Recombinant Factor VIIa Research Articles

Safety and Efficacy of Recombinant Factor VIIa (NovoSeven) Use during ECMO Support in Patients after Cardiac Surgery

Background: Acute postoperative bleeding in cardiac surgical patients is a major cause of morbidity and mortality. Substitution of coagulatory factors may not always provide optimal hemostasis and off label, use of recombinant FVIIa has been proposed. When on ECMO, extra care must be taken during coagulatory substitution as clotting of the system may cause cardiovascular complications and possible ECMO failure, leading to death. In this paper, we examined the safety and efficacy of rFVIIa during ECMO support in postoperative cardio-surgical patients. Methods: We retrospectively examined all patients receiving rFVIIa postoperatively from December 2005 and January 2020. Clinical characteristics, demographics, bleeding, thrombotic complications, mortality, and rFVIIa administration were analyzed. Results: A total of 74 patients received rFVIIa postoperatively due to uncontrollable bleeding after cardiac surgery on our ICU. Of these patients, 23 patients were on ECMO treatment. Twelve patients received rFVIIa during, but not prior to the initiation of ECMO therapy. Six patients (50%) were male; mean age was 46 years (30-72 years). Eleven patients (91.7%) were on venoarterial ECMO, one patient was on central ECMO (8.3%). Dose of administered rFVIIa was corrected for body weight; mean dosage was 82μg/kg. We saw a significant reduction in need for red packed cells, fresh frozen plasma and thrombocyte transfusion after rFVIIa administration. There was no impact on the functionality of the ECMO system, especially regarding the oxygenator after rFVIIa administration. One patient suffered a stroke due thromboembolism (8.3%). One patient developed late thromboembolism in the leg (8.3%), and two cases of pulmonary embolism (16.7%) were recorded. Overall survival was 25% and there was no significant difference in survival between ECMO and non-ECMO patients. Weaning from ECMO could be achieved successfully in 41.7% of our patients. Conclusion: Recombinant Factor VIIa is an effective agent in reducing blood loss during ongoing ECMO therapy in patients with refractory bleeding. Although no direct relation between rFVIIa application and thromboembolic events could be established, its use should be done with the utmost care and in selected patients. However, rFVIIa therapy did not impact ECMO function in our cohort.

Use of a Removable Backbone Modification Strategy to Prevent Aspartimide Formation in the Synthesis of Asp Lactam Cyclic Peptides†

Main observation and conclusionThe synthesis of an Asp lactam derivative of A‐183, a selective inhibitor of Factor 7a with good anticoagulant and antithrombotic activity, is described. Our synthesis depends on the use of a removable backbone modification (RBM) strategy to prevent aspartimide formation, which thwarted all attempts to synthesize this target using direct solid‐phase peptide synthesis. Validation of the RBM strategy in the synthesis of a second Asp lactam derivative was also accomplished. The RBM strategy is therefore proposed as a general method for the synthesis of Asp lactam cyclic peptides.

Pharmacotherapeutic Patterns and Patients’ Access to Pharmacotherapy for Some Rare Diseases in Bulgaria – A Pilot Comparative Study

Provision of the latest innovative and advanced therapies for rare diseases (RDs) patients, following the international therapeutic recommendations, is crucial and necessary for both practitioners and patients. The goal is to assess the access of Bulgarian patients with the most cost-consuming RDs to medicines and to compare the pharmacotherapeutic patterns in Bulgaria and the relevant European professional associations. Pharmaco-therapeutic guidelines for treating the most cost-consuming RDs in Bulgaria were analyzed to assess their compliance with the European ones. Market entrance was evaluated through analysis of the availability of medicines in the Positive Drug List (PDL) and their date of inclusion since marketing authorization. Guidelines’ compliance index was calculated and patient access was analyzed through evaluation of the National Health Insurance Fund (NHIF) standards, which provide additional criteria for treatment initiation. The analyzed guidelines follow the adopted recommendations by the relevant European professional associations. NHIF have exclusion and inclusion criteria for initiating treatment with medicines for rare diseases and for continuation. The average time-lag between centralized procedure approval and inclusion in the Bulgarian PDL for orphan medicinal products (MPs) is 6.75 years (SD = 4.96) with the longest time observed for eptacog alfa (20 years) and the shortest for rurioctocog alfa pegol, octocog alfa and simoctocog alfa (1 year). Bulgarian patients with cystic fibrosis with pulmonary manifestation had a wait time of only 1.6 years to get access to innovative, centrally authorized medicines, whereas the period for access to acromegaly treatment was 8.2 years. The main factors influencing market entrance and patient access are the time to inclusion in the PDL and the NHIF criteria.

Severe haemophilia A in a preterm girl with Turner syndrome: case report \u2013 a diagnostic and therapeutic challenge for a paediatrician (Part 2)

BackgroundHaemophilia A is an X-linked genetic condition which manifests itself mainly in male children in the first 2 years of life, during gross motor skill development. This disorder is rare in females. The clinical manifestation of severe haemophilia in preterm infants poses a great challenge to the therapeutic team. As extreme prematurity is linked to an increased risk of central nervous system or gastrointestinal bleeding, a well-informed and balanced treatment from the first days of life is crucial to prevent long-term damage.Haemophilia is most commonly caused by inheriting defective genes, and can also be linked to skewed X inactivation and Turner syndrome.The coincidental occurrence of haemophilia A and Turner syndrome is extremely rare, with only isolated cases described to date. Hence, a multidisciplinary approach is needed.Case presentationThe authors report on a preterm girl (gestational age 28 weeks) diagnosed with haemophilia and Turner syndrome. The first manifestation of haemophilia was prolonged bleeding from injection sites on the second day of life. Indeterminate aPTT and factor VIII level < 1% confirmed the diagnosis of haemophilia A. Dysmorphic features which did not match the typical clinical picture of haemophilia, the female sex, and a negative paternal family history led to the diagnosis of Turner syndrome. While in hospital, the girl received multiple doses of recombinant factor VIII in response to prolonged bleedings from the injection sites and from a nodule on the girl’s head, and before and after retinal laser photocoagulation. No central nervous system or abdominal cavity bleeding was observed. The substitutive therapy was complicated by the development of factor VIII inhibitor (anti-factor VIII (FVIII) antibodies). Treatment was continued with recombinant factor VIIa. This article aims at demonstrating the complexity of the diagnostics and treatment of a preterm child with two genetic disorders.ConclusionsHaemophilia should always be considered in the differential diagnosis of prolonged bleeding, even in patients with a negative family history. In the case of coinciding atypical phenotypic features, further diagnostics for another genetic disease are recommended. Infant care should follow current care standards, while considering certain individual features.

The effect of emicizumab and bypassing agents in patients with hemophilia – An in vitro study

BackgroundEmicizumab is a nonfactor replacement therapy for hemophilia A (HA) and is a bispecific monoclonal antibody mimicking factor VIII by binding both factors IXa and X. Although it reduces the frequency of bleeding episodes, there is still need for bypassing agents in case of breakthrough bleeds or need for surgery. The HAVEN‐1 study showed an increased risk of thrombotic events and episodes of thrombotic microangiopathic hemolytic anemia with simultaneous treatment with emicizumab and activated prothrombin complex concentrate (aPCC) in high doses (>100 U/kg daily) for more than 1 day, and it is suspected that these drugs have a synergistic hemostatic effect. ObjectivesTo evaluate and compare the hemostatic effect of bypassing agents in vitro in people with HA before and after starting treatment with emicizumab to investigate if dosing should be adjusted to optimize treatment. Patients/MethodsBlood collected before and after start of treatment with emicizumab was spiked with aPCC and recombinant factor VIIa (rFVIIa) at different concentrations. The effect of aPCC and rFVIIa was assessed by thrombin generation assay and thromboelastometry. ResultsSix people with HA were included. The response to aPCC in thrombin generation after starting emicizumab was significantly stronger than before. This synergistic effect was less pronounced for emicizumab and rFVIIa. Furthermore, aPCC shortened thromboelastometry clotting time more effectively after starting emicizumab than before starting this treatment. ConclusionsWe demonstrated a strong synergistic effect of emicizumab and aPCC and a similar but less pronounced effect of rFVIIa in people treated with emicizumab.

In vitro characterization of CT‐001—a short‐acting factor VIIa with enhanced prohemostatic activity

BackgroundTraumatic injury and the associated acute bleeding are leading causes of death in people aged 1 to 44 years. Acute bleeding in pathological and surgical settings also represents a significant burden to the society. Yet there are no approved hemostatic drugs currently available. While clinically proven as an effective pro‐coagulant, activated factor VII (FVIIa) use in acute bleeding has been hampered by unwanted thromboembolic events. Enhancing the ability of FVIIa to quickly stop a bleed and clear rapidly from circulation may yield an ideal molecule suitable for use in patients with acute bleeding. ObjectivesTo address this need and the current liability of FVIIa, we produced a novel FVIIa molecule (CT‐001) with enhanced potency and shortened plasma residence time by cell line engineering and FVIIa protein engineering for superior efficacy for acute bleeding and safety. MethodsTo address safety, CT‐001, a FVIIa protein with 4 desialylated N‐glycans was generated to promote active recognition and clearance via the asialoglycoprotein receptor. To enhance potency, the gamma‐carboxylated domain was modified with P10Q and K32E, which enhanced membrane binding. ResultsTogether, these changes significantly enhanced potency and clearance while retaining the ability to interact with the key hemostatic checkpoint proteins antithrombin and tissue factor pathway inhibitor. ConclusionsThese results demonstrate that a FVIIa molecule engineered to combine supra‐physiological activity and shorter duration of action has the potential to overcome the current limitations of recombinant FVIIa to be a safe and effective approach to the treatment of acute bleeding.

Surgical Evacuation of Intracerebral Hemorrhage: The Potential Importance of Timing.

Surgical Evacuation of Intracerebral Hemorrhage: The Potential Importance of Timing.

Factor V11 deficiency: A rare cause of nasal bleeding

Factor VII deficiency is a rare inherited disorder. Clinically the patient presents with bleeding tendencies. Diagnosis is made by prolonged prothrombin time, normal activated partial thromboplastin time and low functions factor VII assay or factor VII antigen. Therapy involves factor VII concentrates, recombinant factor VII, fresh frozen plasma and fibrinolytic inhibitors. We present a 6 years old boy with nose bleed for six months of whom prothrombin time was prolonged with functional factor VII assay of less than 1% confirming factor VII deficiency. He was managed with fresh frozen plasma, blood transfusion, tranexamic acid. Factor VII deficiency even though rare should be sought out in children presenting with bleeding. Keywords: Factor VII deficiency; coagulation; hemorrhage.

Comparative Glycosylation Mapping of Recombinant and Plasma-Derived Human Factor VIII Reveals Key Biological Differences

Human coagulation factor VIII (FVIII) is a key co-factor in the coagulation pathway. Its functional deficiency is directly linked to Hemophilia A. Both human plasma-derived (pdFVIII) and recombinant FVIII (rFVIII) have been developed as effective reagents for treating the bleeding episodes. However, it remains debatable if these two pharmaceutical products are equivalent in clinical efficiency and safety profile despite of their identical amino acid sequences. Several studies have indicated that rFVIII comes with higher risk of inhibitor antibody development. In this report, we systemically analyzed the site-specific pattern of pdFVIII and rFVIII in glycosylation. A total of 61 N-glycan structures or isomers and 31 O-glycan compositions were identified. The likely immunogenic epitope, N-glycolylneuraminic acid (Neu5Gc), was absent in either recombinant or plasma-derived FVIII. However, potentially immunogenic glycopeptides, including Alpha-Gal epitope, has been identified in rFVIII. The glycosylation site localization between rFVIII and pdFVIII show significant divergence. The specific differences in glycosylation pattern of pdFVIII and rFVIII may be one of the important biological basis leading to their immunological disparity in human patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies on a Branded Recombinant Factor VIIa and Its Generic Version to Demonstrate Bioequivalence

Background: Recombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for the same indication (AryoSeven™, AryoGen, Tehran-Iran). These generic versions of rFVIIa are claimed to be biosimilar to the Novoseven. The purpose of this study is to compare several batches of generic versions of rFVIIa namely, Aryoseven with multiple commercially available batches of branded Novoseven to determine bioavailability.Methods: Four commercially available random lots of Novoseven were obtained through US and European sources and compared to 5 clinical batches of Aryoseven obtained from Aryogen (Tehran, Iran). These drugs were diluted with saline to make a stock solution of 1mg/ml. Further dilutions were made to obtain working concentrations of 100, 10, 1 and 0.1 µg/ml. Molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), immunoblotting studies, and procoagulant activity as measured by clot based method were carried out. Thrombin generation studies were carried out after supplementing with NHP at 0-1.0 ug/ml. The pharmacokinetic profile of one representative batch of Novoseven and Aryoseven was studied in primates after a 50 ug/ml bolus IV in terms of factor VIIa levels.Results: The SELDI mass spectrometric profile of all rFVIIa preparations were comparable. The GEP and immunoblotting studies of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 29 KDa. In the clot based functional assays performed in the citrated whole blood and retrieved plasma the two batches of FVIIa produced comparable procoagulant effects in the PT and aPTT assays. In the 0.25 - 1.0 µg/ml range in comparison to the control with saline supplementation, the aPTT did not exhibit any significant shortening whereas there was a sharpening of PT values in the whole blood which was identical in both the AryoSeven™ and NovoSeven® preparations. In the retrieved plasma both products produced comparable effect on the PT and aPTT assays. In platelet rich plasma (PRP) and platelet poor plasma (PPP) AryoSeven and NovoSeven at graded concentration of 0.125 to 1 µg/ml did not produce any measurable change in both PT and aPTT assays. The correction studies carried out in the FVII deficient plasma at a concentration range of 1 to 0.01 µg/ml did not reveal any differences between 2 groups of drugs. The relative correction was comparable in all concentrations for the 2 tissue factor reagents namely Innovin and Recomboplastin 2G. The results were indistinguishable for the 2 groups of drugs. The effect of NovoSeven® and AryoSeven™ on the reversal of anticoagulation in orally anticoagulated patients' were similar in relative correction of the plasma INR for the 2 groups of FVIIa preparations in the range of 0.03 to 1 µg/ml. In the thrombin generation studies both the Novoseven and Aryoseven produced similar effects as measured by increased thrombin generation. In the primate pharmacokinetic studies both groups exhibited similar pharmacokinetic profile.Conclusions: These results demonstrate that four batches of Novoseven and 5 individual clinical batches of Aryoseven were found to be biosimilar. The pharmacokinetic profile of the Novoseven and Aryoseven were studied in primates measuring the factor VIIa level for 6 hours and were found to be comparable. Thus, the generic Aryoseven is biosimilar to branded Novoseven and warrants further validation studies. DisclosuresNo relevant conflicts of interest to declare.

Assessment of a Novel Dielectric Microsensor for Monitoring Coagulation Factor Therapy in Children with Hemophilia with and without Inhibitors

Introduction: Monitoring coagulation factor or bypassing agent therapy has important clinical implications for the management of children with hemophilia, especially those with inhibitors. For patients with hemophilia, conventional laboratory-based coagulation tests are time-consuming, labor-intensive, and costly. Moreover, no monitoring assay is currently available for use with bypassing agents in patients with hemophilia and inhibitors, except for thromboelastography or rotational thromboelastometry (ROTEM), both of which are not widely available clinically and are labor-intensive. There is a need to develop an easy-to-use, low-cost, portable global assay of coagulation for monitoring therapies in hemophilia. The aim of this study was to assess the clinical utility of monitoring replacement therapy using a new technique based on a novel dielectric microsensor (ClotChip) that can potentially enable global blood coagulation assessment in a miniaturized, portable measurement platform.Methods: Patients accrued for the study were children with severe hemophilia A & B (n = 6), children with hemophilia and inhibitors (n = 4), and normal, healthy children without coagulopathy (n = 22). Blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate anticoagulant. Samples from patients with hemophilia were collected both prior to the initiation of replacement therapy and 15 minutes after administration of an appropriate coagulation factor VIII or IX for severe hemophilia A or B, respectively, or a bypassing agent (FEIBA or recombinant factor VIIa) for hemophilia with inhibitors. Whole blood samples were then tested with the ClotChip within 2 hours of collection, and coagulation was induced with CaCl2 prior to the start of ClotChip measurements. The ClotChip is based on the fully electrical technique of dielectric spectroscopy (DS), and features a three-dimensional, parallel-plate, capacitive sensing structure integrated into a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10µL). The ClotChip curves were calculated as the real part of blood permittivity at 1MHz, and the time to reach a peak in permittivity (Tpeak, Fig 1A) was taken as an indication of coagulation time. Samples from 8 of the 10 hemophilia patients were also tested with ROTEM using the non-activated TEM (NATEM) test according to the manufacturer's direction. The ROTEM clotting time (CT) parameter (Fig 1B) was used in this study as a clinically important parameter to monitor patients with hemophilia.Results: We observed a significant decrease in the ClotChip Tpeak parameter for post-therapy samples compared to baseline (pre-therapy) for children with hemophilia without inhibitors (Fig 1C, n = 6, p = 0.0001) and with inhibitors (Fig 1D, n = 4, p = 0.0024). Interestingly, baseline ClotChip measurements for 2 tolerized patients with inhibitors exhibited Tpeak of 970s and 1,010s, which are within the range of Tpeak for patients without inhibitors (750s - 1,320s), while the baseline ClotChip measurements of 2 non-tolerized patients with inhibitors exhibited higher Tpeak of 1,810s and 2,100s (Fig 1E). Moreover, ClotChip measurements for all samples taken post-therapy exhibited Tpeak that fell within the reference range of Tpeak for normal, healthy children (180s - 720s, Fig 1E). Taken together, these data suggest that the ClotChip Tpeak parameter is sensitive to detection and correction of coagulopathy in children with hemophilia with and without inhibitors, as indicated by Tpeak reaching reference range levels after coagulation factor therapy. Finally, the ClotChip Tpeak exhibited a very strong correlation (Fig 1F, Pearson's r = 0.94) to the clinically important CT parameter of ROTEM.Conclusions: Our studies demonstrate the feasibility of monitoring coagulation factor therapy in children with hemophilia with and without inhibitors, using a novel, microfluidic, dielectric sensor (ClotChip), allowing whole blood assessment of hemostasis in a single disposable sensor. ClotChip has potential to fulfill an important unmet clinical need for monitoring therapy, especially for hemophilia with inhibitors. Further studies are underway to assess the ability of ClotChip to differentiate bleeding phenotypes and monitor outcomes for treatment of bleeds in children with hemophilia. [Display omitted] DisclosuresAhuja:CSL Behring: Honoraria; Bayer: Honoraria; Shire: Honoraria; Shire: Speakers Bureau. Suster:XaTek, Inc.: Patents & Royalties: Michael Suster is an inventor of intellectual property that has been licensed by Case Western Reserve University to XaTek, Inc., and as such, may receive future royalties. Maji:XaTek, Inc.: Patents & Royalties: Denath Maji is an inventor of intellectual property that has been licensed by Case Western Reserve University to XaTek, Inc., and as such, may receive future royalties. Mohseni:XaTek, Inc.: Patents & Royalties: Pedram Mohseni is an inventor of intellectual property that has been licensed by Case Western Reserve University to XaTek, Inc., and as such, may receive future royalties.

Emergency Management of Patients with Immune Thrombocytopenia and Severe Bleeding

BackgroundSevere bleeding is a rare but potentially life-threatening complication of immune thrombocytopenia (ITP). In this study, we describe the management and clinical outcomes of patients with ITP presenting to the emergency room (ER) with thrombocytopenia and severe bleeding over a 9-year period from 3 Canadian tertiary care centers.MethodsPatients with ITP who had severe thrombocytopenia (platelets <20 x109/L) and severe bleeding, and who presented to any of 3 hospital emergency rooms (ER) affiliated with McMaster University in Hamilton, Canada between 2008 and 2016 were included in this retrospective study. We reviewed ER and hospital records and extracted demographic data, platelet counts, and all treatments received until discharge or day 10 of hospital admission. We described all bleeding and thrombotic events. Bleeding was graded from 0 (none) to 2 (severe) at one or more anatomical sites using the ITP Bleeding Score (Page et al, Br J Haematol 2007). Patients with skin or oral mucous membrane bleeding only were excluded. Data from a sample of 41 charts were extracted in duplicate (by SM and NS) to ensure consistency; discrepancies were resolved by the principal investigator (D.M.A.). Data were summarized descriptively as frequencies (percentage), means (standard deviation [SD]), or medians (interquartile range [IQR]). Ethics approval was obtained from the Hamilton Integrated Research Ethics Board. Funding for this study was provided by a grant from the Platelet Disorder Support Association.ResultsThirty-one patients had platelets <20 x109/L with grade 2 bleeding. Mean (SD) age was 56 years (±24) and 16 patients (52%) were female. Twenty-three patients (74%) had primary ITP; others had secondary ITP due to medications (n=2), systemic lupus erythematosus (n=2), malignancy (n=2), hepatitis (n=1), or other infection (n=1). Sixteen patients (52%) had newly-diagnosed ITP; 15 (48%) had known ITP and had previously received a median (IQR) of 3 (3-4) ITP therapies including splenectomy (n=7). Median (IQR) platelet count at presentation to the ER was 4 x109/L (2-8 x109/L).There were 37 ER visits among 31 patients. Sites of grade 2 bleeding at presentation to the ER were: GI (18/37 visits, 49%), epistaxis (10/37 visits, 27%), urinary (6/37 visits, 16%), gynecological (4/37 visits, 11%) and intracranial (4/37 visits, 11%). Patients were given a median (range) of 2 (0-7) treatment modalities in hospital including corticosteroids (30/37 visits, 81%), intravenous immune globulin (25/37 visits, 68%) and platelet transfusions (17/37 visits, 46%). Other treatments were romiplostim (3/37 visits), tranexamic acid (3/37 visits); and emergency splenectomy, recombinant factor VIIa, Rh immune globulin, danazol, azathioprine and mycophenolate (1/37 visits each).Four patients experienced new or recurrent grade 2 bleeding in-hospital, including two deaths: a 76 year-old female with a platelet count of 11 x109/L on warfarin who developed ICH (received 4 treatment modalities); and a 20-year-old male with a platelet count of 8 x109/L and ICH (received 7 treatment modalities). Median (IQR) length of hospital admission was 4 (3-9) days for 29 admissions.ConclusionsSevere bleeding in patients with ITP is a high risk situation that requires prompt intervention. Treatments and outcomes were variable. A standardized approach to the treatment of bleeding emergencies in ITP is needed. DisclosuresArnold:Amgen: Consultancy, Research Funding; Rigel: Consultancy; Dova: Consultancy; Bristol Myers Squibb: Research Funding; UCB: Consultancy; Novartis: Consultancy, Research Funding.

Implementation and Impact of a Multidisciplinary Coagulation Factor Stewardship Program

Introduction: Coagulation factor products are costly and can cause adverse outcomes when used inappropriately. Active stewardship may reduce the risk of inappropriate use, increase the likelihood of safe and effective use, and help control costs. Such goals may be met when these products are dispensed from an inpatient pharmacy. After multidisciplinary discussions of unmet clinical needs and pharmacoeconomic concerns, coagulation factor product guidelines for use were established at our institution and a multidisciplinary stewardship program was implemented.Objective: The purpose of this quality improvement initiative was to implement a multidisciplinary coagulation factor stewardship program and ascertain its impact on the use of coagulation factor products after dispensing them from inpatient pharmacy instead of the blood bank.Methods: We compared the utilization of 4-Factor prothrombin complex concentrate (4F-PCC) and recombinant factor VIIa (rVIIa) from the period of 7/1/16 to 5/31/17, 6 months prior to and 6 months after implementation of the stewardship program. Prior to implementation, pharmacists, hematologists, surgeons, anesthesiologists, and intensivists met to develop hospital-wide guidelines and order sets for 4F-PCC and rVIIa. The guidelines include indications for which 4F-PCC can be dispensed without hematology consultation and the requirement for hematology consultation for approval of requests outside of these guidelines. The Pharmacy & Therapeutics Committee approved all guidelines, order sets and the hematology service stewardship. Data about approval, administration, cost, and outcomes were obtained retrospectively for the 6 months prior and prospectively for the 6 months after implementation of the stewardship program.Results: Pre-implementation, 30 patients received 4F-PCC and 7 received rFVIIa. Warfarin reversal prior to emergent surgery (n=18, 60%) was the most common indication for 4F-PCC, followed by warfarin reversal for major bleeding (n=9, 30%) and direct oral anticoagulant (DOAC) reversal (n=3, 10%). Vitamin K was administered to 78% of the patients requiring warfarin reversal. The indications for rFVIIa included FVII deficiency (n=3, 42%), hemophilia with inhibitors (n=2, 29%), and refractory major bleeding (n=2, 29%). Of all patients, 7 (19%) died before discharge, but not within 72 hours and 1 (3%) had a thrombotic event within 72 hours. The cost of 4F-PCC and rFVIIa during the 6 months preceding the stewardship program was $261,880.After stewardship was implemented, 34 patients received 4F-PCC and 3 received rFVIIa. Indications for 4F-PCC included warfarin reversal prior to emergent surgery (n=21, 62%), warfarin reversal for major bleeding (n=5, 15%), DOAC reversal (n=4, 12%), and peri-operative management of other coagulopathies (n=4, 12%). Vitamin K was administered to 88% of patients requiring warfarin reversal. The pharmacy dispensed the products according to institutional guidelines in 88% of cases, and without hematology consultation when it should have been obtained in 12% of cases. Hematology was consulted for stewardship in 6 cases; 2 were approved and 4 were denied for disseminated intravascular coagulation (DIC) (n=2), factor V deficiency without active bleeding (n=1), and surgical bleeding (n=1). Of all patients, 10 (24%) died before discharge, including the 2 with DIC who were denied product. Three (7%) died within 72 hours and 2 (5%) had thrombotic events within 72 hours. The cost for 4F-PCC and rFVIIa was $160,144 after stewardship was implemented.Conclusions: Dispensing 4F-PCC and rFVIIa from the inpatient pharmacy with stewardship has resulted in similar utilization and efficacy as existed in the pre-implementation period but with cost savings of $101,000 during the first 6 months of stewardship. Furthermore, implementation of a multidisciplinary coagulation factor stewardship program with standardized institutional guidelines, streamlined dispensing from the inpatient pharmacy, as well as use of expert hematologic consultation when indicated, has enhanced the culture of safety in our institution. The experience has also enriched the training of hematology fellows, residents and pharmacists. DisclosuresFongemie:Bristol Myers Squibb: Speakers Bureau. Comenzo:Janssen: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Research Funding.

Splenectomy in a Severe Case of Acquired Factor X Deficiency Secondary to Amyloidosis: Case Discussion and Review of the Literature

Amyloidosis is a protein deposition disorder wherein amyloid fibrils affect soft tissues and visceral organs. One subtype is light-chain (AL) amyloidosis, which is characterized by amyloidogenic immunoglobulins produced generally by clonal plasma cells. Kidney and heart are the most commonly involved organs by AL amyloidosis while liver and sensory/autonomic nervous system can be clinically prominent. In a minority, acquired Factor X (FX) deficiency (related to amyloid binding this factor) can provoke a profound bleeding diathesis.We present a 62-year-old black male who was referred to our institution in May 2016 by a community hematologist after diagnoses of FX deficiency and monoclonal gammopathy. The patient initially presented with ecchymosis, hemearthroses, and blood per rectum. Workup revealed a hemoglobin 7.7g/dL, platelets 62x10³/µL and prolonged PT (35.4s) and aPTT (53.9s). Echocardiogram revealed concentric LV ‘hypertrophy’ and grade 2 diastolic dysfunction. Abdominal ultrasound identified an enlarged spleen of 18 cm. Fibrinogen level was 295mg/dL, PT and PTT mixing studies normalized, and FX activity was measured at 0.02U/mL (normal 0.2-0.4U/mL). Bone marrow biopsy showed plasma cell myeloma involving 50% of the marrow. He was started on therapy with cyclophosphamide, bortezomib, and dexamethasone.Unfortunately, the patient had no measurable response after 3 cycles of therapy at which time he presented with severe and progressive hemarthrosis, mucocutaneous bleeding, symptomatic anemia, thrombocytopenia, and elevations in PT/PTT. He received transfusions with fresh frozen plasma (FFP) around the clock, aminocaproic acid, and supportive transfusion of packed RBC and platelets. Due to active mucosal bleeding, the patient was administered vitamin K and transfused FFP every 4 hours. His coagulation parameters did not improve. Interestingly, despite his mixing studies correcting “in vitro,” he failed to demonstrate any “in vivo” correction despite FFP transfusion and three plasmapheresis treatments. Given his refractory bleeding, the surgical service was consulted regarding the prospect of a splenectomy. In an effort to optimize the patient for surgery, he received prothrombin complex concentrate (PCC) without significant improvement (Table 1). Ultimately, the patient underwent splenectomy (estimated blood loss 200cc) and was managed intra-operatively with FFP and aminocaproic acid infusion. His coagulation studies and platelet count improved immediately post-operatively (Table 1), and repeat FX level the following day demonstrated a normal value of 0.48 U/mL. Surgical pathology of the spleen was consistent with extensive deposition of amyloid (lambda subtype). To date, the patient has had a complete normalization of coagulation studies and FX levels and is clinically responding to daratumumab therapy.The incidence of coagulopathy in AL amyloidosis has been estimated to approach one third of all patients, largely due to acquired coagulation factor deficiency, and most commonly FX. Splenomegaly is present in fewer than 15% of patients with AL amyloidosis, however it is much more frequent in patients with acquired FX deficiency. Amyloid deposition in the spleen is thought to bind FX as well as sequesters platelets leading to potentially life threatening bleeding. Our review of the literature identified five previously reported cases of acquired FX deficiency secondary to AL amyloidosis which completely reversed after splenectomy (Kapoor et. al. American Journal of Hematology 2006), but our patient's presentation was particularly severe. Previous reports have also described the improvement of acquired FX deficiency in AL amyloidosis patients with high-dose melphalan (Choufani et. al. Blood 2001), although it is unclear what degree of clinically significant bleeding these patients were experiencing. Splenectomy is frequently reserved as last-line management in these patients, when more conservative measures such as FFP and plasmapheresis are not effective. This is due to the short half-life of the infused FX (Furie et. al. NEJM 1977), as well as its expedited clearance in the spleen, where it is bound to amyloid. While some cases using recombinant Factor VIIa and PCC have worked, they are more often ineffective. In patients with severe bleeding, splenectomy can produce rapid improvement as evidenced here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Switching Kogenate FS® to Kovaltry® for the Treatment of Severe Haemophilia A Patients: A Successful Single Center Experience

Background: Haemophilia A is an X-linked hereditary disorder caused by deficient levels of the coagulation protein factor VIII (FVIII). Standard therapy for haemophilia A comprises replacement of FVIII with plasma-derived or recombinant FVIII concentrate, which can be administered as on-demand or prophylaxis regimen. The most challenging complication of replacement therapy in hemophilia A is the occurrence of alloantibodies against infused factor FVIII, thus predisposing the patients to an increased morbidity and disability. Historically, first FVIII was isolated from cryoprecipitated plasma concentrates. Thirty years ago, after the cloning of F8 gene, therapy for hemophilia A patients underwent a new exciting era that developed technological strategies aimed to improve haemostatic efficacy and safety of the replacement factor. In this line, a new recombinant FVIII, BAY 81-8973 (octocog alfa; Kovaltry®, Bayer) has recently been introduced onto pharmaceutical market for treatment of these patients. Kovaltry® is an unmodified, full-length, recombinant FVIII concentrate with the same amino acid sequence as Kogenate FS® (Bayer), but produced with innovative manufacturing technologies that include production without addition of human- or animal-derived raw materials from purification or formulation processes, inclusion of a higher degree of sialic acid capping of N-terminal glycans on the molecular surface, coexpression of the human chaperone protein heat shock protein 70, and a 20-nm nanofiltration step to remove small non-enveloped viruses and potential protein aggregates. On this basis we considered beneficial for our patients changing Kogenate FS® therapy for Kovaltry®.Objective: We aimed to demonstrate that switching from Kogenate FS® to Kovaltry® could be easily performed without increasing the risk of bleeding or FVIII consumption in severe haemophilia A patients.Methods: Sixty two adult and pediatric patients with severe haemophilia A, without inhibitors, treated with Kogenate FS® either on on-demand treatment (n=13) or on prophylaxis regimen (n=49), were recruited. On November 2016, we began to switch Kogenate FS® treatment to Kovaltry® in these patients.The trend of prophylaxis with Kovaltry® was evaluated by determining mean injection frequency (per week), mean dose of each injection (IU/kg), mean total dose injected/week (IU), baseline and post-infusion levels of FVIII and annual bleeding rate (ABR). Same data for Kogenate FS® were collected retrospectively from clinical histories of patients.For determining baseline and post-infusion levels of Kovaltry®, whole blood was collected in 3.2% sodium citrate tubes (BD Vacutainer, Becton Dickinson, San José, CA, USA) at pre-dosing and 30 minutes after Kovaltry® infusion. Plasma FVIII:C was measured by chromogenic assay.Results: Switching from Kogenate FS® to Kovaltry® caused no changes, except in two patients, either in mean injection frequency or mean dose of each injection or mean total dose injected/week or baseline and post-infusion levels of FVIII or annual bleeding rate. Increment observed in mean total dose injected/week in two of the pediatric patients was due to an increased physical activity (patient 2) and to weight gain (patient 8). For sake of example, Table 1 shows these variables for 10 pediatric patients under prophylactic treatment.None of the patients treated with Kovaltry® developed inhibitors.Conclusion: Changing severe haemophilia A treatment from Kogenate FS® to Kovaltry® can be performed easily and did not pose any risk to the patient since Kovaltry® proved to be equally effective than Kogenate FS® to prevent bleeding episodes and did not induce the occurrence of inhibitors. [Display omitted] DisclosuresÁlvarez-Roman:Novo Nordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Characterization of the HLA-Class-II (HLAcII)/Therapeutic Factor VIII (FVIII)-Derived Peptidome By FVIII Domain and HLAcII Isomer

Abstract Background: Hemophilia A (HA) is caused by F8 mutations and resultant plasma deficiencies in FVIII activity. While bleeding can be arrested by infusing therapeutic FVIII proteins (tFVIIIs), ~30% of severe HA patients will develop neutralizing tFVIII antibodies (inhibitors) and become resistant to treatment. For inhibitors to develop, foreign peptides must be liberated from a tFVIII and displayed on dendritic cells (DCs) in self HLAcII molecules. Little is known about the inhibitor risk attributable to distinct isomers of individual HLAcII repertoires and different tFVIII domains. Aims: Determine the contributions to FVIII immunogenicity risk of the distinct 1) HLAcII isomers DP, DQ and DR, and 2) tFVIII domains A1, A2, A3, B, C1 and C2. Methods: We analyzed HLAcII/tFVIII peptidomic data generated in DC-protein processing assays (PPAs) that used monocyte-derived DCs from 12 healthy donors to compare the immunogenicity of a full length (FL) and B-domain truncated (BDT) recombinant FVIII (FL- and BDT-FVIII) ± von Willebrand Factor (VWF) and a plasma derived (pd)FVIII + VWF. All tFVIIIs contain 3 small acidic-residue-rich peptides (a1, a2 and a3) that connect A1 to A2, A2 to B, and B to A3. To simplify our analysis, any LC-MS/MS individually sequenced peptide (ISP) with an N-terminal residue in either the (i) C-terminal portion of A1, A2 or B that extends into the adjacent a1, a2 or a3 region, respectively, was placed into that domain, or (ii) a1, a2 or a3 region, whether or not it extends into A3, C1 or C2, was placed into that domain. We then calculated the expected frequency of peptides from each FVIII domain (including, when appropriate, the adjacent acidic regions) based on residue number. Finally, we compared the observed and expected peptide proportions and performed a Z-test on the difference of proportions per domain. We performed this analysis for all ISP data in which the observed frequencies were obtained by a weighting scheme jointly taking into account contributions by tFVIII & by domain (All-data). We also performed this analysis by tFVIII. Results: For the All-data comparisons, we found that A3 had a higher observed than expected proportion (dP = 22%; p = 0.006). For the FL-FVIII comparisons, the B domain had a lower observed than expected proportion (dP = -12.1%; p We also approached these data from the HLAcII perspective. We found that our 12 donors expressed 12 distinct DR molecules based on their DRB1 alleles. After tallying the per allele density of FVIII residues (for all 2332 residues) falling in the DR-bound fraction of peptides, we constructed FVIII residue-by-allele matrices of dimensions 2332 x 12 per tFVIII and performed singular value decompositions that yielded their respective matrices of right singular vectors also known as principal components (PCs). Such PCs are said to span or generate a corresponding vector subspace. Using matrix analytic methods, we can compare the therapeutic-specific subspaces to see if such contrasts reflect differences in their associated density profiles. To compare two subspaces, we compute their projection onto their intersection subspace, and use this projection to compute a subspace distance. We test the null hypothesis of a subspace distance equal to 0 (for statistically similar subspaces) by a permutation test. As an example, we compared the FL-FVIII + VWF and pdFVIII + VWF subspaces, and found that they are significantly different (subspace distance (SD) = 0.96; p Conclusions: Our findings demonstrate that the distinct domains of tFVIIIs and different alleles of individual HLAcII repertoires differentially contribute to inhibitor risk. Download : Download high-res image (175KB) Download : Download full-size image Disclosures Hofmann: CSL Behring: Employment. Escobar: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinh: Haplomics: Employment. Powell: CSL Behring: Employment. Howard: CSL Behring: Honoraria, Research Funding; Haplomics: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Comparison of Different Phenotypes of Bernard-Soulier Syndrome

Introduction. Bernard-Soulier syndrome (BSS) is one of the rare inherited platelet disorder. It is caused by deficiency or defect of the glycoprotein (GP) Ib-IX-V complex on the surface of platelets - the main receptor for von Willebrand factor (vWF). Dissociation of hemostasis in BSS leads to microcirculatory and mixed types of bleeding episodes - mucocutaneous bleedings, spontaneous and posttraumatic hematomas and severe hemorrhagic episodes during surgery.Materials and methods. We diagnosed 2 Caucasian women of age 32 and 29 years (patient 1 and patient 2 with different phenotypes of BSS) using total blood count, clotting time tests, fibrinogen, platelet aggregation with ristomicin, ADP, collagen, arachnid acid, adrenaline, platelet morphology analysis and flow cytometry assay. Flow cytometry assay was performed on isolated from 3 mL of citrated blood platelets. Platelets were loaded with mepacrine (1 mM) and then stimulated with thrombin at 88 nM. Samples from 10 healthy donors were used as controls. We evaluated CD61, PAC1, CD42b, mepacrine, CD62p, annexin V on active and non-active platelets.Results. Both patients had similar results in standard coagulation test: normal or shortened APTT (26-29 sec), normal fibrinogen (1.8-2.5 g/l), increased activity of vWF (209-275%) and FVIII (140-287%). Data of light aggregometry was classic for BSS: decrease of platelet aggregation with ristomicin and normal with other parameters. The peripheral blood smear showed large platelets and absence of neutrophil inclusions.Despite similarity of primary laboratory analysis patient 1 and patient 2 have very different phenotypes of BSS. Patient 1 had first clinical manifestation at age of 2 - large posttraumatic hematomas and then was treated from ITP without any results. At age of 13 menorrhagia had started and was slightly managed by prescribing hormonal therapy. At 16 yrs she had hemorrhage into the abdominal cavity. A therapeutic laparotomy was performed, complicated by the development of bleeding in the early postoperative period. At age of 31, the patient was hospitalized with a diagnosis of acute cerebrovascular accidents of hemorrhagic type with the formation of subdural hematoma with a repeated spontaneous brain hemorrhage in 1 month.Patient 2 was diagnosed with BSS at age of 6 yrs. Clinical pictures is characterized by menorrhagia from age of 14 yrs, that normalized at age of 20. Although she was hospitalized for many times due to severe anemia (Hb 40-50 g/l) of unknown reason. At age of 29 patient 2 was diagnosed with angiectasia of jejunum that appeared to be hidden source of blood loss. After treating it with eptacog alfa (activated) and proscribing gastrointestinal therapy anemia syndrome was managed.We compared the results of flow cytometry of patient 1 and patient 2. СD42b was 0% in both patients. Patient 1 also had a deficiency of dense granules of platelets and elevated levels of CD 61 and CD62. Patient 2 had no other platelet deficiencies and highly elevated parameters of CD61, PAC1, mepacrine, CD62p (up to 3 times).Conclusions. The compensatory high activation of other components of platelets in patient 2 can be evaluated as a result of a mild phenotype. Combined defect of BSS and deficiency of dense granules also can be a result of severe hemorrhage in patient 1. It is important to include flow cytometry of platelets in standard algorithm of diagnostic to evaluate more correctly the phenotype of platelet disorders. DisclosuresNo relevant conflicts of interest to declare.

Rationale for and Design of a Tolerability and Safety Study of FEIBA Reconstituted in Regular or 50% Reduced Volume and Faster Infusion Rates in Patients with Hemophilia A or B with Inhibitors

The absence of FVIII in hemophilia A (HA) and FIX in hemophilia B (HB) leads to spontaneous bleeding episodes and potentially excessive bleeding following trauma or injury. Treatment for HA and HB is complicated by the development of FVIII or FIX neutralizing antibodies in ~30% and ~2-5% of patients with severe HA and HB respectively which preclude the efficacious use of replacement therapy. The available bypassing agents, FEIBA and recombinant FVIIa are utilized in treatment of inhibitor patients, on-demand to control and prevent bleeding episodes, routine prophylaxis to prevent or reduce bleeding episode frequency and as perioperative management. However, the time required for administration of FEIBA reconstituted at standard volume and recommended rate of 2 U/kg/min may be a burden for the patient and the clinician. Mean infusion rates during routine clinical practice were evaluated by Negrier et al in the FEIBA PASS study and seen to be 3.7 U/kg/ min, with a range of 0.9-23.5. Even with a maximum rate being 12 times higher than that recommended in the SmPC of FEIBA (2.0 U/kg per min), a manual analysis of safety listings did not disclose any adverse events associated with a higher infusion rate.In light of data from FEIBA PASS and the objective of reducing burden of infusion with FEIBA, the “FEIBA STAR” study was designed to evaluate the tolerability and safety of infusing a 50% reduced volume of FEIBA at standard (2 U/kg/min) infusion rate and at increased (4 U/kg/min and 10 U/kg/min) infusion rates, as compared with regular volume FEIBA at standard infusion rate of 2 U/kg/min. Occurrence of any adverse events, thromboembolic events, hypersensitivity reactions, and infusion site and infusion related reactions in addition to clinically apparent changes in vital signs and clinical laboratory data will be observed in support. Subject treatment preference and monitoring of pre and post-infusion FII concentrations will also be evaluated.Nonclinical assessments in rats and rabbits of FEIBA reconstituted in 50% reduced volume and administered at increased infusion rates showed comparable efficacy and safety with FEIBA reconstituted and administered at standard volume and infusion rate. A dose-related relative reduction in blood loss was observed with reduced volume FEIBA in rabbits, and no correlation between infusion rate and thrombogenicity was seen. Additionally at infusion rates up to 10-fold the planned clinical infusion rate of the reduced volume reconstitution, there were no concerns regarding adverse clinical signs (Hoebarth 2017).FEIBA STAR is a two part, prospective, open-label, multi-center trial to be conducted in hemophilia A or B subjects with inhibitor titers ≥0.6 Bethesda units requiring the use of bypassing agents. In the 2-way cross-over design of Part 1, subjects will be randomized 1:1 in Sequence A to receive at standard infusion rate either FEIBA reconstituted in 50% reduced volume SWFI or FEIBA reconstituted in regular volume SWFI. After administration of a total of 3 infusions at 48-hour intervals, subjects receiving 50% reduced volume FEIBA will then crossover to receive standard volume FEIBA every 48-hours for a total of 3 infusions and vice versa in Sequence B. Upon completion of Part 1, all subjects will move to Part 2 where all evaluable subjects (receiving 4 out of 6 planned doses in Part 1) will be sequentially treated every 48-hours with FEIBA reconstituted in 50% reduced volume SWFI for 3-infusions at 4 U/kg/min followed by 3-infusions at 10 U/kg/min. Tolerability, safety, clinically apparent changes in vital signs, infusion rate-related events and infusion site reactions will be monitored throughout.The results from FEIBA STAR study aims to provide evidence regarding tolerability and safety to support routine clinical practice where higher infusion rates of FEIBA are used, to reduce burden of infusion in patients. DisclosuresMubarak Ali:Shire: Employment, Other: Stock Options in a publicly traded company. Bajwa:Shire: Employment.

Polyethylene Glycol (PEG) Exposure in Parenteral Therapies Indicated for the Pediatric Population: History and Safety

INTRODUCTIONPolyethylene glycol (PEG) is a chemically inert, amphiphilic polymer used as an excipient in many pharmaceuticals for decades, and as a conjugate with biologicals (ie, PEGylation) since 1990. To date, the US FDA has approved 14 PEGylated therapies with diverse indications. Although it is recognized that such products contain PEG, there may be a lack of awareness regarding the use of large quantities of excipient PEG in factor VIII (FVIII) and factor IX (FIX) therapies for hemophilia A and B, respectively, as well as in other parenteral therapies administered long term in the pediatric population. Due to concern about the potential toxicity of long-term exposure to PEG, particularly in children, based primarily on preclinical observations of cellular vacuolation upon supratherapeutic dosing of PEG molecules ≥ 40kDa (EMA. Safety Working Party's Response to the PDCO Regarding the Use of PEGylated Drug Products in the Pediatric Population; 2012. ), we evaluated the total PEG content of these therapies. Our objective was to estimate annual PEG exposure from each product as commonly prescribed, and to determine whether any adverse events (AEs) reported could be attributed specifically to PEG.METHODSFDA-approved parenteral therapies with pediatric indications were analyzed for PEG exposure per dose and per year. For comparative purposes, we grouped FVIII products together as primary comparators to Antihemophilic Factor (Recombinant), PEGylated (ADYNOVATE®).To quantify excipient PEG, we included free PEG of various molecular weight and excipients with chemical structures containing PEG chains (polysorbate 20 [PS20] and 80 [PS80], and poloxamer 188 [P188]). For FVIII and FIX therapies, PEG exposure was calculated based on a range of dosages recommended in the 2013 World Federation of Hemophilia Guidelines (25-40 IU/kg; Srivastava, et al. Haemophilia . 2013; 19:e1-e47). For extended half-life recombinant (r)FVIII and rFIX products and all other products, exposure was calculated based on prescribing information (PI), including a given dosage range. Three weights were used to cover the pediatric population: 10, 30, and 50 kg, with exceptions pertaining to the PI. The formula used to calculate PEG exposure per dose was:PEGylated biologic: patient weight (kg) × dose (IU or mg/kg) × (PEG MW/total MW)/specific activityExcipient PEG: patient weight (kg) × dose ((IU or mg)/kg)/(PEG concentration) × PEG ratio **Ratio: PEG/PEG=1; for PS80, PEG/PS80=0.645; for P188, PEG/P188=0.69.For this analysis, combinations of vial sizes were pragmatically chosen to minimize factor waste, leading to some variability in dosing. All PEG molecules were treated equally, whether conjugated or present as an excipient and regardless of size or structure.Safety outcomes were investigated by searching the medical literature for any report of clinical PEG-related AEs, and by attempting to identify any salient pattern of AEs listed in the PI or reported to the FDA or other reporting organization that might reflect PEG toxicity.RESULTSFor a child weighing 10 kg and receiving minimal prophylaxis dosages of FVIII, total PEG exposure ranged from <1 to 2,730 mg/year; for prophylaxis with FIX products the range was <1-168 mg/year. For other products, total PEG exposure ranged from 2 to 53,508 mg/year (Table 1). For a child weighing 50 kg and receiving maximum prophylaxis dosages of FVIII, total PEG exposure ranged from 40 to 21,840 mg/year; for prophylaxis with FIX products, the range was 13-1,342 mg/year. For other products, total PEG exposure ranged from 383 to 53,508 mg/year (Figure 1 and 2). Eloctate®, which contains PEG in the form of excipient PS20, had insufficient publicly available information to determine PEG exposure. There were no reports in the medical literature of clinical safety issues or signals specifically attributed to long-term exposure to PEG; and no recognizable patterns or trends of central nervous system, hepatic, allergic, or renal AEs.CONCLUSIONSPediatric exposure to PEG from FVIII, FIX, and other parenteral therapies has occurred for decades. Exposure to PEG as an excipient has been greater than exposure to PEG as a component of a PEGylated therapy. Notably, exposure to PEG from ADYNOVATE, including excipient and conjugated forms, is comparable to or less than that from other FVIII products. There were no discoverable reports of toxicity attributed to PEG accumulation from use of these products. [Display omitted] DisclosuresStidl:Shire: Employment. Schwartz:Shire: Employment. Denne:Shire: Employment. Goldstine:Shire: Employment. Kadish:PAREXEL International: Employment. Korakas:PAREXEL International: Employment. Turecek:Shire: Employment.

Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.Results:As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (KD, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.Conclusions:The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency. DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Wuschko:Octapharma Pharmazeutika Produktionsges.m.b.H.: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.