Heidi L. Holmberg

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Faster onset of effect and greater efficacy of NN1731 compared with rFVIIa, aPCC and FVIII in tail bleeding in hemophilic mice

Summary.  Background: Recombinant factor VIIa (rFVIIa, Novoseven®) is currently used to control bleeding in hemophiliacs with inhibitors. A new rFVIIa variant, NN1731, with increased activity on the surface of activated platelets, has demonstrated a more potent and faster onset of reactivity than rFVIIa in various in vitro models. The present study aimed to investigate whether this translates into greater efficacy and faster promotion of hemostasis in vivo. Method and results: In a severe tail‐bleeding model in hemophilia A mice, NN1731 demonstrated significantly greater efficacy than rFVIIa, plasma‐derived activated prothrombin complex concentrate (pd‐aPCC, FEIBA®) or FVIII (Refacto®). Assessment of the blood loss over time showed that NN1731 significantly and dose‐dependently reduced the blood loss in the first 5‐min observation period, whereas the effect of rFVIIa, FVIII and pd‐aPCC first became evident 5–10 min after injury. Conclusion: This study shows that NN1731 has a greater efficacy and faster resolution of bleeding in a severe bleeding model in hemophilia A mice compared with any of the other agents tested.

Prolonged half-life of glycoPEGylated rFVIIa variants compared to native rFVIIa

INTRODUCTION Bleeding episodes in haemophilia patients with inhibitors are primarily treated with by-passing agents such as recombinant activated FVII (rFVIIa). Prophylactic treatment with rFVIIa has been shown to significantly reduce the number of bleeding episodes as compared to conventional on-demand haemostatic therapy, and a reduced dosing frequency could present an improved treatment option in inhibitor patients. MATERIALS AND METHODS A series of glycoPEGylated rFVIIa derivatives (5-40K PEG) has been produced and their effect and pharmocokinetics have been investigated in several animal species. RESULTS The glycoPEGylated rFVIIa derivatives exhibit significant prolongation of half-life in mice, dogs and pigs as measured by rFVIIa clot activity. The clearance of rFVIIa, rFVIIa-5K PEG, rFVIIa-10K PEG, rFVIIa-20K PEG and rFVIIa-40K PEG in minipigs were estimated to 59, 27, 22, 8.7 and 3.1 ml/h/kg, respectively. Across species a reduction in clearance as a function of the size of the attached PEG was observed. By allometric scaling, the compiled pharmacokinetics predicts a human half-life for rFVIIa-10K PEG and rFVIIa-40K PEG of approximately 7 and 12h, respectively. The rFVIIa-10K PEG and rFVIIa-40K PEG are efficacious in stopping a bleed in the haemophilia A mouse tail-bleeding model after intravenous administration. CONCLUSIONS GlycoPEGylation of rFVIIa significantly increases the rFVIIa exposure in three animal models, glycoPEGylated rFVIIa compounds are effective in vivo and thus, represents a potential prophylactic treatment option for patients with inhibitors.

A novel F8 -/- rat as a translational model of human hemophilia A.

In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed.

Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice.

Blood collection in mice can be a challenge, in particular for samples used for coagulation analysis as initiation of coagulation during the procedures can influence the results. Blood collection from the retrobulbar venous plexus is commonly used but the method remains controversial. Several alternatives exist but not all are applicable to mice with a compromised coagulation system because of subsequently excessive bleeding. We therefore wanted to explore whether blood collection by puncture of the submandibular vein could replace blood collected from the retrobulbar venous plexus during pharmacokinetic and pharmacodynamic studies in mice lacking coagulation factor VIII (FVIII). The plasma concentrations of recombinant activated factor VII were independent of the blood collection method in a pharmacokinetic study. The same applied to the thromboelastographic profile of mice with normal coagulation in a pharmacodynamic study. However, excessive haemorrhages were observed in all FVIII knockout mice after a single puncture of the submandibular vein and 60% of the mice were euthanized 2–4 h after the blood collection. In contrast, no or only slight haemorrhage was observed in animals subjected to blood collection from the retrobulbar venous plexus. No signs of distress determined by blood glucose level or clinical abnormalities of the eye were observed after puncture of the retrobulbar venous plexus. In conclusion, blood collected by puncture of the submandibular vein and retrobulbar venous plexus has a quality which allows it to be used in coagulation assays. However, because of excessive bleedings, puncture of the submandibular vein is not recommended in mice lacking FVIII.

GlycoPEGylated rFVIIa (N7-GP) has a prolonged hemostatic effect in hemophilic mice compared with rFVIIa

Recently, a prospective randomized trial showed that prophylactic treatment with daily intravenous (i.v.) injections of activated coagulation factor VII (rFVIIa; NovoSeven , Novo Nordisk A/S, Maaloev, Denmark) reduced the number of bleeding episodes by approximately 50% in frequently bleeding hemophilia patients with inhibitors [1]. Development of an rFVIIa derivative with a prolonged circulating half-life in plasma could provide hemostatic coverage with less frequent dosing. Modification of proteins with hydrophilic polymers such as polyethylene glycol (PEG) is an established method for reducing the clearance of proteins and has successfully been used on several marketed therapeutic proteins [2]. Extending the plasma half-life for rFVIIa by random PEGylation on the lysine chains has previously been reported [3]. Yet, the coagulation activity of the PEGylated protein using this approach was greatly diminished indicating that non-selective PEGylation at amino groups affects the ability of rFVIIa to interact with macromolecules and cellular receptors that are important for the activity of rFVIIa. Both a tissue factor [4] and platelet surface-mediated [5,6] pathway have been proposed to explain the hemostatic effect of rFVIIa. Evidence is accumulating that the dominant mechanism of action for rFVIIa as a bypassing agent in the treatment of hemophilia is on the platelet surface [7,8]. At pharmacological doses, rFVIIa binds to the surface of activated platelets at the site of injury and is able to directly activate factor (F)X and thereby enhance local thrombin generation, platelet activation and the formation of a localized stable hemostatic plug [9]. The technology utilized to generate glycoPEGylated rFVIIa (N7-GP) allows selective and reproducible PEGylation of a naturally occurring N-glycan in rFVIIa [10] in which the protein structure is unaltered and indistinguishable from native rFVIIa. The positioning of the PEG group on the flexible N-glycan may accommodate for sufficient interaction of N7-GP with tissue factor and FX. This may be the reason why N7-GP retains its catalytic activity in vitro [10,11]. The scope of the present studies was to investigate the hemostatic effect of N7-GP in murine whole blood and the duration of the hemostatic effect in vivo in hemophilic mice. Our data demonstrate that N7-GP retains its efficacy for inducing clot formation in whole blood from hemophilic mice although higher concentrations were required to obtain a maximal effect (Fig. 1A) as compared with rFVIIa. The EC50value for the clot propagation phase was 2.8-fold greater for N7-GP (2.5 lg mL) than for rFVIIa (0.9 lg mL) when the concentration-dependent effect was compared using thromboelastography [12]. Concentrations stated in the current work are based on the protein concentrations and do not take into account the added PEG group. After i.v. injection of an equimolar dose of N7-GP and rFVIIa to mice (10 mg kg; Fig. 1B), a significantly higher plasma FVIIa activity (Cmax) was observed for rFVIIa (74.3 lg mL) than for N7-GP (15.1 lg mL). However, after 90 min and the remainder of the observation period the plasma FVIIa activity in mice dosed with N7-GP was higher than in mice dosed with rFVIIa. This is as a result of decreased clearance of N7-GP, which has a six-fold increased circulating half-life (T1⁄2) compared with rFVIIa (Fig. 1B). Altogether, this results in an increase in the total exposure (AUC0–¥) of N7-GP (83.385 h · lg mL) compared with rFVIIa (58.424 h · lg mL). A similar increase in T1⁄2 has also been reported in other animal species [13] and in humanswhere theT1⁄2ofN7GP was reported to be 15 h [14] compared with 2–4 h for rFVIIa [15]. Thus, we wanted to investigate if the increased duration of activity of N7-GP translated into a longer duration of hemostatic effect. Initially, the duration of the hemostatic effect was studied by thromboelastography using whole blood fromhemophiliamice treated with either rFVIIa or N7-GP (20 mg kg, i.v.). Both molecules significantly increased the clot propagation rate (Fig. 1C) and reduced the clotting time (data not shown). The effect of N7-GP was sustained for the full 24-h observation period on both parameters. In contrast, the effect of rFVIIa was reduced after 4 and 6 h for the clot propagation and clotting time, respectively, and no effect was recorded 24 h after dosing with rFVIIa. Correspondence: Heidi Holmberg, Haemostasis Pharmacology, Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark. Tel.: +45 30 75 26 76; fax: +45 44 49 05 55. E-mail: HLiA@novonordisk.com

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

Significance Coagulation factor VIIa (FVIIa) is an intrinsically poor serine protease that requires assistance from its cofactor tissue factor (TF) to trigger the extrinsic pathway of blood coagulation. TF stimulates FVIIa through allosteric maturation of its active site and by facilitating substrate recognition. The surface dependence of the latter property allowed us to design a potent membrane-triggered activity switch in FVIIa by engineering a disulfide cross-link between an allosterically silent FVIIa variant and soluble TF. These results show that optimization of substrate recognition remote from the active site represents a promising new route to simultaneously enhance and localize the procoagulant activity of FVIIa for therapeutic purposes. Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.