Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.

Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.

Results:

As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (K_D, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.

Conclusions:

The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency.