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