Long-term expression of murine activated factor VII is safe, but elevated levels cause premature mortality.

Intravenous infusion of recombinant human activated Factor VII (FVIIa) has been used for over a decade in the successful management of bleeding episodes in patients with inhibitory antibodies to Factor VIII or Factor IX. Previously, we showed that expression of murine FVIIa (mFVIIa) from an adeno-associated viral (AAV) vector corrected abnormal hemostatic parameters in hemophilia B mice. To pursue this as a therapeutic approach, we sought to define safe and effective levels of FVIIa for continuous expression. In mice transgenic for mFVIIa or injected with AAV-mFVIIa, we analyzed survival, expression levels, in vitro and in vivo coagulation tests, and histopathology for up to 16 months after birth/mFVIIa expression. We found that continuous expression of mFVIIa at levels at or below 1.5 microg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 microg/ml or higher were associated with thrombosis and early mortality, with pathologic findings in the heart and lungs that were rescued in a low-factor X (low-FX) mouse background, suggesting a FX-mediated effect. The findings from these mouse models of continuous FVIIa expression have implications for the development of a safe gene transfer approach for hemophilia and are consistent with the possibility of thromboembolic risk of continuously elevated FVIIa levels.

Administration of recombinant FVIIa (rFVIIa) to concizumab-dosed monkeys is safe, and concizumab does not affect the potency of rFVIIa in hemophilic rabbits.

Essentials Hemophilia patients on concizumab prophylaxis may need rFVIIa to treat breakthrough bleeds. Effect and safety of concizumab + rFVIIa were tested in vitro and in vivo. Concizumab + rFVIIa had no additive effects on bleeding in hemophilic rabbits. High steady-state levels of concizumab did not affect the safety of rFVIIa in cynomolgus monkeys. SUMMARY: Background Concizumab is a monoclonal antibody (mAb) against tissue factor pathway inhibitor (TFPI), currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A/B with and without inhibitors. In patients with inhibitors, the treatment choice for breakthrough bleeding will comprise bypassing agents, e.g. activated recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrates. Objectives To explore the effect and safety of concizumab and rFVIIa when they are simultaneously present. Methods Human blood made hemophilic with a FVIII antibody was spiked with increasing concentrations of concizumab, rFVIIa, or concizumab and rFVIIa in combination, and this was followed by thrombin generation test or thromboelastography. Blood loss in hemophilic rabbits was measured when concizumab, rFVIIa or concizumab + rFVIIa was administered either before or during cuticle bleeding. In a safety study, cynomolgus monkeys were exposed to high steady-state concizumab concentrations and given three doses of rFVIIa, and then subjected to full necropsy and histopathological examination. Results In human blood, concizumab + rFVIIa had more pronounced procoagulant effects under hemophilic conditions than the sum of individual responses. In contrast, concizumab + rFVIIa had no additional effects on blood loss in hemophilic rabbits as compared with rFVIIa or concizumab alone. In cynomolgus monkeys, the macroscopic and microscopic pathological examinations revealed no thrombi or other signs of excessive coagulation activation. Both rFVIIa and concizumab caused increases in thrombin-antithrombin and D-dimer concentrations; this effect tended to be additive with concomitant administration. Conclusions Concizumab did not affect the potency or safety of rFVIIa in vivo. These results support a clinical evaluation of rFVIIa at standard dose (90 µg kg-1 ) to treat breakthrough bleeds in concizumab clinical trials.

Preclinical toxicity biomarkers for combination treatment with clotting factors rFXIII and rFVIIa.

Combination treatment with the clotting factors recombinant activated factor VII (rFVIIa), serine protease, and recombinant factor XIII (rFXIII), protransglutaminase, is being explored for haemostatic therapy. We performed a single-dose toxicology study in the cynomolgus monkey, with four dose groups receiving 0.1 + 0.34 mg kg(-1) (group 1), 0.33 + 1.12 mg kg(-1) (group 2), 1.67 + 5.60 mg kg(-1) (group 3) and 5.00 + 16.80 mg kg(-1) (group 4) of a rFVIIa + rFXIII combination. In the three lower dose groups, no clinical, histopathological or blood chemistry changes were observed. In group 4, the animals died at 4 h post-dosing, with histopathology revealing a systemic coagulopathy resembling, but distinct from, disseminated intravascular coagulation. Due to the absence of toxicity warning signs, toxicity biomarkers were identified by a Western blot-based screening of approximately 20 plasma proteins known to be involved in the clotting cascade. Three of the examined proteins were specifically affected by rFVIIa + rFXIII treatment. Fibronectin and fibrinogen exhibited dose-dependent reductions from less than 10% reduction (group 2) to more than 90% reduction (group 4). These reductions were reversible, and specific. For vitronectin, a dose-dependent conversion to the 65-kDa form was found to occur in groups 3 and 4. Thus, fibrinogen, fibronectin and vitronectin represent the first biomarkers for clotting factor toxicity.

A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.

BACKGROUND AND OBJECTIVES: Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone. MATERIALS AND METHODS: The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model. RESULTS: The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA. CONCLUSIONS: The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.

Recombinant factor VIIa for patients with inhibitors to factor VIII or IX or factor VII deficiency.

Inhibitors to factor VIII (FVIII) or IX (FIX) in patients with haemophilia A or B create a challenging problem for the treatment of these patients. Recombinant FVIIa (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a realistic treatment option, owing to its specific mode of action and lack of immunogenicity. This was a multicentre, open-label, compassionate-use trial in patients with severe haemophilia A (FVIII:C < 1%) or B (FIX:C < 1%) with inhibitors, acquired antibodies to FVIII or FIX, or FVII deficiency (FVII:C < 5%), for whom alternative therapies had failed or were contraindicated. Patients received rFVIIa treatment for life- or limb-threatening bleeding episodes or for coverage during essential surgery. The mean rFVIIa dose was approximately 90 microg kg-1 for haemophilia A/B and acquired inhibitor patients, and 25 microg kg-1 for FVII-deficient patients. Efficacy data for 67 treatment episodes (45 bleeding episodes, 22 surgical procedures) are presented; seven patients were treated for a concurrent serious bleeding episode and surgical procedure. At the end of treatment, rFVIIa was effective or partially effective in 85% of serious bleeding episodes. During surgery, bleeding was assessed as none or less than or equivalent to normal in 91% of surgical procedures; postoperatively, 91% of procedures were associated with no or minimal oozing. During 60 separate treatment episodes, 26 adverse events (22 nonserious, four serious) were reported in 15 patients, during 17 bleeding episodes or surgical procedures. Only 10 were considered as having a possible, probable, or unknown relationship with rFVIIa; of these, fever (n=2) and thrombophlebitis (n=3) were the most common. There was no evidence of disseminated intravascular coagulation. In conclusion, rFVIIa is an effective, well-tolerated treatment for serious bleeding episodes and bleeding associated with surgical procedures in patients with severe haemophilia A/B with inhibitors, acquired inhibitors, or FVII deficiency.