Novel Von Willebrand Factor (vWF) Fragment Supports Subcutaneous Administration of Factor VIII: Pharmacokinetic Data from Hemophilia A Mouse Model

Patients with hemophilia A are currently treated by intravenous (IV) injections of factor VIII (FVIII) since other routes of administration do not provide sufficient FVIII bioavailability. If given subcutaneously (SC), FVIII will not be efficiently absorbed from the subcutis into vascular circulation but proteolytically degraded. vWF functions as a chaperone protecting FVIII from proteolysis and from binding to phospholipid membranes. However, FVIII complexed with high molecular weight and adhesive vWF would also likely be trapped in connective tissues. Therefore, low-molecular weight vWF fragments consisting of engineered vWF domains were generated and analyzed for their capability to support FVIII subcutaneous administration in hemophilia A mouse. Dimeric forms of vWF fragments consisting of domains D'D3 (OCTA12) and D'D3-A1-A2-A3 (OCTA13) were transiently expressed in HEK293 cell line with a C-terminal Strep-Tag and purified by Strep-tactin affinity chromatography. The fragments were characterized by SDS-PAGE to confirm dimer formation. FVIII binding affinity and ability to inhibit the interaction between FVIII and phospholipid monolayer was investigated by surface plasmon resonance (SPR). F8^-/- mice received single doses of recombinant human (rh) FVIII (simoctocog alfa, Octapharma) either 200 IU/kg IV or rhFVIII 1000 IU/kg SC in the presence of OCTA12 or OCTA13. Plasma samples were collected 1, 3, 6, 9, 24, 32, and 48 hours after injection for pharmacokinetic analysis. Plasma levels of FVIII:C and FVIII:Ag were determined by chromogenic and ELISA assay, respectively. One hour after IV administration, the mean peak FVIII:C activity (C_max) was 133.4 ± 78.1 IU/dl. FVIII:C decreased quickly with a half-life (t_1/2) of 2.5 hours. After SC administration of rhFVIII with OCTA12 or OCTA13, FVIII:C reached a maximum at 6 hours C_max = 49.6 ± 36.7 IU/dl and 26.1 ± 14.4 IU/dl, respectively. FVIII:C was detectable up to 32 h in circulation with OCTA12. FVIII:Ag and activity data were very similar for both IV and SQ administration. Estimated bioavailability of rhFVIII SC with OCTA12 and OCTA13 was 18.4% and 7.2%, respectively. Engineered vWF fragments enabled absorption of FVIII after SC administration into the blood circulation. Pharmacokinetic profile of rhFVIII with a dimeric D'D3 fragment (OCTA12) was particularly promising demonstrating bioavailability of 18.4% and FVIII:C detectable for up to 32 h after a single subcutaneous administration.