Preclinical Pharmacology of Albumin-Free B-Domain Deleted Recombinant Factor VIII

Abstract

A second-generation recombinant factor VIII molecule was developed with an albumin-free formulation. In this modified form of factor VIII, the N- and C-terminal sections of the B-domain are retained and fused at serine 743 and glutamine 1638, resulting in a B-domain deleted factor VIII protein known as ReFacto (Genetics Institute, Andover, MA). Preclinical studies of ReFacto have focused on efficacy of the product for the hemophilia A patient population. The efficacy and pharmacokinetic profiles of ReFacto were similar to plasma-derived factor VIII in correcting the hemostatic defect of hemophilia A dogs. Both ReFacto and plasma-derived human factor VIII (Octonativ-M7, Pharmacia, Stockholm, Sweden) were found to associate with von Willebrand factor (vWF) after infusion into hemophilia A dogs as demonstrated by size exclusion chromatography. Infusion of either ReFacto or Octonativ-M7 quickly corrected factor VIII coagulant activity (FVIIIc), whole blood clotting time (WBCT), and activated partial thromboplastin time (aPTT). No obvious differences were seen between ReFacto and Octonativ-M7. Both ReFacto and Octonativ-M7 treatment reduced secondary bleeding time to less than 6 minutes. The clearance was faster and the volume of distribution at steady state was larger for plasma-derived factor VIII compared with ReFacto. The half-life was similar between Octonativ-M7 and ReFacto. These data predict that ReFacto will be effective in correcting human factor VIII deficiency states.

Recombinant canine B-domain–deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model

“…Together these data demonstrate that cFVIII-BDD is safe and efficacious in inducing sustained hemostasis in vivo and has a protein half-life comparable with the pharmacokinetics of hFVIII-BDD in HA dogs and from clinical experience in humans. 20 The use of these outbred immunocompetent HA dogs provides an ideal model to test the immunogenicity of cFVIII-BDD protein in both naive neonates and adult dogs previously exposed to plasma-derived cFVIII. These dogs do not develop antibodies to cFVIII on infusion of plasma-derived cFVIII.…”

“…These data are in contrast to the strong immune responses of adult HA dogs to hFVIII characterized by long-lasting antibody to hFVIII after exposure to the protein or after delivery of hFVIII gene-or cell-based therapies. 1,17,20,21 Thus, cFVIII-BDD presents no immunogenicity in this pivotal HA dog model, which is essential for determining long-term efficacy and safety of novel therapeutic strategies for HA.…”

A review of current methods for assessing hemostasis in vivo and introduction to a potential alternative approach

“…Pre-clinically, conventional in vivo tests to assess the hemostatic efficacy of therapeutic agents include the primary saline bleeding time (1°SBT) in VWD dogs and the primary cuticle bleeding time [19,20], the gingival biopsy bleeding time [21], and the secondary cuticle bleeding time (2° CBT) [22–24] in hemophilia A dogs. Several variations of the primary bleeding time have been used to detect disorders of primary hemostasis such as von Willebrand disease (VWD) [25,26] and monitor the response to therapy.…”

ARFI ultrasound for in vivo monitoring of soft-tissue bleeding and hemostasis in a dog model of hemophilia

“…Such a method is critical for documenting hemostatic efficacy of gene therapy and infused replacement products and conversely detecting treatment failure at bleeding sites that would justify additional interventions. The cuticle bleeding time has historically been used as an assessment of the hemostatic potential of infused products in animal bleeding models [3][4][5], but it is very difficult to reproduce in a given dog and can vary greatly among normal and hemophilic dogs. Ex vivo plasma-based assays can detect the presence or absence of clotting factor activity but do not measure hemostasis in vivo.…”