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Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Background:Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations.Methods:Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined.Results:Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents.Conclusions:The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues.Funding:Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Intravenous (IV) administration in mice is predominantly performed via the lateral tail veins. The technique requires adequate training before it can be used safely and routinely. A novel anaesthesia induction chamber has been developed to simplify the treatment and to facilitate IV injection in mice, particularly for untrained personnel. We have assessed the benefits of the chamber in refining IV injection in isoflurane-anaesthetized mice in direct comparison with the common restrainer method on conscious animals. The body weight, nesting behaviour and concentrations of faecal corticosterone metabolites were taken as indicative of distress induced by the various procedures. The results suggest that both methods of tail-vein injection induce similar levels of momentary stress in the animals, revealed by a short-term increase in the levels of stress hormone metabolites in faeces. A temporary reduction of body weight was observed after IV injection under isoflurane anaesthesia but not for conscious mice injected in the common restrainer. We conclude that the severity of tail-vein injection in mice is ‘mild’ for both methods. There was no evidence that refining the procedure by using isoflurane anaesthesia in the induction chamber was associated with any benefit.

Treatment of hemophilia A is complicated by development of FVIII neutralizing antibodies, which occurs in ∼30% of patients with severe hemophilia A. Acute bleeds in inhibitor patients can be treated with the available bypassing agents, activated prothrombin complex concentrate (APCC, “FEIBA”) or recombinant factor FVIIa (rFVIIa). However, in 10-20% of bleeding events, the patient becomes refractory or non-responsive to monotherapy. Sequential combined bypass therapy (SCBT) or concomitant combined bypass therapy (CCBT) of rFVIIa and APCC are last resorts to stop bleeding. Using a translational in vitro-to-in vivo approach, we studied the combined effect of APCC and rFVIIa (“BAX 817”) in a global hemostasis assay and the efficacy and acute toxicity of CCBT in a hemophilia A mouse model. The combined hemostatic effect of rFVIIa and APCC was assessed by calibrated automated thrombography (CAT) in FVIII-inhibited normal human plasma under optimized conditions. Clinically relevant plasma concentrations of rFVIIa (0.11-5.25 µg/mL) and APCC (20-1000 mU/mL) alone and in combination showed a concentration-dependent response. The total effect of combined rFVIIa/APCC on thrombin generation was always lower or equal to the sum of individual effects, suggesting an additive effect for the joint action of rFVIIa/APCC. Several combinations reached the target activity equivalent to 1000 mU/mL APCC of which we selected 200 mU/mL APCC + 0.88 µg/mL rFVIIa as the starting point for the efficacy study. In vitro plasma concentrations were translated to doses for the mouse tail clip model, based on previous efficacy data from rFVIIa and APCC monotherapy studies. For the efficacy study, FVIII knockout (ko) mice (n=16 per group) received a single intravenous injection of 200-2700 µg/kg rFVIIa, 60-250 U/kg APCC, concomitantly administered combinations of 60 U/kg APCC and 200-2000 µg/kg rFVIIa, or buffer. Tail clip was performed and blood loss measured over 60 min. Statistical analysis of the total blood loss determined that the minimally effective doses were 2000 µg/kg rFVIIa, 250 U/kg APCC, and 1200 µg/kg rFVIIa + 60 U/kg APCC for the combination therapy. Importantly, treatment with 1200 µg/kg rFVIIa and 60 U/kg APCC alone was not significantly different to treatment with buffer control. Thus, low doses of rFVIIa and APCC in combination were shown to correct a bleeding phenotype. Safety and thrombogenicity of rFVIIa/APCC in FVIII ko mice were evaluated in an acute toxicity study. Eight mice per treatment group received a single intravenous dose of a minimally effective combination (1200 µg/kg rFVIIa and 60 U/kg APCC), or a combination of the minimally effective stand-alone doses (2000 µg/kg rFVIIa and 270 U/kg APCC), or buffers only. No clinical signs were observed in any group for 24 h after treatment. No changes in hematological parameters were measured except for a dose-dependent decrease in platelet count, indicating some activation of the coagulation system. There were also no changes in blood chemistry including levels of lactate dehydrogenase, an indicator of toxic effects on the liver or heart and hemolysis. In addition, potential target organs, including the brain, heart, lungs, were macroscopically inspected for lesions, and paraffin embedded and stained tissue samples histologically examined for microscopic abnormalities. To our knowledge, these are the first in vivo studies assessing CCBT with low doses of rFVIIa and APCC in a hemophilia animal model. We show that combined doses of rFVIIa/APCC – not efficacious as stand- alone – reduce blood loss after tail clip and appear to be safe in FVIII ko mice. Interestingly, the minimally effective combination dose in mice translates to human doses of 40 µg/kg rFVIIa + 20 U/kg APCC which falls into the clinically applied dose ranges of CCBT. We further provide in vitro evidence that the joint action of rFVIIa/APCC is additive rather than synergistic. Disclosures: Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Knappe:Baxter Innovations GmbH, Vienna, Austria: Employment. Leidenmuehler:Baxter Innovations GmbH, Vienna, Austria: Employment. Piskernik:Baxter Innovations GmbH, Vienna, Austria: Employment. Tippl:Baxter Innovations GmbH, Vienna, Austria: Employment. Resch:Baxter Innovations GmbH, Vienna, Austria: Employment. Verdino:Baxter Innovations GmbH, Vienna, Austria: Employment. Bauer:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Abstract 2216 Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for potential prophylaxis and treatment of thrombotic thrombocytopenic purpura (TTP). We established a disease model in ADAMTS13 ko mice (B6.129-ADAMTS13tm1Dgi), in which the animals simultaneously and consistently develop TTP-like symptoms by challenge with a high dose of human recombinant von Willebrand factor (rVWF) containing ultra-large VWF multimers. To test the prophylactic efficacy of Baxter's rADAMTS13, groups of 10 ADAMTS13 ko mice received single doses of rADAMTS13 (1, 5, 40, 80 or 200U/kg) before challenge with rVWF. Doses of 1 and 5mL/kg fresh frozen plasma corresponding to 1 and 5U/kg ADAMTS13 served as positive controls. In a second study, the prophylactic and therapeutic efficacy of Baxter's rADAMTS13 was tested over time. Groups of 10 ADAMTS13 ko mice received a single dose of 200 FRETS-U/kg rADAMTS13 5min-120h before, or 15–180min after challenge with rVWF. In both studies, buffer was used as a negative control. Efficacy was defined as the degree of prevention of platelet drop and LDH increase. Schistocytosis and organ damage were also assessed. Morbidity in negative controls was 100%: all animals receiving rVWF containing ultra-large VWF multimers were severely thrombocytopenic, showed increased LDH levels, schistocytosis and organ damage. Prophylactic treatment with rADAMTS13 prevented LDH increase (p Efficacy of rADAMTS13 was treatment interval-dependent in both studies. Platelet count at termination of all rADAMTS13-treated animals was statistically higher than that of buffer-treated controls (p≤0.0001). However, animals receiving prophylactic treatment 120h before administration of rVWF showed severe thrombocytopenia; clinically relevant protection was only seen with treatment intervals ≤72h. Therapeutic treatment with rADAMTS13 stabilized platelet count and prevented further thrombocytopenia. LDH, schistocytosis and organ damage confirmed the treatment interval-dependent improvement in platelet count. In summary, Baxter's rADAMTS13 was effective in an rVWF-induced animal model closely mimicking the situation in patients with hereditary TTP. Comparison with FFP, the current standard of treatment of TTP, showed Baxter's rADAMTS13 to have similar or slightly improved efficacy. Disclosures: Schiviz: Baxter Innovations GmbH: Employment. Resch:Baxter Innovations GmbH: Employment. Farnleitner:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Abstract 4659 Baxter has developed a recombinant FVIIa (rFVIIa) product for the treatment of patients with hemophilia A and hemophilia B with inhibitors. The aim of the presented preclinical studies was to evaluate the efficacy of Baxter's rFVIIa. Four animal models that are considered to relevantly reflect the conditions in different patient populations were used: hemophilia A (FVIII ko) mice, hemophilia B (FIX ko) mice, factor VIII (FVIII)-inhibited rabbits and warfarin-pretreated rats. In all studies, Baxter's rFVIIa was tested at different doses to obtain dose-effect curves. A commercially available rFVIIa served as the active control item, buffer served as the negative control item. All test and control items were administered intravenously. Mice received prophylactic treatment 5 minutes prior to start of the experiment, rabbits and rats received therapeutic treatment after the injuries were inflicted. Twenty FVIII ko mice (B6;129S4-F8tm1Kaz; 10m/10f) and 20 FIX ko mice (B6;129P2-F9tm1Dws; 10m/10f) per group were used in a tail-tip bleeding model. Baxter's rFVIIa was tested at doses of 0.6, 1.2 and 2.7 mg/kg, the active control item was tested at a dose of 2.7 mg/kg. Ten FVIII ko mice (B6;129S4-F8tm1Kaz; 5m/5f) per group were used to assess the influence of rFVIIa on the thrombelastogram of hemophilic mice. Both items were tested at doses of 0.1, 0.6 and 1.2 mg/kg. Six rabbits (New Zealand White; 6m) per group were used in a nail-cut model. The animals were pretreated with a goat polyclonal FVIII-antibody (34,000 BU/kg i.v., 45 minutes prior to start of the experiment), leading to deficiency of endogenous FVIII. Baxter's new rFVIIa was tested at doses of 0.1, 1.0, 2.0, 2.5 and 3.0 mg/kg, the active control item at 2.0 mg/kg. Six rats (Sprague Dawley; 6m) per group were used in a tail-tip bleeding model. The animals were pretreated with warfarin (11 mg p.o., 1 day prior to start of the experiment), leading to a deficiency of vitamin K-dependent coagulation factors II, VII, IX and X, protein C and protein S. Baxter's new rFVIIa was tested at 0.25, 1.0 and 2.0 mg/kg, the active control item at 2.0 mg/kg. A dose-dependent hemostatic effect of Baxter's new rFVIIa could be shown in all primary pharmacodynamic studies performed. Furthermore, statistical evaluation of the efficacy of Baxter's new rFVIIa did not reveal any statistically significant differences from the commercially available rFVIIa product after treatment at the same doses. In summary, the results of our studies show that Baxter's new rFVIIa is prophylactically and therapeutically effective in animal models of efficacy closely reflecting the conditions in hemophiliacs with inhibitors and patients suffering from FVII-deficiency. Disclosures: Schiviz: Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Resch:Baxter Innovations GmbH: Employment. Leidenmuehler:Baxter Innovations GmbH: Employment. Bischetsrieder:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.