To determine whether the extension in half-life translates into prolonged activity, mice were intravenously dosed with equimolar levels of rFIXFc or rFIX that translate into 50 IU/kg and 100 IU/kg, respectively, and whole blood samples collected at various times were analyzed using the NATEM assay (Fig 5)

To determine whether the extension in half-life translates into prolonged activity, mice were intravenously dosed with equimolar levels of rFIXFc or rFIX that translate into 50 IU/kg and 100 IU/kg, respectively, and whole blood samples collected at various times were analyzed using the NATEM assay (Fig 5). is reflected in the absorbance at 405 nm as a result of the cleavage of an FXa chromogenic substrate.(EPS) pone.0148255.s002.eps (547K) GUID:?F55DC685-ADA0-4DA2-B24C-F56A8802461C S1 Text: Supplemental Methods. (DOCX) pone.0148255.s003.docx (18K) GUID:?3FE5BAAB-2699-4EBB-961A-B1D6F461C382 S2 Text: ARRIVE Guidelines Checklist. (PDF) pone.0148255.s004.pdf (1.0M) GUID:?0CEEB2F9-CC38-4738-9A9A-BE56D28AAF5B Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Introduction Hemophilia B is an inherited Rabbit polyclonal to FAK.This gene encodes a cytoplasmic protein tyrosine kinase which is found concentrated in the focal adhesions that form between cells growing in the presence of extracellular matrix constituents. X chromosomeClinked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans. Materials and Methods Procoagulant properties of rFIXFc and rFIX (BENEFIX?) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential Azathioprine compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in hemophilia B mouse bleeding models. Results and Conclusions The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, Azathioprine and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice. Introduction Blood clotting in response to vessel and capillary damage is essential for normal hemostasis. Hemophilia B is an Azathioprine inherited X chromosomeClinked disorder characterized by the inability to clot blood and is due to the absence or diminished levels of coagulation factor IX (FIX) [1]. In its severe form (FIX activity 1%), patients may experience bleeding either spontaneously or following an injury; over time, repeated bleeding episodes in the joints and muscles can result in severe arthropathy. The current standard of care for patients with hemophilia B is replacement therapy through infusion of recombinant or highly purified plasma FIX concentrates either on demand to treat bleeding or prophylactically to prevent bleeding [1,2]. In on-demand therapy, patients receive FIX concentrates in response to injury, hemorrhage, or prior to surgery and may require multiple infusions to achieve and maintain protective factor levels. In prophylaxis, a preferred regimen for severe hemophilia B, patients infuse replacement factor (rFIX or plasma-derived FIX) two to three times per week with the goal of maintaining adequate levels of FIX to prevent bleeding [3,4]. The frequency of infusions required for prophylactic treatments (generally 2C3 times per week) is a limiting aspect of treatment and due in part to the relatively short half-life of conventional FIX products [5C7]. To extend the circulating half-life of FIX and, therefore, reduce the dosing frequency for hemophilia B therapy, we have developed a recombinant factor IX Fc fusion protein (rFIXFc) that is composed of a single molecule of rFIX covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening linker sequence [8,9]. Fc fusion leverages an endogenous pathway utilized by IgG to extend the half-life of rFIXFc by enabling interactions intracellularly with the neonatal Fc receptor (FcRn). IgG catabolism is normally prevented by FcRn in vascular endothelium and monocytes, which contributes to the long half-life of antibodies [10C14]. We previously reported that in this monomeric configuration, the elimination half-life of rFIXFc is approximately 3- to 4-fold longer than that of recombinant FIX (rFIX) in multiple animal models including normal mice, rats and monkeys and in FIX-deficient mice and dogs [8]. In addition, a prolonged half-life of rFIXFc in comparison with rFIX was also observed in humans in phase 3 clinical trials (geometric mean, 82.1 hours for rFIXFc vs 33.8 hours for rFIX; 0.001 in a phase 3 trial) [15]..