Abstract

Introduction: Congenital human blood coagulation factor XI (FXI) deficiency is associated with bleeding that is less severe than that observed in patients with other coagulation factor deficiencies, whereas severe FXI deficiency confers the decreased risk of deep vein thrombosis. FXI knockout mice show a reduction in venous thrombus formation with no bleeding. Therefore, FXI is considered to be a promising drug target for treatment and prevention of venous thromboembolism without increasing bleeding risk. Recently, we discovered a novel, potent, selective and injectable small molecule inhibitor of activated FXI (FXIa), ONO-IG-012, and evaluated the in vitro and in vivo pharmacological profiles of the compound.

Methods: In anin vitro study, inhibitory effects of ONO-IG-012 on enzyme activities of human FXIa, other blood coagulation factors, and fibrinolytic factors were evaluated. Anticoagulant effects of ONO-IG-012 were also evaluated in human and rabbit plasma. In in vivo studies, the antithrombotic and hemorrhagic effects of ONO-IG-012 were compared to those of enoxaparin in rabbit models of deep venous thrombosis and femur hemorrhage. In the thrombosis model, under ketamine and xylazine anesthesia, inferior vena cava was isolated and partially ligated to reduce blood flow. The vein at the distal site was wrapped with filter paper saturated with ferric chloride (FeCl3) solution for 15 minutes to induce endothelial injury and subsequent thrombus formation. Sixty minutes after the application of FeCl3, the thrombus wet weight was measured. In the bleeding model, a puncture wound was made into the medullary canal at the epiphysis of femur using a drill under isoflurane anesthesia and mechanical ventilation. Blood was continuously collected with absorbent cotton for one hour, and the blood loss volume was calculated from its specific gravity. Intravenous administration of ONO-IG-012 or enoxaparin was initiated as a loading dose an hour before applying FeCl3 or producing a puncture wound, followed by their maintenance dose infusion. Blood was collected to measure APTT and PT just before the administration of the compounds and applying FeCl3 or producing a puncture wound.

Results: ONO-IG-012 competitively inhibited human FXIa with a Ki value of 0.0019 μmol/L. Although ONO-IG-012 moderately inhibited human plasma kallikrein with a Ki value of 0.15 μmol/L, it had little effect on other human blood coagulation factors, and fibrinolytic factors [thrombin, FVIIa, FIXa, FXa, FXIIa, tPA, urokinase, and plasmin (Ki value >100 μmol/L)]. ONO-IG-012 prolonged APTT, and the concentration required to double the APTT was 0.098 μmol/L in human plasma and 0.30 μmol/L in rabbit plasma. However, prolongation of PT was not observed even at 33 μmol/L. ONO-IG-012 inhibited thrombus formation even at a dose as low as 0.1 mg/kg/h and achieved maximum antithrombotic effect at greater than or equal to 0.3 mg/kg/h. The ex vivo APTT was increased from baseline by 1.7 ± 0.0-fold at 0.1 mg/kg/h, by 2.8 ± 0.3-fold at 0.3 mg/kg/h, and by 5.4 ± 0.4-fold at 1 mg/kg/h, while PT showed no changes at any of the concentrations tested. Enoxaparin also inhibited thrombus formation at a dose of 10 IU/kg/h, and the antithrombotic effect at 30 IU/kg/h was comparable to that of ONO-IG-012 at 0.3 mg/kg/h (−87% vs. −80%). ONO-IG-012 did not affect the blood loss volume at all even at 10 mg/kg/h, which is 33-fold higher than the dose showing maximum antithrombotic effect (0.3 mg/kg/h). At 10 mg/kg/h, the blood loss volume was 0.7 ± 0.1 mL which is not statistically significant as compared to 1.2 ± 0.3 mL in the vehicle group, and the APTT ratio was 9.0 ± 1.1-fold. In contrast, enoxaparin increased the blood loss volume dose-dependently with the values of 4.5 ± 1.8 mL (not statistically significant) at 10 IU/kg/h, 8.4 ± 2.2 mL (P <0.01) at 30 IU/kg/h, and 23.1 ± 5.0 mL (P <0.001) at 100 IU/kg/h.

Conclusions: ONO-IG-012 demonstrated a competitive, highly selective and potent inhibitory effect on FXIa among proteases involved in blood coagulation or fibrinolysis and a potent anticoagulant effect on APTT. ONO-IG-012 did not affect the blood loss volume at all even at 33-fold higher dose than the dose showing the maximum antithrombotic effect comparable to enoxaparin. ONO-IG-012 is expected to be a novel potent anticoagulant without an increased risk of bleeding for the treatment and prevention of venous thromboembolism.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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