Glycopegylated Factor IX Enables Prolonged Efficacy After Subcutaneous Injection But Requires a Higher Than Expected Plasma Activity To Prevent Bleeding In Hemophilia B Mice

Reduced frequency of administration as well as subcutaneous (s.c.) injection would improve the treatment of Hemophilia B. Conjugation to polyethylene glycol (PEG) has been shown to increase the half-life of i.v. dosed Factor IX (FIX), but s.c. dosing of PEGylated FIX was not previously evaluated. Because s.c. dosing is limited by volume and bioavailability, we evaluated the combination of PEGylation with the increased specific activity variant R338A to reduce the amount of protein needed to provide therapeutic levels of FIX.

FIX-R338A was PEGylated on N-linked glycans in the activation peptide by periodate oxidation of sialic acid residues followed by conjugation to amino-oxy functionalized PEG. Pharmacokinetic (PK) profiles were determined in hemophilia B mice and cynomologous monkeys. Allometric scaling was used to predict dose regimens in humans. Prophylactic efficacy was determined in a Hemophilia B mouse tail bleeding model.

60kDaPEG-R338A had prolonged terminal half-life in mice (3-fold) and monkeys (5-fold) and the s.c. bioavailability was 44% and 35%, respectively. The volume of distribution was reduced 5-fold. To achieve a trough level of 3% FIX activity, s.c. dosing at weekly, bi-monthly and monthly intervals was predicted to require doses of 7, 25 and 220 IU/kg in patients. However, in a tail vein transection injury model, approximately 10-fold higher plasma FIX activity levels of PEGylated proteins were found to be needed to protect hemophilia B mice against bleeding than was required for i.v. dosed un-PEGylated recombinant FIX. This difference was observed for PEGylated wild-type and R338A proteins, dosed i.v or s.c. We hypothesize that this is related to the reduced distribution of PEGylated FIX to the extravascular compartment. Trough levels of 30% FIX activity were predicted to be achievable in humans after weekly and bi-monthly s.c. dosing at 70 and 260 IU/kg.

The PEGylation of FIX led to a significant improvement in both i.v. and s.c. PK. Unexpectedly, a 10-fold higher plasma activity was needed for PEGylated FIX to provide protection against bleeding in Hemophilia B mice, suggesting that trough levels of 10 to 30% of PEGylated FIX activity may be needed in patients to provide efficacy equivalent to current therapy of recombinant or plasma derived FIX. Nevertheless, 60kDaPEG-R338A has the potential to treat hemophilia B patients with once weekly or twice monthly subcutaneous injection.

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