At Equal Doses FIXFc (Alprolix) Is Not Significanly Better in a Saphenous Vein Bleeding Model Than Recombinant FIX (BeneFix) Seven Days Post-Infusion

The use of extended half-life coagulation factors is predicated on the use of less frequent infusions to maintain a therapeutic plasma level.  Factor IX presents a problem to this approach because much of the FIX is sequestered in the extravascular space in significantly greater quantities than that found within the circulation. Extravascular FIX may be important for either extended clotting activity, enhanced catalytic activity, or both. We previously demonstrated that: 1) FIX binds tightly to Collagen IV; 2) variant FIX with increased Collagen IV affinity (FIX_K5R) protects mice from bleeding better than wild-type (wt) FIX; 3) knock-in mice expressing a variant FIX with decreased Collagen IV affinity (FIX_K5A) have a bleeding diathesis. This is despite the fact that plasma activity of FIX_K5A expressed as a knock-in the Hem B mouse, is not only normal and functional, it is present in the plasma of these animals at slightly greater levels than FIX in normal mice.

Here, we report a direct comparison of the hemostatic efficacies of FIXFc (Alprolix) and recombinant FIX_wt (BeneFix) in a murine saphenous vein bleeding model in which hemostasis was tested at 7 days post-infusion. Although the terminal half-life of FIXFc is significantly longer than BeneFix, Alprolix at equivalent doses is not significantly better at correcting bleeding (Fig. 1a). This unexpected result could be explained if the larger size, and/or cycling through the Fc receptor, renders FIXFc less accessible to the extravascular space.

If binding to Collagen IV is physiologically relevant, we posited that more BeneFix would be required to achieve hemostasis in mice expressing a Collagen IV-binding FIX that is catalytically defective, compared to mice with complete FIX deficiency. This is because the infused, catalytically active FIX must compete with the endogenous, catalytically defective FIX for binding to Collagen IV. We confirmed this hypothesis using the saphenous vein bleeding model in a knockout mouse expressing only human FIX_R333Q, which binds Collagen IV normally but is catalytically defective (Figure 1b). Clearly more FIX is required to achieve an equivalent clotting effect in mice expressing defective FIX that binds collagen IV normally.

The extravascular space contains approximately 4 to 5 times more FIX than is present in the circulation. Thus, it would be expected that at least 4-5 times more infused FIX than current dosing regimens (40-60 IU/kg) would be required to obtain the maximum hemostatic effect and duration. To this end, we compared the hemostatic effect of 50, 100, 150, 200, and 250 IU/kg of BeneFix, 7 days post-infusion. The median value for the number of times that the saphenous vein can re-clot increases as the dose increases from 50 to 150 IU/kg with noticeable leveling off after this (Figure 2); at least 16 mice were used for each dose.

Little or no consideration is given to the amount of extravascular FIX and the role of FIX's binding to Collagen IV in current dosing regimens. Based upon the results presented here, we predict that considerably greater doses of FIX will contribute to a longer protective effect. Furthermore, given the apparent importance of FIX's binding to Collagen IV, it is likely that still higher doses will be required for patients whose defective FIX binds normally to type IV Collagen. Finally, at equivalent doses, in mice, Benefix is not statistically different from Alprolix in bleeding protection 7 days post-injection. This observation may be explained by a different physical location of the two molecules.