A next generation FVIII mimetic bispecific antibody, Mim8, the impact on non-factor VIII related haemostasis assays.

INTRODUCTION AND AIMS: Mim8 is a next generation bispecific antibody developed for the treatment of haemophilia A (HA). Mim8 has an increased potency compared to first generation molecules. The impact on Mim8 on non-FVIII measuring haemostasis assays was assessed in plasma containing Mim8. METHODS: Congenital severe HA plasma was spiked with increasing concentrations of Mim8 (0-20 µg/mL). 28 routine and specialist haemostasis assays were used to measure activities. These included tests for prothrombin time (PT), fibrinogen, thrombin, D-dimer, anti-Xa, heparin induced thrombocytopenia (HIT), clotting factors II-XII, factor XIII, von Willebrand factor (VWF), thrombophilia and DRVVT. RESULTS AND CONCLUSIONS: Less than 10 % difference was calculated between plasma without Mim8 and plasma spiked to 15 µg/mL Mim8 in all assays except thrombin time (-10.5%), APTT-based factor IX, XI and XII, Werfen VWF:RCo (10.6%) and Siemens LA1 (-26.4%) and LA2 (-16.9%). At the expected therapeutic steady state levels of Mim8 (5-8 µg/mL), less than 10% difference was calculated for thrombin time and Werfen VWF:RCo. APTT-based assays of FIX, XI and XII are significantly elevated in the presence of Mim8 and should not be performed. A chromogenic FIX assay could be used to accurately measure FIX activity in the presence of Mim8. There was some interference in the DRVVT method we used so local assessment of other DRVVT methods is advised. Differences in all other tests would not be predicted to affect patient management.

In vitro effects of combining Mim8 with factor VIII, FVIIa, and activated prothrombin complex concentrates in thrombin generation assays.

BACKGROUND: Mim8 is a novel antifactor IXa/antifactor X bispecific antibody in clinical development for prophylactic treatment of hemophilia A with and without inhibitors. Patients treated with Mim8 may need supplementary bleed treatment under certain conditions such as surgery or major trauma. OBJECTIVES: This study aimed to better understand the response of Mim8 in thrombin generation assays (TGAs) alone or in combination with other hemostatic proteins. METHODS: We used TGAs with different activators (tissue factor (TF) and activated factor XI) to better understand the similarities and differences between the mode of action of Mim8 and factor VIII (FVIII). Following this, we investigated the effects of mixing Mim8 with the main bleed treatment options for persons with hemophilia A with or without inhibitors: FVIII, activated factor VII (FVIIa), and activated prothrombin complex concentrates (aPCC). RESULTS: The results indicated that for patients without inhibitors, Mim8 does not interfere with FVIII's mode of action. For patients with inhibitors, Mim8 mixed with aPCC results in a strong synergistic effect causing thrombin generation far exceeding the normal levels. Contrary to this, mixing Mim8 with FVIIa results in a more controlled additive effect, visible only when using TF as a trigger, which does not exceed the normal level of thrombin generation. CONCLUSION: These findings support the use of approved clinical doses of FVIIa for bleed treatment of patients with FVIII inhibitors treated with Mim8. Additionally, the findings suggest that concomitant use of FVIII and Mim8 is safe for managing breakthrough bleeds.

The effect of a next generation factor VIII mimetic bispecific antibody (Mim8) on assays of factor VIII activity and thrombin generation.

BACKGROUND: Mim8 is a next generation bispecific antibody developed for the prophylactic treatment of hemophilia A. The sensitivity of activated plasma thromboplastin time (APTT), assays measuring factor VIII activity (FVIII:C) and thrombin generation to plasma containing Mim8 was assessed. METHODS: Congenital severe hemophilia A plasma was spiked with Mim8 at 0 µg/mL to 20 µg/mL. APTT was measured with 4 reagents, one-stage FVIII with 9 reagents, and chromogenic FVIII with 6 assays. Thrombin generation was assessed in a low tissue factor system. RESULTS: At 1-µg/mL Mim8, the APTT was shortened to within normal limits and one-stage FVIII:C was >1.00 IU/mL with all APTT reagents. Modified one-stage assays calibrated with Mim8 reference material calibrated recovered within 20% of the target Mim8 concentration with most APTT reagents. Factor VIII:C of bovine only chromogenic assays was <0.04 IU/mL at all Mim8 concentrations. Factor VIII:C of human only chromogenic assay was >4.00 IU/mL at 20-µg/mL Mim8. Factor VIII:C of hybrid bovine FX, human FIXa chromogenic assays ranged from 0.076 to >3.00 IU/mL at 20-µg/mL Mim8. Normal thrombin generation was restored at 5-µg/mL Mim8. CONCLUSIONS: APTT-based assays are sensitive to Mim8 and should not be performed in the presence of the drug. Chromogenic assays containing human proteins or hybrid human/bovine proteins demonstrated variable sensitivity to Mim8. Bovine only chromogenic assays were largely insensitive to the presence of Mim8. Thrombin generation normalized at increased Mim8 concentrations. Modified one-stage and chromogenic assays could be used to quantify the Mim8 concentration in plasma.

Measuring factor VIII activity in samples from patients treated with N8-GP (Esperoct® ; turoctocog alfa pegol) during the pathfinder clinical trials programme.

FVIII activity in samples taken at various time points from 21 patients treated with N8-GP (Esperoct® ; turoctocog alfa pegol) during the pathfinder clinical trial programme was assessed and compared using different assay methods. FVIII activity measurements in samples from patients treated with N8-GP were similar using chromogenic assays, regardless of calibration method or kit/analyser combination. FVIII activity measurements using one-stage aPTT-based assays were slightly lower when calibrated using normal human plasma (NHP) compared with a product-specific standard; this difference may be partially attributable to differences in the aPTT reagent/analyser combinations used to perform the measurements. Overall, these results confirm the accuracy of FVIII activity measurements using N8-GP-treated patient samples and routine clinical laboratory methods with NHP calibration.

An overview of turoctocog alfa pegol (N8-GP; ESPEROCT® ) assay performance: Implications for postadministration monitoring.

Factor replacement therapy with factor VIII (FVIII) concentrates is the current standard of care for patients with haemophilia A. Postadministration monitoring of FVIII activity during on-demand or prophylactic treatment is important, for example to guide a suitable dosing regimen. While the use of two-stage chromogenic substrate (CS) assays is increasing, activated partial thromboplastin time (APTT)-based one-stage clotting (OSC) assays are most commonly used to measure FVIII activity in clinical laboratories. Substantial variations in activity measurements have been observed in association with some OSC assay reagents when assessing extended half-life FVIII molecules. Certain silica-based APTT reagents have previously been shown to underestimate FVIII activity with the polyethylene glycol (PEG)-conjugated product turoctocog alfa pegol (N8-GP [ESPEROCT® ]; Novo Nordisk A/S). As a wide range of assay reagents are used in clinical laboratories worldwide, it is essential to establish which can be used to accurately measure activity with modified FVIII concentrates. Here, we describe the approach taken by Novo Nordisk to determine the suitability and accuracy of assays and reagents to measure FVIII activity in samples that contain N8-GP. While accurate activity measurements were possible with all tested CS assays and most of the OSC APTT reagents tested, three APTT reagents that contain silica as a contact activator were found to underestimate N8-GP recovery (APTT-SP, TriniCLOT™, STA® PTT-Automate). The data demonstrate the importance of characterizing the accuracy of each FVIII activity assay. Any limitations should be communicated to treating physicians and the clinical laboratories that test samples containing N8-GP.

Evaluation of N8-GP Activity Using a One-Stage Clotting Assay: A Single-Center Experience.

N8-GP (ESPEROCT®; turoctocog alfa pegol; Novo Nordisk A/S, Bagsvaerd, Denmark) is an extended half-life recombinant factor VIII (FVIII) molecule. FVIII-deficient plasma spiked with N8-GP can be accurately measured using many activated partial thromboplastin time (aPTT)-based one-stage clotting assay reagents with normal human plasma calibrators. To date, there are few data on the measurement accuracy of samples from patients treated with N8-GP. Here, we measure patient samples during routine treatment monitoring. Three previously treated patients with severe hemophilia A (HA) without inhibitors (baseline FVIII activity <0.01 IU/mL) received 50 IU/kg N8-GP every fourth day or twice weekly over 5 years as part of the pathfinder2 trial. Patient samples were monitored using the Pathromtin® SL aPTT reagent (Siemens Healthcare GmbH, Erlangen, Germany), a BCS® XP System analyzer (Siemens), and Standard Human Plasma (Siemens) or product-specific calibrators. Patient age ranged from 36 to 62 years. Overall, measurements performed using product-specific or Standard Human Plasma calibrators were in good agreement, with ratios randomly distributed around 1.0. Peak ratios tended to be closer to 1.0 than trough samples. Pathromtin® SL with Standard Human Plasma calibrator consistently and accurately measured FVIII activity in samples from severe HA patients receiving N8-GP prophylaxis.

A field study evaluating the activity of N8-GP in spiked plasma samples at clinical haemostasis laboratories.

AIM: N8-GP (turoctocog alfa pegol) is a glycoPEGylated, extended half-life human recombinant factor VIII (FVIII) shown to be an efficacious treatment for patients with haemophilia A. Accurate monitoring of replacement therapy helps ensure proper dosing, leading to better patient care. The objective of this field study was to evaluate the accuracy and intra- and inter-laboratory variabilities of N8-GP and rAHF (Advate® ) FVIII activity (FVIII:C) measurements in clinical laboratories using their routine methods and reagents. METHODS: Laboratories measured plasma samples spiked with 0.03, 0.2, 0.6 and 0.9 IU/mL N8-GP or rAHF. Samples were blinded, and laboratories were instructed to perform evaluations using their routine FVIII activity assays and calibrators. RESULTS: Of the 67 participating laboratories from 25 countries, 60 used a one-stage assay, 36 used a chromogenic assay, and 29 used both one-stage and chromogenic assays. Participating laboratories used nine different activated partial thromboplastin time (aPTT) reagents, the most common being SynthASil® and Actin® FS. Most aPTT reagents recovered N8-GP close to target. Three silica-based aPTT reagents (APTT-SP, TriniCLOT™ and STA® PTT-Automate) underestimated N8-GP, recovering 40%-83% of target concentration. For chromogenic assays, N8-GP and rAHF recoveries were comparable at all concentrations, with overall mean recoveries for both products close to 130%. Assay variability was similar for both assay types and both products; inter-laboratory variability was greater than intra-laboratory variability and highest at 0.03 IU/mL. CONCLUSIONS: Most clinical laboratories accurately measured N8-GP and rAHF when using their in-house one-stage or chromogenic FVIII:C assays. However, three silica-based aPTT reagents underestimated N8-GP recovery.

Prophylactic administration of glycoPEGylated factor IX provides protection and joint outcome superior to recombinant factor IX after induced joint bleeding.

BACKGROUND: Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. OBJECTIVE: We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). METHODS: Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. RESULTS: In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. CONCLUSIONS: These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

FIXing postinfusion monitoring: Assay experiences with N9-GP (nonacog beta pegol; Refixia® ; Rebinyn® ).

N9-GP (nonacog beta pegol; Refixia® ; Rebinyn® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated extended half-life recombinant factor IX (rFIX) that exhibits efficacy and potency comparable to unmodified FIX molecules in non-clinical models. Phase 3 clinical trials have confirmed the efficacy and tolerability of N9-GP for the prevention and on-demand treatment of bleeding episodes in patients with haemophilia B. Recent studies have shown that PEGylation affects clotting times in activated partial thromboplastin time (aPTT)-based one-stage activity assays due to interaction between the FIX molecule and certain aPTT reagents. In recognition of the challenges surrounding FIX activity assessment, the identification of consistent, reproducible and accurate assays to measure FIX activity has been a priority for Novo Nordisk, running in parallel to the clinical development program for N9-GP. N9-GP activity can be reliably measured using chromogenic substrate assays and specific aPTT reagents. The conjugation of the PEG moiety to the FIX molecule may affect one-stage aPTT-based clotting assays in a reagent-dependent manner. Many aPTT reagents that use silica as the contact activator dramatically overestimate N9-GP activity due to premature activation. On the other hand, the contact activator in some other aPTT reagents negatively affects the enzymatic activity of FXIa, causing the underestimation of N9-GP activity. While N9-GP activity cannot be measured consistently with all available aPTT reagents, accurate N9-GP measurements can be achieved with certain aPTT reagents. Here, we review the studies that led to these findings and summarize the current options for accurate measurement of N9-GP in patient samples.

Evaluating the thrombin generation profiles of four different rFVIII products in FVIII-deficient plasma using FIXa and FXIa activation.

INTRODUCTION: The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%. AIMS: To evaluate the sensitivity of the TGA when FIXa (5 nmol/L) and FXIa (0.22 nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays. METHODS: Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight® , Advate® , Eloctate® , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa. RESULTS: Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV) < 10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R2  = 0.9912) and between the one-stage and chromogenic assays (R2  = 0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays. CONCLUSIONS: Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.

Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis.

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Turoctocog alfa (NovoEight®)--from design to clinical proof of concept.

Turoctocog alfa (NovoEight®) is a recombinant factor VIII (rFVIII) with a truncated B-domain made from the sequence coding for 10 amino acids from the N-terminus and 11 amino acids from the C-terminus of the naturally occurring B-domain. Turoctocog alfa is produced in Chinese hamster ovary (CHO) cells without addition of any human- or animal-derived materials. During secretion, some rFVIII molecules are cleaved at the C-terminal of the heavy chain (HC) at amino acid 720, and a monoclonal antibody binding C-terminal to this position is used in the purification process allowing isolation of the intact rFVIII. Viral inactivation is ensured by a detergent inactivation step as well as a 20-nm nano-filtration step. Characterisation of the purified protein demonstrated that turoctocog alfa was fully sulphated at Tyr346 and Tyr1664, which is required for optimal proteolytic activation by thrombin. Kinetic assessments confirmed that turoctocog alfa was activated by thrombin at a similar rate as seen for other rFVIII products fully sulphated at these positions. Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd ) for binding to von Willebrand factor (VWF). Half-lives of 7.2 ± 0.9 h in F8-KO mice and 8.9 ± 1.8 h haemophilia A dogs supported that turoctocog alfa bound to VWF after infusion. Functional studies including thromboelastography analysis of human haemophilia A whole blood with added turoctocog alfa and effect studies in mice bleeding models demonstrated a dose-dependent effect of turoctocog alfa. The non-clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.

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.

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.

Vatreptacog alfa from conception to clinical proof of concept.

Vatreptacog alfa is a genetically engineered variant of recombinant factor VIIa (rFVIIa) containing three amino acid changes. Aspartic acid, valine, and glutamine residues replace valine, glutamic acid, and methionine at positions 158, 296, and 298, respectively. These substitutions result in considerable enhancement of the intrinsic (tissue factor-independent) capability to activate factor X and the downstream hemostatic events are consequently augmented. The beneficial effects of vatreptacog alfa have been demonstrated in numerous in vitro systems attempting to mimic hemophilia and corroborated in in vivo models. Vatreptacog alfa has successfully passed through phase 1 and 2 clinical trials and the molecule is currently being explored in phase 3 clinical trial for the treatment of bleedings in hemophilia patients with inhibitors. This article describes the proposed mechanism behind the increased activity and action of vatreptacog alfa and reviews available data, which suggest that vatreptacog alfa could be a valuable addition to the existing portfolio of treatment options for hemophilia patients with inhibitors.

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.

Overcoming delayed in-vitro response to rFVIIa: effects of rFVIIa and rFVIIa analogue (vatreptacog alfa) concentration escalation in whole blood assays.

In a previous pharmacokinetic/pharmacodynamic study in nonbleeding hemophilia patients, variability in laboratory response to recombinant factor VIIa (rFVIIa) 90 µg/kg was noted, and the patients were described as delayed or rapid laboratory responders based on time to clot formation. The current study determined whether in-vitro experiments could reproduce previous in-vivo findings; whether the delayed laboratory response to rFVIIa 90 µg/kg is improved by spiking with high-dose rFVIIa or rFVIIa analogue (vatreptacog alfa); whether a dose-response is observed with our method. In-vitro experiments were conducted in our previous patient cohort using rFVIIa 1.28 and 3.84 µg/ml and vatreptacog alfa 0.28 and 0.56 µg/ml. Whole blood studies were conducted using the Hemodyne Hemostasis Analysis System (platelet contractile force, clot elastic modulus, force onset time) and rotational thromboelastometry (clotting time, maximum clot firmness). Spiking with rFVIIa 1.28 µg/ml showed the same distribution of delayed and rapid laboratory response as observed previously. Increasing in-vitro rFVIIa concentrations improved the coagulation parameters; however, there remained delayed and rapid responders. Vatreptacog alfa improved the coagulation parameters at all concentrations tested, and the 0.56 µg/ml concentration normalized the force onset time, platelet contractile force, clot elastic modulus and clotting time parameters. A dose-response was observed with both assays. There was good agreement between the laboratory responses obtained after intravenous administration of rFVIIa 90 µg/kg and in-vitro spiking studies. Escalating rFVIIa and vatreptacog alfa concentrations improved coagulation parameters in all patients compared to rFVIIa 1.28 µg/ml. Vatreptacog alfa produced more pronounced coagulation effects at lower concentrations than rFVIIa; and the 0.56 µg/ml concentration completely normalized responses in all patients.

Recombinant factor VIIa analog NN1731 (V158D/E296V/M298Q-FVIIa) enhances fibrin formation, structure and stability in lipidated hemophilic plasma.

INTRODUCTION: The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298Q-FVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice. METHODS: We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis. RESULTS: Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeak(tPA)) and increased the area under the clot formation curve (AUC(tPA)). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeak(tPA), and AUC(tPA) as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa. CONCLUSIONS: These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity.

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.

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, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈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.