Objective:
TFPI is a potent inhibitor of the tissue factor (TF)-induced extrinsic pathway of coagulation. Inhibition of TFPI with antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option for non-i.v. treatment of patients with hemophilia including those with inhibitors.
Method:
We developed a TFPI inhibitory fusion peptide (FP) consisting of a linear and a cyclic peptide connected by an optimized linker. The two peptides bind to different epitopes on TFPI and synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitro inhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia.
Results:
HL-FP bound to and efficiently inhibited TFPI in several in vitro test systems. The binding affinity of < 1nM correlated well with inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. HL-FP efficiently inhibited plasma TFPI, which improved all thrombin generation (TG) parameters in hemophilia A and B patient plasma (EC50s of 6 to 20nM). HL-FP increased peak thrombin levels of hemophilia plasma to a range established for individual normal plasma. Assays were also carried out at flTFPI concentrations up to 10nM, which is 40- to 50- fold above normal. Increased TFPI levels may occur locally upon platelet activation. HL-FP efficiently neutralized elevated TFPI, raising TG to levels observed for inhibition of physiologic TFPI concentrations. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by TG correlated well with HL-FP plasma concentrations. In a repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, indicating that HL-FP did not interfere with TFPI clearance. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma TG, even at low peptide plasma concentrations (25-55nM).
Conclusion:
To summarize, we developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently binding to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia, did not interfere with TFPI clearance receptor interactions, and efficiently neutralized elevated TFPI. Our HL-FP appears to be useful in preventing bleeding in hemophilia, and provides a FVIII and FIX independent approach for non-i.v. treatment.