Bleeding Episodes In Hemophilia Research Articles

Design of a Superior Factor VIIIa Mimetic By Coupling a Membrane Binding Domain to a Factor IXa Binding Antibody Fragment

Emicizumab is a bispecific antibody that augments Factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Clinical use of this FVIIIa mimetic significantly reduces bleeding episodes in Hemophilia A (HA) patients. Membrane dependent assembly of FVIIIa-FIXa-FX is an absolute requirement for the intrinsic tenase activity. In contrast, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation even in the absence of membranes. This major distinction likely allows FVIIIa to function at sub-nanomolar concentrations to greatly accelerate the activation of FX while only much lower enhancements of rate are achieved at several 100-fold higher concentrations of emicizumab. We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity obtained with the bispecific antibody. We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (V H) with the variable light chain (V L) to produce V H9V L. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, V H9V L, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, V H9V L, was used as a template to introduce membrane binding properties. The Factor V C2-domain, which binds membrane, was fused at the C-terminus of V H9V L via a repeating Gly 4Ser flexible linker of ~ 5.7 nm to allow the V H and V L domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct V H9V LC2 showed a substantial amplification in cofactor mimetic activity as compared to V H9V L. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by V H9V LC2 as compared to V H9V L. Remarkably, despite its inability to bind FX, cofactor mimetic activity of V H9V LC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by V H9V LC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). V H9V LC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM). The role of C2 domain-mediated increase in cofactor activity of V H9V LC2 was further assessed by using an antibody fragment (E 9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E 9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of V H9V LC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E 9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of V H9V LC2. To evaluate whether V H9V LC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of V H9V LC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that V H9V LC2 can bypass FVIII inhibitor activity. The engineered scFv with a factor V C2 domain fusion, V H9V LC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.

A review of usage of telemedicine for management of acute bleeding episodes in haemophilia

Introduction and background: Haemophilia is an X-linked inherited genetic disorder that impairs the body's ability to make blood clots. It results in spontaneous uncontrolled bleeding. Maximum of its manifestation are acute and require immediate intervention. Therefore this study is aimed at focusing on impact of telemedicine in emergency management of haemophilia in remote areas. Materials and methods: At Multicentric HIH centres the patients are prescribed with homoeopathic medicines as per the symptom similarity which is the base for homoeopathic treatment, and given constitution medicines in Fleiss kappa manner, on monthly basis. Patients are provided with emergency kits which consist of around 30 medicines which have been formulated considering haemorrhagic diathesis, and articulation problems, oedema, ecchymosis, pain, inflammation, bleeding, tenderness etc. This tool employed in telemedicine in remote areas to manage acute conditions on the proper time. Result: Total number of interactions of these patients with a team of HIH is 22926, out of which 43.59% was telephonically managed emergencies. In Thane, Surat and Amravati, around 75% of interactions are dealt with the use of social media that is17722 emergencies have been managed telephonically. Conclusion: Telemedicine has efficiently helped to manage and control acute episodes of people with haemophilia and thereby increasing their quality of life as well as the quality of healthcare with homoeopathic medicines.

Cost-effectiveness of emicizumab vs bypassing agents in the prevention of bleeding episodes in haemophilia A patients with anti-FVIII inhibitors in France.

The development of an anti-FVIII inhibitor is the most serious complication of haemophilia A occurring in up to 30% of severe haemophilic patients. The current management of haemophilia A with inhibitor uses bypassing agents (BPA) and represents a significant therapeutic burden together with a limited adherence to prophylactic treatment. Emicizumab is the first monoclonal antibody developed in haemophilia A approved for the prevention of bleeding episodes in patients with anti-FVIII inhibitor. The purpose of this study is to evaluate the incremental cost-effectiveness ratio (ICER) of emicizumab versus BPAs. A Markov model was developed over a five-year time horizon to estimate the comparative costs and benefits of the different therapeutic approaches in this rare disease. Model inputs were clinical, including annual bleeding rate and quality of life, and economical including mainly costs of prophylaxis, bleeds and adverse events. Emicizumab treatment is dominant, ie lest costly and more effective, in the base-case analysis saving 234191 € for a gain of 0.88 QALY. This is confirmed by both the deterministic and probabilistic sensitivity analyses. The main limit of the study remains the absence of long-term clinical data allowing to relate treatment consumption to clinical benefit, especially in the progression of haemophilic arthropathy. Our results show that emicizumab is a cost-effective treatment allowing to consider an easy to implement prophylactic treatment for haemophilia A patients with anti-FVIII inhibitors.

Thrombin Generation Assay in Plasma from Hemophilia a Patients with or without Inhibitors on Concizumab Prophylaxis: Spiking with rFVIIa or aPCC

Introduction: Concizumab is a humanized monoclonal antibody that inhibits tissue factor pathway inhibitor (TFPI). Concizumab is currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A and B patients with and without inhibitors. Breakthrough bleeding episodes experienced by inhibitor patients while on concizumab prophylaxis may be treated with the bypassing agents recombinant activated factor VII (rFVIIa; NovoSeven®) or activated prothrombin complex concentrate (aPCC; FEIBA®). Aim: To investigate the in vitro effect of rFVIIa and aPCC on hemophilia A plasma containing concizumab using a thrombin generation assay and pooled plasma spiked with concizumab or samples from patients treated prophylactically with concizumab. Methods: Pooled hemophilia A plasma was spiked with concizumab at 1, 3 and 10 nM and patient plasma samples from explorer4 (n=16; hemophilia with inhibitors; NCT03196284) and explorer5 (n=30; hemophilia A; NCT03196297) before and during concizumab prophylaxis at steady state exposure levels were assessed. Samples were spiked with rFVIIa (25 or 75 nM) or aPCC (0.25, 0.5 or 1 U/mL), and analyzed using a thrombin generation assay initiated with tissue factor (PPP-Low, Thrombinoscope). The effects of rFVIIa or aPCC in the absence or presence of concizumab were compared using ANOVA methodology. Results: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation both in the absence and presence of concizumab. A significant additional effect of rFVIIa and aPCC was observed for all concizumab concentrations spiked to the plasma pool. Overall, the effects of the combination of concizumab and rFVIIa or aPCC were mainly additive; however, a small but statistically significant drug-drug interaction was observed for rFVIIa (25 nM or 75 nM) and aPCC (0.5 U/ml or 1 U/mL) in the presence of 10 nM concizumab. At this concizumab concentration, the additive effect of aPCC corresponded to 68% of the total observed effect and the additive effect of rFVIIa to 85% of the total observed effect. At lower concizumab concentrations (1 and 3 nM), statistically significant drug-drug effects were only observed in combination with aPCC. No excessive thrombin generation above the level obtained with 1 IU/mL recombinant factor VIII (rFVIII) was observed at 1 nM concizumab combined with either rFVIIa (25 and 75 nM) or aPCC 0.5 U/mL. However, addition of 1 U/mL aPCC to 1 nM concizumab resulted in a thrombin peak modestly above the upper 95% confidence interval of the rFVIII range. In the experiments using plasma from patients treated with concizumab, the increase in thrombin peak upon addition of rFVIIa was within or below the range observed by spiking with 1 IU/mL rFVIII. The increase in thrombin peak upon addition of aPCC was within or above the rFVIII range. The effects of concizumab and rFVIIa or aPCC were mainly additive; however, a small, statistically significant contribution caused by drug-drug interaction was observed for concizumab and rFVIIa (75 nM) in both plasma from patients with and without inhibitors, and for 1 U/mL aPCC in plasma from patients with inhibitors. The additive effects of concizumab and rFVIIa corresponded to between 60% (25 nM rFVIIa, plasma without inhibitors) and 75% (75 nM rFVIIa, inhibitor plasma), and the additive effects of concizumab and 1 U/mL aPCC corresponded to 77% of the total observed effects. Conclusions: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation as expected both in the absence and presence of concizumab. Thus, the bypassing agents function as expected in plasma containing concizumab. The effects of concizumab and rFVIIa or aPCC were mainly additive. A small but statistically significant contribution was synergistic in accordance with the concizumab mechanism of action (Hilden I et al, Blood, 2012). These in vitro results support the concomitant use of bypassing agents to treat breakthrough bleeding episodes in hemophilia with inhibitor patients on concizumab prophylactic treatment. Disclosures Kjalke: Novo Nordisk A/S: Employment, Honoraria. Kjelgaard-Hansen:Novo Nordisk A/S: Employment, Equity Ownership. Andersen:Novo Nordisk A/S: Employment, Equity Ownership, Honoraria. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.

Efficacy of EHL N9-GP for on-demand treatment of bleeding episodes in hemophilia B: analysis of pivotal trial data.

Efficacy of EHL N9-GP for on-demand treatment of bleeding episodes in hemophilia B: analysis of pivotal trial data.

Effect of Prophylactic Management of Hemophilia on Bleeding Episodes.

Hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency) are the most common and serious congenital coagulation factor deficiencies with repeated hemarthroses leading to development of target joints. Continuous prophylaxis is regular infusion of factor concentrates at fixed dose at regular interval to prevent hemorrhages. The study was designed to assess the outcome of continuous prophylaxis in hemophilia on bleeding episodes. It was conducted from November, 2017 to April, 2018 in the Paediatrics Department of Midnapore Medical College, Paschim Medinipur, West Bengal on 33 boys from 4 to 18years of age suffering from Hemophilia with frequent bleeding episodes. Prior to starting continuous prophylaxis all the patients' target joint(s) were assessed based on Gilbert Score and bleeding episodes in the last 6months were assessed based on Annualized Bleeding Rate and ISTH-BAT Score. All the children were provided prophylaxis therapy with plasma derived Factor concentrate twice per week at a dose of 20 + - 2IU/Kg. All bleeding episodes of the children during prophylaxis were recorded. Bleeding episodes showed significant improvement only in children who could sufficiently adhere to continuous prophylactic therapy. Continuous prophylaxis reduces bleeding episodes in Hemophilia in twice weekly protocol provided the patients sufficiently adhere to continuous prophylaxis regimen.

The Tissue Factor Pathway Inhibitor Antibody, PF-06741086, Increases Thrombin Generation in Rare Bleeding Disorder and Von Willebrand Factor Deficient Plasmas

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration.The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. DisclosuresRakhe:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA).Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. DisclosuresRakhe:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Synergistic Effects of a Procoagulant Bispecific Antibody and Rescue Therapies on Thrombin Generation- a Potential Safety Risk

Background: Investigational non-factor products, such as ACE 910 (Emicizumab), offer potential new treatment options for hemophilia patients with inhibitors. However, their uncertain and unregulated mechanisms of action raise multiple concerns around safety and efficacy in specific clinical contexts. As an antibody to FIXa and FX, ACE 910 lacks the inherent regulatory characteristics that are present in replacement factor and bypassing agents in the context of hemostasis.FEIBA is a plasma-derived, activated prothrombin complex concentrate developed to prevent and treat bleeding episodes in hemophilia A and B patients with inhibitors. There are more than 40 years of clinical experience with FEIBA. Extensive prospective clinical study data, as well as post-approval pharmacovigilance data demonstrated the product to be safe and highly effective. In an ongoing phase III study (NCT02622321), hemophilia A patients with inhibitors are treated with Emicizumab. To evaluate the treatment options for ACE910-treated patients experiencing breakthrough bleeding, we studied, in-vitro, the thrombin generation profile of various combinations of FEIBA with a biosimilar version of ACE910.Methods: A biosimilar antibody to Emicizumab (BS-Em) was expressed in mammalian cells, purified and extensively biochemically characterized. Therapeutic doses of BS-Em (200-600nM) in combination with several concentrations of FEIBA (0.1-1IU/ml) were analyzed in standard in-vitro thrombin generation (TG) experiments (1pM TF trigger) using 3 inhibitor patient plasma. A normal range of TG for the experimental conditions used was established using plasma from 34 healthy individuals.Results: The combination of FEIBA and BS-Em resulted in peak thrombin values of >500nM (600nM BS-Em/1IU/ml FEIBA) while the normal range was established to span peak thrombin levels of ~50-120nM thrombin. Titration experiments established that at 600nM BS-Em, FEIBA concentrations of >0.2IU/ml led to peak thrombin values 4- 10-fold higher than the normal range.Conclusions: Our in-vitro experiments demonstrate an excessive thrombin generation potential for the combination of BS-Em and FEIBA at concentrations expected to be generated in patients participating in this study. Caution and clinical judgement will be required when considering the use of ACE910 in hemophilia A patients with inhibitors as options to treat breakthrough bleeds if they occur might be reduced or lead to potential AE. DisclosuresKnappe:Shire: Employment. Hartmann:Shire: Employment. Goldstein:Shire: Employment. Ewenstein:Shire: Employment. Valentino:Shire: Employment. Scheiflinger:Shire: Employment.

A Comparison of Efficacy Between Recombinant Activated Factor VII (ARYOSEVEN TM) and NOVOSEVEN® in Patients with Hereditary FVIII Deficiency with Inhibitor

Introduction: Concentrated factor VIII replacement therapy in severe hemophilia prevents disabling arthropathy, life-threatening bleeding, improves health related quality of life and increase life expectancy. Patients with neutralizing antibodies (inhibitors) did not respond to usual doses of FVIII and they need higher doses of FVIII or bypassing agents (eg. rFVIIa, FEIBA) to control bleeding. The aim of this study was to compare the efficacy of a new biosimilar recombinant factor VIIa (Aryoseven ™) with Novoseven® in controlling bleeding episodes in hemophilia A patients with inhibitors.Methods: A randomized double blind clinical trial study was conducted in eight comprehensive hemophilia care centers in Iran. We randomized 66 male patients in two groups, with 4 consecutive block randomization when bleeding occurred. All patients entered the study with only one bleed. Informed consent signed by all patients or their parents. Group A (31 patients, 47%) received Aryoseven ™ and group B (35 patients, 53%) received Novoseven®. rFVIIa dosage was 90 -120 μg/kg intravenously every 2 hours.Results: During the study 66 patients (All male) were enrolled in two arms. The mean age was 20.5±10.9 years. Comparison of baseline data between the two showed no significant difference. The distributions of these variables were similar between the two groups. Other factors, such as vital signs, coagulation tests, liver, renal function tests, and blood count were not significantly different between the two groups. Median plasma level of FVII clotting activity (FVII: C) in group A and B was 103.8±38.4 IU/ dl and 98.6±26.7 IU/dl before injection respectively. Median plasma level of FVIII inhibitor in group A and B was 15.0 BU (IQR: 8.8-58.0) and 19.0 BU (IQR: 11.0-31.0). The average time from onset of bleeding to start treatment were 1246± 1104 and 2301 ± 1693 minutes (p = 0.311) in group A and B respectively.This study results showed that increased levels of factor VII were comparable between two groups after rFVIIa injection. A comparison of the Kavakli global response scores after injection and the treatment success rate in terms of achieving to score 6 or higher showed that both groups were comparable in treatment success rates. The global treatment response rate was 96.8% in group A and 91.4% in group B. Administration of either Aryoseven™ or Novoseven® had comparable effect on controlling pain and joint mobility. Reported side effects were minor (headache, nausea, and rash) and occurred in similar frequency.Discussion: The present study showed increased FVII levels and clinical efficacy in the control of the bleeding episodes in Hemophilia A patients with inhibitor for both drugs. Some similarities exist between our findings and the previous clinical trials designed for Novoseven®. In other studies, the treatment success rate by using the global response scoring system has been 70% -86% by different rFVIIa dosage regimen, in our study was above 90% in both treatment groups. In most studies, it was reported more than 90% achieving to cessation of bleeding at 9 hours after first injection of rFVIIa as response time which was also similar to our study.Conclusion: This study showed non-inferiority of Aryoseven ™ compared to Novoseven® in cessation of bleeding episodes in hemophilia A patients with inhibitor. There was no evidence of significant difference between the efficacy and safety of two drugs. There were no clinically meaningful differences between the biosimilar product and the original product in terms of the safety, purity, and efficacy. Biosimilar rFVIIa is effective in treatment of acute joint bleeding in patients with inhibitor in comparison to Novoseven®. DisclosuresMehrvar:Aryogen Zist Darou: Employment. Khoein:Aryogen Zist Darou: Employment. Kaymar:Aryogen Zist Darou: Employment. Mahbodi:Aryogen Zist Darou: Employment. Vaziri:Aryogen Zist Darou: Employment.

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

Use of recombinant factor VIIa in treatment of bleeding episodes for patients with refractoriness to platelet transfusion therapy

Abstract 4382Recombinant factor VIIa (rVIIa) is currently used for treatment and prevention of bleeding episodes in hemophilia with inhibitors, congenital factor VII deficiency, and acquired hemophilia. We report a series of 3 cases of immune mediated refractoriness to platelet transfusion therapy that were successfully treated with rVIIa, an off-label use. Data was obtained retrospectively over a 6 month period. All patients were known to be platelet refractory with presence of significant titers of plasma HLA antibodies. Two patients had undergone matched unrelated donor HSCT and the third patient suffered from Myelodysplastic syndrome (MDS). Two of the patients presented with GI bleeding and the third with intracranial bleeding. Due to limited availability of HLA matched platelets and negligible increment in platelet count from ABO matched platelets, use of factor VIIa was considered. Transfusion with PRBCs to maintain a Hemoglobin (Hb) > 8.0g/dl and close monitoring of coagulation parameters were performed. Patients received boluses of rFVIIa at 90mcg/kg, every 3–12 hrs. The dose of rFVIIa ranged from 10800–28,000 mcg/day. All three episodes of bleeding achieved clinical or radiologic improvement. No toxicities or arteriothrombotic events were observed with rFVIIa use. Our experience indicates that rFVIIa might be an effective treatment option for patients with refractoriness to platelet transfusion therapy. The optimal dosing schedule in this clinical situation needs to be studied.Patient demographics and outcomesPatientDiagnosisPre-intervention Hb/plateletrFVIIa used (microgms)PRBC/Platelet transfused (units)Clinical outcomeSV – GI bleedALL-MUD, BMT, day +102.7.9/400028000/d × 4 days6/3Bleeding stopped and Hemoglobin stabilized on day 4CS- GI bleedAML-MUD,BMT, day + 787.8/300025200/d × 2 days2/2Bleeding stopped and Hemoglobin stabilized on day 2MW-Intracranial hemorrhageMDS – IPSS risk group Intemediate-27.6/600010800/d × 2 days2/2Improvement in Neurological exam and stabilization on CT scan seen by day 3 Disclosures:Off Label Use: Recombinant factor VIIa for refractoriness to platelet transfusion therapy.

Pharmacokinetic study of minipooled solvent/detergent‐filtered cryoprecipitate factor VIII

Eighteen cryoprecipitate minipools, each made of 30 units of low volume, concentrated cryoprecipitate, have been treated by solvent-detergent and filtration (S/D-F) in a single-use CE-marked bag system. The S/D-F cryoprecipitate contained a mean of 10.5 IU mL⁻¹ factor VIII (FVIII), 17 mg mL⁻¹ clottable fibrinogen, and >10 IU mL⁻¹ von Willebrand factor ristocetin co-factor, and anti-A and anti-B isoagglutinins were undetectable. The products have been infused in 11 severe (FVIII <1%) haemophilia A patients (mean age: 17.4 years; mean weight: 57.6 kg) at a dose close to 40 IU kg⁻¹. Patients were hospitalized for at least 36 h to determine FVIII recovery, half-life and clearance. They were also closely monitored for possible adverse events. None of the infused patients demonstrated reactions or adverse events even though they did not receive anti-allergic drugs or corticosteroids prior to infusion. The mean recovery of FVIII 10 min postinfusion was 69.7%. Mean FVIII half-life was 14.2 h and clearance was 2.6 mL h⁻¹ kg⁻¹. All patients had a bleeding-free interval of 8-10 days postS/D-F cryoprecipitate infusion. The data show that S/D-F cryoprecipitate FVIII presents a normal pharmacokinetics profile, and support that it could be safely used for the control of acute and chronic bleeding episodes in haemophilia A patients.

Experience with a single dose of recombinant activated factor VII for the management of mild-to-moderate bleeds in haemophilia

Experience with a single dose of recombinant activated factor VII for the management of mild-to-moderate bleeds in haemophilia

Pulmonary hemorrhage: A novel mode of therapy

Major hemoptysis a potentially life-threatening condition in pulmonology and can originate from both identifiable and unidentifiable sites. Identifiable bleeding sites can be controlled locally by iced saline, vasopressors, laser, electrocautery and balloon tamponade. Bleeding from an unidentifiable source, on the other hand, is much more difficult to control as the bleeding site is not accessible by the bronchoscope. Tranexamic acid (TA), a synthetic anti-fibrinolytic agent, is approved for treatment or prophylaxis of bleeding episodes in hemophilia or following major operative procedures via intravenous or oral routes. Its efficacy in controlling bleeding from mucosal tissue led us to apply it to patients with pulmonary bleeding. Six patients with significant hemoptysis, two who bled during bronchoscopy biopsy and four with spontaneous bleeding (lung cancer, diffuse alveolar hemorrhage, idiopathic pulmonary bleeding, metastatic thyroid carcinoma) were treated with TA. For the two who bled during bronchoscopy, we used a bolus of 500mg/5mL through the bronchoscope working channel, while the latter four received aerosolized TA 500mg/5ml 3-4 times a day. In all cases, the bleeding stopped with the first dose of TA, and the treatment was well tolerated without adverse events. While limited due to the small number of patients, these data show that TA administered either as a bolus through the bronchoscope or via inhalation seems to be effective in controlling severe hemoptysis from both identifiable and unidentifiable bleeding sites. Further clinical studies are needed to evaluate the use of the TA in this set-up.

The Cost Effectiveness of a New Formulation of Recombinant Factor VIIa in the Home Treatment of Mild to Moderate Bleeding Episodes in Haemophilia a Patients with Inhibitors in the United Kingdom.

The introduction of room temperature stable recombinant Factor VIIa (rFVIIa) (eptacog alfa, activated, [NovoSeven®]) in the United Kingdom (UK) provides a step forward in care for haemophilia A patients with inhibitors through enabling greater patient independence and freedom. It allows patients to treat bleeds earlier which has been shown to be associated with a greater rate of success and fewer injections being required1. Delayed treatment resulting in prolonged bleeding may result in longer hospitalization, and in some patients, a subsequent need for surgical procedures which results in an increase in direct medical costs. The objective of this analysis was to assess whether the benefits of room temperature stable rFVIIa would offset the small price premium associated with the new formulation compared to the original formulation as well as remain cost-effective compared to the other bypassing agent, activated prothrombin complex concentrate (aPCC). The study used a decision analytic approach, similar to a published economic modeling study2, to model the expected costs and outcomes of room temperature stable rFVIIa, original rFVIIa and aPCC in the home treatment of a mild to moderate bleeding episode in haemophilia A patients with inhibitors. The mean weight of patients was assumed to be 70kg. Each treatment regimen comprised first-, second- and third- line treatment:original rFVIIa - original rFVIIa - original rFVIIa ;room temperature stable rFVIIa - room temperature stable rFVIIa – room temperature stable rFVIIa; andaPCC – aPCC – room temperature stable rFVIIa.The efficacy and dosing of aPCC and original rFVIIa were obtained from the published literature. The efficacy of room temperature stable rFVIIa was assumed to be slightly higher than that for original rFVIIa and the dose of room temperature stable rFVIIa was assumed to be slightly less than that for original rFVIIa. Patients whose mild to moderate bleeding episodes were not resolved with first-line home treatment were admitted to hospital for second- and third-line treatment. All bleeding episodes resolved with third-line treatment. The outcome used in the analysis was resolved mild to moderate bleeding episode. Only the costs of the haemostatic agents were included in the base-case analysis. The different treatment regimens for the treatment of a mild to moderate bleeding episode of haemophilia A patients with inhibitors in the UK resulted in different costs attributable to resolving a mild to moderate bleeding episode:£9,644 (EUR 12,151);£9,458 (EUR 11,917); and£14,097 (EUR 17,762).Sensitivity analysis showed the results to be robust to changes in key parameters. The results show that both original rFVIIa and room temperature stable rFVIIa are cost-effective compared to aPCC, in the home treatment of a mild to moderate bleeding episode, in haemophilia A patients with inhibitors. In addition, the analysis highlights the fact that price alone does not determine overall treatment costs, and that even small improvements in efficacy and dosing associated with earlier treatment may reduce overall treatment costs.

Evaluation of the effectiveness of DDAVP in surgery and in bleeding episodes in haemophilia and von Willebrand's disease. A study on 43 patients.

1-deamino-8-D-arginine-vasopressin (DDAVP), was used in a wide spectrum of clinical situations employing two different dosages (0.3 and 0.4 microgram/kg b.w.) for the management of 43 patients with factor VIII deficiencies--mild and moderate haemophilia A and von Willebrand's disease (vWD). In most instances, the drug was given in association with antifibrinolytics. Twenty-five dental extractions were carried out with two different protocols: one based upon a single infusion and the other based upon three infusions. Bleeding occurred in three patients regardless of the protocol used. The vasopressin analogue promptly stopped bleeding in 12 'spontaneous' open bleeds (haematuria, epistaxis, menometrorrhagia, gum bleeding) and it appears to be also effective in closed bleeds. DDAVP was used to minimize blood loss during surgical interventions and to avoid haemorrhage in the postoperative period. Nine surgical procedures were carried out in six vWD patients and three haemophiliacs. Bleeding occurred late in the postoperative period on one occasion only. No difference was demonstrated between the two doses of the drug either in terms of clinical benefit or rise of factor VIII coagulant activity. The efficacy of DDAVP and the absence of side-effects make this vasopressin analogue worthy of consideration as a reliable alternative to factor VIII concentrates in a wide variety of clinical situations.

Polyphosphate Shortens the Clotting Time of Hemophilic and Anticoagulated Plasma.

Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain abundant polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway, promoting factor V activation, and abrogating tissue factor pathway inhibitor function. We now report that polyP shortens the clot time of hemophilic plasmas and plasmas containing a variety of anticoagulant drugs.Methods: Citrated plasmas were obtained from normal donors, patients receiving coumadin, and individuals with severe hemophilia A or B. In some experiments, clinically relevant concentrations of unfractionated heparin, enoxaparin, or argatroban were added to normal plasma. In other experiments, therapeutic concentrations of FIX, FVIII, or FVIIa were added to hemophilic plasma. Clotting was initiated by dilute thromboplastin.Results: Adding 33 uM polyP to normal plasma shortened clot times at all doses of heparin (by 37–55%), enoxaparin (by 35–57%), and argatroban (by 28–59%; see Figure). PolyP shortened clot times of plasmas from coumadin patients (by 29–39%) with INR values ranging from 1.7 to 4.8. PolyP also shortened the clot times of plasma from patients with hemophilia A (by 54–75%) and B (by 51–79%). PolyP alone shortened clot times of hemophilia A or B plasma equivalent to adding 6 to 15 nM recombinant FVIIa, and the polyP effect was additive to that of FVIIa, FVIII or FIX.Conclusions: PolyP shortened the in vitro clot time for all plasmas tested and was additive with replacement of missing clotting factors or recombinant FVIIa for hemophilic plasmas. These results suggest that treatment with polyP may be useful for reversal of anticoagulation in patients treated for thrombophilia, or for treatment of bleeding episodes in patients with hemophilia. Furthermore, polyP could potentially reduce the dose of replacement clotting factors or recombinant FVIIa needed to treat bleeding episodes in hemophilia. [Display omitted]

Reduction of Anti-FIX Inhibitor Titers in a Hemophilia B Patient with Continuous Use of FEIBA.

Background: Inhibitor formation is a severe complication of hemophilia B associated with poor response to factor replacement and uncontrolled bleeding, although an inhibitor formation in hemophilia B is a rare event compared to hemophilia A. Currently, immune tolerance is the only proven method for inhibitor eradication. However, immune tolerance induction in patients with hemophilia B is a rare occurrence. We report a case of a hemophilia B patient who had a reduction of inhibitor titer during continual use of FEIBA.Case report: Patient is an 11-year-old boy with severe FIX deficiency and high titer inhibitor (historical peak: 65 BU/mL) since age 3 years. Since inhibitor formation, the patient had received FEIBA or FVIIa on demand. FVIIa became therapeutically not effective in 1999 October, and bleeding frequency increased as he developed a target joint. In 1999 November, 2002 February, and 2004 September, he received high dose of FIX concentrate for neutralization of inhibitor and then cyclophosphamide for 8 weeks. We achieved only a transient effect in reducing the inhibitor titer. The inhibiter titer fluctuated between 20 and 60 BU/mL. Since 2005 January, subsequently, he had received FEIBA 1,000–2,000 IU per dose (30–50 units/kg) on demand. He needed FEIBA twice or thrice a week, and continued to receive this dose till now. The inhibitor titer was gradually getting lower after a transient rise up to 39 BU/mL on 2005 February. On 2006 February, the inhibitor titer became below 1.0 BU/mL, and finally undetectable on 2006 March, despite the longevity of the inhibitor (8 years). Factor IX recovery was normalized, and bleeding frequency dramatically decreased. Elimination of the inhibitor by the continued administration of FEIBA was observed. Anaphylactic reaction and the development of nephrotic syndrome were not seen.Conclusion: The continuing use of FEIBA safely and effectively might decrease the inhibitor titer and the frequency of bleeding episodes in hemophilia B patients. This case may provide us the optimization of the FEIBA dose, and duration of treatment for inducing tolerance by using FEIBA.

The use of recombinant activated factor VII in congenital and acquired von Willebrand disease

Recombinant activated factor VII (NovoSeven), a novel hemostatic agent originally developed for the treatment of bleeding episodes in hemophilia A or B patients with inhibitors, has been recently employed with benefit for the management of hemorrhages in other nonhemophilic congenital and acquired hemostatic abnormalities. This review focuses on the use of this drug in acquired and congenital von Willebrand disease. The analysis of the literature data shows that recombinant activated factor VII is an effective agent for the treatment of refractory bleeding in von Willebrand disease patients and for the treatment or prevention of bleeding in those patients with alloantibodies or autoantibodies against von Willebrand factor. Further studies are needed, however, to assess its safety and to optimize the dosages and regimens of therapy in such patients.