Pre-Clinical Characteristics of a Recombinant Fusion Protein Linking Activated Coagulation Factor VII with Albumin (rVIIa-FP)

Abstract 1115

Recombinant factor VIIa (rFVIIa) is approved to control bleeding in hemophilia A and B patients who have developed inhibitory antibodies to replacement therapy and in acquired hemophilia. rFVIIa is rapidly eliminated with a terminal half-life of approximately 2.5 hours in humans. This short half life necessitates frequent injections and considerably limits prophylactic use. A recombinant fusion protein linking activated factor VII with albumin (rVIIa-FP) was engineered to extend the half life of rFVIIa.

The present studies were conducted to gather knowledge about the pharmacokinetic and pharmacodynamic (PK/PD) properties of rVIIa-FP in preclinical animals models to enable a more precise estimate of the procoagulant activity of rVIIa-FP for the future clinical use in haemophilia patients with inhibitors to FVIII and FIX. Single intravenous doses of either rVIIa-FP or the licensed rFVIIa comparator, NovoSeven® were administered to mice, rabbits and monkeys. Subsequently, plasma samples were harvested at different time-points after study drug administration and systemic levels of rVIIa-FP or NovoSeven® were assessed either by FVIIa activity measurements using the Staclot® assay system or by FVII antigen measurements using an enzyme linked immunoabsorbance assay (ELISA). When investigating dose responses or duration of hemostatic activity, rVIIa-FP and NovoSeven® were administered to mice or rabbits before tail clip or induction of venous stasis, respectively. Following a single intravenous bolus application the procoagulant activity of both study drugs was assessed in both models at different time points after administration up to 24 hours to proof prolonged procoagulant effects of rVIIa-FP compared to NovoSeven®.

Intriguingly, in all animal species tested the systemic bioavailability, clearance and in addition the terminal half-life (t_1/2β), of rVIIa-FP were significantly better in comparison to NovoSeven®. Based on FVIIa activity measurements in plasma samples derived from monkeys the 11 fold higher area under the curve (AUC) was accompanied with a 4-fold longer t_1/2β, while in vivo recovery (IVR) of rVIIa-FP exceeded that of NovoSeven® by a 70 %. In rabbits the 20 fold higher AUC was associated with a 5-fold longer t_1/2β and a 4-fold higher IVR when measuring specific FVIIa activity. In mice measurements of FVII antigen revealed a 11 fold higher AUC, while t_1/2β was 4 times longer and IVR was found to be doubled in comparison to NovoSeven®. The assessment of pharmacodynamic activity after dosing based on specific FVIIa activity adjusted by the Staclot^® assay revealed, that the procoagulant effect of rVIIa-FP was not different from NovoSeven®, when measuring total blood loss under acute bleeding conditions after tail clip. However, when assessing late time-points after treatment the prolonged systemic availability of rVIIa-FP translated into a longer and sustained hemostatic activity of rVIIa-FP compared to NovoSeven® in both animal models. Intriguingly, ex vivo studies in hemophilia A mice showed that the 4 fold prolonged systemic availability of rVIIa-FP in plasma translates into sustained FVIIa activity when recorded as thrombin generation activity compared to NovoSeven® at 16 hours after treatment. Furthermore in rabbits, the 5 fold longer half-life translated into a significantly greater procoagulant activity as measured by thrombus formation in both jugular veins after venous stasis.

In conclusion, the recombinant albumin fusion technology was successfully applied to human recombinant FVIIa for a significantly improved of PK/PD profile as observed in different pre-clinical animal species. Future clinical studies can proof whether the observed improved PK/PD characteristics will also translate into a half-life extension and a longer hemostatic effect in hemophilia patients with inhibitors to FVIII and FIX.

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