Introduction/Objective: Subcutaneous (SC) drug delivery represents a convenient method of administration compared to intravenous (IV) delivery. However, due to very low bioavailability this route is not yet applicable for the administration of FVIII as a therapy for hemophilia A (HA). von Willebrand factor (VWF), in particular the D'D3 domains, is known to protect FVIII from proteolytic degradation, rapid clearance and binding to membranes. Therefore VWF represents a promising candidate for a strategy to increase FVIII bioavailability after SC treatment. Different VWF fragments containing at least the D'D3 domains were engineered in order to achieve high FVIII binding affinity, prolonged half-life of the VWF fragment, and consequently high bioavailability of FVIII.
Materials/Methods: A modified VWF fragment (OCTA12) or an unmodified D'D3 domain, both with a C-terminal Strep-Tag, was transiently expressed in HEK293 cells and purified by Strep-Tactin® affinity chromatography. Recombinant human FVIII (rhFVIII, simoctocog alfa) complexed with OCTA12, D'D3 or full-length plasma-derived VWF (flVWF, Wilate®) was administered via IV route to FVIII/VWF double knockout (FVIII/VWF DKO) mice at 240 IU FVIII/kg in order to test the ability of the fragments to stabilize FVIII in circulation. Blood samples were taken at 5 minutes and 1, 4, 8, 12, 24, 36 and 48 hours after treatment and analyzed for FVIII activity. Subsequently, IV and SC administration of the complex of rhFVIII and OCTA12 or rhFVIII alone was evaluated in a HA dog model. For this purpose, HA dogs were treated according to the scheme presented in Table 1. Blood samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 32, 48, 72 and 94 hours after treatment and analyzed for FVIII activity.
Results: Five minutes after IV administration of rhFVIII complexed with different VWF variants to FVIII/VWF DKO mice, peak activity levels were determined (expressed in % mean ± SEM, n = 15): rhFVIII alone 78.9 ± 6.1; rhFVIII/D'D3 351.2 ± 22.3; rhFVIII/OCTA12 295.9 ± 17.4; rhFVIII/flVWF 404.4 ± 20.5. The FVIII activity levels were significantly higher (statistically evaluated by Student's t-test) when rhFVIII was administered with any VWF variant in comparison to rhFVIII administered alone. The three co-administered VWF species D'D3, OCTA12 or flVWF did not differ significantly in terms of measured FVIII levels. rhFVIII administered alone was rapidly cleared from the circulation (T1/2 = 0.07 h). Co-administration with flVWF or D'D3 resulted in a 4.5- or 6.4-fold prolongation of the half-life respectively (T1/2 = 0.32 h and T1/2 = 0.45 h respectively). The highest half-life prolonging effect (14.7-fold) was measured when rhFVIII was co-administered with OCTA12 (T1/2 = 1.03 h). The IV administration of rhFVIII alone (200 IU/kg) or complexed with OCTA12 (100 IU/kg) in the HA dog resulted in peak FVIII activity level of 215% or 134% respectively at 30 minutes post-infusion (mean of two injections). The SC administration of rhFVIII complexed with OCTA12 resulted in a 10.8% activity level at 30 minutes post-infusion and reached a peak level of 24.6% at 8 h post-infusion (mean of three injections). For calculation of bioavailability, the area under the curve (AUC) of FVIII activity after IV administration of rhFVIII alone was defined as 100%. The AUC of IV-administered rhFVIII/OCTA12 complex at 100 IU/kg (half the dose) was 90%. Bioavailability of FVIII after SC administration of the rhFVIII/OCTA12 complex was 37.7%.
Conclusions: The co-administration of rhFVIII with the novel engineered VWF fragment OCTA12 results in high FVIII bioavailability after SC administration in HA dogs. The bioavailability of FVIII was also increased after IV administration of the FVIII/OCTA12 complex in FVIII/VWF DKO mice and HA dogs. Moreover, when complexed with OCTA12, FVIII exhibits prolonged half-life in FVIII/VWF DKO mice.
Solecka-Witulska: Octapharma Biopharmaceuticals GmbH: Employment. Kannicht: Octapharma Biopharmaceuticals GmbH: Employment.
Asterisk with author names denotes non-ASH members.