Jeffrey S. Bostwick

Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y1 antagonists.

Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.

Inhibition of Repetitive Thrombus Formation in the Stenosed Canine Coronary Artery by Enoxaparin, But Not by Unfractionated Heparin

Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg + 5 microg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg + 10 microg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (P<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg + 10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (P<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.

Conformationally Constrained ortho-Anilino Diaryl Ureas: Discovery of 1-(2-(1′-Neopentylspiro[indoline-3,4′-piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a Potent, Selective, and Bioavailable P2Y1 Antagonist

Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.

Effect of the direct factor Xa inhibitor apixaban in rat models of thrombosis and hemostasis.

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 μM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparins prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.

Pharmacodynamic activity and antithrombotic efficacy of RPR120844, a novel inhibitor of coagulation factor Xa.

These studies were designed to examine the pharmacodynamic profile and antithrombotic efficacy of RPR120844, a competitive inhibitor of coagulation factor Xa, with a K(i) of 7 nM against human factor Xa. In vitro, RPR120844 doubled activated partial thromboplastin time (APTT) at concentrations of 1.54, 1.48, and 0.74 microM in plasma obtained from humans, dogs, and rats, respectively. Intravenous bolus administration of RPR 120844 at 0.3, 1, and 3 mg/kg to rats resulted in maximal increases in APTT of 1.8-, 2.6-, and 8.4-fold over baseline, respectively. The effect on prothrombin time (PT) was less pronounced, resulting in a 4.4-fold increase at 3 mg/kg. These effects were rapidly reversible; APTT and PT returned to control values by 30 min after dosing. Intragastric administration to rats at 50, 100, and 200 mg/kg resulted in modest increases in APTT and PT of 1.5- and 1.3-fold over baseline at the highest dose. Plasma levels were estimated by anti-Xa activity by using an amidolytic, chromogenic assay. Plasma levels were 0.65, 1.29, and 2.45 microM at 30 min after dosing at 50, 100, and 200 mg/kg, respectively. Intravenous administration to dogs at 0.1 and 0.3 mg/kg produced maximal increases in APTT of 1.7- and 2.4-fold over baseline, respectively. Intragastric administration to dogs at 50 mg/kg resulted in maximal increases in APTT and PT of 1.7- and 1.1-fold over baseline, with peak plasma levels of 3.9 microM observed at 15 min after dosing. In a rat model of FeCl2-induced carotid artery thrombosis, RPR120844 (3 mg/kg, i.v. bolus + 300 microg/kg/min constant infusion; n = 4) significantly increased time-to-occlusion from 18+/-1 min (vehicle, n = 4) to 60 min (maximal observation time) and reduced thrombus mass from 5.5 +/- 0.2 mg (vehicle) to 1.4 +/- 0.2 mg. These results indicate that RPR120844 is a potent, selective inhibitor of Xa that exhibits oral activity and is efficacious in a standard model of arterial thrombosis.

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC50 values of 3.7, 4.6, 6.3, 126, 136 and 1600 microM, respectively. RG13965 (3, 10, and 30 mg/kg, i.v.) decreased the incidence of collagen/epinephrine-induced thrombosis in mice from 90% in untreated animals to 63, 37, and 0%, respectively. In hamsters, RG13965 (10 and 30 mg/kg, i.v.) prolonged the time required for formation of a hemostatic plug in severed mesenteric arteries by 1.6- and 3.6-fold, respectively. In a canine model of repetitive platelet thrombus formation in the coronary artery, RG13965 (0.1, 0.3, and 1 mg/kg, i.v.) reversibly inhibited cyclic flow reductions (CFRs) and inhibited ADP-induced ex vivo platelet aggregation by 29, 57, and 77%, respectively. RG13965 (1 mg/kg) completely inhibited CFRs for at least 40 min. Platelet count was not altered at any dose and template bleeding time was prolonged modestly (1.8-fold) at only the highest dose. RG13965 dose-dependently and reversibly inhibited thrombus formation at doses which did not completely inhibit ex vivo platelet aggregation and only modestly prolonged template bleeding time.

Platelet aggregometry and receptor binding to predict the magnitude of antithrombotic and bleeding time effects of clopidogrel in rabbits

Target levels of ex vivo inhibition of platelet aggregation (IPA) induced by adenosine diphosphate (ADP) that produce clinically relevant effects of clopidogrel, a P2Y12 antagonist, are unclear. We examined standard and modified IPA and P2Y12 receptor occupancy as predictors of antithrombotic (% thrombus weight reduction) and bleeding time (BT, fold-increase over control) effects of clopidogrel in rabbit models of carotid artery thrombosis and cuticle bleeding, respectively. Standard and modified IPA with 20 μM ADP were measured in the absence and presence of partial P2Y1 blockade, respectively. Clopidogrel maximally produced standard IPA of 57% ± 5%, antithrombotic effect of 85% ± 1%, BT increase of 6.0 ± 0.4-fold and P2Y12 receptor occupancy of 87% ± 5%. Surprisingly, a clopidogrel dose that produced a low standard IPA of 17% ± 4% and P2Y12 receptor occupancy of 39% ± 5% achieved a significant antithrombotic activity of 55% ± 2% with a moderate increase in BT of 2.0 ± 0.1-fold. This underestimation of clopidogrel efficacy by standard IPA was improved by measuring either modified IPA or P2Y12 receptor occupancy. These results suggest that in clopidogrel-treated rabbits, low standard IPA is associated with significant antithrombotic effects. Moreover, modified IPA and P2Y12 receptor occupancy appear to better predict the magnitude of clopidogrels efficacy compared with standard IPA, which may be a better predictor of BT.