Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura.

OBJECTIVE: The metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted. APPROACH AND RESULTS: We have developed a new rat model mimicking various aspects of acquired TTP to investigate the therapeutic efficacy of human recombinant ADAMTS13. A polyclonal antibody against ADAMTS13 completely blocked endogenous rat ADAMTS13 activity in Sprague-Dawley rats. When TTP was triggered using recombinant von Willebrand factor, the animals displayed severe TTP-like symptoms, such as thrombocytopenia, hemolytic anemia, and von Willebrand factor-rich thrombi in the kidneys and brain. Subsequent injection of 400, 800, or 1600 U/kg recombinant ADAMTS13 prevented full development of these symptoms. Analysis of plasma samples confirmed that recombinant ADAMTS13 was able to override circulating anti-ADAMTS13 inhibitory antibodies, resulting in restoration of ADAMTS13 activity and degradation of ultralarge von Willebrand factor multimers. CONCLUSIONS: Recombinant ADAMTS13 was shown to be effective in averting severe acquired TTP-like symptoms in rats and holds promising value for the treatment of this severe and life-threatening disease in humans.

Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes.

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.

Hemostatic efficacy of a novel, PEG-coated collagen pad in clinically relevant animal models.

PURPOSE: Currently available hemostatic pads are effective in treating oozing bleeds, but otherwise ineffective in more severe bleeding. This study investigates the hemostatic efficacy of a new hemostatic pad with advanced sealing properties using protein-reactive polyethylene glycol-coated collagen (PCC, Hemopatch) versus an oxidized regenerated cellulose (ORC, Tabotamp/Surgicel Original) in a leporine arterial bleeding model of vascular reconstruction and a porcine hepatic model of general surgery. METHODS: In both models, paired lesions were created and treated according to a randomized scheme and evaluated up to 10 min after application (40 lesions/group/model). Arterial needle holes were created in the femoral arteries of anesthetized rabbits and hepatic lesions were created into hepatic parenchyma of anesthetized pigs. Both models were heparinized to mimic clinical comorbidity. RESULTS: In the leporine vascular surgical model, PCC provided superior hemostatic success compared to ORC at 2 min (Odds Ratio of Success: 85, 95% CI: 25.8-282) and similar hemostatic success at 10 min. In the porcine hepatic model, PCC provided superior hemostatic success compared to ORC at 2 (98 vs 55%, P < 0.001), 3 (93 vs 65%, P < 0.001), 4 (98 vs 68%, P < 0.001) and 5 min (95 vs 80%, P < 0.001), but similar hemostatic success at 8 and 10 min. DISCUSSION: PCC provided 75.4% greater hemostatic success at 2 min in the arterial model and was at least 100 times more likely to be hemostat effective at 2 min in the hepatic model than ORC. CONCLUSIONS: PCC provided faster hemostasis than ORC in a vascular and hepatic surgical model with impaired coagulation.

Randomized, controlled comparison of advanced hemostatic pads in hepatic surgical models.

Blood loss during hepatic surgery leads to poor patient outcomes. This study investigates the hemostatic efficacy of a novel sealing hemostatic pad (polyethylene glycol-coated collagen, PCC) and a fibrin sealant pad (fibrin-thrombin coated collagen, FTC) in a leporine hepatic segmentectomy and a porcine hepatic abrasion model. A segmentectomy was used to compare hemostatic success and hematoma incidence in 20 rabbits (10/group). Hepatic abrasions were used to compare hemostatic success up to 10 min after application in six pigs (42 lesions/group). In the segmentectomy model, PCC achieved 100% hemostatic success within 2 min (95% CI: 72.3% to 100%) and FTC achieved 80% hemostatic success within 3 min (49.0% to 94.3%). PCC had lower hematoma incidence at 15 min (0.0 versus 11.1%) and 24 h (20.0 versus 66.7%). In the abrasion model, PCC provided superior hemostatic success at 3 (odds ratio: 24.8, 95% CI: 8.86 to 69.2, P < 0.001), 5 (66.3, 28.5 to 153.9, P < 0.001), 7 (177.5, 64.4 to 489.1, P < 0.001), and 10 min (777.6, 148.2 to 4078, P < 0.001) leading to statistically significant less blood loss. The novel sealing hemostat provides faster and more sustained hemostasis than a fibrin sealant pad in a leporine hepatic segmentectomy and a porcine hepatic abrasion model of hepatic surgery.

Preclinical safety and efficacy of a new recombinant FIX drug product for treatment of hemophilia B.

Baxter has developed a new recombinant factor IX (rFIX) drug product (BAX326) for treating patients with hemophilia B, or congenital FIX deficiency. An extensive preclinical program evaluated the pharmacokinetics, efficacy, and safety of BAX326 in different species. The efficacy of BAX326 was tested in three mouse models of primary pharmacodynamics: tail-tip bleeding, carotid occlusion, and thrombelastography. The pharmacokinetics was evaluated after a single intravenous bolus injection in mice, rats, and macaques. Toxicity was assessed in rats and macaques, safety pharmacology in rabbits and macaques, and immunogenicity in mice. BAX326 was shown to be efficacious in all three primary pharmacodynamic studies (P ≤ 0.0076). Hemostatic efficacy was dose related and similar for the three lots tested. Pharmacokinetic results showed that rFIX activity and rFIX antigen concentrations declined in a bi-phasic manner, similar to a previously licensed rFIX product. BAX326 was well tolerated in rabbits and macaques at all dose levels; no thrombogenic events and no adverse clinical, respiratory, or cardiovascular effects occurred. BAX326 was also shown to have a similar immunogenicity profile to the comparator rFIX product in mice. These results demonstrate that BAX326 has a favorable preclinical safety and efficacy profile, predictive of a comparable effect to that of the previously licensed rFIX in humans.

Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice.

Coronary heart disease is a major cause of death in the western world. Although essential for successful recovery, reperfusion of ischemic myocardium is inevitably associated with reperfusion injury. To investigate a potential protective role of ADAMTS13, a protease cleaving von Willebrand factor multimers, during myocardial ischemia/reperfusion, we used a mouse model of acute myocardial infarction. We found that Adamts13(-/-) mice developed larger myocardial infarctions than wild-type control mice, whereas treatment of wild-type mice with recombinant human ADAMTS13 (rhADAMTS13) led to smaller infarctions. The protective effect of ADAMTS13 was further confirmed by a significant reduction of cardiac troponin-I release and less myocardial apoptosis in mice that received rhADAMTS13 compared with controls. Platelets adherent to the blood vessel wall were observed in few areas in the heart samples from mice treated with vehicle and were not detected in samples from mice treated with rhADAMTS13. However, we observed a 9-fold reduction in number of neutrophils infiltrating ischemic myocardium in mice that were treated with rhADAMTS13, suggesting a potent anti-inflammatory effect of ADAMTS13 during heart injury. Our data show that ADAMTS13 reduces myocardial ischemia/reperfusion injury in mice and indicate that rhADAMTS13 could be of therapeutic value to limit myocardial ischemia/reperfusion injury.

A new mouse model mimicking thrombotic thrombocytopenic purpura: correction of symptoms by recombinant human ADAMTS13.

Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.

T cell-independent restimulation of FVIII-specific murine memory B cells is facilitated by dendritic cells together with toll-like receptor 7 agonist.

Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4(+) T cells. However, CD11c(+) dendritic cells facilitate the T cell-independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell-dependent restimulation, the antibody response after T cell-independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor-mediated effector functions, both are required for effective immune responses against pathogens.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model.

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Retinal cone topography of artiodactyl mammals: influence of body height and habitat.

This study addresses the correlation of retinal topography with factors such as the visual environment, life style, and behavior for a major mammalian group, the artiodactyls. To provide a broader basis for semiquantitative comparison, short-wavelength-sensitive (S)- and middle-to-long-wavelength-sensitive (M)-opsin cone receptor populations from 25 species from five artiodactyl families and of the African elephant were labeled and sampled. The resulting topographic maps were analyzed with respect to the position and extension of high-density regions. For better parameter differentiation, systematic relationships were statistically normalized. In all species examined, two classes of cones have been detected. In most species, the S-cone maxima were located in the temporodorsal retina, but there are exceptions such as the roe deer with accumulation in the ventral retina. For M-cones, as a consequence of their role in terrain/food assessment and predator detection, the standard topography is L-shaped: a horizontal visual streak including a temporal area centralis is extended by a temporal rim. Its extension is correlated with the animal's body height (P = 0.0017): small species (pudu, mouse deer) tend to have a visual streak only, whereas the giraffe shows a complete dorsal arch of elevated densities. Furthermore, a size-independent habitat correlation was revealed for a similar M-cone pattern (P < 0.0001): mountainous species show a striking specialization around the dorsal retina, pointing to the importance of the inferior visual field in precipitous terrain.

Independent variation of retinal S and M cone photoreceptor topographies: A survey of four families of mammals.

In mammals, cone photoreceptor subtypes are thought to establish topographies that reflect the species-relevant properties of the visual environment. Middle- to long-wavelength-sensitive (M) cones are the dominant population and in most species they form an area centralis at the visual axis. Short-wavelength-sensitive (S) cone topographies do not always match this pattern. We here correlate the interrelationship of S and M cone topographies in representatives of several mammalian orders with different visual ecology, including man, cheetah, cat, Eurasian lynx, African lion, wild hog, roe deer, and red deer. Retinas were labeled with opsin antisera and S and M cone distributions as well as S/M cone ratios were mapped. We find that species inhabiting open environments show M cone horizontal streaks (cheetah, pig, deer). Species living in structured habitats (tiger, lynx, red deer) have increased S cone densities along the retinal margin. In species with active vision (cheetah, bear, tiger, man), S cone distributions are more likely to follow the centripetal M cone gradients. Small species show a ventral bias of peak S cone density which either matches the peak of M cone density in a temporal area centralis (diurnal sciurid rodents, tree shrews) or not (cat, manul, roe deer). Thus, in addition to habitat structure, physical size and specific lifestyle patterns (e.g. food acquisition) appear to underlie the independent variations of M and S cone topographies.