Abstract-A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg ⅐ kg Ϫ1 ⅐ h Ϫ1 ), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl 3 -patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U ⅐ kg Ϫ1 ⅐ min Ϫ1 ), whereas the latter rendered the blood incoagulable (aPTTϾ1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18Ϯ0.49 to 1.80Ϯ0.3 mg, PϽ0.001), increasing vessel patency time (from 14.9Ϯ0.9 minutes to 58.3Ϯ1.7 minutes, PϽ0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle-versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (PϽ0.01), extended vessel patency time (PϽ0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8Ϯ0.7 to 3.3Ϯ0.6 mL (PϽ0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.

Summary Background: A significant need exists for new chronic oral anticoagulation therapies to replace warfarin. Previous studies have shown that b-D-xylosides, which prime glycosaminoglycan (GAG) synthesis, have antithrombin and antithrombotic activity. In the following report, a new orally active b-D-xyloside (odiparcil) has been characterized in a rat model of venous thrombosis and its efficacy and bleeding liability compared to warfarin. Additionally, studies were conducted to investigate odiparcil's ex vivo antithrombin and antiplatelet activity, and also to explore the potential utility of protamine sulfate as a neutralizing agent. Methods and results: In vivo thrombosis studies were conducted in a rat inferior vena cava model, and bleeding studies in a rat tail transection model. Following oral dosing, warfarin and odiparcil produced doserelated suppression of thrombus formation. A therapeutically relevant dose of warfarin in this model (international normalized ratio; INR 3.0) achieved 65% inhibition of thrombus formation. Warfarin caused dose-related significant increases in bleeding indices. Odiparcil antithrombotic activity was limited by its mechanism to a maximum suppression of thrombus formation of 65-70%, and did not prolong bleeding indices. Additionally, odiparcil-induced heparin cofactor II (HCII)-dependent antithrombin activity was shown to be a function of dermatan sulfate-like GAG production. Other than thrombinrelated effects, no odiparcil effects on platelet function were observed. In antidote studies, it was demonstrated that odiparcil-induced antithrombotic activity could be partially neutralized by protamine sulfate. Conclusions: These experiments suggest that an antithrombotic approach based upon xyloside induction of circulating GAGs may have the potential to approximate the efficacy of warfarin and yet with a reduced risk to hemostasis.

Background and Purpose-Although used clinically to prevent stroke, there are few examples of anticoagulant investigations in the treatment of acute thromboembolic stroke in animal models. The treatment of thromboembolic stroke in experimental models has been investigated almost exclusively around the use of tissue plasminogen activator (tPA). In this study, using a rat thromboembolic stroke model, we investigated the use of an inhibitory anti-factor IX(a) monoclonal antibody (SB 249417) for the treatment of thromboembolic stroke and compared its efficacy to that of tPA. Methods-Stroke was initiated by delivering 6 clots into the internal carotid artery. After 2, 4, or 6 hours, rats received either intravenous vehicle, 10.0 mg/kg tPA, or 1.0, 2.0, or 3.0 mg/kg SB 249417. At 24 hours after stroke, infarct volumes and neurological deficits were assessed. Results-Treatment with tPA 2, 4, or 6 hours after stroke reduced infarct volumes by 35% (PϭNS), 45%, and 39%, respectively. tPA treatment did not improve neurological deficits at any time point. Treatment with SB 249417 (3.0 mg/kg) 2, 4, or 6 hours after stroke reduced infarct volumes by 44%, 50%, and 13% (PϭNS), respectively. Neurological deficits were reduced by 49%, 42%, and 13% (PϭNS), respectively. Neither mortality nor hemorrhage was affected by either treatment. Conclusions-The data indicate that the inhibition of factor IX(a) within 4 hours of thromboembolic stroke produced a more favorable outcome than tPA. When treatment was initiated 6 hours after stroke, the benefits of factor IX(a) inhibition were lost, whereas tPA continued to suppress lesion development, albeit without a corresponding improvement in functional deficits. This study suggests that cerebral ischemia and the resultant perfusion deficit are exacerbated by the activation of blood coagulation and that anticoagulants like SB 249417 may find utility in the treatment of ischemic stroke. (Stroke. 2002;33:578-585.)

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SummaryAn inhibitory anti-factor IX/IXa antibody (BC2) has been investigated as an anti-thrombotic agent in a rat venous thrombosis model. The treatment of rats post-injury with a single bolus dose of BC2 (3mg/kg, iv.) resulted in an ~4 fold reduction in venous thrombus mass (P = 0.043). This efficacy was matched by a minimal (<2.5 fold) prolongation of the aPTT and had no effect on the prothrombin time (PT). Heparin by comparison, given as a bolus followed by continuous infusion, at doses comparable in efficacy at reducing thrombus formation, prolonged the aPTT >50 fold. These results demonstrate that the anti-factor IX/IXa antibody (BC2), when compared to heparin, can effectively reduce venous thrombosis with less disruptive consequences on blood clotting.