Carol A. Watson

Favorable therapeutic index of the direct factor Xa inhibitors, apixaban and rivaroxaban, compared with the thrombin inhibitor dabigatran in rabbits

Summary.u2002 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 EC50 of 65, 33 and 194u2003nm, respectively. At doses for 80% reduction of control thrombus, apixaban, rivaroxaban and dabigatran prolonged BT by 1.13u2003±u20030.02‐, 1.9u2003±u20030.1‐* and 4.4u2003±u20030.4‐fold*, respectively (*Pu2003<u20030.05, 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.

A small-molecule factor XIa inhibitor produces antithrombotic efficacy with minimal bleeding time prolongation in rabbits.

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 1xa0h before thrombus induction or cuticle transection to the end of the experiment. In vitro, BMS-262084 prolonged activated partial thromboplastin time (aPTT) with EC2x (concentration required to double aPTT) of 10.6xa0μM 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 ED50 (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 1.5xa0mg/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 10xa0mg/kg/h of BMS-262084 were 1.17xa0±xa00.04 and 1.52xa0±xa00.07*, respectively (*Pxa0<xa00.05 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.

Arterial antithrombotic and bleeding time effects of apixaban, a direct factor Xa inhibitor, in combination with antiplatelet therapy in rabbits

Summary.u2002 Background:u2002Optimal treatment of arterial thrombosis may include a combination of antiplatelet and anticoagulant drugs. We evaluated apixaban, a direct and highly selective factor Xa inhibitor, in combination with clinically relevant doses of aspirin and/or clopidogrel for prevention of arterial thrombosis in rabbits. Methods:u2002Studies were conducted in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding time (BT). Apixaban 0.04 and 0.3u2003mgu2003kg−1u2003h−1 or aspirin 1u2003mgu2003kg−1u2003h−1 was infused intravenous (i.v.) continuously from 1u2003h before artery injury or cuticle bleed until the end of the experiment. Clopidogrel at 3u2003mgu2003kg−1 was dosed orally once daily for threeu2003days, with the last dose given 2u2003h before injury. Results:u2002Control thrombus weight and BT averaged 8.6u2003±u20030.9u2003mg and 181u2003±u200312u2003s, respectively (nu2003=u20036 per group). Effective doses of apixaban that reduced thrombus weight by 20 and 50% (ED20 and ED50) were 0.04 and 0.3u2003mgu2003kg−1u2003h−1 i.v., respectively. Addition of aspirin to apixaban ED20 and ED50 significantly reduced the thrombus weight from 7.4u2003±u20030.5 to 5.3u2003±u20030.3 and 3.6u2003±u20030.3u2003mg, respectively, with no significant increases in BT (190u2003±u20037u2003s vs.181u2003±u20039 and 225u2003±u200311u2003s, respectively). Addition of aspirin and apixaban (ED20 dose) to clopidogrel produced a further significant reduction in thrombus weight from 5.3u2003±u20030.3 to 0.7u2003±u20030.1u2003mg. This combination of clopidogrel and aspirin with apixaban (ED20 dose) produced a significant but moderate BT increase of 2.1 times control. Conclusions:u2002The combination of apixaban and aspirin or apixaban, aspirin and clopidogrel can reduce formation of occlusive arterial thrombosis without excessive increases in BT in rabbits.

Razaxaban, a direct factor Xa inhibitor, in combination with aspirin and/or clopidogrel improves low-dose antithrombotic activity without enhancing bleeding liability in rabbits

Coactivation of platelets and the blood coagulation cascade contributes to the pathophysiology of arterial thrombosis. Combination therapy with antiplatelet and anticoagulant drugs may be needed for maximizing the prevention and treatment of arterial thrombosis. Few studies have thoroughly investigated the combined antithrombotic and bleeding effects of these antithrombotic agents. We, therefore, evaluated the antithrombotic and bleeding profiles of dual and triple therapy with razaxaban, a direct factor Xa inhibitor, plus aspirin and/or clopidogrel in rabbit models of electrolytic injury-induced carotid artery thrombosis and cuticle bleeding time, respectively. Compounds were infused either IV or into the portal vein from 1xa0h before arterial injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. We first evaluated the antithrombotic potency of razaxaban, and examined its ex vivo effects on coagulation parameters to confirm its selectivity. Antithrombotic ED50 of razaxaban averaged 0.22xa0±xa00.05xa0mg/kg/h (nxa0=xa06). Razaxaban at 3xa0mg/kg/h IV produced full antithrombotic efficacy, increased significantly ex vivo activated partial thromboplastin time and prothrombin time by 2.2xa0±xa00.1- and 2.3xa0±xa00.1-fold, respectively, and inhibited ex vivo factor Xa activity significantly by 91xa0±xa05% (nxa0=xa06, Pxa0<xa00.05) without affecting ex vivo thrombin activity. Razaxaban at concentrations up to 10xa0μM did not alter in vitro platelet aggregation responses to ADP, γ-thrombin or collagen. To identify additive or synergistic antithrombotic effects of the various combination therapies, we purposefully used marginally effective doses of razaxaban at 0.1xa0mg/kg/h, aspirin at 0.3xa0mg/kg/h and clopidogrel at 1xa0mg/kg/h. Dual combination of threshold doses of razaxaban and aspirin or clopidogrel produced an enhanced antithrombotic effect without further increases in bleeding time. When compared with dual therapy with aspirin and clopidogrel (38xa0±xa05% increase in blood flow), addition of razaxaban increased blood flow to 75xa0±xa05% without additional bleeding time effects (nxa0=xa06/group, Pxa0<xa00.05). In summary, razaxaban was an effective antithrombotic agent in a rabbit model of arterial thrombosis. Low-dose razaxaban was useful in combination with sub-optimal doses of aspirin and/or clopidogrel for the prevention of occlusive arterial thrombosis without excessive bleeding.

Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding

PAR4 inhibition with a reversible small-molecule antagonist is a promising antiplatelet therapy. This antiplatelet agent is just right Antiplatelet agents are common drugs that are used to prevent thrombotic events such as stroke in patients at high risk. Unfortunately, a frequent side effect of these drugs is excessive bleeding because they indiscriminately suppress thrombotic function. To improve on the safety margin of antiplatelet agents, Wong et al. identified a new target for treatment, a platelet receptor called PAR4. The authors developed a small-molecule drug against this target and evaluated its efficacy and safety in animal models. In head-to-head comparisons, the new drug was no less effective than clopidogrel, an antiplatelet agent widely used in the clinic, but it had a much larger therapeutic window. 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.

In vitro, antithrombotic and bleeding time studies of BMS-654457, a small-molecule, reversible and direct inhibitor of factor XIa

BMS-654457 ((+) 3′-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5′-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90xa0min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37xa0mg/kgxa0+xa00.27xa0mg/kg/h produced almost 90xa0% preservation of iCBF compared to its vehicle (87xa0±xa010 and 16xa0±xa03xa0%, respectively, nxa0=xa06 per group) and increased BT by 1.2xa0±xa00.04-fold (Pxa0<xa00.05). At a higher dose (1.1xa0mg/kgxa0+xa00.8xa0mg/kg/h), BMS-654457 increased BT by 1.33xa0±xa00.08-fold. This compares favorably to equivalent antithrombotic doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) which produced four- to six-fold BT increases in the same model. In summary, BMS-654457 was effective in the prevention of arterial thrombosis in rabbits with limited effects on BT. This study supports inhibition of FXIa, with a small-molecule, reversible and direct inhibitor as a promising antithrombotic therapy with a wide therapeutic window.

Discovery of diarylurea P2Y1 antagonists with improved aqueous solubility

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

The P2Y1 receptor antagonist MRS2500 prevents carotid artery thrombosis in cynomolgus monkeys

Adenosine diphosphate directly induces platelet aggregation via the G-protein coupled P2Y1 and P2Y12 receptors. P2Y12, but not P2Y1, receptor antagonists are available in the clinic. The relevance of the P2Y1 receptor as an antiplatelet target has been studied in rodents, but not in higher species. We therefore examined effects of the pharmacological blockade of the P2Y1 receptor with its selective antagonist MRS2500 in monkey models of electrolytic-mediated arterial thrombosis (ECAT) and kidney bleeding time (KBT). Abciximab, a GPIIb-IIIa antagonist, and cangrelor, a P2Y12 antagonist, were utilized to validate these monkey models. Compounds were given IV at 15–60xa0min before thrombosis initiation in anesthetized monkeys. Scanning electron microscopy showed the luminal surface of thrombotic artery covered with platelet aggregates and fibrin network. Administration of abciximab at 0.25 and 0.7xa0mg/kg IV significantly reduced thrombus weight by 71xa0±xa01 and 100xa0±xa00xa0%, and increased KBT by 10.0xa0±xa00.1- and 10.1xa0±xa00-fold, respectively (nxa0=xa03/dose). Likewise, cangrelor at 0.6 and 2xa0mg/kg/h IV significantly reduced thrombus weight significantly by 72xa0±xa09xa0% and 100xa0±xa00xa0% and increased KBT by 2.1xa0±xa00.1- and 9.8xa0±xa00.2-fold, respectively (nxa0=xa03/dose). MRS2500 [mg/kgxa0+xa0mg/kg/h IV] at 0.09xa0+xa00.14 and 0.45xa0+xa00.68 significantly reduced thrombus weight by 57xa0±xa01xa0% and 88xa0±xa01xa0% and increased KBT by 2.1xa0±xa00.3- and 4.9xa0±xa00.6-fold, respectively (nxa0=xa04/dose). In summary, MRS2500 prevented occlusive arterial thrombosis at a dose that moderately prolonged KBT, indicating a role of P2Y1 receptors in arterial thrombosis and hemostasis in monkeys. Thus P2Y1 receptor antagonism provides a suitable target for drug discovery.