Activity of Xase complex toward FX in the presence of non-activated and activated platelets.

<p>Activity of Xase complex toward FX in the presence of non-activated and activated platelets.</p

Interactions of FXIa-activated rFIXFc and rFIX with FVIIIa in the presence of different phospholipid sources.

<p>Interactions of FXIa-activated rFIXFc and rFIX with FVIIIa in the presence of different phospholipid sources.</p

Recombinant Factor IX Fc Fusion Protein Maintains Full Procoagulant Properties and Exhibits Prolonged Efficacy in Hemophilia B Mice

<div><p>Introduction</p><p>Hemophilia B is an inherited X chromosome–linked 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.</p><p>Materials and Methods</p><p>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 compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX <i>in vivo</i> in hemophilia B mouse bleeding models.</p><p>Results and Conclusions</p><p>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, 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.</p></div

Activity of Xase complex toward FX in the presence of synthetic phospholipids (25% PS/75% PC).

<p>Activity of Xase complex toward FX in the presence of synthetic phospholipids (25% PS/75% PC).</p

Activation of rFIXFc by FXIa, active Xase complex formation, and rFIXa levels in non-activated rFIXFc.

<p>rFIXFc, recombinant factor IX Fc fusion protein; FXIa, activated factor XIa; rFIXa, recombinant activated factor IXa; rFIX, recombinant factor IX; FVIIIa, activated factor VIIIa; PL, phospholipids; FXa, activated factor Xa; SD, standard deviation. (A) Cleavage of rFIXFc by FXIa. rFIXFc (left) or rFIX (right) was activated with FXIa in the presence of calcium chloride at 37°C. Aliquots were collected over 5 minutes at the indicated times and subjected to non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis to resolve the cleaved products. (B) Quantitation of non-activated rFIXFc or rFIX (%) remaining in samples at different times following FXIa activation (mean ± standard deviation (SD); <i>n</i> = 3). (C) Formation of active Xase complexes with rFIXaFc requires FVIIIa, PL, and prior activation. Rates of FXa generation were derived from reactions containing rFIXFc or rFIX prior to activation (i) or following activation (ii) by FXIa. Also, rates of FXa generation were calculated for rFIXaFc in the absence of a source of PL (iii, cephalin, rabbit brain extracts) or FVIIIa (iv) (mean ± SD; <i>n</i> = 4). (D) Rates of FXa generation (mean ± standard deviation) were determined using rFIXFc or rFIX that were either activated (1 nM) with FXIa or nonactivated (125–1000 nM) to compare the levels of FIXa in rFIXFc and rFIX (<i>n</i> = 4).</p

Interaction of rFIXaFc with AT and with phospholipids.

<p>rFIXFc, recombinant factor IX Fc fusion protein; AT, antithrombin III; FXIa, activated factor XIa; rFIX, recombinant factor IX; rFIXa, recombinant activated factor IXa; FXa, activated factor Xa; SD, standard deviation; FVIIIa, activated factor VIIIa. (A) Inhibition of rFIXaFc by AT. AT was titrated in 100 nM FXIa-activated rFIXFc or rFIX. The activity of rFIXaFc or rFIXa was monitored using an endpoint FXa generation assay. The amount of FXa generated is reflected in the absorbance at 405 nm as a result of the cleavage of an FXa chromogenic substrate. For each molecule, the data at AT concentrations above or below 100 nM were grouped and fitted (two linear fits per molecule). The point of intersection (inflection point) of both fits was mathematically determined and reflected the concentration of rFIXaFc (89.1% ± 0.55%) and rFIXa (97.2% ± 1.15%) following 5 minutes of FXIa activation (mean ± SD; <i>n</i> = 3). (B) Interaction of rFIXaFc with phospholipid vesicles. rFIXaFc or rFIXa was incubated with FVIIIa in the presence of varying concentration of phospholipid vesicles composed of 25% L-α-phosphatidylserine and 75% L-α-phosphatidylcholine to form active Xase complexes. The formed complexes were then evaluated in FXa generation to derive the rates of FXa formation (<i>n</i> = 4).</p

rFIXFc prophylactic efficacy in hemophilia B mice.

<p>rFIXFc, recombinant factor IX Fc fusion protein; TVT, tail vein transection; rFIX, recombinant factor IX; rFIXFc_72h, recombinant factor IX Fc fusion protein dosed 72 hours prior to injury; rFIX_24h, recombinant factor IX dosed 24 hours prior to injury. (A) TVT study design in hemophilia B mice. Dosing of rFIXFc was performed 72 hours prior to injury, whereas that of rFIX was 24 hours prior to injury. (B) Percent survival following TVT. Mice were dosed with vehicle (grey), rFIXFc_72h (black dashed line, open symbols), rFIX_24h (black solid line, closed symbols) (120 IU/kg, 40 IU/kg, 13 IU/kg or 4 IU/kg; <i>n</i> = 20/dose except for 40 IU/kg dose; <i>n</i> = 30). Following tail vein transection (TVT), mice were monitored over 24 hours and the survival curve of time to re-bleed was generated. The survival curves for mice treated with similar doses of rFIXFc_72h or rFIX_24h were comparable using a log-rank (Mantel-Cox) test (<i>P</i> = 0.4886, 0.9203, 0.7574 and 0.5210 for the 4-, 13-, 40- and 120-IU/kg treatment groups, respectively). (C) Dose-response curves at 24 hours following TVT. Survival rates were plotted for both rFIXFc_72h (open circles) and rFIX_24h (closed squares) treatments. The ED₅₀ values were derived for rFIXFc_72h treatments (17.8 IU/kg) and rFIX_24h (15.4 IU/kg).</p

rFIXFc efficacy in treating acute bleeds in hemophilia B mice.

<p>rFIXFc, recombinant factor IX Fc fusion protein; rFIX, recombinant factor IX. A tail clip mouse bleeding model was used to evaluate the acute efficacy of rFIXFc. Hemophilia B mice were given rFIXFc (720, 240, 120, 80 or 40 IU/kg, <i>n</i> = 15/dose) or rFIX (360, 120 or 40 IU/kg; <i>n</i> = 15/dose). (A) Tail clip was performed as described in methods, and the blood loss was quantified. Symbols indicate blood loss from individual mice, horizontal bar median blood loss per group. (B) The linear regression curve of median blood loss versus the log (base 10) of dosing concentration was plotted. Both unpaired <i>t</i> test with Welch’s correction and Mann-Whitney test indicated no significant difference between the rFIXFc and rFIX curves (<i>P</i> = 0.9315 and 1.0, respectively). (C) The linear regression curve of the percentage of protection versus the log (base 10) of dosing concentration was plotted. Mice were considered protected if they bled ≤ mean + 2 standard deviation of the blood loss in normal C57BL/6 mice following the same procedure. The <i>t</i> tests indicated that the two curves derived from rFIXFc and rFIX treatment groups were comparable with <i>P</i> values of 0.9385 and 0.8801, respectively.</p

Thrombin generation profile of FVIII variants.

<p>Activity determination was based on equal chromogenic activity. Representative curves with selected concentrations are shown in (A) 1 IU/mL (B) 0.25 IU/mL (C) 0.0625 IU/mL. Select parameters are shown in (D) as peak thrombin and (E) as ETP.</p

Summary of PK parameters for SC rFVIIIFc and rFVIIIFc.

<p>Summary of PK parameters for SC rFVIIIFc and rFVIIIFc.</p