No abstract

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.

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 microM. 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. Heparin's 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.

The antithrombotic and bleeding effects of a low-molecular-weight heparin (LMWH, fragmin) and a thrombin active-site inhibitor (argatroban) were determined in anesthetized rats. Occlusive thrombi were produced in the vena cava, either by partial stasis of blood flow or transmural vessel injury, and in the carotid artery by transmural vessel injury. Bleeding time was measured by puncturing small mesenteric arteries. Each drug was tested in multiple intravenous (i.v.) doses and inhibited venous and arterial thrombosis when the activated partial thromboplastin time (APTT) was increased as much as or more than twofold, although greater APTT increases were required with fragmin and against arterial thrombosis. Fragmin and argatroban decreased to an equivalent extent the weight of venous thrombi induced by stasis (> or = 99%) or vessel injury (90 and 96%, respectively). The maximum inhibition of arterial thrombosis was less with fragmin (69%) and argatroban (65%) and required higher doses of each drug relative to venous thrombosis. At doses that were just optimal against arterial thrombosis, bleeding time was increased moderately by fragmin (32%) and was unaffected by argatroban. These studies demonstrate that doses of fragmin and argatroban that exert comparable antithrombotic activity in large arteries and veins have only moderate effects on bleeding time in small arteries.

We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl 2 . Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y 12 receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED 50 ) for thrombus reduction was 2.4 Ϯ 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction.The ED 50 for bleeding was 10.5 Ϯ 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED 50 s for peak platelet aggregation to 10 M ADP (11.9 Ϯ 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 Ϯ 1.3 mg/kg) approximated the higher ED 50 for bleeding. ED 50 s for ligand binding (3.0 Ϯ 0.3 mg/kg) and late aggregation (5.1 Ϯ 0.4 mg/kg) better matched the lower ED 50 for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.The thienopyridine clopidogrel is an ADP receptor antagonist that, upon conversion to an active metabolite, irreversibly blocks the platelet P2Y 12 receptor (Savi and Herbert, 2005). The structure of the active metabolite and the identity of the platelet receptor for clopidogrel were not known before phase III clinical trials had been completed (CAPRIE Steering Committee, 1996). In fact, the P2Y 12 receptor was referred to as the thienopyridine receptor in the original publication that detailed its molecular identification (Hollopeter et al., 2001). The primary activity biomarker for tracking the effects of clopidogrel has been ex vivo inhibition of ADPinduced platelet aggregation (IPA) (Nguyen et al., 2005).It is well recognized that the standard 75-mg maintenance dose of clopidogrel that reduces clinical cardiovascular events exerts a submaximal effect on ex vivo IPA (Thebault et al., 1999). The antiplatelet effect of clopidogrel in man requires oral bioavailability of parent drug, its bioactivation to the active metabolite, and productive blockade by that metabolite of platelet P2Y 12 receptors. Its effects will not prevent ADP-mediated platelet stimulation through P2Y 1 receptors. Our goal was to understand whether IPA is in fact a good predictor of clopidogrel's efficacy and bleeding ...