Phase 1/2 Trial of Subcutaneously Administered Factor IX Variant CB2679d/ISU304: Pharmacokinetics and Activity

Marketed factor IX (FIX) products require intravenous (IV) administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous (SQ) administration is a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity, which result in 22-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared with recombinant wild-type FIX dosed at the same mass and may allow SQ administration to provide prophylaxis.

Methods: The trial design is provided below. IV pharmacokinetics (PK) (antigen and activity) was sampled at predose, 0, 0.25, 0.5, 1, 3, 6, 9, 24, 48 and 72 hours. SQ PK was sampled at predose, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours. Cohort 5 has PK sampled before each injection, 6 hours after first and 6^th injection and 24 hours after 6^th daily injection. Hematology, chemistry and coagulation was measured at Seoul Clinical Laboratories (Yongin-si, South Korea). FIX antigen and FIX activity, anti-drug antibody to BeneFIX and ISU304 and neutralizing antibody were measured at Haematologic Technologies Inc (Essex Junction, VT). A safety follow-up was done 2 weeks after last visit. FIX antigen was measured using VisuLize^TM Factor IX Antigen KitAG (Affinity Biologicals, Inc, Ancaster, ON, Canada) and FIX activity was measured using a one-stage clotting assay using ACL TOP 700 and Instrumentation Laboratories (Bedford, MA) reagents. The calculation of AUC was based on the trapezoidal rule. To calculate the additional area for AUC0-inf, the log-linear regression line for the last three time points was fit and extrapolated to the x-intercept. The calculation of half-life was based on the use of Demitasse 2000 (version 1.1.3, M. Lee, 2000) which uses an iterative piecewise fitting algorithm based on a robust (M-regression) log-linear model (Lee, 1990, 1997). All activity data were adjusted for baseline before analysis, assuming exponential falloff after IV administration and a half-life of 20 hours. Bioavailability was calculated from the AUC0-t for the IV and SQ data using FIX activity data.

Subject safety was reviewed by an external Data Safety Monitoring Board and also an internal Data Monitoring Committee.

Results: PK and activity of ISU304 demonstrate the 22-fold greater potency over BeneFIX and longer mean residence time (Cohort 1 figure, Table 1). Bioavailability of 18.2-23.6%, SQ beta half-life 66-103 hours and Tmax 6-24 hours (Cohort 2 figure, Table 2) One subject reported transient fever and a mild SQ injection site reaction.

Conclusion: Interim study results support the aim of achieving normal or high mild hemophilia FIX levels in individuals with hemophilia B with repeated SQ dosing. Complete PK and activity and steady-state levels after 6 daily doses will be reported from this phase 1/2 subcutaneous dosing study.

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