Evaluation of Nonacog Beta Pegol Long-term Safety in the Immune-deficient Rowett Nude Rat (Crl:NIH-Foxn1rnu).

Nonacog beta pegol is a 40-kDa polyethylene glycosylated (PEGylated) human recombinant coagulation factor IX, intended for the treatment of hemophilia B. Human coagulation factors are immunogenic in animals; therefore, to evaluate the long-term toxicity of nonacog beta pegol, an immune-deficient, athymic rat (Rowett nude; Crl:NIH-Foxn1(rnu)) was used. Rats (n = 216) were given intravenous nonacog beta pegol 0, 40, 150, 600, or 1,200 IU/kg every 5th day for 26 weeks. To avoid infections, the animals were housed in a full-barrier environment with sterilized food and bedding. Standard toxicity end points were unaffected by treatment. All treated animals were exposed to nonacog beta pegol throughout the study, and no animals developed antidrug antibodies. Immunohistochemical staining revealed PEG in choroid plexus epithelial cells in a dose-dependent manner. Transmission electron microscopy showed that PEG was distributed in cytoplasmic vesicles of these cells, with no apparent effect on cellular organelle structures. Fourteen (6.5%) animals were euthanized or died prematurely due to nontreatment-related infections in the urogenital system and skin. In conclusion, the athymic rat is a suitable model for testing chronic toxicity of human proteins that are immunogenic in animals. Nonacog beta pegol was generally well tolerated, with no adverse effect of PEG on choroid plexus epithelial cells.

Evaluation of the toxicology, pharmacokinetics, and local tolerance of recombinant factor IX Fc fusion protein in animals.

INTRODUCTION: Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. MATERIALS AND METHODS: Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. RESULTS: There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. CONCLUSIONS: rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Thrombotic safety of prothrombin complex concentrate (Beriplex P/N) for dabigatran reversal in a rabbit model.

INTRODUCTION: In vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding. MATERIALS AND METHODS: Anesthetized rabbits were treated with initial 0, 75, 200 or 450 µg kg(-1) dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15 min after the start of dabigatran administration, PCC doses of 0, 50 or 300 IU kg(-1) were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model. RESULTS: At the suprapharmacologic dose of 300 IU kg(-1), PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75 µg kg(-1) dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300 IU kg(-1) PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p=0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding. CONCLUSIONS: In this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis.

Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model.

In earlier work, we showed that adeno-associated virus-mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.

Myocardial fibrosis in mice with overexpression of human blood coagulation factor IX.

Elevated circulatory levels of many blood coagulation factors are known to be a risk factor for deep vein thrombosis in humans. Here we report the first direct demonstration of a close association between elevated circulatory factor IX levels in mice with thrombosis as well as myocardial fibrosis. Transgenic mice overexpressing human factor IX at persistently high levels died at much younger ages than their cohorts expressing lower levels, or nontransgenic control animals. The median survival age of animals was inversely related to the circulatory levels of human factor IX. Prematurely dying animals had focal fibrotic lesions predominantly present in the left ventricular myocardium, and vasculatures in these lesions showed fibrin deposition. Thromboemboli were also present in other organs, including lung and brain. These observations support the hypothesis that persistently high circulatory levels of factor IX are a risk factor not only for thrombosis and/or thromboembolism, but also for myocardial fibrosis mimicking human myocardial infarction.

Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity.

We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 x 10(10) to 1 x 10(11) plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.

Effect of sodium pentosan polysulphate on the thrombogenicity of prothrombin complex concentrates.

The addition of pentosan polysulphate sodium (NaPPS) to thrombogenic prothrombin complex concentrates (PCC) dose-dependently reduces or abolishes thrombus formation in rats in the stasis model acc. to Wessler. However, no reduction of thrombogenicity was found in PCC preparations manufactured in the presence of NaPPS.

Toxicity of factor IX concentrates in mice.

Mice to which Factor IX concentrates are administered intraperitoneally at high doses (approximately 1000 u/kg) die between 4 and 18 hours thereafter. Histologic examination reveals hemorrhage in the lung and liver. Death is prevented by reduction of the concentrate with dithiothreitol but not by treatment of the preparation with enzyme inhibitors (diisopropyperosphorofluoridate, soybean trypsin inhibitor, heparin). Prothrombin, devoid of Factors VII, IX and X, also is lethal to mice. Activation of the Factor IX concentrate by insolubilized Russel's Viper Venom (NAPTT 1/5000 less than 20'') resulted in decreased mortality when compared to controls (4/30 vs. 11/20). These experiments indicate that some of the adverse effects of Factor IX concentrates may result from high levels of zymogens rather than the presence of small amounts of active enzymes. Intensive animal testing might be indicated for routine quality control of Factor IX concentrates.