotid blood flow (percent control) at 45 min to 10 Ϯ 4, 24 Ϯ 6, and 74 Ϯ 7, respectively (n ϭ 6/group). Cuticle bleeding times (percent change over control) determined at the maximum antithrombotic dose were 88 Ϯ 12 for argatroban, 69 Ϯ 13 for heparin, 4 Ϯ 3 for enoxaparin, 5 Ϯ 4 for DPC423, and Ϫ3 Ϯ 2 for the vehicle (n ϭ 5-6/group), suggesting dissociation of antithrombotic and bleeding time effects for DPC423 and enoxaparin. The combination of aspirin and DPC423 at ineffective antithrombotic doses produced significant antithrombotic effect. Therefore, these results suggest that DPC423 is a clinically useful oral anticoagulant for the prevention of arterial thrombosis.