Recombinant FVIII formulated in PEGylated liposomes (rFVIII-PEG-Lip) was reported to increase the bleed-free days from 7 to 13 days (at 35 IU/kg rFVIII) in severe hemophilia A patients. To understand the underlying mechanism, we sought to recapitulate its efficacy in hemophilia A mice. Animals treated with rFVIII-PEG-Lip achieved approximately 30% higher survival relative to rFVIII after tail vein transection inflicted 24 hours after dosing. The efficacy of rFVIII-PEG-Lip represents an approximately 2.5-fold higher "apparent" FVIII activity, which is not accounted for by its modestly increased (13%) half-life. The enhanced efficacy requires complex formation between rFVIII and PEG-Lip before the administration. Furthermore, PEG-Lip associates with the majority of platelets and monocytes in vivo, and results in increased P-selectin surface expression on platelets in response to collagen. Rotational thromboelastometry (ROTEM) analysis of whole blood from rFVIII-PEG-Lip-treated animals at 5 minutes up to 72 hours after dosing recapitulated the 2-to 3-fold higher apparent FVIII activity. The enhanced procoagulant activity is fully retained in plasma unless microparticles are removed by ultracentrifugation. Taken together, the efficacy of rFVIII-PEG-Lip is mediated mainly by its sensitization of platelets and the generation of procoagulant microparticles that may express sustained high-affinity receptors for FVIII.

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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.

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Prophylactic therapy with rFVIII has been shown to have a significant positive impact on the treatment of Hemophilia A. One of the impediments to effective prophylaxis is the requirement for frequent injections necessitated by the 12–14 hour circulating half-life of FVIII. We have evaluated a number of approaches to modify FVIII to reduce the need for frequent injections. In one approach, the active form of FVIII was stabilized by addition of a disulfide linkage between the A2 and A3 domains. As previously reported1, these molecules exhibited prolonged FVIII activity in a number of in vitro assays following activation. In vivo characterization of these molecules is in progress. Other approaches focused on increasing the circulating half-life of FVIII. In an attempt to reduce FVIII clearance by the liver receptor LRP, site-directed mutations in the reported LRP binding region of the FVIII A2 domain were generated. Twenty mutants, with single amino acid changes, were analyzed in mouse and rabbit recovery studies and no significant increase in recovery was observed. Since the point mutants may not have covered a large enough surface of the LRP binding domains, polyethlylene glygol (PEG) modification was used to disrupt a larger surface area. Previously, attachment of PEG moieties to FVIII had been shown to lead to an increase in plasma half-life in animal models; however, standard pegylation chemistries result in significant product heterogeneity and thus may not be suitable for commercial production. We have developed a novel method, based on protein engineering, to introduce PEG to specific cysteine residues on the surface of FVIII. Using this method, different molecular weight PEGs have been conjugated to sites in the A1, A2 or A3 domains of FVIII. Analysis of the pegylated proteins confirms that the attachment of PEG is highly specific to the engineered site. The pegylated products retain activity based on the two stage chromogenic assay, but exhibit reduced activity when analyzed by the one stage coagulation assay. Pharmacokinetic studies, performed in mouse and rabbit, show that pegylation increases half-life in a manner that is proportional to PEG molecular weight. Using a number of injury models in hemophilic mice, the pegylated molecules have been shown to be efficacious in stopping bleeds. The prospects for using site-specific pegylation of FVIII to produce a therapeutic for treatment of hemophilia A will be discussed.

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