Factor VIII Half-life Research Articles

Soy Phosphatidylinositol Containing Nanoparticle Prolongs Hemostatic Activity of B-Domain Deleted Factor VIII in Hemophilia A Mice

Factor VIII (FVIII) replacement therapy in hemophilia A (HA) is complicated by a short half-life and high incidence of inhibitory antibody response against the protein. Phosphatidylinositol (PI) containing lipidic nanoparticles have previously been shown to reduce the immunogenicity and prolong the half-life of full length FVIII. It has not been established whether this prolongation in half-life improves hemostatic efficacy and whether this approach could be extended to the B-domain deleted form of FVIII (BDD FVIII). In the current study, we evaluated the pharmacokinetics (PK), hemostatic efficacy, and immunogenicity of BDD FVIII associated with PI nanoparticles (PI-BDD FVIII) in HA mice. Comparative human PK was predicted using an "informed scaling" approach. PI-BDD FVIII showed an approximate 1.5-fold increase in terminal half-life compared with free BDD FVIII following i.v. bolus doses of 40 IU/kg. PI-BDD FVIII-treated animals retained hemostatic efficacy longer than the free FVIII-treated group in a tail vein transection model of hemostasis. PI association reduced the development of inhibitory and binding antibodies against BDD FVIII after a series of i.v. injections. The combined improvements in circulating half-life and hemostatic efficacy could significantly prolong the time above clinically established therapeutic thresholds of prophylactic FVIII replacement therapy in humans.

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A.

In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7h, with a median of 10.0h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0h vs. 10.4h, P=0.018). Age was significantly associated with FVIII half-life (r=0.32, P=0.035). Besides age, also VWF antigen (r=0.52, P<0.001) and blood group (r=-0.37, P=0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

Reduced Polyethylene Glycol-Conjugated B-Domain–Deleted Factor VIII (PEG-BDD-FVIII) Clearance: Selective Peg Steric Modulation without Affecting Potency

Prophylactic factor VIII (FVIII) replacement therapy in hemophilia A requires intravenous administration up to every other day due to the short half-life of FVIII in plasma. Plasma half-life extension of FVIII by polyethylene glycol (PEG) conjugation is thought to be mediated by decreasing hepatic clearance of FVIII. BAY 94-9027 is a rationally designed B-domain–deleted (BDD) FVIII molecule, in which a single 60-kDa PEG molecule was attached to a specific amino acid (1804) to increase its circulating half-life and reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain while preserving full biological function. BAY 94-9027 is currently in clinical trials and has prolonged half-life and improved efficacy in animal models and humans.As a first step in determining whether the half-life extension with BAY 94-9027 is related to steric hindrance exerted by PEG, we investigated whether PEG impacts BAY 94-9027 binding interactions. Direct binding of HKB11-derived FVIII, BAY 94-9027 or BDD-FVIII, was assessed by measuring the ability of a panel of immobilized monoclonal antibodies directed toward different FVIII domains to capture FVIII. Interactions with more physiologic partners were indirectly assessed by thrombin generation assay (TGA) and by an in vitro hepatocyte clearance assay. TGA monitored FVIII-dependent thrombin generation, while the hepatocyte clearance assay assessed the ability of primary human hepatocytes to remove FVIII from the incubation medium.Our results indicate that the presence of the A3-directed PEG reduced BAY 94-9027 capture by immobilized antibodies directed toward the FVIII regions at or near the site of conjugation. Capture by antibodies directed toward the A3 and C2 domains were most impacted, while those directed toward A1 and A2 still bound BAY 94-9027. The A3-specific C7F7 antibody showed ~50% lower capture of BAY 94-9027 vs BDD-FVIII at 20 ng/mL of FVIII. C7F7 capture of PEG-BDD-FVIII was further reduced when a di-PEG conjugate of BDD-FVIII was subjected to the same assay, again confirming that PEG sterically modulates PEG-BDD-FVIII reactivity to the antibody. To determine whether the steric effects observed with PEG may impact FVIII function globally, TGA was performed with BAY 94-9027 spiked into FVIII-deficient plasma and subjected to 1 pM tissue factor initiation. By TGA, both BDD-FVIII and BAY 94-9027 generated comparable peak thrombin levels, with EC50 values of 3.9 and 3.2 nM for BDD-FVIII and BAY 94-9027, respectively. As thrombin generation is a consequence of activated FVIII amplification of factor X activation by activated factor IX, these results indicate that the PEG did not disrupt activated PEG-BDD-FVIII interactions with its partners in the factor Xase enzyme complex, consistent with published PEG-BDD-FVIII efficacy. By hepatocyte clearance assay, PEG-BDD-FVIII clearance was reduced ~30-40% compared with BDD-FVIII, regardless of whether von Willebrand factor was present. This reduction in hepatocyte clearance is likely to contribute to the prolonged plasma half-life reported for BAY 94-9027 (Mei B, et al. Blood. 2010;116(2):270-279; Coyle TE, et al. Journal of Thrombosis and Haemostasis. 2014;12(4):488-496). DisclosuresBlasko:Bayer Healthcare: Employment. Leong:Bayer Healthcare: Employment. Sim:Bayer Healthcare: Employment. Tang:Bayer Healthcare: Employment. Ho:Bayer Healthcare: Employment. Wu:Bayer Healthcare: Employment. Kauser:Bayer Healthcare: Employment. Subramanyam:Bayer Healthcare: Employment.

Targeting Clotting Factor VIII Plasma Values in the Perioperative Setting in Hemophilia a: “Tackling a Moving Target” (“OPTI-CLOT” Studies)

Targeting Clotting Factor VIII Plasma Values in the Perioperative Setting in Hemophilia a: “Tackling a Moving Target” (“OPTI-CLOT” Studies)

Design of the INHIBIT trial: preventing inhibitors by avoiding ‘danger’, prolonging half-life and promoting tolerance

Inhibitor formation is among the most serious complications of hemophilia treatment. With the US FDA licensure of the novel long-lasting recombinant factor VIII (FVIII) Fc fusion protein, Eloctate, which prolongs FVIII half-life, we propose an innovative approach to prevent inhibitor formation. In this paper, we describe a multicenter, Phase II, single-arm, 48-week trial, the INHIBIT trial, to determine if Eloctate, begun before a bleed and continued as once weekly prophylaxis, will reduce inhibitor formation in children with hemophilia A. We hypothesize that avoiding ‘danger,’ that is, immune activation by a bleed at first factor exposure and prolonging FVIII half-life will prevent inhibitors and promote FVIII-specific T-cell tolerance. If successful, this approach will suggest a new paradigm in clinical practice.

Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias.

Prophylaxis with regular infusions of factor VIII (FVIII)- or factor IX (FIX)- containing products is the mainstay of modern hemophilia care. However, this therapeutic regimen is inconvenient, requiring repeated intravenous injections from childhood. Approaches meant to prolong the half-life of FVIII and FIX in plasma have been developed in order to improve the feasibility and acceptability of replacement therapy, extending protection from bleeding, reducing infusion frequency and hence the need for venous access devices in young children. Several strategies have been implemented to enhance the pharmacokinetics of clotting factors, including conjugation with polyethylene glycol and the production by genetic engineering of fusion proteins containing the coagulation factors linked to a long-lived plasma protein such as albumin or the Fc fragment of immunoglobulin (Ig)G. The latter technology is one of the most promising, since the prolongation of FVIII and FIX half-life is obtained by exploiting the physiological binding of the Fc domain to the neonatal Fc receptor. Fc fusion monomers have been obtained with both recombinant FVIII (rFVIIIFc) and FIX (rFIXFc), and data from preclinical and clinical studies showed improved pharmacokinetics for both factors, which are produced in human embryonic kidney (HEK) 293 cells, thus ensuring full human post-translational modifications. In Phase I/IIa studies, rFVIIIFc and rFIXFc showed 1.5–1.7 fold and 3.0–4.0 fold longer elimination half-life, respectively. Similar data have been obtained in the Phase III clinical studies with rFVIIIFc and rFIX-Fc published recently. Both drugs were satisfactorily safe, particularly with respect to immunogenicity, and no serious adverse event was observed.

Von Willebrand Disease

Von Willebrand Disease

Evidence For Flexible Tethering Of Fc To FVIII In Recombinant FVIII-Fc Fusion Protein rFVIIIFc

IntroductionHemophilia A is characterized by a deficiency in factor VIII (FVIII) activity, resulting in failure of blood to clot properly, leading to traumatic and spontaneous bleeding, particularly into joints and soft tissues. Current treatment strategies rely on intravenous administration of FVIII, but require frequent dosing owing to the relatively short half-life of FVIII. We previously reported the development of a recombinant FVIIIFc fusion protein (rFVIIIFc), that shows an approximate 1.5-fold increase in half-life relative to otherwise unmodified FVIII, potentially enabling less frequent dosing compared to rFVIII. Both FVIII and rFVIIIFc exhibit comparable specific activities and similar affinities for von Willebrand factor (VWF). Furthermore, hydrogen-deuterium exchange (H/Dx) analysis established structural comparability of rFVIII and the FVIII component of rFVIIIFc, while small angle x-ray scattering (SAXS) results suggested that the Fc is flexibly tethered to the C-terminus of FVIII, leaving the regions of FVIII necessary for interaction with phospholipid, VWF, FIXa, and FX binding sterically accessible. Consistent with these findings, the crystal structure of rFVIIIFc shows that the FVIII structure is unaffected by the appended Fc domain, while the Fc domain itself is not visible, suggesting conformational freedom. AimsThe goal of this study is to further compare the structure of rFVIII with that of the FVIII component of rFVIIIFc by evaluating the affinities of a panel of anti-FVIII antibodies having structurally distinct epitopes. Additionally, we assess the conformational flexibility of the Fc component of rFVIIIFc using negative stain electron microscopy. MethodsThirty-nine anti-FVIII mouse monoclonal antibodies, 14 of which are being reported here for the first time, were evaluated by surface plasmon resonance (SPR) to determine their affinities for both rFVIII and rFVIIIFc. Affinities were then compared to assess whether the respective epitopes, which collectively span the 5 structural domains of FVIII, are conformationally conserved between FVIII and rFVIIIFc. Subsequently, Fab fragments were generated for two of these antibodies, GMA-8015 and ESH8, and the structures of the complexes formed between these Fabs and FVIII were visualized by negative stain EM. The structure of rFVIIIFc was determined by negative stain EM to determine the positional variability of the Fc element with respect to the FVIII component of rFVIIIFc. Results and ConclusionThe affinities of all 39 antibodies for rFVIII and rFVIIIFc, expressed in terms of KD, spanned the picomolar to nanomolar range and each showed comparable affinities for rFVIII and rFVIIIFc. Among these are 13 antibodies which are known to compete with VWF for FVIII binding, providing further evidence that the structural determinants of VWF binding are the same in both molecules. As expected, the GMA-8015 and ESH8 Fabs bound to the A2 and C2 domains of FVIII, respectively, and were clearly visible in EM images. For rFVIIIFc, particles were grouped into 100 classes, and the resulting class averages revealed a high degree of conformational freedom between FVIII and the appended Fc domain, while the gross morphology of the FVIII component remained unchanged. Notably, rFVIII and rFVIIIFc exhibited similar affinities for ESH8, despite the close proximity of the ESH8 epitope and the Fc fusion site in the C2 domain at the C-terminus of FVIII, indicating flexible tethering between FVIII and the Fc domain. These findings provide further mechanistic insight into the observed functional similarity of FVIII and rFVIIIFc with respect to cofactor activity and VWF binding. Disclosures:Leksa:Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Goodman:Biogen Idec: Employment, Equity Ownership. Chiu:Biogen Idec: Research Funding. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment, Equity Ownership. Kulman:Biogen Idec: Employment, Equity Ownership.

Natural History Of Inhibitor Recurrence Following Successful Immune Tolerance Induction

Introduction The formation of Factor VIII (FVIII) inhibitory antibodies is a major complication of hemophilia A. Currently immune tolerance induction (ITI) is successful in up to 70% of patients. Outside of the International Immune Tolerance Registry, where 6 of 128 patients had a recurrent inhibitor between 1 and 6 years, little is known about the probability of inhibitor recurrence following successful ITI. Objective To determine the probability of inhibitor recurrence and the influence of adherence to post-ITI prophylaxis on inhibitor recurrence following successful ITI. Methods All persons with hemophilia A (FVIII level 6 hours and/or FVIII recovery > 66% in addition to inhibitor titer 80% of prescribed infusions vs. < 80% of prescribed infusions) and inhibitor recurrence was assessed using the chi-square test. Results Eighty-three male subjects were enrolled. The median age at start of ITI was 3.3 years (range: 0.08 - 39). The majority of the subjects were white (73%) and non-Hispanic (73.5%). Seventy-one (85.6%) had severe hemophilia. The median peak inhibitor titer was 8.5 BU/ml (range: 0.6 - 950). Four subjects (5%) had a prior unsuccessful course of ITI. FVIII alone was used in 85% of subjects. Sixty-seven (80.7%) met criteria for tolerance and 64 had follow-up data available, with a median follow up time of 3.4 years (range: 0.08-12.4). Forty-four subjects (68.7%) remained tolerant without a recurrent inhibitor titer after a median 4.7 years (range: 0.25-12.4) of follow-up. Twenty subjects (31.3%) had at least one inhibitor titer ≥ 0.6 BU/ml after a median of 1.6 years (range 0.08-5.7). The probability of recurrent inhibitor at 1 year is 0.15 (95% CI: [0.05, 0.20]); at 3 years is 0.30 (95% CI: [0.2, 0.4]) and 5 years is 0.35 (95% CI: [0.2, 0.5]) ([Figure 1][1]). Four subjects discontinued post-ITI prophylaxis anywhere from 6 months to greater than 6 years after tolerance was achieved, of whom 2 (50%) developed a recurrent inhibitor. Of those that remained on post-ITI prophylaxis, 41 subjects (64.1%) were adherent (took >80% of prescribed infusions) to their post-ITI prophylaxis regimen, of whom 13 (31.7%) developed a recurrent inhibitor. Twenty-three (35.9%) who were non-adherent (took <80% of the prescribed infusions) of which 7 (30.4%) subjects developed a recurrent inhibitor; no statistically significant association was found between adherence and inhibitor-free status (p=0.92). ![Figure 1][2] Figure 1 Conclusion ITI is currently the most effective treatment to eradicate FVIII inhibitors, however 5 years after completion, 30-35% of patients will have at least one inhibitor titer ≥ 0.6 BU/ml. A recurrent inhibitor is unlikely after 5 years. Adherence to post-ITI prophylaxis does not appear to be a major driver of inhibitor recurrence. It is imperative to elucidate the factors that influence the durability of successful ITI to improve quality of life and cost of treatment in these patients. Disclosures: Monahan: Baxter: Consultancy, Honoraria, Research Funding, travel support, travel support Other; Bayer: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria; Prolor Biotech: Research Funding; Asklepios: Consultancy, Research Funding, travel support Other. Manco-Johnson: Eisai: Research Funding; Novo Nordisk: Membership on an entity’s Board of Directors or advisory committees; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees; Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Carpenter: Novo Nordisk: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Grifols: Honoraria, Research Funding. Kruse-Jarres: Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy; Baxter Healthcare: Consultancy. Ragni: Novo Nordisk: Research Funding; Merck: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Baxter: Research Funding; Tacere Benitec: Consultancy; Smith Kline Glaxo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Kempton: Novo Nordisk: Research Funding; Baxter Healthcare: Membership on an entity’s Board of Directors or advisory committees. [1]: #F1 [2]: pending:yes

Comparative pharmacokinetics of factor VIII and recombinant factor IX: for which coagulation factors should half-life change with age?

The half-life of factor VIII (FVIII) increases with the age of the patient, while studies on recombinant factor IX (rFIX) and factor VIIa (rFVIIa) have not demonstrated corresponding age-related changes. The purpose of this analysis was to relate the changes in FVIII and rFIX pharmacokinetics (PK) with age to developmental changes in body size and fluid volumes and explain why the elimination half-life of FVIII, but not of rFIX, would change with age, and to consider how the findings could be applied prospectively to other coagulation factors. Published PK data for FVIII from 186 patients aged 1-74 years and for rFIX from 56 patients aged 4-56 years were used. The relationships of FVIII and rFIX clearance (CL) with body weight could be described by allometric expressions. Relative changes in CL with age or weight were similar for FVIII and rFIX. The age-related change in volume of distribution at steady state (V(ss)) of rFIX was parallel to the change in CL in the children while for FVIII the change was much less pronounced. Elimination half-life was clearly age-dependent for FVIII while only a very weak trend could be seen for rFIX. Limited data suggest that rFVIIa in this respect resembles rFIX, with parallel changes in CL and V(ss) producing insignificant change in half-life. To what extent the elimination half-life of a coagulation factor would show a correlation with age can in principle be predicted from the characteristics of its CL and distribution.

Generation of a Novel Bispecific Antibody (ACE910) Against Activated Factor IX and Factor X Mimicking the Function of Factor VIII Cofactor Activity

AbstractAbstract 1126Exogenous factor VIII (FVIII) is used to reduce bleeding complications in patients with severe hemophilia A. However, there are two drawbacks of current routine prophylaxis by FVIII. One is the requirement of frequent intravenous administration due to its short half-life and low subcutaneous bioavailability of FVIII. Second is the development of anti-FVIII antibodies (inhibitors) in approximately 30% of the severe patients which deprives the patients from routine prophylaxis by FVIII. To overcome these drawbacks, bispecific IgG antibody against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII by placing these two factors into spatially appropriate positions, was screened from approximately 40,000 bispecific antibodies recognizing FIXa by the one arm and FX by the other arm.The therapeutic potential of the bispecific antibody identified from the screening was marginal due to insufficient FVIII-mimetic activity and poor pharmacokinetics, and moreover, large scale purification of recombinant bispecific IgG antibody was challenging. Therefore, the lead bispecific antibody, after humanization, was subjected to multidimensional optimization process in order to improve both the therapeutic potential and the manufacturability of the bispecific antibody. FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulty of manufacturing bispecific antibody was overcome by identifying common light chain for anti-FIXa and FX heavy chain through framework/complementarity determining region shuffling, and by isoelectric point engineering of the two heavy chain variable regions to facilitate ion exchange chromatography purification of the bispecific antibody. Engineering to overcome low solubility and deamidation was also performed to enable stable high concentration liquid formulation for clinical use.ACE910, multidimensionally optimized bispecific antibody, exhibited potent FVIII-mimetic activity in human FVIII deficient plasma (more than 10% of FVIII activity at 300 nM in thrombin generation assay), and half-life of approximately 3 weeks with high subcutaneous bioavailability in cynomolgus monkey, enabling effective prophylaxis by subcutaneous administration with long dosing interval. In silico immunogenicity prediction analysis suggested that ACE910 was minimally immunogenic in human, in contrast to high immunogenicity of FVIII in human. Importantly, the activity of ACE910 was not affected by the presence of inhibitors, while polyclonal anti-ACE910 antibody did not inhibit FVIII activity, allowing the use of ACE910 without considering the development or presence of inhibitors. Furthermore, ACE910 could be purified in a large scale manufacturing, and formulated into patient-friendly subcutaneously injectable liquid formulation for clinical use.We believe that ACE910, with its multidimensionally optimized profile, would significantly improve the quality of life of hemophilia A patients by reducing not only bleeding but also the burden on the patients themselves, their parents, and all medical staff. Disclosures:Igawa:Chugai Pharmaceutical Co.,Ltd: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co.,Ltd: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Moriyama:Chugai Pharmaceutical Co.,Ltd: Employment. Wakabayashi:Chugai Pharmaceutical Co.,Ltd: Employment. Tanaka:Chugai Pharmaceutical Co.,Ltd: Employment. Muto:Chugai Pharmaceutical Co., Ltd.: Employment. Kojima:Chugai Pharmaceutical Co.,Ltd: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihashi:Chugai Pharmaceutical Co.,Ltd: Employment. Harada:Chugai Pharmaceutical Co.,Ltd: Employment. Funaki:Chugai Pharmaceutical Co.,Ltd: Employment. Haraya:Chugai Pharmaceutical Co.,Ltd: Employment. Tatsuhiko:Chugai Pharmaceutical Co.,Ltd: Employment. Suzuki:Chugai Pharmaceutical Co.,Ltd: Employment. Esaki:Chugai Pharmaceutical Co.,Ltd: Employment. Nabuchi:Chugai Pharmaceutical Co.,Ltd: Employment. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

AbstractReplacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

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.

Genetic Characterization of Hemophilia and Implications for Novel Therapies

AbstractAbstract SCI-9The genes encoding factor VIII (FVIII) and factor IX (FIX) were cloned 25 years ago. Since then, substantial translational application of this knowledge has been witnessed. First, genetic analysis of hemophilia has enabled a detailed understanding of the types and patterns of mutation responsible for these conditions. This knowledge has provided important biological insights into the mechanisms underlying mutation generation and has also formed the basis for precise molecular diagnosis for hemophilia carriers and potentially affected fetuses. In addition, the hemophilic genotype is the best-characterized and strongest predictor for the development of inhibitor generation in previously untreated patients. The second benefit deriving from the genetic characterization of the hemophilias has been the production of recombinant coagulation factor concentrates. Over the past 20 years, the use of recombinant factor concentrates has increased dramatically and we are currently witnessing a flurry of activity to develop second-generation, enhanced concentrates. The major objective of these projects is to facilitate clotting factor prophylaxis through the production of concentrates whose half-lives are extended, thus reducing the frequency of factor administration. These endeavors are utilizing a variety of approaches, but most are focused on either chemical modification of the factors through polymer conjugates or the generation of fusion proteins with immunoglobulin or albumin to take advantage of the recycling function of the neonatal Fc receptor. The first wave of these new products is now undergoing early phase clinical studies, and while substantial benefits for FIX half-life extension appear achievable, FVIII half-life modification may be more challenging. Finally, molecular genetic knowledge of the hemophilias has resulted in the pursuit of strategies to utilize genetic approaches to effect long-term “cures” of the disease. These initiatives have resulted in several small phase I/II trials of viral vector-based gene transfer but have also formed the basis of mutation-specific therapies such as a translational read-through approach being undertaken in patients with nonsense mutations. Currently, two phase I/II trials are in progress with liver-directed AAV FIX gene transfer in which two different AAV serotypes are being evaluated. These studies follow on from two previous AAV clinical trials in which the major obstacle to clinical benefit appears to have been the host immune response to the vector. There is considerable interest in the outcomes of these new studies. Disclosures:No relevant conflicts of interest to declare.

Commentary on Brackmann and Gormsen (Lancet 1977; ii: 933): Massive factor‐VIII infusion in haemophiliac with factor‐VIII inhibitor, high responder

This letter, describing a curative treatment for inhibitor development by induction of immune tolerance, was published one-third of a century ago. This became the basis for the ‘Bonn protocol’, a high-dose regimen designed to induce lifelong tolerance towards substituted factor VIII (FVIII) [1]. Brackmann himself has described the birth of the Bonn protocol [2]. A patient, aged 1.5 years, who had experienced severe bleeding episodes in the right shoulder, right upper arm and right chest and an inhibitor titre of >500 Bethseda units had presented to the Bonn centre. Brackmann knew of the report by Kurczinsky and Penner in 1974 which described the successful treatment of bleeding episodes in patients with an inhibitor using activated prothrombin complexes (APCCs) [3]. However, this product was not available on the German market at that time and therefore Brackmann used high dosages of FVIII with a regular prothrombin concentrate. The regimen was given twice daily to the patient and the bleeding was controlled. After 3 weeks, the patient recovered and the inhibitor titre reduced to 40 BU. The treatment was evaluated in further patients and although an initial booster of the inhibitor titre was observed in some patients, in all cases, the inhibitor titre decreased after some weeks. It was therefore decided not to stop but to continue the treatment. The success of the treatment was shown by the disappearance of the inhibitor and the normalization of the half-life of FVIII. The initial Bonn protocol had two treatment phases – the first used 100 IU FVIII kg−1 and 50 IU APCC given twice daily until the inhibitor titre was <1 BU and the 30-min recovery was measurable. In the second phase, treatment was continued with 150 IU FVIII kg−1 twice daily until the inhibitor disappeared and the half-life normalized. Later modification used 150 IU FVIII kg−1 twice daily. FEIBA was used to treat patients with increased bleeding [1, 4]. To a certain extent, the management of inhibitors was left in abeyance in the 1980s and 1990s because the overwhelming attention was to transfusion transmitted disease, particularly HIV and HCV, and the development of virally safe concentrates. The widespread availability of safe recombinant and plasma-derived concentrates has brought the problem of inhibitors to the forefront again and attention has been given to the assessment of eradication regimens. The international prospective randomized immune tolerance study was recently terminated [5]. This study, commenced in 1992, compared a high-dose regimen, 200 IU FVIII kg−1 daily and a low-dose regimen, 50 IU FVIII kg−1 three times weekly. Preliminary results show equal efficacy of the high-dose and low-dose regimens with 76% patients reaching tolerance at study endpoint. However, the study was terminated because there were significantly more bleeds in the low-dose arm. Thus in 2010, we may be reverting to the regimen pioneered by Brackmann in 1977. This early publication is a great tribute to the clinical observation together with the brave pioneering spirit of Hans Brackmann. C. A. Lee is a co-editor for Haemophilia.

Immuntoleranzinduktion mit hoch dosiertem FVIII und intravenösen Immunglobulin-Pulsen

SummaryThe development of neutralizing allo-antibodies against factor VIII (FVIII) or FVIII inhibitors is a severe complication in the treatment of haemophilia A. About 25% of the children with severe haemophilia A develop FVIII inhibitors. Here we report on a boy with severe haemophilia A and intron 22 inversion of the FVIII gene who was diagnosed at ten months of age. After 16 exposure days to FVIII (81 days after initial exposure) he developed a FVIII inhibitor (maximum: 9.76 BU/ml). Therapy: We started immune tolerance induction (ITI) according to the Bonn protocol with high dose plasma derived FVIII concentrate (100 IU per kg body weight) twice daily. For additional inhibitor elimination treatment the patient received intravenous immunoglobulin (ivIg) at a dose of 1–2 g/kg body weight every 4 to 6 weeks. After start of treatment a rapid decline of the inhibitor level was observed, nevertheless low FVIII inhibitor levels persisted (&lt;5 BU/ ml). Furthermore, the FVIII half-life was still accelerated. However, after every course of ivIg the inhibitor level declined and FVIII half-life was prolonged. Currently, the FVIII half-life is approaching normal values after more than seven months of ITI duration. Conclusion: Additional application of immunoglobulin is beneficial for immune tolerance induction.

Models for Prediction of Factor VIII Half-Life in Severe Haemophiliacs: Distinct Approaches for Blood Group O and Non-O Patients

BackgroundVon Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life.MethodologyStandardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15–44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated.Principal FindingsFVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%).ConclusionOur approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

Inhibitor treatment by rituximabin congenital haemophilia A

The development of neutralizing alloantibodies (inhibitors) to factor VIII (FVIII) is one of the most serious complications in the treatment of haemophiliacs. Inhibitors occur in approximately 20 to 30% of previously untreated patients (PUPs), predominantly children, with severe haemophilia A within the first 50 exposure days (ED). Immune tolerance induction (ITI) leads to complete elimination of the inhibitor in up to 80% of the patients and offers the possibility to restore regular FVIII prophylaxis. However, patients with high titre inhibitors, in whom standard ITI fails, usually impose with high morbidity and mortality and therefore prompting physicians to alternate therapy regimens. Rituximab, an anti-CD 20 monoclonal antibody has been successfully used in children and adults for the management of B-cell mediated disorders. We report on the use of a new protocol including rituximab in two adolescents with severe haemophilia A and high titre inhibitors, severe bleeding tendency and high clotting factor consumption after failing standard ITI. Both patients received a concomitant treatment with FVIII according to the Bonn protocol, cyclosporine A and immunoglobulin. Treatment with rituximab resulted in a temporary B-cell depletion leading to the disappearance of the inhibitor. FVIII recovery and half-life turned towards normal ranges. In patient 1 the inhibitor reappeared 14 months after the last rituximab administration. In patient 2 complete immune tolerance could be achieved for 60 months. Bleeding frequency diminished significantly and clinical joint status improved in both patients. In patient 1 the treatment course was complicated by aspergillosis and hepatitis B infection. Rituximab may be favourable for patients with congenital haemophilia, high-titre inhibitors and a severe clinical course in whom standard ITI has failed. Prospective studies are required to determine safety, efficacy and predictors of success.

Extending half-life in coagulation factors: where do we stand?

The human coagulation factor VIII (FVIII) and von Willebrand factor (VWF) are two distinct glycoproteins that circulate in the plasma as a non-covalently bound complex (VWF/FVIII complex). Deficiencies or structural defects in FVIII and VWF are responsible for the most common inherited plasma bleeding disorders haemophilia A and von Willebrand disease (VWD), respectively. Current therapies for the treatment of haemophilia have favourable efficacy, tolerability and safety profiles. However, multiple, frequent infusions are usually required to manage a bleeding episode, owing to the short half-life of FVIII. This makes treatment inconvenient and impacts patient quality of life. Several strategies are currently being pursued in an attempt to reduce the number of infusions required per bleeding episode. One of the more promising approaches involves prolonging the half-life of FVIII. This article summarizes the methods that are being used to extend FVIII half-life.

Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation

The development of factor VIII (FVIII) inhibitors remains the major hurdle in the clinical management of patients with hemophilia A. FVIII uptake by professional antigen-presenting cells (APC) is the first step involved in initiation of immune responses to FVIII. Studies on FVIII catabolism have highlighted the role played by CD91/LRP as a potential target for increasing FVIII half-life in patients and prolonging treatment efficiency. We investigated the involvement of CD91 in FVIII endocytosis by human dendritic cells (DC), a model of professional APC. Immature DC were generated from circulating monocytes from healthy donors. Surface expression of CD91 was assessed by flow cytometry. Uptake of fluorescein isothiocyanate-conjugated ligands by immature DC was studied in the presence of various blocking agents. CD91 was expressed on approximately 20% of DC and mediated the internalization of its model ligand, alpha2-macroglobulin. DC internalized FVIII and activated a human FVIII-specific T-cell clone in a dose-dependent manner. FVIII uptake by DC and subsequent T-cell activation were not inhibited by receptor-associated protein. Our results indicate that CD91 and other members of the LDL receptor family are not strongly implicated in FVIII internalization by monocyte-derived DC, and suggest the involvement of alternative divalent ion-dependent endocytic receptors.