Concomitant Administration Of Activated Prothrombin Complex Concentrate and Recombinant Activated Factor VII At Low Doses Are Efficacious and Safe In Hemophilic Mice
Authors: Dockal Michael , Knappe Sabine , Leidenmuehler Peter , Piskernik Christina , Tippl Sylvia , Resch Marlene , Verdino Dagmar , Bauer Alexander , Hoellriegl Werner , Scheiflinger Friedrich 
Published: October 2019

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.