Evaluation of PEG-FVIII Molecules with Prolonged Half-Lives in a Murine FVIII-Dependent Bleeding Model.

Abstract Current treatment of patients with hemophilia A often requires the frequent infusion of Factor VIII (FVIII) due to its short circulating half-life. A longer-acting FVIII molecule could profoundly impact patients’ lives by extending bleeding protection with a reduced frequency of infusions. Several strategies to prolong plasma concentrations of FVIII have been attempted. In particular, targeting domains on FVIII that bind to LRP, the putative clearance receptor, has been a popular strategy. We have investigated the use of site-directed pegylation of B-domain deleted (BDD) FVIII to evaluate the utility of PEG as a method to decrease FVIII clearance through steric hindrance of LRP binding, or other unknown clearance mechanisms, while minimizing decreases in vWF binding and in vivo activity. The evaluation of novel constructs required the development of in vivo pharmacokinetic models and a FVIII-dependent bleed model. We describe the development of an acute bleed model following uniform tail transection in the hemophilia A mouse that is FVIII dependent and allows the evaluation of the acute pharmacologic effects of FVIII or variants in vivo. Pharmacokinetic analysis of recombinant FVIII (rFVIII) and its variants was performed in rabbits over 32-hours and rFVIII or variants were measured using a modified Coatest® to differentiate endogenous rabbit FVIII from the administered human FVIII. For efficacy evaluations, hemophilia A mice were anesthetized with isoflurane and their pre-warmed tail was cut by a scalpel and placed into a new tube of warmed saline (37–40°C). Blood was collected over 40 minutes and blood loss was measured gravimetrically. Three modes of treatment were evaluated: prevention of bleeding (drug was administered 5 minutes before injury), treatment of an acute bleeding event (drug was administered 5 minutes after injury), and a delayed injury model (tail cut occurred at 20 or 24 hours after the drug administration). Over the course of 40 minutes control (C57BL6) mice demonstrated negligible bleeding (approximately 41 ± 8 μL) compared to 919 ± 26 μL in hemophilia A mice. A dose response curve was constructed for doses ranging from 0.1 to 5.0 IU of human rFVIII per mouse. Hemophilia A mice treated with 200 IU/kg of human rFVIII (5 IU/mouse) lost a similar volume of blood as control mice. The protective effect was rFVIII dose dependent over a range of 4–200 IU/kg (0.1–5 IU/mouse). In contrast, more rFVIII was required to stop an acute bleeding event when administered after the injury. In the delayed injury model, mice injured 24 hours after drug administration had a significantly larger mean blood volume loss compared to mice injured 20 hours post drug administration. Pegylated rFVIII constructs with longer half-lives also had increased activity over time compared to non-pegylated rFVIII in this mouse model. These results describe a superior hemophilia A tail bleed model that demonstrates FVIII-dependent bleeding reduction in response to acute hemorrhage over a 40 minute time course. This is the first demonstration of a hemophilia A mouse model in which all untreated animals uniformly bleed and all control animals demonstrate negligible bleeding. This model was used to evaluate the in vivo hemostatic efficacy of new rFVIII molecules that were designed to have superior pharmacologic and/or pharmacokinetic properties compared to rFVIII.