Pharmacokinetics and Pharmacodynamics of Recombinant FIX Variants in the FIX Knockout Mouse Tail Clip Bleeding Model

Introduction: The recessive X-linked bleeding disorder Hemophilia B is caused by a mutation in the coagulation factor (F) IX gene leading to partial or total loss of its function. Preventive treatment with replacement long-acting FIX is an attractive option for patients to reduce administration frequency and prevent bleeding. New recombinant FIX therapeutics like the albumin-fused FIX (rFIX-FP) or the Fc-fused FIX (rFIX-Fc) enable longer half-life in circulation and thus less frequent administration, as compared to non-fused FIX (rFIX). Studies in FIX knockout (KO) mice were conducted to characterize the effect of the modifications on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the different recombinant FIX products.

Methods: Pharmacokinetics: Recombinant FIXs were administered intravenously at doses of 25 nmol/kg (corresponding to ~175-400 IU/kg FIX clotting activity) to FIX KO mice. Blood samples were collected starting at 5 min, and up to 336 h. FIX plasma levels were measured with an ELISA-based assay with anti-human FIX paired antibodies. PK was evaluated by non-compartmental analysis. Biodistribution: ³H-labeled recombinant FIXs were administered intravenously at doses of 200 IU/kg to FIX KO mice. Plasma levels and organ distribution were quantified starting at 15 min, and up to 240 h. Pharmacodynamics: FIX KO mice were treated intravenously with 50 IU/kg FIX clotting activity (nominal or labeled potency) of different rFIX products at 24, 72, 120 168 and 336 h prior to determination of bleeding time and total blood loss in a tail clip bleeding model. Immediately upon lesion, the tail tip was submerged in isotonic saline (0.9 %), kept at the mice physiological body temperature. Time to hemostasis is quantified as the time until bleeding stops for a minimum of 2 min. The volume of total blood loss was calculated by measuring the hemoglobin present in the isotonic saline solution at the end of the experiment.

Results: Distinct PK profiles were observed for the three FIX molecules, where rFIX and rFIX-Fc exhibit an initial rapid distribution phase from plasma, while rFIX-FP showed a monophasic elimination profile up to 120 h post administration (p.a.). In the terminal phase, rFIX levels were quantifiable for up to 48 h p.a., while both; rFIX-FP and rFIX-Fc were measurable in plasma up to 240 h p.a. In line with this, the overall exposure AUC _0-inf is ranked in the following order: rFIX-FP > rFIX-Fc > rFIX.

In the biodistribution study, a similar plasma PK profile was determined. Given the sensitivity of the radioactive method, an exposure plateau was observed for rFIX-Fc, and at lower levels for rFIX, whereas rFIX-FP continued to exhibit monophasic plasma clearance behavior. rFIX-FP exposure in the extravascular space (EVS) was lower than for the other FIX products. This is in line with volumes of distribution (Vss and Vz) which were highest for rFIX-Fc (AUC ranking rFIX-Fc > rFIX > rFIX-FP).

FIX hemostatic efficacy in tail clip model was comparable for all 3 FIXs at the early time points but diverged at later time points post dosing. The blood loss and bleeding time measurements returned to baseline within 168 h for rFIX and rFIX-Fc, while the rFIX-FP group maintained robust hemostatic activity for up to 336 h. In contrast to lowest tissue exposure of rFIX-FP, the plasma AUC for rFIX-FP was highest, compared to FIX-Fc or FIX. In line, efficacy over time was also highest for rFIX-FP, suggesting that tissue exposure might not be the main driver for hemostatic activity.

Conclusion: Different FIX products exhibit divergent PK and PD behaviors. rFIX-FP plasma PK profile suggests somewhat lower tissue distribution in comparison to rFIX-Fc and rFIX, which was confirmed in the tissue biodistribution study. Despite its limited extravasation into tissue, rFIX-FP exhibits superior and prolonged hemostatic activity in the FIX KO mouse tail clip model. rFIX and rFIX-Fc show comparable tissue biodistribution behavior, with robust extravasation into the EVS. Despite having the longest half-life and overall (plasma and tissue) exposure in the mouse, rFIX-Fc lost hemostatic activity in the tail clip model significantly faster than rFIX-FP. As a result, hemostatic efficacy was highest for the FIX-FP, the product with the lowest distribution volumes. The results therefore suggest that EVS is not the main determining factor for FIX efficacy in vivo.