The antithrombotic effects of CI-1031 (ZK-807834) and enoxaparin in a canine electrolytic injury model of arterial and venous thrombosis

Abstract

Factor Xa is a serine protease positioned at the convergence point of the intrinsic and extrinsic coagulation pathways and is therefore an attractive target in the development of novel anticoagulant drugs. The objective of this study was to evaluate the efficacy of CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective inhibitor of Factor Xa, in a canine electrolytic injury model of arterial and venous thrombosis. Enoxaparin (enoxaparin sodium), a low molecular weight heparin currently approved for treatment and prevention of deep vein thrombosis and unstable angina, was also tested for efficacy in this model. CI-1031 was administered intravenously to anesthetized dogs at three doses: 1.25, 2.5 and 5 μg/kg/min (n=5 for each group) as a continuous infusion for 5.5 h. The control group (n=5) received a continuous infusion of vehicle (3.69 mmol citric acid and 0.9% sodium chloride solution) at a rate of 1 ml/kg/h. Ninety minutes after administration of CI-1031 prothrombin times increased 1.2-, 1.6- and 2.0-fold over baseline values in the 1.25, 2.5 and 5 μg/kg/min groups, respectively. The time to formation of an occlusive thrombus in the femoral arteries averaged 69±5 min in the control group compared to 127±19, 192±33 and 219±15 min in the low-, mid- and high-dose CI-1031 groups. In the femoral veins, occlusion time in the controls averaged 56±11 min compared to 153±22, 137±30 and 214±26 min in the three treatment groups. Thrombus weights in the control arteries averaged 51±4 mg compared to 45±5, 28±10 and 15±3 mg in the CI-1031 treated groups. On the venous side, control thrombus weights averaged 96±18 mg compared to 75±16, 51±16 and 25±4 mg in the low-, mid- and high-dose CI-1031 groups. A plasma CI-1031 concentration of approximately 400 ng/ml was associated with a 50% reduction in thrombus weight relative to control animals. Enoxaparin was administered intravenously at a loading dose of 50, 100 or 200 IU/kg for 1 h followed by a maintenance infusion of 25, 50 or 100 IU/kg/h for 4.5 h. The most dramatic changes in coagulation parameters were observed in thrombin time with virtually no changes in prothrombin time. Enoxaparin elicited a dose-dependent increase in time to thrombotic occlusion and a dose-dependent decrease in thrombus weight similar to that observed with CI-1031. Time to occlusion in the enoxaparin-treated groups averaged 117±33, 188±32 and 217±22 min in the low-, mid- and high-dose groups in the femoral arteries and 84±22, 171±31 and 133±33 min in the femoral veins. Thrombus weights averaged 33±10, 12±5 and 10±4 mg in the arteries and 32±9, 13±2 and 21±6 mg in the veins in the low-, mid- and high-dose groups. Blood loss with CI-1031 tended to be less than enoxaparin at doses that provided comparable efficacy. These results demonstrate that CI-1031, like enoxaparin, is an effective antithrombotic agent in an established canine model of arterial and venous thrombosis. CI-1031 provided dose-dependent efficacy with minimal changes in ex vivo coagulation parameters, suggesting it may be a safe and effective antithrombotic agent for both arterial and venous indications.

Introduction

The activated serine protease Factor Xa plays an important role in the blood coagulation cascade because of its central location at the point where the extrinsic and intrinsic pathways converge. Factor Xa inhibitors prevent the generation of thrombin, thereby disrupting the thrombin feedback loop which amplifies thrombin production Gardell and Sanderson, 1998, Al-Obeidi and Ostrem, 1999. By reducing thrombin generation, inhibitors of Factor Xa may also attenuate platelet activity and clot stabilization. Several preclinical studies have demonstrated an enhanced efficacy-to-bleeding ratio with Factor Xa inhibitors compared to other antithrombotic strategies Lynch et al., 1994, Nicolini et al., 1996, Lefkovits et al., 1996, Sato et al., 1998a, Vlasuk et al., 1991. Therefore, the development of new antithrombotic agents that target Factor Xa may provide greater efficacy and reduced bleeding risk than current therapeutic approaches to thrombosis.

Low molecular weight heparins mediate inhibition of Factor Xa by enhancing antithrombin III activity and have been shown to provide more reliable anticoagulation with fewer side effects than standard unfractionated heparin (Zed, 1999). Enoxaparin is a low molecular weight heparin that is currently approved for the prevention and treatment of deep vein thrombosis and, more recently, to prevent ischemic events in patients with unstable angina (Brener, 2000). A number of limitations with low molecular weight heparins, including the inability to inactivate fibrin-bound thrombin and the risk of heparin-induced thrombocytopenia and osteoporosis, leave significant room for the development of safe, effective anticoagulants (Weitz, 1997).

Direct inhibitors of Factor Xa, in contrast to low molecular weight heparin, bind directly to the active site of Factor Xa and have been proven to be effective anticoagulants in experimental models of thrombosis (Weitz and Hirsh, 1998). CI-1031 (N-[2-[5-amidino-2-hydroxyphenoxy]-6-[3-(1-methyl-1H-imidazolin-2-yl)-phenoxy]-3,5-difluoropyrid), a potent and selective Factor Xa inhibitor with a K_i of 0.11 nM Phillips et al., 1998, Subramanyam et al., 1998, has previously been shown to attenuate thrombus progression in a rabbit model of thrombosis (Abendschein et al., 2000). The objective of this study was to evaluate CI-1031 and establish a dose–response relationship for antithrombotic efficacy versus bleeding risk. The study also compares the efficacy of CI-1031 with enoxaparin in an established canine model of arterial and venous thrombosis.