Safety Aspects of NN1731, a New Variant of Recombinant Factor VIIa with Increased Intrinsic Activity, in a Venous Stasis Model in Rabbits.

Variants of recombinant factor VIIa (rFVIIa) with enhanced enzymatic activity have recently been developed. Some variants, including NN1731, show more rapid Fxa (and consequently thrombin) generation on the phospholipid surface of activated platelets, independent of TF, and possess increased reactivity towards inhibitors as compared to rFVIIa (1). The increased potency of these FVIIa variants has been shown in haemophilia A mouse models (2) and in human in vitro systems. A potential concern with such analogues could be increased risk of systemic activation. Therefore, we tested the effects of NN1731 and rFVIIa on thrombus formation and coagulation activation in a venous stasis model in rabbits.

NN1731, 0.9 mg/kg (HD) and 0.09 mg/kg (LD) were compared to rFVIIa 1 mg/kg and vehicle. There was 6 NZ female rabbits in each group. Under pentobarbital anaesthesia, catheters were inserted in the carotid artery for blood sampling and monitoring of blood pressure, and in the femoral vein for injection of the test solutions. Blood was drawn before injection and at 5 min, 1, 2, and 3 hours. The facial veins were ligated for 30 min starting 10 min after the injection. The thrombi were removed and weighed. Platelet counts, aPTT, PT, fibrinogen, thrombin-antithrombin complex (TAT), and fibrin(ogen) degradation products (FDP) were measured. NN1731 and rFVIIa in plasma were determined by ELISA. A non-compartmental approached was used to estimate the half-life and clearance rate. The thrombi weights were analysed by Kruskal-Wallis test. Other parameters were analysed by ANCOVA, and the results are reported as the mean estimates adjusted for the baseline level with the 95% C.I., To obtain normality and variance homogeneity the data were log transformation followed by back-transformation.

The average thrombi weights ± SE (mg) were 9.2±1.6, 15.5±4.4, 1.1±0.4, and 1.6±1.0 after NN1731HD, rFVIIa, NN1731LD, and vehicle, respectively (P< 0.01). There was no statistically significant difference between NN1731HD and rFVIIa, and between NN1731LD and vehicle. There was no difference in platelet counts between groups. TAT levels (pmol/L) averaged over time increased about 2-fold from baseline, reaching 1733 (1109–2709) and 1561 (959–2541) in the NN1731HD and rFVIIa groups (P< 0.33). Fibrinogen and FDP levels were not affected by the treatments. At 5 min, the aPTT (sec) was 169 (142–201) in the vehicle group. It decreased to 34 (28–41) and 22 (18–26) in the NN1731HD and rFVIIa groups (P< 0.005), respectively. The shortening in aPTT was sustained for a longer period of time in the animals treated with FVIIa. A similar difference between NN1731HD and rFVIIa was observed in PT, reflecting the shorter plasma half-life of NN1731 as compared to FVIIa, being 1.0 hr vs. 1.4 hr, respectively. The clearance rates were found to be 3.1 vs. 1.0 ml/min/kg for NN1731 and rFVIIa. No differences were found in blood pressure between groups.

In conclusion, the study demonstrated the expected procoagulant effect of NN1731 and rFVIIa as assessed by their ability to generate local stasis thrombi in the facial veins of rabbits. The overall results of the study suggest that neither NN1731 nor rFVIIa in the doses used causes uncontrollable activation of the coagulation system in spite of the increased thrombin generation potential of NN1731. Future studies will include higher doses of NN1731 and rFVIIa and incorporate histopathology on relevant tissues.