Activated Blood Coagulation Factor X (FXa) Contributes to the Development of Traumatic PVR Through Promoting RPE Epithelial-Mesenchymal Transition.

Purpose: Uncontrolled coagulation reactions contribute to pathological fibroproliferation in several organs, and yet their role in proliferative vitreoretinopathy (PVR) remains to be elucidated. In this study, we evaluated the profibrotic effects of FXa in RPE cells and in a mouse model of PVR. Methods: FXa levels in the eyes of traumatic PVR patients and rabbit models of mechanical ocular trauma was measured by ELISA and immunohistochemistry. FXa-induced RPE EMT was assessed by examining cell proliferation, migration, tight junction changes, and expression of fibrotic markers. For in vivo study, FXa was injected into dispase-injured eyes, then intraocular fibrosis was evaluated by histological analysis and Western blotting. The therapeutic effect of FXa inhibitor was also examined in PVR mouse models. Results: Vitreous FXa were higher in patients with traumatic PVR compared to patients with macular hole. Moreover, expressions of FXa and PAR1 were found in the epiretinal membranes from traumatic PVR patients. Vitreous FXa were markedly increased after mechanical ocular trauma in rabbits. In vitro, FXa stimulated RPE EMT characterized as ZO-1 disruption, compromised cell polarity, and increased fibronectin expressions. Co-injection of FXa and dispase in mice induced more severely damaged retinal structures, and increased α-SMA expressions than FXa or dispase treatment alone. Oral FXa or thrombin inhibitors significantly blocked intraocular fibrosis in PVR mouse models. FXa promoted phospho-activation of p38 in ARPE19 cells, which was dependent on PAR1. Moreover, TGF-ßR inhibitor also significantly alleviated FXa-induced intraocular fibrosis in mice. Conclusions: FXa promotes intraocular fibrosis in mice via mechanisms involving RPE activation.

The carboxyl-terminal region of human coagulation factor X as a natural linker for fusion strategies.

Fusion with human serum albumin (HSA), which represents a well-established technique to extend half-life of therapeutic proteins, commonly exploits intervening peptide linkers as key components. Here, we explored the human coagulation factor X (FX) carboxyl-terminal region, previously demonstrated by us to be dispensable for secretion and coagulant activity, as a natural linker for fusion purposes. To test our hypothesis, we compared direct FX-HSA fusion with the designed FX-HSA fusion proteins mimicking the recombinant activated factor VII (rFVIIa)-HSA or factor IX (FIX)-HSA chimeras, both strongly dependent from artificial linkers. Three constructs were produced by direct tandem fusion (FX-HSA) and through flexible (glycine/serine; FX-GS-HSA, mimicking rFVIIa-HSA) or cleavable (incorporating the FX activation site; FX-CL-HSA, mimicking FIX-HSA) linkers. The FX-HSA was efficiently secreted and displayed prolonged plasma persistence in mice. All chimeras possessed remarkable pro-coagulant activity, comparable to FX for FX-HSA (88.7 ±â€¯6.0%) and FX-CL-HSA (98.0 ±â€¯16.4%) or reduced for FX-GS-HSA (55.8 ±â€¯5.4%). Upon incubation with activators, FX-HSA and FX-CL-HSA displayed a correct activation profile while the FX-GS-HSA activation was slightly defective. In fluorogenic-based assays, FX-HSA showed normal activity over time and a specific amidolytic activity (1.0 ±â€¯0.12) comparable to that of FX. Overall, the FX-HSA features indicate that the FX carboxyl-terminal region represents an intrinsic sequence allowing direct tandem fusion. Our results provide the first experimental evidence for i) a coagulation factor fusion protein with biological properties independent from artificial linkers, ii) the suitability of FX carboxyl-terminal region as a natural linker for fusion purposes.

Prophylactic treatment of bleeding episodes in children <12 years with moderate to severe hereditary factor X deficiency (FXD): Efficacy and safety of a high-purity plasma-derived factor X (pdFX) concentrate.

BACKGROUND: Hereditary factor X (FX) deficiency (FXD) affects 1:500 000-1:1 000 000 people worldwide. A novel, high-purity plasma-derived FX concentrate (pdFX) is available in the United States and European Union as replacement therapy for FXD, but data are scarce on pdFX use in children <12 years. AIM: This prospective, open-label phase 3 study assessed the safety, efficacy and pharmacokinetics of pdFX in children <12 years with moderate/severe FXD. METHODS: Subjects aged <12 years with basal plasma FX activity (FX:C) <5 IU/dL received pdFX as prophylactic and on-demand treatment, with doses adjusted to maintain FX:C > 5 IU/dL. After ≥26 weeks and ≥50 exposure days, investigators rated pdFX efficacy for preventing/decreasing bleeds. Secondary endpoints included number and severity of bleeds, trough FX:C and incremental recovery. Safety parameters were adverse events (AEs), inhibitor development and changes in laboratory parameters. RESULTS: The study enrolled 9 subjects (0-5 years, n = 4; 6-11 years, n = 5) with severe (n = 8) or moderate (n = 1) FXD. At end of study, investigators rated pdFX efficacy excellent for all subjects. Ten bleeds occurred (n = 3 subjects; 6 major, 3 minor, 1 unassessed for severity). Trough FX:C levels remained >5 IU/dL for all subjects after the last dose adjustment study visit. Mean incremental recovery was significantly lower for younger vs older subjects (1.53 vs 1.91 IU/dL per IU/kg; P = .001). All AEs were unrelated to treatment; no inhibitor development or clinically significant changes in laboratory parameters were observed. CONCLUSIONS: These results demonstrate the efficacy and safety of pdFX for treating children <12 years with moderate/severe hereditary FXD.

Efficacy, safety and pharmacokinetics of a new high-purity factor X concentrate in women and girls with hereditary factor X deficiency.

Essentials Plasma-derived factor X concentrate (pdFX) is used to treat hereditary factor X deficiency. pdFX pharmacokinetics, safety and efficacy were assessed in factor X-deficient women/girls. Treatment success rate was 98%; only 6 adverse events in 2 subjects were possibly pdFX related. On-demand pdFX 25 IU kg-1 was effective and safe in women/girls with factor X deficiency. SUMMARY: Background A high-purity, plasma-derived factor X concentrate (pdFX) has been approved for the treatment of hereditary FX deficiency, an autosomal recessive disorder. Objective To perform post hoc assessments of pdFX pharmacokinetics, safety and efficacy in women and girls with hereditary FX deficiency. Patients/Methods Subjects aged ≥ 12 years with moderate/severe FX deficiency (plasma FX activity of < 5 IU dL-1 ) received on-demand or preventive pdFX (25 IU kg-1 ) for ≤ 2 years. Results Of 16 enrolled subjects, 10 women and girls (aged 14-58 years [median, 25.5 years]) received 267 pdFX infusions. Mean monthly infusions per subject were higher among women and girls (2.48) than among men and boys (1.62). In women and girls, 132 assessable bleeding episodes (61 heavy menstrual bleeds, 47 joint bleeds, 15 muscle bleeds, and nine other bleeds) were treated with pdFX, with a 98% treatment success rate versus 100% in men and boys. Mean pdFX incremental recovery was similar in the two groups (2.05 IU dL-1 versus 1.91 IU dL-1 per IU kg-1 ), as was the mean half-life (29.3 h versus 29.5 h). Of 142 adverse events in women and girls, headache was the most common (12 events in six subjects). Six events (two infusion-site erythema, two fatigue, one back pain, one infusion-site pain) in two subjects were considered to be possibly pdFX-related. Following the trial, pdFX was used to successfully maintain hemostasis in two subjects undergoing obstetric delivery. Conclusions pdFX was well tolerated and effective in women and girls with FX deficiency. Although women and girls had different bleeding symptoms and sites than men and boys, their pdFX pharmacokinetic profile was comparable.

Plasma-derived human factor X concentrate for on-demand and perioperative treatment in factor X-deficient patients: pharmacology, pharmacokinetics, efficacy, and safety.

INTRODUCTION: Hereditary factor X (FX) deficiency is a rare autosomal recessive bleeding disorder characterized mainly by mild-to-severe bleeding into the mucous membranes, muscles or joints. Previously, treatment options for hereditary FX deficiency were limited mostly to products that may not specify FX content (i.e. fresh frozen plasma and prothrombin complex concentrates) and that have associated safety concerns. To meet the need for a single-factor replacement therapy specifically for use in FX-deficient patients, a high-purity, high-potency, human plasma-derived FX concentrate (pdFX; Coagadex®; Bio Products Laboratory, Elstree, UK) has been developed and approved for treatment of perioperative bleeding and on-demand treatment in FX-deficient patients. Areas covered: The pharmacology, efficacy, and safety of pdFX are discussed, with a review of preclinical studies and clinical trial data that led to regulatory approval of pdFX in the United States and Europe. Expert opinion: As the first single-factor replacement therapy indicated for hereditary FX deficiency, pdFX is a safe and efficacious treatment option in patients aged ≥12 years with hereditary FX deficiency. Clinical studies of pdFX provide a dosing regimen for use in cases of bleeding episodes, surgery, and prophylaxis. Further studies are ongoing regarding use of pdFX long term and in patients aged ≤12 years.

Efficacy, safety and pharmacokinetics of a new high-purity factor X concentrate in subjects with hereditary factor X deficiency.

INTRODUCTION: Hereditary factor X (FX) deficiency is a rare bleeding disorder affecting 1:500 000 to 1:1 000 000 of individuals. Until recently, no specific replacement factor concentrate was available. AIM: The aim of this study was to assess safety and efficacy of a new, high-purity plasma-derived FX concentrate (pdFX) in subjects with hereditary FX deficiency. METHODS: Subjects aged ≥12 years with moderate or severe FX deficiency (plasma FX activity <5 IU dL(-1) ) received 25 IU kg(-1) pdFX as on-demand treatment or short-term prophylaxis for 6 months to 2 years. Subjects assessed pdFX efficacy for each bleed; at end-of-study, investigators assessed overall pdFX efficacy. Blood samples for pharmacokinetic analysis were obtained at baseline and ≥6 months. Safety was assessed by adverse events (AEs), inhibitor development and changes in laboratory parameters. RESULTS: Sixteen enrolled subjects (six aged 12-17 years; 10 aged 18-58 years) received a total of 468 pdFX infusions. In the 187 analysed bleeds, pdFX efficacy was categorized as excellent, good, poor or unassessable in 90.9%, 7.5%, 1.1% and 0.5% of bleeds respectively; 83% of bleeds were treated with one infusion. For pdFX, mean (median; interquartile range) incremental recovery and half-life were 2.00 (2.12; 1.79-2.37) IU dL(-1) per IU kg(-1) and 29.4 (28.6; 25.8-33.1) h respectively. No serious AEs possibly related to pdFX or evidence of FX inhibitors were observed, and no hypersensitivity reactions or clinically significant trends were detected in laboratory parameters. CONCLUSION: These results demonstrate that a dose of 25 IU kg(-1) pdFX is safe and efficacious for on-demand treatment and short-term prophylaxis in subjects with moderate or severe hereditary FX deficiency.

Pharmacokinetics of a high-purity plasma-derived factor X concentrate in subjects with moderate or severe hereditary factor X deficiency.

INTRODUCTION: Hereditary factor X (FX) deficiency affects 1:500 000 to 1:1 000 000 of individuals. There are few published data on the pharmacokinetics (PK) of FX for existing treatments for FX deficiency, and no specific replacement factor concentrate exists. A high-purity plasma-derived FX concentrate (pdFX) has been developed for use as replacement therapy in subjects with hereditary FX deficiency. AIM: This analysis assessed pdFX PK after a single 25 IU kg(-1) bolus dose in subjects with hereditary moderate or severe FX deficiency (plasma FX activity [FX:C] <5 IU dL(-1) ). METHODS: For a baseline PK assessment, blood samples were taken predose and at intervals up to 144 h (7 days) post dose. After ≥6 months of on-demand pdFX treatment and treatment of ≥1 bleed with pdFX, subjects underwent repeat PK assessment. Samples were assayed for plasma FX:C (measured using the clotting and chromogenic assays) and FX antigen. RESULTS: FX:C peaked at 0.4-0.5 h and subsequently declined over the course of 144 h with a biphasic decay curve. PK parameters observed at the baseline (n = 16) and repeat (n = 15) assessments were equivalent, therefore summary PK values were obtained by combining data from both visits (n = 31). The mean terminal half-life and incremental recovery of pdFX was 29.4 h and 2.00 IU dL(-1) per IU kg(-1) respectively. CONCLUSION: This is the most comprehensive PK study to date in subjects with hereditary FX deficiency. These results are consistent with the observed haemostatic efficacy of pdFX and provide the PK data required for the treatment of hereditary FX deficiency using pdFX replacement therapy.

A Phase II clinical trial of a mixture of plasma-derived factor VIIa and factor X (MC710) in haemophilia patients with inhibitors: haemostatic efficacy, safety and pharmacokinetics/pharmacodynamics.

MC710, a mixture of plasma-derived activated factor VII and factor X at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In a Phase II trial, we evaluated the haemostatic efficacy and safety of single doses of MC710, and investigated pharmacokinetic and pharmacodynamic parameters in nine joint bleeding episodes in six male haemophilia patients with inhibitors. This trial was a multi-centre, open-label, non-randomized study of two doses (60 and 120 µg kg(-1) as FVIIa dose), allowing the re-administration of different MC710 dosages to the same subjects. Haemostatic efficacy was assessed by evaluating reduction in pain and swelling, as well as increase in range of motion in a bleeding joint. The results of the study showed that in nine bleeding episodes, seven treatments were rated as 'excellent' or 'effective' according to investigator's rating system of efficacy at 8 h after administration. No serious or severe adverse events were observed after administration; furthermore, measurement of several diagnostic markers revealed no signs or symptoms of disseminated intravascular coagulation (DIC). The haemostatic potential of MC710 was confirmed at doses of 60 and 120 µg kg(-1) in this trial. MC710 is thus expected to be a safe and efficacious novel bypassing agent for controlling bleeding in haemophilia patients with inhibitors.

Coagulation factor X interaction with macrophages through its N-glycans protects it from a rapid clearance.

Factor X (FX), a plasma glycoprotein playing a central role in coagulation has a long circulatory half-life compared to closely related coagulation factors. The activation peptide of FX has been shown to influence its clearance with two N-glycans as key determinants of FX's relatively long survival. To decipher FX clearance mechanism, organ biodistribution and cellular interactions of human plasma FX (pd-FX), recombinant FX (rFX), N-deglycosylated FX (N-degly-FX) and recombinant FX mutated at both N-glycosylation sites (rFX(N181A-N191A)) were evaluated. Biodistribution analysis of (125)I-labelled FX proteins after administration to mice revealed liver as major target organ for all FX variants. Liver tissue sections analysis showed an interaction of pd-FX and N-degly-FX to different cell types. These findings were confirmed in cell binding studies revealing that FX and FX without N-glycans interact with macrophages and hepatocytes, respectively. N-degly-FX appeared to be degraded in hepatocytes while interestingly pd-FX was not by macrophages. Furthermore, the chemical inactivation of macrophages by gadolinium chloride resulted in a significant decrease of circulating pd-FX into mice and not of N-degly-FX. Altogether our data lead to the conclusion that FX interaction with macrophages through its N-glycans protects it from a rapid clearance explaining its relatively long circulatory half-life.

Pharmacokinetics, distribution, and excretion of 125I-labeled human plasma-derived-FVIIa and -FX with MC710 (FVIIa/FX mixture) in rats.

INTRODUCTION: MC710 is a mixture agent consisting of plasma-derived activated factor VII (FVIIa) and factor X (FX) at a weight ratio of 1:10 developed as a novel bypassing agent for the management of the bleeding of hemophilia patients with inhibitors. The pharmacokinetics, distribution, and excretion of (125)I-labeled-FVIIa ((125)I-FVIIa) and -FX ((125)I-FX) were studied in male rats after a single intravenous administration of (125)I-FVIIa or (125)I-FX combined with MC710. METHODS: (125)I-FVIIa or (125)I-FX was administered intravenously with MC710 to male rats in a single dosage (FVIIa 0.4 mg and FX 4 mg/kg body weight) and radioactivity and antigen levels in plasma were quantified for 24h. Urine and feces were sampled to study the excretion of radioactivity during 168 h after dosing. Whole-body autoradiography was performed to evaluate the qualitative distribution of radioactivity 168 h after dosing. RESULTS AND CONCLUSIONS: The half-life (t(1/2)α and t(1/2)ß) of radioactivity and FVIIa antigen were 0.704 and 6.27 h, and 0.496 and 1.66 h, respectively and the area under the plasma concentration-time curve (AUC(0-∞)) of radioactivity and FVIIa antigen were 17,932 and 8671 ng·h/mL, respectively. The t(1/2) of radioactivity and FX antigen were 4.06 and 3.05 h, respectively, and the AUC(0-∞) of radioactivity and FX antigen were 320,143 and 395,794 ng·h/mL, respectively. About 80% of the administered dose of radioactivity was excreted in urine and feces by 168 h after administration. Tissue distribution experiments showed that FVIIa- and FX-related (125)I accumulated in bone and bone marrow, and disappeared slowly.

Clinical pharmacological study of a plasma-derived factor VIIa and factor X mixture (MC710) in haemophilia patients with inhibitors--phase I trial.

MC710, a combined product of plasma-derived activated factor VII (FVIIa) and factor X (FX) at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In this study, pharmacokinetic (PK), pharmacodynamic (PD) parameters and safety of single doses of MC710 were investigated in 11 male haemophilia patients with inhibitors in a non-bleeding state. This was a multi-centre, open-labelled, non-randomized, active controlled crossover, dose-escalation study of five doses (20-120 µg kg(-1) of FVIIa) with re-administration of different MC710 dosages to the same subjects. The active controls were NovoSeven (120 µg kg(-1)) and/or FEIBA (50 and 75 U kg(-1)) which were used to compare PD parameters. The area under the curve (AUC) and maximum plasma concentration (C(max)) of MC710 active ingredients increased dose-dependently within the range of 20 and 120 µg kg(-1). After administration of MC710, activated partial thromboplastin time (APTT) was dose-dependently improved and prothrombin time (PT) was shortened to approximately 6 s at 10 min, and APTT improvement and PT shortening effects were maintained until 12 h after administration of MC710 at all doses. No serious or severe adverse event was observed after administration of MC710; furthermore, several diagnostic marker values and those changes did not indicate any signs of disseminated intravascular coagulation (DIC). These results suggest that MC710 would have haemostatic potential equal to or greater than NovoSeven and FEIBA and was be tolerable when given at doses up to 120 µg kg(-1).

Activation peptides prolong the murine plasma half-life of human factor VII.

Coagulation factors VII (FVII), IX (FIX), X (FX), and protein C share the same domain organization but display very different plasma half-lives. It is plausible that the half-life is influenced by the activation peptide, differing in length and glycosylation and missing in FVII. To test this hypothesis, the influence of activation peptides on the plasma half-life of human FVII was studied by administering human FVII variants containing activation peptide motifs to mice. Insertion of the activation peptide from FX gave 4-fold longer terminal half-life (5.5 hours vs 1.4 hours for FVII), whereas the activation peptide from FIX and protein C resulted in half-lives of 4.3 and 1.7 hours, respectively. Using FX's activation peptide we identified the N-linked glycans as structural features important for the half-life. The peptide location within the FVII molecule appeared not to be critical because similar prolongation was obtained with the activation peptide inserted immediately before the normal site of activation and at the C-terminus. However, only the latter variant was activatable, yielding full amidolytic activity and reduced proteolytic activity with preserved long half-life. Our data support that activation peptides function as plasma retention signals and constitute a new manner to extend the half-life of FVII(a).

Pharmacokinetics of Beriplex P/N prothrombin complex concentrate in healthy volunteers.

Prothrombin complex concentrates (PCCs) are widely administered for emergency oral anticoagulation reversal and for coagulation defects in liver disease. Pharmacokinetic data may help to optimize treatment. The objective of this study was to characterize the pharmacokinetics of a PCC (Beriplex P/N) containing coagulation factors II (FII), VII (FVII), IX (FIX) and X (FX) and anticoagulant proteins C and S. Fifteen healthy volunteers received a single rapid 50 IU/kg infusion of PCC and underwent frequent blood sampling until 144 hours (h) after infusion. Coagulation factors and anticoagulant protein pharmacokinetic parameters were estimated by non-linear regression. The mean infusion rate of PCC was 7.9 ml/min, equivalent to 196.4 IU/min. By the earliest post-infusion sampling point at 5 minutes (min), plasma FIX concentration increased by a median of 73%. Median increases in FII, FVII and FX at 5 min were 122%, 62% and 158%, respectively. Proteins C and S also increased rapidly. The median terminal half-life of FIX was 16.7 h, FII 59.7 h, FVII 4.2 h and FX 30.7 h. The median in-vivo recovery of FIX was 1.57 %/IU/kg and that of the other three coagulation factors > 2 %/IU/kg. Plasma concentration of thrombogenicity marker D-dimer did not increase, and there was no clinical evidence of thrombosis. Through up to 12 weeks follow-up there were no laboratory findings indicating PCC-related viral exposure. Rapid PCC infusion produced prompt sustained increases in coagulation factors and anticoagulant proteins with no clinical evidence of thrombosis or viral transmission.

Pharmacokinetics, thrombogenicity and safety of a double-treated prothrombin complex concentrate.

The pharmacokinetic profile, the thrombogenicity and the virus safety of Preconativ, a PCC subjected both to virus removal procedure and dry-heat treatment were studied. Preconativ is produced from plasma pool, negative both for HBsAg and for antibodies to HIV. To further reduce the risk of virus transmission, the manufacturing process includes hydrophobic gel chromatography and dry-heat treatment at +68 degrees C for 48 hours. Nine patients with hemophilia B participated in a single dose, pharmacokinetic study. The decay curves of factor IX clotting activity were evaluated by model-independent methods. The Clearance and the Mean Residence Time were very similar to those previously reported for untreated PCC. The Volume of Distribution Area and In Vivo Recovery resulted inversely correlated and respectively larger and smaller than those of untreated PCC. A slight fall in platelet count and Antithrombin III level and an increase of Beta-Thromboglobulin and Fibrinopeptide A concentration were found, indicating a clear-cut activation of the coagulation process during the first hours following Preconativ administration. Seven patients (2 of the ones enrolled in the pharmacokinetic study) were completely fulfilling the SSC-ISTH criteria for virus safety prospective study. The follow up of these patients did not show any transaminases elevation or seroconversion against HBV, HCV or HIV. These findings did not change over a 3-5 year follow up in 3 out of 7 patients, repeatedly infused with Preconativ.

Recurrent, isolated factor X deficiency in myeloma: repeated normalization of factor X levels after cytostatic chemotherapy followed by late treatment failure associated with the development of systemic amyloidosis.

We describe the case of a 64-year-old woman with isolated severe factor X deficiency associated with kappa light chain myeloma. At the time of diagnosis there was no evidence for amyloidosis. Complete remission (CR) of myeloma as well as normalization of factor X levels were achieved after cytostatic chemotherapy. Subsequently, factor X deficiency recurred twice without any evidence for relapse of myeloma. The first time factor X normalized again following cytostatic treatment, the second time, however, factor X deficiency was refractory to chemotherapy. Finally, relapse of myeloma became evident associated with rapidly progressing, systemic amyloidosis, which was fatal within a few months. Initially, factor X infusion studies showed a normal recovery, but when amyloidosis became overt the recovery decreased to 0%. We assume that factor X deficiency was due to a binding of factor X to kappa light chains associated with the proliferation of the malignant myeloma cell clone.