Ditte M. Karpf

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.

Plasma elimination kinetics for factor VII are independent of its activation to factor VIIa and complex formation with plasma inhibitors.

The mechanism for the elimination of factor VII (FVII) from the circulation is unknown, just as it is unclear how activation of FVII to FVIIa and subsequent complex formation with antithrombin III (AT) or alpha2-macroglobulin (alpha2M) affects clearance. The possibility that the clearance mechanism involves activation and inhibitor complex formation as obligatory intermediate reactions is examined in this study. Human and murine sera were spiked with human FVIIa in the absence and presence of heparin and analysed for complex formation. Complex formation in vivo was studied after intravenous injection of (125)I-VIIa in mice; and the pharmacokinetics (PK) of human and murine FVIIa was studied in normal mice. Furthermore, comparative PK studies were performed with FVII, FVIIa, active site blocked FVIIa and a preformed FVIIa-AT complex in normal and alpha2M-deficient mice. The data demonstrated that FVIIa-AT complexes and to a much lesser extent FVIIa-alpha2M-complexes accumulated in vivo after FVIIa administration. FVIIa-AT accounted for about 50% of total FVIIa antigen left in the circulation after 3 hours. All FVII derivatives studied including FVII, FVIIa and FVIIa-AT were cleared with similar rates suggesting an elimination kinetics which is unaffected by FVII activation and subsequent inactivation by plasma inhibitors.

Pharmacokinetics and pharmacodynamics of a new recombinant FVIII (N8) in haemophilia A mice

Summary.  N8 is a new recombinant factor VIII (rFVIII) compound produced and formulated without human‐ or animal‐derived protein. The aims of the present studies were to evaluate the pharmacokinetics and pharmacodynamics properties of N8 and to compare with a commercially available rFVIII product (Advate®) in haemophilia A mice. The pharmacokinetics were evaluated after single i.v. administration of 80, 120 and 280 IU kg−1 of N8 and Advate® and measurements of FVIII blood concentrations as a function of time. The efficacy and dose response curves of N8 and Advate® (1–200 IU kg−1) were evaluated in a tail bleeding model. Furthermore, the effects in a newly developed haemophilia knee joint haemarthrosis model were investigated. No significant differences were found in the pharmacokinetic parameters between N8 and Advate®. The clearances were 11 ± 1 vs. 10 ± 2 mL h−1 kg−1 (P = 0.14) and the half‐lives 7.2 ± 0.9 vs. 7.7 ± 1.4 h (P = 0.31) after administration of N8 and Advate® respectively. Dose‐independent pharmacokinetics was shown, and comparable efficacy and potency were shown between N8 and Advate® in the tail bleeding model. Both compounds normalized the bleeding at the dose of 200 IU kg−1, and for blood loss ED50 values of 27 IU kg−1 (N8) and 28 IU/kg (Advate®) were found (P = 0.97). In the haemarthrosis model, treatment with N8 and Advate® at 200 IU kg−1 reduced the mean increase in the joint diameter significantly from 1.23 ± 0.19 to 0.32 ± 0.08 mm (P < 0.01) and 0.25 ± 0.08 mm (P < 0.001) respectively. Pharmacokinetics and pharmacodynamics of N8 and Advate® were comparable after i.v. administration to haemophilia A mice.

Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa‐ and FVIIa‐AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t½ ≈ 1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t½ ≈ 0·5 h) pro‐coagulant response, whereas rFVIIa induced a lower, longer lasting (t½ ≈ 1·1 h) response. Western Blot and FVIIa‐AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.

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.

Pharmacokinetics and ex vivo whole blood clot formation of a new recombinant FVIII (N8) in haemophilia A dogs

Summary.  N8, a new recombinant factor VIII (rFVIII) compound developed for the treatment of haemophilia A, is produced in Chinese hamster ovary (CHO) cells and formulated without human‐ or animal‐derived materials. The aim of the present study was to compare the pharmacokinetics (PK) and the procoagulant effect, measured by ex vivo whole blood clot formation, of N8 and a commercial rFVIII in a cross‐over study in haemophilia A dogs. N8 and Advate® (100 IU kg−1) were administered intravenously to three haemophilia A dogs. Blood was sampled between 0 and 120 h postdose and FVIII:C analysed. PK parameters maximum plasma concentration, area under the curve, half‐life (t½), clearance, mean residence time (MRT) and volume of distribution and incremental recovery were calculated. Whole blood clotting time (WBCT) and thromboelastography (TEG®) were used to determine the haemostatic potential. No adverse reactions were observed with N8 or Advate®. N8 and Advate® exhibited similar PK parameters, with t½ 7.7–11 h and MRT 11–14 h. Both rFVIII compounds corrected the prolonged WBCT (>48 min) to the range of normal dogs (8–12 min), i.e. N8 to 7.5–10.5 min and Advate® to 7.5–11.5 min. N8 and Advate® also normalized the whole blood clot formation according to TEG®. The native whole blood clotting assays (WBCT, TEG®) appeared to be more sensitive to low concentrations of FVIII than assays in citrated plasma samples. In conclusion, comparison of N8 and Advate® in haemophilia A dogs revealed similar safety, similar PK and similar effects in whole blood clot formation assays.

In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver

INTRODUCTION Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes. MATERIALS AND METHODS A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB). RESULTS Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2M). CONCLUSIONS Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2M.

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

Clearance of rFVIIa and NN1731 after intravenous administration to Beagle dogs

AIM NN1731 is a recombinant activated factor VII (rFVIIa) analogue with enhanced activity. The objective of the present study was to evaluate the clearance mechanisms of rFVIIa and NN1731 after intravenous administration to Beagle dogs. METHODS The study was performed in Beagle dogs administered with a single dose of 5.4 nmol/kg rFVIIa or NN1731 intravenously. Plasma samples collected up to 12-h post-administration were analysed using three different assays to determine FVIIa clot activity (FVIIa:C), total FVIIa antigen, and levels of FVIIa-antithrombin (AT) complexes. Pharmacokinetic parameters were determined by use of standard non-compartmental and non-linear mixed effects methods. RESULTS For both compounds, complex formation with AT accounted for the observed difference between the activity and the antigen curves and constituted 60-70% of the total clearance. The clearance of rFVIIa and NN1731 was estimated to be 73 and 214 mL/h/kg, respectively, accordingly, AT complex formation occurred around three times faster for NN1731. The difference in activity observed in the initial phase, resulting in distribution half-lives of 0.71 and 0.22 h for rFVIIa and NN1731, was mainly caused by the 3-fold difference in clearance. The terminal half-life of rFVIIa and NN1731 was estimated to be 2.1 and 2.5 h, respectively. The non-compartmental analysis resulted in almost identical parameters. CONCLUSION The present study demonstrates that the difference between the activity and the antigen profiles of rFVIIa and NN1731 in Beagle dogs is the result of complex formation with AT which constitutes a major pathway for the clearance of rFVIIa activity.

Activation peptides prolong the murine plasma half-life of human factor VII.

Coagulation factors VII (FVII), IX (FIX), X (FX), and protein C share the same domain organization but display very different plasma half-lives. It is plausible that the half-life is influenced by the activation peptide, differing in length and glycosylation and missing in FVII. To test this hypothesis, the influence of activation peptides on the plasma half-life of human FVII was studied by administering human FVII variants containing activation peptide motifs to mice. Insertion of the activation peptide from FX gave 4-fold longer terminal half-life (5.5 hours vs 1.4 hours for FVII), whereas the activation peptide from FIX and protein C resulted in half-lives of 4.3 and 1.7 hours, respectively. Using FXs activation peptide we identified the N-linked glycans as structural features important for the half-life. The peptide location within the FVII molecule appeared not to be critical because similar prolongation was obtained with the activation peptide inserted immediately before the normal site of activation and at the C-terminus. However, only the latter variant was activatable, yielding full amidolytic activity and reduced proteolytic activity with preserved long half-life. Our data support that activation peptides function as plasma retention signals and constitute a new manner to extend the half-life of FVII(a).