A TFPI Inhibitory Half Life Extended Fusion Peptide Proves Efficacy in FVIII Knock out Mice and Marmoset Monkeys

Introduction

Tissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia.

Methods

HL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies.

Results

HL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of < 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. 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 CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM).

Summary

We developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. 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.