Brian Lauritzen

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

Summary.  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.

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

Summary.  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.

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

Summary.  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.

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

Summary.  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.

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.

INTRODUCTION A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab. MATERIALS AND METHODS Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats. RESULTS AND CONCLUSIONS Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.

Recombinant factor VIIa reduces bleeding after blunt liver injury in coagulopathic, hypofibrinogenaemic pigs

BACKGROUND Recombinant factor VIIa (rFVIIa) has been successfully used in various clinical conditions to treat severe coagulopathy, but its efficacy may be affected by the underlying conditions. We therefore investigated the efficacy of rFVIIa treatment under conditions of hypofibrinogenaemia in a pig model of blunt liver injury. METHODS Severe haemodilution was instigated in four groups of seven anaesthetized pigs. Before inflicting liver injury, animals were assigned to receive either 70 mg kg(-1) fibrinogen (fibrinogen group) or placebo (control group). Thirty seconds after injury, rFVIIa (180 µg kg(-1)) (rFVIIa and fibrinogen+rFVIIa groups) or vehicle (control and fibrinogen groups) was administered. Haemodynamic variables, coagulation parameters, and blood loss were monitored for 2 h. Histology was examined to evaluate the presence of thrombi and the consistency of liver injury. RESULTS At the end of the observation period, total blood loss [median (range)] decreased in all intervention groups [fibrinogen: 1275 (1221-1439) ml, P=0.036; rFVIIa: 966 (923-1136) ml, P=0.008; fibrinogen+rFVIIa: 678 (475-756) ml, P=0.008] when compared with control animals [blood loss: 1752 (1735-2221) ml]. The mortality rate in the control group was 100%, whereas only 42% of fibrinogen-substituted animals died (P=0.023). All animals treated with rFVIIa or fibrinogen+rFVIIa (P<0.001) survived and no signs of thromboembolism were observed. CONCLUSIONS rFVIIa under conditions of hypofibrinogenaemia exhibited a positive impact on coagulation parameters and a reduction in blood loss. These effects were significantly improved after prior substitution with fibrinogen.

Factor XIII Transglutaminase Supports the Resolution of Mucosal Damage in Experimental Colitis.

The thrombin-activated transglutaminase factor XIII (FXIII) that covalently crosslinks and stablizes provisional fibrin matrices is also thought to support endothelial and epithelial barrier function and to control inflammatory processes. Here, gene-targeted mice lacking the FXIII catalytic A subunit were employed to directly test the hypothesis that FXIII limits colonic pathologies associated with experimental colitis. Wildtype (WT) and FXIII-/- mice were found to be comparable in their initial development of mucosal damage following exposure to dextran sulfate sodium (DSS) challenge. However, unlike FXIII-sufficient mice, FXIII-deficient cohorts failed to efficiently resolve colonic inflammatory pathologies and mucosal damage following withdrawal of DSS. Consistent with prior evidence of ongoing coagulation factor activation and consumption in individuals with active colitis, plasma FXIII levels were markedly decreased in colitis-challenged WT mice. Treatment of colitis-challenged mice with recombinant human FXIII-A zymogen significantly mitigated weight loss, intestinal bleeding, and diarrhea, regardless of whether cohorts were FXIII-sufficient or were genetically devoid of FXIII. Similarly, both qualitative and quantitative microscopic analyses of colonic tissues revealed that exogenous FXIII improved the resolution of multiple colitis disease parameters in both FXIII-/- and WT mice. The most striking differences were seen in the resolution of mucosal ulceration, the most severe histopathological manifestation of DSS-induced colitis. These findings directly demonstrate that FXIII is a significant determinant of mucosal healing and clinical outcome following inflammatory colitis induced mucosal injury and provide a proof-of-principle that clinical interventions supporting FXIII activity may be a means to limit colitis pathology and improve resolution of mucosal damage.

Automated registration of tail bleeding in rats

An automated system for registration of tail bleeding in rats using a camera and a user-designed PC-based software program has been developed. The live and processed images are displayed on the screen and are exported together with a text file for later statistical processing of the data allowing calculation of e.g. number of bleeding episodes, bleeding times and bleeding areas. Proof-of-principle was achieved when the camera captured the blood stream after infusion of rat whole blood into saline. Suitability was assessed by recording of bleeding profiles in heparin-treated rats, demonstrating that the system was able to capture on/off bleedings and that the data transfer and analysis were conducted successfully. Then, bleeding profiles were visually recorded by two independent observers simultaneously with the automated recordings after tail transection in untreated rats. Linear relationships were found in the number of bleedings, demonstrating, however, a statistically significant difference in the recording of bleeding episodes between observers. Also, the bleeding time was longer for visual compared to automated recording. No correlation was found between blood loss and bleeding time in untreated rats, but in heparinized rats a correlation was suggested. Finally, the blood loss correlated with the automated recording of bleeding area. In conclusion, the automated system has proven suitable for replacing visual recordings of tail bleedings in rats. Inter-observer differences can be eliminated, monotonous repetitive work avoided, and a higher through-put of animals in less time achieved. The automated system will lead to an increased understanding of the nature of bleeding following tail transection in different rodent models.

Recombinant factor VIIa reduces rebleed hemorrhage volume in a swine aortotomy model: a randomized double-blinded study.

Noncompressible hemorrhage requires hypotensive resuscitation until definitive measures can be taken to prevent rebleeding by sustaining blood pressure at subphysiological levels. Previous studies have demonstrated that a 180- or 720-μg kg−1 dose of recombinant factor VIIa (rFVIIa) increases the MAP at which rebleeding occurs in a swine aortotomy model. The purpose of the current study was to determine the efficacy of a lower dose of 90 μg kg−1 given prophylactically to prevent or reduce rebleeding in a prospective, randomized, blinded study using a porcine model of uncontrolled hemorrhage and resuscitation. Fourteen female 40-kg Yorkshire-cross pigs were splenectomized and instrumented with venous and arterial catheters. The infrarenal aorta was exposed, and suction catheters were placed along the right and left paracolic gutters. After a 10-min baseline, 90 μg kg−1 (i.v.) of either rFVIIa (n = 6) or vehicle (n = 8) was administered. Five minutes later, an aortotomy was created using a 2.5-mm biopsy punch. The weight of the shed blood was continuously recorded. Lactated Ringers was given (100 mL kg−1 min−1) 10 min after aortotomy until rebleeding occurred. The MAP at rebleed and the subsequent rebleed hemorrhage volume was recorded over the 2-h study period. After rebleed occurred, lactated Ringers sufficient to maintain MAP at baseline levels was given. Initial hemorrhage volume and rebleed MAP (P = 0.31) did not differ significantly between groups. Rebleed hemorrhage volume was reduced by 54% in the rFVIIa group from 79 ± 4 mL kg−1 in the vehicle group to 43 ± 6 mL kg−1 in the rFVIIa group (mean ± SEM; P < 0.005). The MAP at which rebleed occurred was not different between the groups, 71 ± 4 mmHg in the rFVIIa group versus 59 ± 5 in the vehicle group. Prophylactic administration of rFVIIa at 90 μg kg−1, a dose similar to the recommended dose in hemophilia patients with inhibitors, reduced rebleed hemorrhage volume, suggesting that this dose is effective in this swine aortotomy model.ABBREVIATIONS H and E-hematoxylin and eosin; LR-lactated Ringer; PT-prothrombin time; PTAH-phosphotungstic acid hematoxylin; PTT-partial thromboplastin time; RFVIIa-recombinant factor VIIa; TEG-thrombelastograph

rFVIIa and NN1731 reduce bleeding in hydroxyethyl starch hemodiluted rabbits.

BACKGROUND Colloid plasma expanders are used to maintain blood pressure and ensure tissue perfusion during hypovolemia, e.g., caused by traumatic bleeding. Although colloids stabilize the cardiovascular system, they can also potentially cause coagulopathy. Consequently, bleeding tendency may increase, as well as the associated risk of morbidity and mortality. Thus, there is a need for hemostatic treatment options for these patients. rFVIIa (NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a hemostatic agent that effectively controls bleedings in patients with inhibitor-complicated hemophilia. rFVIIa works by enhancing thrombin generation on the activated platelet surface at the site of injury, leading to the formation of a stable fibrin clot. NN1731 is an rFVIIa analog with increased hemostatic potential and is currently under clinical development. METHODS In this study, the effect of rFVIIa and NN1731 on cuticle bleeding in rabbits 50% hemodiluted with hydroxyethyl starch (molecular weight ∼ 200,000) was tested. Cuticle bleeding was induced after a two-stage hemodilution procedure. After 5 minutes, the animals were treated with rFVIIa (2, 5, or 10 mg/kg), NN1731 (1 or 2 mg/kg), or vehicle, followed by 30 minutes of observation. RESULTS Hemodilution caused a significant increase in bleeding time and blood loss. rFVIIa dose-dependently reduced bleeding time and blood loss, reaching statistical significance at 10 mg/kg. However, 2 mg/kg NN1731 reduced bleeding time and blood loss significantly and to a similar extent as 10 mg/kg rFVIIa. This increased hemostatic potential of NN1731 compared with rFVIIa and was confirmed by findings using thromboelastography on ex vivo hemodiluted whole blood. CONCLUSION In summary, rFVIIa and NN1731 significantly and dose-dependently reduced bleeding in extensively hemodiluted rabbits.