Recombinant Human Factor VIIa Research Articles

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits

BackgroundPlaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AimsWe aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. Methods and resultsWe assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5′-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. ConclusionsIncreased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

Eptacog beta for the management of patients with haemophilia A and B with inhibitors: A European perspective.

Eptacog beta (activated), a recombinant human factor VIIa (rFVIIa), was approved by the US Food and Drug Administration (FDA) in 2020 (SEVENFACT®, LFB & HEMA Biologics) and the European Medicines Agency (EMA) in 2022 (CEVENFACTA®, LFB). In Europe, eptacog beta is indicated for the treatment of bleeds and the prevention of bleeds during surgery or invasive procedures in adults and adolescents (≥12 years old) with congenital haemophilia A or B with high-titre inhibitors (≥5 BU) or with low-titre inhibitors who are expected to have a high anamnestic response to factor VIII or factor IX, or to be refractory to increased dosing of these factors. The efficacy and safety of eptacog beta were evaluated in three Phase III clinical studies, PERSEPT 1, 2 and 3. For the EMA filing dossier, the analysis of data from PERSEPT 1 and 2 differed from the analysis used to support the filing in the US. In this review, we summarise current data regarding the mode of action, clinical efficacy and safety of eptacog beta for the management of haemophilia A and B in patients with inhibitors from a European perspective. In addition to providing a valuable summary of the analyses of the clinical data for eptacog beta conducted for the EMA, our review summarises the potential differentiators for eptacog beta compared with other current bypassing agents.

Maladaptive lymphangiogenesis is associated with synovial iron accumulation and delayed clearance in factor VIII–deficient mice after induced hemarthrosis

BackgroundMechanisms of iron clearance from hemophilic joints are unknown. ObjectivesTo better understand mechanisms of iron clearance following joint bleeding in a mouse model of hemophilia. MethodsHemarthrosis was induced by subpatellar puncture in factor VIII (FVIII)–deficient (FVII−/−) mice, +/− periprocedural recombinant human FVIII, and hypocoagulable (HypoBALB/c) mice. HypoBALB/c mice experienced transient FVIII deficiency (anti-FVIII antibody) at the time of injury combined with warfarin-induced hypocoagulability. Synovial tissue was harvested weekly up to 6 weeks after injury for histological analysis, ferric iron and macrophage accumulation (CD68), blood and lymphatic vessel remodeling (αSMA; LYVE1). Synovial RNA sequencing was performed for FVIII−/− mice at days 0, 3, and 14 after injury to quantify expression changes of iron regulators and lymphatic markers. ResultsBleed volumes were similar in FVIII−/− and HypoBALB/c mice. However, pronounced and prolonged synovial iron accumulation colocalizing with macrophages and impaired lymphangiogenesis were detected only in FVIII−/− mice and were prevented by periprocedural FVIII. Gene expression changes involved in iron handling (some genes with dual roles in inflammation) and lymphatic markers supported proinflammatory milieu with iron retention and disturbed lymphangiogenesis. ConclusionAccumulation and delayed clearance of iron-laden macrophages were associated with defective lymphangiogenesis after hemarthrosis in FVIII−/− mice. The absence of such findings in HypoBALB/c mice suggests that intact lymphatics are required for removal of iron-laden macrophages and that these processes depend on FVIII availability. Studies to elucidate the biological mechanisms of disturbed lymphangiogenesis in hemophilia appear critical to develop new therapeutic targets.

COMPARING THE EFFECTIVENESS OF CONCENTRATE OF ALL PROTHROMBIN COMPLEX FACTORS WITH RECOMBINANT HUMAN COAGULATION FACTOR VIIa IN THE TREATMENT OF BLEEDING AFTER CARDIAC SURGERY

Aim: The authors evaluated the effectiveness of treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors in patients with massive postoperative bleeding that could not be controlled with traditional therapy. Materials and Methods: In the period from 2020 to 2021, recombinant human coagulation factor VIIa was administered to 18 patients after cardiac surgery (group I), while the concentrate of all prothrombin complex factors was administered to 16 patients postoperatively (group II). During this period, 647 patients were operated on. The patients had normal coagulation screening tests (APTT, INR, TT, fibrinogen level, and PLT level) before surgery. Mean blood loss before and after administration of eptacog alfa and the total prothrombin complex concentrate was assessed. The mean dose of eptacog alfa was 30.95 mcg/kg b.w., and the total prothrombin complex factor concentrate dose was 14.17 mcg/kg b.w. After transfusion with red blood cell concentrate, fresh frozen plasma, and platelet concentrate, in the absence of improvement in the dynamics of postoperative drainage, it was decided to include recombinant human coagulation factor VIIa or a concentrate of all prothrombin complex factors in the treatment. Results: After administration of recombinant human coagulation factor VIIa at a dose of 30.95 mcg/kg b.w., bleeding stopped in 12 patients, but the remaining 6 patients required reoperation due to persistently high drainage. The decision to perform a rethoracotomy was made by a team of cardiothoracic surgeons and anesthesiologists, taking into account the dynamics of drainage (bleeding) and the hemodynamic stability of the patient. After the administration of concentrate of all prothrombin complex factors at a dose of 14.17 U/kg b.w., bleeding stopped in 12 patients. Four patients required reoperation due to persistent bleeding. Conclusions: Treatment with recombinant human coagulation factor VIIa and concentrate of all prothrombin complex factors is effective and safe for cardiac surgery patients.

Prophylactic Recombinant Factor VIIa for Preventing Massive Transfusion During Orthotopic Liver Transplantation.

RecombinanthumanactivatedfactorVIIahas been usedprophylactically to mitigate requirements for transfusion in liver transplant. We explored its effectiveness andrisks amonglivertransplantrecipients at high risk for massive transfusion. We performed a retrospective study of recipients who underwent liver transplant from 2012 to 2015. Patients considered at risk for massive transfusion received up to two 20 μg/kg doses of recombinant human activated factor VIIa, with rescue use permitted for other patients. We used propensity matching to determine the average treatment effectson patients who received recombinant human activated factor VIIa prophylactically to prevent massive transfusion. We determined thromboembolic events from medical record review. Of 234 liver transplant recipients, 38 received prophylactic and 2 received rescue recombinant human activated factor VIIa. We used a prediction model to readily identify those who would receive prophylactic recombinant human activated factorVIIa (C statistic = 0.885; 95% CI, 0.835-0.935). Propensity matching achieved balance, particularly for massive transfusion. Twenty-three of 38 patients (60.5%) who received recombinant human activated factorVIIa and 47 of 76 matched controls (61.8%) experienced massive transfusion. The coefficient for the average treatment effect of prophylactic administration was - 0.013 (95% CI, -0.260 to 0.233; P = .92). The cohorts exhibited no difference in number ofthromboembolic events (P > .99), although fatal events occurred in 1 patient who had prophylactic and 1 patient who had rescue recombinant human activated factor VIIa. Prophylactic recombinanthumanactivated factor VIIa use in patients at elevated risk of massive transfusion did not affect incidence of massive transfusion and was not associated with an increase in thromboembolic events overall. The lack of clinical benefit and the potential for fatal throm-boembolic events observed with recombinant human activated factor VIIa precluded its prophylactic use in liver transplant recipients.

FVIII regulates the molecular profile of endothelial cells: functional impact on the blood barrier and macrophage behavior.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII.

Extra-Splenic Role of B Cell Activating Factor Blockade in Prevention of Factor VIII Inhibitors

The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) remains the most challenging complication of protein-based replacement therapy in hemophilia A (HA). Elevated levels of the B cell survival cytokine B cell activating factor (BAFF) are associated with inhibitors in HA patients and BAFF levels decrease with successful immune tolerance induction (Doshi BS et al, J Clin Invest 2021). In HA mice, localization of labeled FVIII to the spleen and subsequent depletion of splenic leukocytes has been shown to reduce and/or temporarily prevent FVIII inhibitors. In contrast, transient depletion of BAFF prevented inhibitors upfront and promoted long-term tolerance to FVIII in HA mice. Given BAFF's ability to modulate B cell pools in secondary lymphoid organs, here we compared the splenic versus extra-splenic role of BAFF in regulating the FVIII immune response in order to localize its mechanism of action.As BAFF could regulate survival of innate-like splenic-resident marginal zone B cells, initial experiments compared depletion of BAFF or marginal zone B cells in HA mice and showed more complete inhibitor prevention with BAFF depletion (median 0 vs 16.9 Bethesda Units [BU]/ml, respectively, p < 0.01). These data supported our hypothesis that BAFF's regulatory effect is not solely reliant on its modulation of the splenic-resident marginal zone B cell compartment in HA mice. To further understand the extra-splenic role of BAFF in FVIII immunogenicity, wild-type (WT) or severe HA mice (with F8 exon 16 knockout) on the C57Bl/6 background, were treated according to three groups (n=9-11/group): 1) sham controls, 2) splenectomy alone, and 3) anti-BAFF monoclonal antibody (2mg/kg IP once) 4 days prior to splenectomy (“combined therapy”). Following splenectomy, mice were injected with recombinant full length human FVIII at 80 IU/kg weekly for six weeks. Bethesda titers were measured one week following the final immunization. In order to provide hemostasis in HA mice without FVIII exposure prior to splenectomy, mice were given liver-directed gene therapy with AAV8-mFVIIa at 5e11 vg/mouse two weeks prior to splenectomy. Circulating mFVIIa levels of 5.8 ± 2.4 ug/ml at day 10 were observed. These levels normalized hemostasis allowing successful splenectomy in 85% of animals, which is similar to our experience in WT mice.In the combined therapy group, only a single HA mouse developed a high titer inhibitor (7.4 BU/ml) and no WT mice developed high titer inhibitors. As the presence of endogenous FVIII antigen in WT mice does not seem to significantly alter BAFF's role in preventing the alloantibody response to xenoantigen (human FVIII protein), data for WT and HA mice were combined for analysis. Although splenectomy decreased inhibitors titers compared to sham controls (23.1 vs 78.7 BU/ml), the addition of anti-BAFF antibody therapy to splenectomy significantly reduced the risk of high titer inhibitors (RR 0.15, 95% CI 0.02-0.66, p < 0.01). The median Bethesda titer in HA and WT splenectomized mice was 23.1 (0-139.4) versus 0 (0-7.4) with addition of BAFF antibody therapy. Of note, inhibitor prevention with combination splenectomy and anti-BAFF antibody therapy seen here is similar to our prior data using just anti-BAFF antibody monotherapy for FVIII inhibitor prevention in HA mice (RR 0.23, 95% CI 0.08-0.57) suggesting that splenectomy does not significantly improve FVIII inhibitor prevention in the setting of BAFF inhibition.Our data, using a challenging HA mouse model, suggest that anti-BAFF therapy may prevent FVIII inhibitors by inhibiting BAFF's function in non-splenic lymphoid compartments. As certain B cell subtypes, including marginal zone B cells, reside in non-splenic compartments in humans, our model mimics the anticipated human immune response to FVIII. Ongoing investigations into the B and T cell compartments in lymph nodes and bone marrow will provide further insights into the location of BAFF's regulatory role in the FVIII immune response and may provide insights into potential alternative pathways in the initial FVIII immune response. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau.

Real-World Data of the Hemostatic Efficacy of Recombinant Human Factor VIIa Eptacog Beta for Acute Bleeding Events in Patients with Hemophilia a and B with Inhibitors

Introduction: Activated prothrombin complex concentrate (FEIBA, Takeda) and recombinant factor VIIa (rFVIIa, Novoseven, NovoNordisk) remain the primary bypassing agents (BPA) available for bleeding management in patients with hemophilia and inhibitors. Eptacog beta [rFVIIa-B, Coagulation factor VIIa (recombinant)-jncw, Sevenfact, HEMA Biologics & LFB] is a recombinant human FVIIa approved by the FDA in 2020 for the treatment of acute bleeding events in adult and adolescent patients ≥12 years old with hemophilia A or B with inhibitors. rFVIIa-B demonstrated hemostatic efficacy of 86% in a phase 3, randomized cross-over study of 465 mild/moderate bleeding events in 27 patients with hemophilia A and B with inhibitors. The purpose of this study is to report real-world data on the hemostatic efficacy of rFVIIa-B for acute bleeding management in patients with hemophilia A and B with inhibitors.Methods: This is a retrospective chart-review of individuals ≥12 years of age with severe hemophilia A (factor VIII <1%) or hemophilia B (factor IX <1%) with an active inhibitor from 3 U.S. hemophilia treatment centers who utilized rFVIIa-B for acute bleed management.Results: Seven bleeds were treated among the 3 patients identified. Patient characteristics and bleeding events are summarized in Table 1. The 3 patients were 12, 13, and 31 years of age. Two patients had hemophilia A and 1 patient had hemophilia B. The 2 patients with hemophilia A were on emicizumab prophylaxis per standard dosing regimens for bleeding prevention. The individual patient with hemophilia B had a history of anaphylaxis at inhibitor development and received on-demand BPA for acute bleeding events. All 7 bleeds consisted of hemarthroses with the knee being the primary site in 57% of the bleeds. Five of the 7 bleeds (71%) received a severe bleeding dose of 210-225 microgram per kilogram (mcg/kg) for the initial rFVIIa-B dose followed by 70-75 mcg/kg for subsequent doses. Final dosing regimens were dependent on available vial sizes. rFVIIa-B resulted in complete resolution of all bleeds (100%) at a median of 4 doses (range 1-8 doses) and a median of 24 hours (range 3-48 hours). There were no failures in bleeding resolution following treatment. No adverse events with infusions were reported including infusion-related reactions, hypersensitivity reactions, thrombosis, or thrombotic microangiopathy.Conclusions: Administration of rFVIIa-B in the real-world setting appears to demonstrate hemostatic efficacy in a cohort of pediatric and adult patients with hemophilia A and B with inhibitors and may serve as an alternative human rFVIIa therapy for the management of acute bleeds in this population. Further post-marketing studies are warranted to expand the indication to younger children (<12 years of age) and to continue to monitor drug efficacy and adverse events in a larger population of patients over time including individuals on non-factor therapies such as emicizumab. [Display omitted] DisclosuresBatsuli: Bio Product Laboratory: Honoraria; Kedrion: Honoraria. Tran: HEMA Biologics: Honoraria. Young: Apcintex, BioMarin, Genentech/Roche, Grifols, Novo Nordisk, Pfizer, Rani, Sanofi Genzyme, Spark, Takeda, and UniQure: Consultancy; Genentech/Roche, Grifols, and Takeda: Research Funding. Sidonio: Guardian Therapeutics: Consultancy; Octapharma: Consultancy, Research Funding; Catalyst: Consultancy; Pfizer: Consultancy; Takeda: Consultancy, Research Funding; Biomarin: Consultancy; Novo Nordisk: Consultancy; Bayer: Consultancy; Genentech: Consultancy, Research Funding.

Novel Von Willebrand Factor (vWF) Fragment Supports Subcutaneous Administration of Factor VIII: Pharmacokinetic Data from Hemophilia A Mouse Model

Patients with hemophilia A are currently treated by intravenous (IV) injections of factor VIII (FVIII) since other routes of administration do not provide sufficient FVIII bioavailability. If given subcutaneously (SC), FVIII will not be efficiently absorbed from the subcutis into vascular circulation but proteolytically degraded. vWF functions as a chaperone protecting FVIII from proteolysis and from binding to phospholipid membranes. However, FVIII complexed with high molecular weight and adhesive vWF would also likely be trapped in connective tissues. Therefore, low-molecular weight vWF fragments consisting of engineered vWF domains were generated and analyzed for their capability to support FVIII subcutaneous administration in hemophilia A mouse. Dimeric forms of vWF fragments consisting of domains D'D3 (OCTA12) and D'D3-A1-A2-A3 (OCTA13) were transiently expressed in HEK293 cell line with a C-terminal Strep-Tag and purified by Strep-tactin affinity chromatography. The fragments were characterized by SDS-PAGE to confirm dimer formation. FVIII binding affinity and ability to inhibit the interaction between FVIII and phospholipid monolayer was investigated by surface plasmon resonance (SPR). F8-/- mice received single doses of recombinant human (rh) FVIII (simoctocog alfa, Octapharma) either 200 IU/kg IV or rhFVIII 1000 IU/kg SC in the presence of OCTA12 or OCTA13. Plasma samples were collected 1, 3, 6, 9, 24, 32, and 48 hours after injection for pharmacokinetic analysis. Plasma levels of FVIII:C and FVIII:Ag were determined by chromogenic and ELISA assay, respectively. One hour after IV administration, the mean peak FVIII:C activity (Cmax) was 133.4 ± 78.1 IU/dl. FVIII:C decreased quickly with a half-life (t1/2) of 2.5 hours. After SC administration of rhFVIII with OCTA12 or OCTA13, FVIII:C reached a maximum at 6 hours Cmax = 49.6 ± 36.7 IU/dl and 26.1 ± 14.4 IU/dl, respectively. FVIII:C was detectable up to 32 h in circulation with OCTA12. FVIII:Ag and activity data were very similar for both IV and SQ administration. Estimated bioavailability of rhFVIII SC with OCTA12 and OCTA13 was 18.4% and 7.2%, respectively. Engineered vWF fragments enabled absorption of FVIII after SC administration into the blood circulation. Pharmacokinetic profile of rhFVIII with a dimeric D'D3 fragment (OCTA12) was particularly promising demonstrating bioavailability of 18.4% and FVIII:C detectable for up to 32 h after a single subcutaneous administration. DisclosuresVollack:Octapharma Biopharmaceuticals GmbH: Research Funding. Werwitzke:Octapharma Biopharmaceuticals GmbH: Research Funding. Solecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment. Tiede:Shire: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding.

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.

Recombinant VWF fragments improve bioavailability of subcutaneous factor VIII in hemophilia A mice

AbstractConventional treatment of hemophilia A (HA) requires repetitive IV injection of coagulation factor VIII (FVIII). Subcutaneous administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PLs), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII-stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (1) no significant impact on rhFVIII recovery or half-life after a single IV administration; (2) enhanced bioavailability (up to 18.5%) of rhFVIII after subcutaneous administration; and (3) slow absorption (peak concentration, 6 hours) and prolonged half-life (up to 2.5-fold) of rhFVIII after subcutaneous administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 subcutaneously compared with rhFVIII IV. A single subcutaneous dose of rhFVIII/VWF-12 provided protection in the HA tail-bleeding model for up to 24 hours. In summary, recombinant VWF fragments support FVIII delivery through the subcutaneous space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after subcutaneous administration highlight the potential application of VWF fragments for subcutaneous FVIII prophylaxis in HA.

Perioperative application of recombinant human coagulation factor Ⅶa combined with prothrombin complex in two hemophilia A patients with high titer inhibitor

Perioperative application of recombinant human coagulation factor Ⅶa combined with prothrombin complex in two hemophilia A patients with high titer inhibitor

Separate Effect of Perioperative Recombinant Human Factor VIIa Administration and Packed Red Blood Cell Transfusions on Midterm Survival in Lung Transplantation Recipients

ObjectiveThe purpose of this study was to determine the relationship between blood product transfusion, with or without recombinant human activated factor VIIa, and survival after lung transplantation. DesignRetrospective analysis of a single center with follow-up out to 6 years post-transplantation. SettingSingle-center academic lung transplantation program. ParticipantsThe study comprised 265 adult patients who underwent single or bilateral sequential lung transplantation from March 2011 to June 2017. InterventionsOverall survival using Kaplan-Meier curves was compared among the following 3 cohorts: those not transfused with blood products, those transfused with blood products, and those given blood products and recombinant human activated factor VIIa. Cox proportional hazards regression was used to estimate hazard ratios (HRs), confidence intervals (CIs), and p values. Measurements and Main ResultsSeventy-eight patients received no packed red blood cell transfusions, 149 received packed red blood cell transfusions, and 38 received both packed red blood cell transfusions and recombinant human activated factor VII. Packed red blood cell transfusion was associated with an increased risk of mortality that did not reach statistical significance (HR 2.168, CI 0.978-4.805; p = 0.057). Additional packed red blood cells beyond 15 U were associated with worsened survival (HR 1.363, CI 1.137-1.633; p = 0.001), but recombinant human activated factor VIIa did not increase the risk of mortality. ConclusionBlood product transfusion during and after lung transplantation is associated with decreased survival, especially with large-volume transfusions. Survival is not worse with recombinant human activated factor VIIa administration, but additional studies are needed to determine whether recombinant human activated factor VIIa administration reduces the need for blood product transfusions.

Medication-Use Evaluation of Recombinant Human Factor VIIa.

Medication-use evaluation (MUE) is a performance improvement method used to achieve optimal patient outcomes. The recombinant human factor VIIa (rFVIIa) (NovoSeven) is an expensive agent approved by the U.S. Food and Drug Administration (FDA) for specific indications. However, in clinical practice, rFVIIa is often used for conditions unrelated to the one approved, with limited evidence. The use of rFVIIa has been associated with expenditures of more than Saudi riyal (SR)30 million ($8 million) annually at King Abdul-Aziz Medical City-Western Region (KAMC-WR). Therefore, we planned a MUE of rFVIIa. The primary purpose was to determine the off-label use of rFVIIa, and the secondary purpose was to evaluate the cost impact of off-label use of rFVIIa at KAMC-WR. This was an observational retrospective cohort study conducted to assess the off-label usage pattern and the direct cost of rFVIIa for one year. A total of 27 patients who received rFVIIa were included. Two out of the 27 patients had hemophilia A with inhibitors (7%), and 23 of the 27 patients received rFVIIa with off-label indications (85%). The total cost associated with the use of rFVIIa was SR18.61 million ($4.96 million). The cost of the rFVIIa used for the appropriate purpose was SR17.83 million ($4.75 million), which represented 95.8% of the expenditures. Recombinant FVIIa is one of the most expensive medications in our hospital. It has been used mostly in patients having hemophilia A with inhibitors.

91-Year-Old Man With Upper Extremity Ecchymoses

91-Year-Old Man With Upper Extremity Ecchymoses

The severe spontaneous bleeding phenotype in a novel hemophilia A rat model is rescued by platelet FVIII expression

AbstractPrevious studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII−/− rat model in a Dahl inbred background (Dahl-FVIII−/−) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII−/− rats. Sixty-one percent developed anti-FVIII inhibitors after ≥2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII−/− background, none of the resulting 2bF8tg+FVIII−/− rats (with platelet FVIII levels of 28.26 ± 7.69 mU/108 platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8tg bone marrow (BM) was transplanted into FVIII−/− rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII−/− rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8tg BM transplantation recipients were significantly improved compared with FVIII−/− control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8tg BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII−/− rats with no anti-FVIII antibody development.

Anti-FVIII Antibody and Inhibitor Development Patterns in Hemophilia A Mouse Models: A Systematic Review

Background: Management of hemophilia A with exogenous factor VIII (FVIII) infusions can result in the development of alloantibodies that neutralize FVIII activity (inhibitors). The presence of FVIII inhibitors is associated with high morbidity and a decreased quality of life. Hemophilia A mouse models are a valuable tool for studying inhibitor development and hemophilia A. These models have been shown to develop inhibitor upon treatment with human FVIII. However, there are numerous hemophilia A mouse models, each with potentially varying patterns of inhibitor development. Objective: To identify existing hemophilia A mouse models and characterize anti-FVIII antibody and inhibitor development patterns. Methods: A systematic literature review was conducted using Ovid MEDLINE and EMBASE. The Databases were searched from inception through May 2019 for full-length publications, published abstracts, and conference proceedings that reported use of mouse models of hemophilia A to investigate total anti-FVIII antibody levels and/or inhibitor titers. The search strategy used the categorical search terms: "hemophilia A" AND "mouse" AND "animal model" AND "inhibitor" (Figure 1). Results: Our review consisted of a total of 88 studies which reported the use of 11 different hemophilia A mouse models. Out of the 88 studies, 19 studies only reported total anti-FVIII antibody data without reporting inhibitor data. In 30% of the studies that reported both total anti-FVIII antibody and inhibitor data, not all mice that developed anti-FVIII antibodies had positive inhibitor titres. Our systematic review identified the E16 - C57BL/6 model as the most frequently used model, as 27 studies (31%) reported using this model. The E16 - C57BL/6, E16 - Balb/c, E16 - C57BL6/129, E17 - C57BL/6, E17 - C57BL6/129, and FVIII total knockout (TKO) models demonstrate high immunogenicity to exogenous FVIII and were used in 65% of the studies. These mice all consistently develop anti-FVIII antibodies and inhibitors at &gt;95% incidence following treatment with at least 4 doses of full-length recombinant human FVIII (rhFVIII). The E16 - R593C model and the E17 - HLA-DRB1*1501 model both display anti-FVIII antibody and inhibitor development patterns that are representative of the human clinical scenario (~30% inhibitor development incidence). The only model completely immunotolerant to rhFVIII is the E17 - subline E model generated by van Helden et al. (2011) which is a transgenic mouse line on the hemophilic E17 background that expresses human full-length F8 cDNA. Treatment with 8 weekly doses of native human FVIII resulted in no anti-FVIII antibody or inhibitor development in this model. Conclusions: This systematic review demonstrated that mice of the most frequently used models almost always develop inhibitors when treated with full-length rhFVIII. Humanized models may be ideal for use in studies that aim to replicate human inhibitor development patterns. In addition, immunotolerant models may be best used for in vivo studies in hemophilia A that aim to avoid both antibody and inhibitor development. Lastly, this systematic review emphasises that it is important to report both anti-FVIII antibodies and neutralizing activity in Bethesda Units since in 30% of the studies that reported both values, the incidence of inhibitor development was less than the incidence of anti-FVIII antibody development in mice. Disclosures Matino: Bioviiix: Honoraria; Roche: Research Funding; Sanofi: Honoraria; Sobi: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

PRO17 A BUDGET IMPACT ANALYSIS OF ANTI-INHIBITOR COAGULANT COMPLEX USE IN PEDIATRIC HEMOPHILIA A PATIENTS WITH INHIBITORS ACROSS KINGDOM OF SAUDI ARABIA.

Hemophilia is rare genetic disorder affecting blood’s ability to clot due to clotting factor deficiency. Hemophilia A (HA), the most common form of Hemophilia, is due to factor VIII (FVIII) deficiency. HA treatment options include plasma-derived or recombinant-FVIII concentrates. Approximately 30% of HA patients develop inhibitors against FVIII concentrates. Patients with inhibitors are usually managed using bypassing agents, e.g., Anti-Inhibitor Coagulant Complex (ACC) or recombinant human coagulation Factor VIIa. (rFVIIa). In 2016, the Kingdom of Saudi Arabia (KSA) reported 31 pediatric HA inhibitor patients, among whom 26 patients were treated in the ministry of health (MOH) facilities. To assess the budget impact (BI) of switching MOH pediatric HA patients with inhibitors from-rFVIIa to ACC both as prophylaxis and an on-demand therapy. A global BI excel model was adapted from payer’s perspective. Model inputs were retrieved from literature and validated by key experts through face-to-face interviews. The model was adapted for two scenarios: Switching patients from (1) rFVIIa prophylaxis therapy to ACC prophylaxis therapy and (2) rFVIIa on-demand therapy to ACC on-demand therapy. The model considered direct costs such as drug acquisition, drug administration and bleeding episodes management. A sensitivity analysis was performed to assess the robustness of the results. The BI analysis showed that switching patients from rFVIIa prophylaxis to ACC prophylaxis would result in three times (-112 million Saudi Riyal [SAR]) and eight times (-418 million SAR) savings in the annual payers’ budget using rFVIIa standard dose and megadose. Similarly, switching patients from rFVIIa on-demand to ACC on-demand therapy would result in approximately 1.5 times (-15 million SAR) savings in the payers’ budget. Switching pediatric HA patients with inhibitors from rFVIIa to ACC as prophylaxis and on-demand therapies could be cost saving options for KSA payers thereby freeing more resources for other diseases management.

Coagulant activity of recombinant human factor VII produced by lentiviral human F7 gene transfer in immortalized hepatocyte-like cell line.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into hepatocyte-like cells, indicating that these cells may be the new target cell of interest to produce biopharmaceuticals. Our group recently established a hMSC-derived immortalized hepatocyte-like cell line (imHC) that demonstrates several liver-specific phenotypes. However, the ability of imHC to produce coagulation factors has not been characterized. Here, we examined the potential for imHC as a source of coagulation protein production by investigating the ability of imHC to produce human factor VII (FVII) using a lentiviral transduction system. Our results showed that imHC secreted a low amount of FVII (~22 ng/mL) into culture supernatant. Moreover, FVII from the transduced imHC (0.11 ± 0.005 IU/mL) demonstrated a similar coagulant activity compared with FVII from transduced HEK293T cells (0.12 ± 0.004 IU/mL) as determined by chromogenic assay. We demonstrate for the first time, to the best of our knowledge, that imHC produced FVII, albeit at a low level, indicating the unique characteristic of hepatocytes. Our study suggests the possibility of using imHC for the production of coagulation proteins.

The endothelial lectin clearance receptor CLEC4M binds and internalizes factor VIII in a VWF‐dependent and independent manner

Essentials CLEC4M is an endocytic receptor for factor FVIII. CLEC4M interacts with FVIII in a VWF-dependent and independent manner. CLEC4M binds to mannose-containing glycans on FVIII. CLEC4M internalization of FVIII involves clathrin coated pits. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex, and the majority of FVIII is likely to be cleared by VWF-dependent pathways. Clearance of VWF-free FVIII is rapid and underlies the pathological basis of some quantitative FVIII deficiencies. The receptor pathways that regulate the clearance of VWF-bound and VWF-free FVIII are incompletely uncharacterized. The human liver-expressed endothelial lectin CLEC4M has been previously characterized as a clearance receptor for VWF, although its influence on FVIII is unknown. Objective The interaction between FVIII and CLEC4M was characterized in the presence or absence of VWF. Methods FVIII interactions with CLEC4M were evaluated by invitro cell-based and solid phase binding assays. Interactions between FVIII and CLEC4M or liver sinusoidal endothelial cells were evaluated invivo by immunohistochemistry. Results CLEC4M-expressing HEK 293 cells bound and internalized recombinant and plasma-derived FVIII through VWF-dependent and independent mechanisms. CLEC4M binding to recombinant FVIII was dependent on mannose-exposed N-linked glycans. CLEC4M mediated FVIII internalization via a clathrin-coated pit-dependent mechanism, resulting in transport of FVIII from early and late endosomes for catabolism by lysosomes. In vivo hepatic expression of CLEC4M after hydrodynamic liver transfer was associated with a decrease in plasma levels of endogenous murine FVIII:C in normal mice, whereas infused recombinant human FVIII was associated with sinusoidal endothelial cells in the presence or absence of VWF. Conclusions These findings suggest that CLEC4M is a novel clearance receptor that interacts with mannose-exposed glycans on FVIII in the presence or absence of VWF.