Mette B. Hermit

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8−/−) did not rescue the embryonic death of TFPI null (Tfpi−/−) mice. Tfpi+/− did not alter the bleeding phenotype of F8−/− mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8−/− mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi+/−;F8−/− mice with hematopoietic Tfpi−/− cells compared with Tfpi+/−;F8−/− mice with Tfpi+/+ hematopoietic cells. Additionally, following femoral vein injury, Tfpi+/−;F8−/− mice with Tfpi−/− hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi+/+ hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

Pharmacokinetics of an anti-TFPI monoclonal antibody (concizumab) blocking the TFPI interaction with the active site of FXa in Cynomolgus monkeys after iv and sc administration

INTRODUCTION Concizumab (mAb 2021) is a monoclonal IgG4 antibody (mAb) that binds to the Kunitz-type protease inhibitor (KPI) 2 domain of TFPI thereby blocking the interaction of this domain with the active site of FXa. The objective of the present study was to characterize the pharmacokinetics of concizumab in Cynomolgus monkeys after intravenous (iv) and subcutaneous (sc) administration. METHODS Data from two studies were included in the modelling, all in all data from 52 monkeys distributed into 9 groups. Three groups received three escalating sc doses of concizumab with a one week dosing interval, two groups were administered a single dose, and four groups received multiple doses over 13 weeks of concizumab. The plasma concentration was measured using a standard ELISA, and pharmacokinetic data were analysed using NONMEM. RESULTS The pharmacokinetics of concizumab were characterised by a high bioavailability (93%) after sc administration. The time course of the elimination of concizumab from the circulation was well described by the proposed target mediated drug disposition (TMDD) model. The clearance of concizumab was estimated to be 0.14 ml/h/kg, the target clearance was characterized by a 50% saturation level of 0.54 μg/ml (Km), and the clearance at target saturation was estimated to be 11 μg/h/kg. CONCLUSION Concizumab displays a typical TMDD profile with important implications for a putative treatment regime in haemophilia patients. Compared to current standard haemophilia treatment, concizumab has a high bioavailability after sc administration and may provide a viable alternative to intravenous dosing for the treatment of haemophilia.

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.

Prolonged effect of GlycoPEGylated rFVIIa (40k-PEG-rFVIIa) in rabbits correlates to activity in plasma

The pharmacokinetics and pharmacodynamics of 40k-PEG-rFVIIa, a GlycoPEGylated derivative of recombinant wild-type FVIIa, were compared with rFVIIa in rabbits. The procoagulant effect was determined as the weight of the clot formed in a defined segment of a facial vein. A time course study was conducted where ligation was made 10 minutes, 12 or 24 hours after i.v. injection of equimolar doses of 40k-PEG-FVIIa or rFVIIa (2 mg/kg). This dose was selected based on a dose response study and a duration of effect study with rFVIIa. The clot weight increased with increasing doses of rFVIIa, and the duration of effect correlated with the plasma FVIIa clot activity. The plasma half-life of 40k-PEG-rFVIIa measured as FVIIa clot activity was found to be 25 hours, which was 5-6 times longer than rFVIIa. The aPTT and PT were reduced, and the measured increase in thrombin-antithrombin correlated to the effect on clot formation. Thus, the effect was similar at ligation 10 minutes after administration of 40k-PEG-rFVIIa or rFVIIa. At 12 hours, the effect of rFVIIa was absent while significant effect was seen 12 and 24 hours post dosing with 40k-PEG-rFVIIa. No consumption of platelets or fibrinogen was found and no thrombi formation was seen in histological examination of various organs. In conclusion, 40k-PEG-rFVIIa has shown prolonged duration of effect that correlated to various plasma markers and FVIIa clot activity. In perspective, the data support further clinical development of 40k-PEG-rFVIIa to potentially become a long-acting recombinant treatment option for prophylaxis in haemophilia patients with inhibitors.

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.

Hyperglycosylation prolongs the circulation of coagulation factor IX.

Mutations abolishing or substantially reducing coagulation factor IX (FIX) function lead to hemophilia B, a hereditary bleeding disorder that can be managed by replacement therapy using plasma-derived or recombinant FIX. For prophylaxis, 2weekly infusions are traditionally recommended. Thus, a modified FIX protein with prolonged circulatory half-life allowing less frequent infusions may improve patient convenience and compliance. Several strategies for reducing clearance of FIX have been described. Recombinant FIX compounds prolonged by glycoPEGylation [1], genetic fusion to albumin [2] or genetic fusion to the IgG Fc domain [3] are currently in clinical development. In the present study, we investigated the possibility of prolonging FIX by introducing new N-glycans. Previous studies suggest a possible role for N-glycans in determining the clearance of FIX. Enzymatic removal of N-glycans increased clearance of wild-type recombinant FIX [4,5], and a correlation between N-glycan sialic acid content and terminal half-life of recombinant FIX has been reported [6]. N-glycosylation is anintracellular processcarriedoutbythe oligosaccharyl transferase complex, which glycosylates asparagine residues in N-X-S/T motifs of nascent proteins. Thus, N-glycans can be added to a recombinant protein by introducing mutations that establish N-glycosylation motifs in the amino acid chain. This strategy has previously proven efficient in prolonging the circulatory half-lives of erythropoietin, follicle stimulating hormone, interferon alpha and growth hormone [7].

Evaluating IL-21 as a Potential Therapeutic Target in Crohn’s Disease

Background and Aim Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohns Disease (CD) and could be a potential new therapeutic target in CD. Methods In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R−/− T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. Results In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2−/− mice receiving CD4+CD45RBhighIL-21R−/− T cells developed less severe colitis compared to Rag2−/− mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. Conclusion Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.