Development of a tail vein transection bleeding model in fully anaesthetized haemophilia A mice - characterization of two novel FVIII molecules.

INTRODUCTION: The tail tip bleeding model and the tail vein transection survival model in mice are important tools for assessment of in vivo effect in haemostasis research. While the tail vein transection model exhibits the best sensitivity to pharmacological intervention it uses death or near-death as endpoint which is fully avoided in the tail tip bleeding model. AIM: The aim of this study was to develop a new tail bleeding model maintaining the sensitivity of the previous survival model but avoiding death/near-death as endpoint. METHODS: Combining the two existing tail bleeding models we developed an optimized version of the survival model with full anaesthetic coverage and short duration of experiments. Using this model, we characterized the effect of turoctocog alfa, a B-domain truncated FVIII molecule (NovoEight(®) ), as well as the prolonged half-life version of the same molecule (turoctocog alfa pegol, N8-GP). RESULTS: Data showed that the model was sensitive to clinically relevant doses of both turoctocog alfa as well as N8-GP when dosed for 'on demand' treatment. The model also correctly identified a longer duration of effect for N8-GP compared with turoctocog alfa. Moreover, the model allowed the use of mice of both genders and was reproducible over time. CONCLUSION: The optimized tail vein transection bleeding model is sensitive to standard as well as half-life prolonged FVIII molecules and should be a valuable alternative to both the tail tip bleeding model, enhancing sensitivity to pharmacological intervention, as well as to the previously used tail vein transection survival model, avoiding death or near-death as endpoint.

Prolonged effect of a new O-glycoPEGylated FVIII (N8-GP) in a murine saphenous vein bleeding model.

Prophylaxis in severe haemophilia significantly increases health-related quality of life for patients, but the dosing frequency still constitutes a challenge. Thus, there is a need for new treatment options, utilizing compounds with longer duration of action, while still maintaining potency. The objective of this study was to evaluate the acute and prolonged effects of a new glycoPEGylated recombinant factor VIII (rFVIII) (N8-GP) in a venous bleeding model in haemophilia A mice and to compare the efficacy and potency to turoctocog alfa (rFVIII). Following intravenous administration of turoctocog alfa or N8-GP to normal and FVIII-deficient mice, bleeding time and blood loss from a saphenous vein incision were evaluated in an acute dose-response study and a duration of action study. In the acute setting, N8-GP dose dependently reduced the number and duration of bleeding episodes as well as blood loss compared to FVIII-deficient mice, reaching statistical significance at doses as low as 5-10 U kg(-1) . In the duration of action study, a significantly prolonged and maintained effect of N8-GP was found for up to 48 h after dosing, whereas the effect of rFVIII was no longer present for any end-points 24 h after dosing. Seventy-two hours after dosing, no significant effect of either compound was found. This study shows a prolonged haemostatic effect of N8-GP compared to rFVIII supporting other recent studies that N8-GP may hold a potential to increase the quality of life for patients with haemophilia A by reducing dosing frequency.

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

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 ED(50) 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.

A sensitive venous bleeding model in haemophilia A mice: effects of two recombinant FVIII products (N8 and Advate(®)).

Haemostatic effect of compounds for treating haemophilia can be evaluated in various bleeding models in haemophilic mice. However, the doses of factor VIII (FVIII) for normalizing bleeding used in some of these models are reported to be relatively high. The aim of this study was to establish a sensitive venous bleeding model in FVIII knock out (F8-KO) mice, with the ability to detect effect on bleeding at low plasma FVIII concentrations. We studied the effect of two recombinant FVIII products, N8 and Advate(®), after injury to the saphenous vein. We found that F8-KO mice treated with increasing doses of either N8 or Advate(®) showed a dose-dependent increase in the number of clot formations and a reduction in both average and maximum bleeding time, as well as in average blood loss. For both compounds, significant effect was found at doses as low as 5 IU kg(-1) when compared with vehicle-treated F8-KO mice. Normalization of maximum bleeding time was found at doses equal to or above 10 IU kg(-1) N8 or Advate(®), corresponding to plasma concentrations of approximately 10% of the level in wild type mice. The present study adds a new model to the armamentarium of bleeding models used for evaluation of pro-coagulant compounds for treatment of haemophilia. Interestingly, the vena saphena model proved to be sensitive towards FVIII in plasma levels that approach the levels preventing bleeding in haemophilia patients, and may, thus, in particular be valuable for testing of new long-acting variants of e.g. FVIII that are intended for prophylaxis.

Pharmacokinetics, pharmacodynamics and safety of recombinant canine FVIIa in a study dosing one haemophilia A and one haemostatically normal dog.

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 µg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans.

A ferric chloride induced arterial injury model used as haemostatic effect model.

A number of experimental bleeding models have been applied to animal models of haemophilia in order to evaluate the acute haemostatic effect of procoagulant compounds. In contrast, in vivo thrombosis models (including the FeCl(3) induced injury model) have mainly been used to study antithrombotic pharmacological intervention. However, as there are limitations to existing bleeding models and as new recombinant FVIII, FIX, and FVIIa variants with increased and prolonged activity are generated there is an increasing need for new and optimized in vivo animal models for testing the efficacy of these haemostatic drug candidates. This led us to look at existing thrombosis models in a new perspective. We have studied the effect of a FeCl(3) induced arterial injury in both F8-KO and F9-KO mice using optimized conditions where exposure to FeCl(3) induces occlusion within 4.2 +/- 0.2 min in wild type mice with a normal coagulation system. In contrast, no occlusion was observed in haemophilic mice providing a therapeutic window in the model making it suitable for pharmacological testing of therapeutic intervention. We demonstrate that replacement therapy with a clinical relevant dose of rFVIII (Advate 20-80 U kg(-1)) and rFIX [(0.75 mg kg(-1) BeneFIX) approximately 50 IU kg(-1)] restored coagulation and normalized the time to occlusion following FeCl(3) induced injury in F8-KO mice and restored coagulation and nearly normalized the time to occlusion in F9-KO mice. In conclusion, we have demonstrated that under optimized conditions the FeCl(3) induced arterial injury model provides a therapeutic window that makes it an useful effect model for evaluation of the haemostatic potential of procoagulant drugs.

IL-1 beta, IL-6, KC and MCP-1 are elevated in synovial fluid from haemophilic mice with experimentally induced haemarthrosis.

The hallmark of haemophilia is the joint morbidity resulting from haemarthrosis that accounts for the majority of the bleeds. The exact mechanisms underlying changes are not fully elucidated. Cytokines are speculated to be involved in the progression and in vitro studies have confirmed the presence of elevated levels of cytokines in synovial tissue and cartilage from patients with haemophilic synovitis. In this study, the presence of selected cytokines in synovial fluid from haemophilia A mice with experimentally induced haemarthroses treated with rFVIII, rFVIIa and an rFVIIa analogue were investigated. Ten cytokines previously shown to be involved in arthritic syndromes were evaluated. Interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10, IL-17, Tumor Necrosis Factor-alpha (TNF- alpha), keratinocyte-derived chemokine (KC), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) were included. In this article, we demonstrate, for the first time, that bleeding in knee joints of haemophilia A mice resulted in correlated increased levels of the pro-inflammatory cytokines: IL-1 beta, IL-6, KC and the MCP-1 in synovial fluid. These results suggest an important role of MCP-1 in the recruitment of monocytes and furthermore that the inflamed synovium releases IL-1 beta, IL-6 and KC, which in turn might contribute to further progression of the inflammatory process.

Prevention of haemarthrosis in a murine model of acute joint bleeding.

Preservation of normal joint function in patients with haemophilia is a goal of modern therapy. Regular injections of anti-haemophilic factor concentrate reduce the risk of joint bleeding, the optimal regimen for which remains under investigation. The goals of the experiment described here are: (i) to assess the capacity of a murine model of severe haemophilic arthropathy to predict the likelihood of success of a test product to prevent joint bleeding and the complications that follow and (ii) to compare the effectiveness of recombinant human activated factor VII (rFVIIa) to recombinant human factor VIII (rFVIII) to prevent acute joint bleeding in the mouse model of haemarthrosis. Mice lacking expression of FVIII received a single intravenous injection of human rFVIII (280 U kg(-1)), rFVIIa (10 mg kg(-1)) or vehicle prior to blunt trauma injury to the knee joint. Mice receiving rFVIII and rFVIIa developed less injury-induced joint bleeding, swelling and loss of range of motion compared to mice pretreated with vehicle. Despite the reduction in clinical symptoms, synovial hyperplasia was evident in all groups after 7 days although less pronounced in mice receiving rFVIII and rFVIIa. The data under these experimental conditions demonstrate: (i) that this model can be used to evaluate novel therapies designed to prevent joint bleeding (prophylaxis) and (ii) both rFVIII and rFVIIa reduced acute haemarthrosis but did not completely prevent synovitis, the sequelae of blood induced joint injury.

Thrombin generation and platelet activation induced by rFVIIa (NovoSeven) and NN1731 in a reconstituted cell-based model mimicking haemophilia conditions.

Replacement therapy with factor VIII (FVIII) and factor IX (FIX) is routinely used in haemophilia patients with haemophilia A and B, respectively, while recombinant activated FVII (rFVIIa) has proven to induce haemostasis in haemophilia patients with inhibitors. To evaluate the effect of therapeutic intervention in patients with residual factor activities, the effects of increasing concentrations of rFVIIa or NN1731 on thrombin generation and platelet activation were measured in a cell-based model system mimicking severe, moderate and mild haemophilia A or B. Purified monocytes stimulated to express tissue factor and non-activated platelets from peripheral blood of healthy donors were incubated with a mixture of purified human coagulation factors in the absence or presence of increasing concentrations of FVIII or FIX. Sub-samples were analysed for thrombin activity and platelet activation measured as exposure of P-selectin by flow cytometry. Dose-dependent increases in thrombin generation and platelet activation were observed following increasing concentrations of rFVIIa or NN1731 in both haemophilia A- and B-like conditions. At 25 nm rFVIIa, which nears the peak levels in patient plasma after 90 microg kg(-1) intravenous dosing, the effects on maximum thrombin generation rate (maxTG) at 1-10% FVIII were comparable to those at 100% and 200% FVIII in the absence of rFVIIa. Normalization of maxTG required 500 nm rFVIIa and 25 nm NN1731 or 25-100 nm rFVIIa and 5 nm NN1731 in severe or moderate/mild haemophilia A and haemophilia B, respectively. This suggests that NN1731 holds its promise as a future bypassing agent for haemophilia patients with and without inhibitors.

In vivo models of haemophilia - status on current knowledge of clinical phenotypes and therapeutic interventions.

Animal models have contributed immensely to the understanding of and the improvement in treatment of haemophilia A and B. First, establishment of haemophilic dog colonies provided an invaluable opportunity to investigate the diseases and later, the advances in gene technologies resulting in small haemophilic animal models were a milestone in the preclinical research making it possible to address some of the many unanswered questions. This review provides an overview of animal models used in the study of haemophilia as well as a short overview of the contributions resulting from studies in these models.

Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular coagulation.

BACKGROUND: There is considerable variation in the coagulation profile of dogs with disseminated intravascular coagulation (DIC), making it difficult to assess overall hemostatic function. OBJECTIVES: To characterize the overall hemostatic state in dogs with DIC, by use of tissue factor-activated thromboelastography (TF-TEG), and to determine whether there is an association between hemostasis and outcome. ANIMALS: 50 dogs with DIC. METHODS: Dogs admitted to the intensive care units, with an underlying disease known to predispose to DIC, were prospectively assessed with TF-TEG. Citrated blood samples were collected daily during hospitalization and an extended coagulation panel and TF-TEG were performed. Diagnosis of DIC was based on expert opinion. RESULTS: Hemostatic dysfunction was observed on the TF-TEG profile in 33/50 of the dogs, of which 22/50 were hypercoagulable and 11/50 were hypocoagulable based on the TF-TEG G value alone. There were significant differences in k, alpha, and MA values (P < .0001) among hypo-, normo-, and hypercoagulable dogs. There was a significant difference in case fatality rate between hypo- (64%) and hypercoagulable (32%) dogs (relative risk = 2.38; P= .04). Dogs that died had significantly lower antithrombin activity (P= .03) and higher d-dimer concentration (P= .03) than survivors. CONCLUSIONS: The most common overall hemostatic abnormality in dogs diagnosed with DIC was hypercoagulability, and there was significant difference in survival between hyper- and hypocoagulable dogs. The results suggest TF-TEG is valuable in the assessment of hemostatic function in dogs diagnosed with DIC.

Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A.

BACKGROUND: Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. OBJECTIVES: Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. METHODS: Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. RESULTS: In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. CONCLUSIONS: Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response.

Expression of tissue factor in canine mammary tumours and correlation with grade, stage and markers of haemostasis and inflammation.

Tissue factor (TF) expression in human cancers has been associated with a procoagulant state and facilitation of metastasis. This study was conducted in order to evaluate if TF was expressed in canine mammary tumours. Forty epithelial mammary tumours from 28 dogs were included. TF expression of the tumours was evaluated by immunohistochemistry using a polyclonal antibody against recombinant canine TF. In addition, thromboelastography, haemostatic and inflammatory parameters were evaluated in the patients. TF was recognized in 44% of benign and 58% of malignant tumours. TF localized to the cytoplasmic membrane of neoplastic luminal epithelial cells and/or diffusely in the cytoplasm. No association was found between TF expression and stage or grade of disease. A significant association between TF expression and antithrombin and plasminogen was found, and extensive TF expression was seen in a lymph node metastasis classified as anaplastic mammary carcinoma from a dog with concomitant disseminated intravascular coagulation (DIC).

The F8(-/-) rat as a model of hemophilic arthropathy.

UNLABELLED: Essentials Validating the F8 rat as a new intermediate-size animal model of hemophilic arthropathy. Factor VIII (FVIII) treated F8(-/-) rats suffered induced hemarthrosis analyzed by histopathology. F8 (-/-) animals develop hemophilic arthropathy upon hemarthrosis, preventable by FVIII treatment. The F8 (-/-) rat presents as a new pharmacologic model of hemophilic arthropathy. SUMMARY: Background Translational animal models of hemophilia are valuable for determining the pathobiology of the disease and its co-morbidities (e.g. hemophilic arthropathy, HA). The biologic mechanisms behind the development of HA, a painful and debilitating condition, are not completely understood. We recently characterized a F8(-/-) rat, which could be a new preclinical model of HA. Objectives To establish the F8(-/-) rat as a model of HA by determining if the F8(-/-) rat develops HA resembling human HA after an induced joint bleed and whether a second joint bleed causes further disease progression. Methods Wild-type and F8(-/-) rats were treated with vehicle or recombinant human factor VIII (rhFVIII) prior to a needle-induced joint bleed. Joint swelling was measured prior to injury, the following 7 days and upon euthanasia. Histologic sections of the joint were stained, and athropathic changes identified and scored with regard to synovitis, bone remodelling, cartilage degradation and hemosiderin deposition. Results Vehicle-treated F8(-/-) rats experienced marked joint swelling and developed chronic degenerative joint changes (i.e. fibrosis of the subsynovial membrane, chondrocyte loss and excessive bone remodeling). Treatment with rhFVIII reduced or prevented swelling and degenerative joint changes, returning the F8(-/-) animals to a wild-type phenotype. Conclusion The hemophilic phenotype of the F8(-/-) rat resulted in a persistent hemarthrosis following an induced joint bleed. This caused development of HA resembling human HA, which was prevented by rhFVIII treatment, confirming the potential of the F8(-/-) rat as a model of HA.

Novel, high incidence exercise-induced muscle bleeding model in hemophilia B mice: rationale, development and prophylactic intervention.

INTRODUCTION: Muscle hematomas are the second most common complication of hemophilia and insufficient treatment may result in serious and even life-threatening complications. Hemophilic dogs and rats do experience spontaneous muscle bleeding, but currently, no experimental animal model is available specifically investigating spontaneous muscle bleeds in a hemophilic setting. AIM: The objective of this study was to develop a model of spontaneous muscle bleeds in hemophilia B mice. We hypothesized that treadmill exercise would induce muscle bleeds in hemophilia B mice but not in normal non-hemophilic mice and that treatment with recombinant factor IX (rFIX) before treadmill exercise could prevent the occurrence of pathology. METHODS: A total of 203 mice (123 F9-KO and 80 C57BL/6NTac) were included in three separate studies: (i) the model implementation study investigating the bleeding pattern in hemophilia B mice after treadmill exercise; (ii) a study evaluating the pharmacokinetics of recombinant FIX (rFIX) in hemophilia B mice and based on these data; (iii) the treatment study, which tested therapeutic intervention with rFIX. At termination of the treadmill studies the presence of bleeds was evaluated. RESULTS: Treadmill exercise resulted in a high incidence of muscle bleeds in F9-KO mice but not in C57BL/6NTac mice. Treating hemophilia B mice with rFIX before treadmill exercise prevented muscle bleeds. CONCLUSION: A novel model of muscle bleeds in hemophilia B mice, responsive to rFIX, has been developed.

Buprenorphine does not impact the inflammatory response in haemophilia A mice with experimentally-induced haemarthrosis.

Haemarthrosis is the most common clinical manifestation of haemophilia and is responsible for significant morbidity in haemophilic patients. The murine experimentally-induced knee bleeding model is an important model in haemophilia research but it is currently unknown if the use of analgesia in this model might impact on the inflammatory response. The aim was to investigate the inflammatory response after a needle induced knee bleed in haemophilia A mice treated with buprenorphine or saline. One hundred and sixty mice were randomized into two groups to blindly receive buprenorphine or saline. All the mice were anaesthetized and knee injury was induced by inserting a 30 G needle into the right knee joint. At t = 6, 24, 48 and 72 h, 20 mice from each group were terminated and the following parameters were assessed: change in body weight and joint diameter, visual bleeding score (VBS), white blood counts, haematocrit, platelet concentrations, haemoglobin, plasma haptoglobin and plasma and synovial fluid levels of 23 cytokines. Twenty mice were terminated at t = 0 receiving no injury or treatment to provide baseline measures. Twenty-one cytokines in plasma and 22 cytokines in synovial fluid, joint diameter change, VBS and blood parameters were not significantly altered by the administration of buprenorphine. Slight alterations of plasma haptoglobin at t = 48 h, body weight, plasma and synovial eotaxin and plasma G-CSF were found in buprenorphine-treated mice. We demonstrated that buprenorphine does not overall impact on the inflammatory response, and the use of buprenorphine in the knee bleeding model in haemophilic mice should be continued.

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

BACKGROUND: In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed. OBJECTIVES: Here, we describe the generation and characterization of a novel genetically engineered F8(-/-) rat model. METHODS: The rats were produced on a Sprague Dawley background with the zinc finger nuclease technique. A founder with a 13-bp deletion in exon 16 causing a premature translational stop in the C-terminal part of the A3 domain of factor VIII was selected, and a breeding colony was established. RESULTS: Seventy per cent of the homozygous rats had clinically manifest spontaneous hemorrhagic episodes that needed treatment. The F8(-/-) rats had no detectable FVIII activity, and had a significantly prolonged activated partial thromboplastin time (APTT) and clot formation time as compared with wild-type (WT)/WT rats. In vitro spiking of rat plasma with human recombinant FVIII resulted in dose-dependent normalization of the APTT. CONCLUSION: On the basis of the targeted deletion in F8, and the distinct physical and analytic characteristics of the rat, we conclude that an FVIII-deficient rat strain has been generated that has the potential to contribute greatly to translational research.

rFVIIa and a new enhanced rFVIIa-analogue, NN1731, reduce bleeding in clopidogrel-treated and in thrombocytopenic rats.

BACKGROUND: The pharmacological effect of rFVIIa occurs at the surface of activated platelets by enhancing thrombin generation at the site of vascular damage. It is therefore important to study the effects of rFVIIa in platelet-related bleeding situations. We examined the effect of rFVIIa and an rFVIIa-analogue, NN1731, on clopidogrel-induced and thrombocytopenic bleeding in rats. METHODS AND RESULTS: Clopidogrel [10 mg kg(-1); per oral (p.o.)] severely inhibited platelet aggregation and increased blood loss after tail-transection four hours after administration. Treatment with rFVIIa (5, 10, 20 mg kg(-1)) or NN1731 (1, 5, 10 mg kg(-1)), administered five minutes after induction of bleeding, reduced blood loss significantly and dose-dependently. NN1731 had an increased hemostatic potential compared with rFVIIa, reducing blood loss to the control level, whereas this was not even achieved with the highest dose of rFVIIa. Antibody-induced thrombocytopenia reduced platelet numbers by more than 90% and increased the blood loss after tail-transection. Treatment with 10 and 20 mg kg(-1) rFVIIa significantly reduced blood loss, whereas 10 mg kg(-1) NN1731 reduced the bleeding to control levels. CONCLUSIONS: The hemostatic effect of rFVIIa and NN1731 was demonstrated in thrombocytopenic and clopidogrel-treated rats, showing efficacy in situations with decreased platelet number or functionality. Our findings are consistent with the hypothesis that rFVIIa/NN1731 contribute to hemostasis by thrombin generation even in situations with platelet disorders. Furthermore, NN1731 demonstrated a higher hemostatic potential than rFVIIa.

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

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.

Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats.

BACKGROUND: Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. OBJECTIVES: To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. METHODS: Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. RESULTS: rFVIIa (5, 10 and 20 mg kg(-1)) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg(-1) (n = 8) and 500 IU kg(-1) (n = 9), respectively. Similarly, 10 mg kg(-1) NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg(-1) tinzaparin, 10 mg kg(-1) NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg(-1) rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. CONCLUSIONS: This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.