Recombinant Factor VIIa Research Articles

Perioperative continuous infusions of factor VIII versus factor IX for patients with hemophilia A or B undergoing major surgery.

Continuous factor VIII (FVIII) or factor IX (FIX) infusions are commonly used for patients with hemophilia A (HA) or B (HB) undergoing surgery to secure perioperative hemostasis. To describe differences between the initial recovery and subsequent FIX and FVIII levels, and describe clinical outcomes among HB and HA patients receiving perioperative continuous infusion (CI) of recombinant FVIII and FIX concentrates. Retrospective chart review was conducted on 8 consecutive patients with HB and 7 consecutive patients with HA who underwent major surgery between 2014 and 2018 and received continuous infusions of standard half-life factor concentrate. Median initial bolus dose per kilogram was higher for HB compared to HA patients [90.8 (IQR 78.0-98.7) vs. 52.1 (IQR 48.6-55.6) IU/kg], while initial CI dose-rates were similar [4.3 (IQR 3.8-4.6) vs. 4.2 (IQR 3.8-4.4) IU/kg/h]. Median post-bolus recovery was higher for FVIII compared to FIX [1.70 (IQR 1.23-1.75) vs. 0.88 (IQR 0.75-1.00) IU/mL]. Median factor levels also were higher for FVIII on post-operative days 1 to 3. HB patients had greater mean intraoperative estimated blood loss [285.7 (range 0-1000) vs. 142.8 (range 0-400) mL] and longer median length of hospital stay [9 (IQR 8-12) vs. 5 (IQR 4-6.5) days]. Our initial evidence suggests greater in vivo yield of rFVIII compared to rFIX in the perioperative setting. We identified poorer clinical outcomes in this small cohort of perioperative HB patients indicating that they may benefit from a higher CI rate for adequate surgical hemostatic coverage.

Biological activity of a new recombinant human coagulation factor VIII and its efficacy in a small animal model

Deficiency in human coagulation factor VIII (FVIII) causes hemophilia A (HA). Patients with HA may suffer from spontaneous bleeding, which can be life-threatening. Recombinant FVIII (rFVIII) is an established treatment and prevention agent for bleeding in patients with HA. Human plasma-derived FVIII (pdFVIII), commonly used in clinical practice, is relatively difficult to prepare. In this study, we developed a novel B-domain-deleted rFVIII, produced and formulated without the use of animal or human serum-derived components. rFVIII promoted the generation of activated factor X and downstream thrombin, and, similar to that of other available FVIII preparations, its activity was inhibited by FVIII inhibitors. In addition, rFVIII has ideal binding affinity to human von Willebrand factor. Activated FVIII (FVIIIa) could be degraded by activated protein C and lose its procoagulant activity. In vitro, commercially available recombinant FVIII (Xyntha) and pdFVIII were used as controls, and there were no statistical differences between rFVIII and commercial FVIII preparations, which demonstrates the satisfactory efficacy and potency of rFVIII. In vivo, HA mice showed that infusion of rFVIII rapidly corrected activated partial thromboplastin time, similar to Xyntha. Moreover, different batches of rFVIII were comparable. Overall, our results demonstrate the potential of rFVIII as an effective strategy for the treatment of FVIII deficiency.

Prothrombin complex concentrate in cardiac surgery for the treatment of coagulopathic bleeding.

Prothrombin complex concentrate in cardiac surgery for the treatment of coagulopathic bleeding.

Hemorrhagic and Thrombotic Adverse Events Associated with Emicizumab and Extended Half-Life FVIII Replacement Drugs in Patients with Hemophilia a: Data from the Eudravigilance Database

Introduction Safety evaluation of novel drugs for treatment of hemophilia A is challenging due to the small number of patients involved in clinical trials. Concerns of increased risk of thrombotic complications against bleeding in patients with hemophilia A have been discussed, particularly with non-replacement treatment and concomitant bypassing agents. Surveillance with Roche Global Database reporting adverse events in emicizumab has been discontinued on May 2021 and reporting was transferred directly to regulators. Aim To evaluate the reporting rate for hemorrhagic and thrombotic adverse drug reactions (ADR) with non-replacement (emicizumab) and replacement extended half-life (EHL) FVIII products as retrieved from the EudraVigilance database during the post-marketing surveillance phase. Methods Total ADR reported during treatment with emicizumab or with EHL FVIII products (rurioctocog alfa pegol, efmoroctocog alfaturoctocog alfa pegol and danoctocog alfa pegol) retrieved from January 1st to December 31st, 2021 from EudraVigilance database were collected. The proportional reporting ratios (PRR), i.e., the ratio of the proportion of all reported cases of event of interest among people exposed to a particular drug compared with the corresponding proportion among people exposed to other drugs was calculated. Moreover, the reporting odds ratio (ROR), i.e,. a derived as a case-control measure where cases of interest such as hemorrhagic or thrombotic ADR represent the case series and the remaining ADR represent the control series, with their relative 95% confidence intervals (CI) was also calculated to express the hemorrhagic and thrombotic ADR reporting frequency ratio between emicizumab and EHL-FVIII products. Results We retrieved from the EudraVigilance surveillance database 406 ADR for emicizumab and 376 for EHL FVIII products. Overall, 232 and 275 hemorrhagic ADR were reported for emicizumab and for EHL FVIII products, respectively. Thrombotic ADR were 24 for emicizumab (i.e. 1 disseminated intravascular coagulation, 1 microangiopathy, 8 venous and 14 arterial thrombosis) and 9 for EHL FVIII products (i.e. 1 disseminated intravascular coagulation for danoctocog alfa pegol, 3 venous and 4 arterial thrombosis for efmoroctocog alfa and 1 arterial thrombosis for turoctocog alfa pegol).On the whole, about 25% of thrombotic ADR were reported in concomitance with eptacog alfa (activated). The reporting rates of hemorrhagic and thrombotic ADR are shown in the Table. The reporting odds of hemorrhagic ADR was smaller for emicizumab than for EHL FVIII products, with a ROR of 0.49 (95% CI 0.36-0.66). On the other hand, excluding 1 thrombotic microangiopathy and 2 disseminated intravascular coagulation events and including only thrombosis in large vessels, the reporting odds of thrombotic ADR was higher in emicizumab than in EHL products, with a ROR of 2,56 (95%CI 1,18-5,59). Conclusion In conclusion, the analysis of pharmacovigilance reports of 2021 detects a signal of increased reporting rate of thrombotic ADR in emicizumab (25% in association with eptacog alfa (activated) and 8% with FVIII) despite a lower reporting rate of hemorrhagic ADR when compared to what observed in EHL FVIII products. We maintain pharmacovigilance is pivotal in monitoring novel drugs in hemophilia. However, this signal requires further validation by mean of cohort studies to estimate the magnitude of the adverse effect minimizing possible. Moreover, the treatment benefit-risk balance should be personalized according to the different hemostatic power of replacement and non-replacement drugs alongside with the individual bleeding and thrombotic risk factors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CT-001, a Rapid Clearing Factor VIIa, Provides Pro-Hemostatic Activity to Reduce Blood Loss in Coagulopathic Conditions Induced By Activated Protein C

Background Acute bleeding leads to significant morbidity and mortality. Coagulopathy during acute bleeding further increases the risk of death. Recent studies have shown that the increase in activated protein C (APC) is one of the major contributors to coagulopathic conditions. Recombinant Factor VIIa (rFVIIa) had been reported to have some therapeutic effects in some clinical trials, however, its use was associated with thromboembolic events. Therefore, we sought to develop a novel FVIIa molecule (CT-001) with enhanced activity and lower thrombogenicity to address these bleeding situations. CT-001 has 4 N-glycans (T106N/N145/V253N/N322) with terminal sialic acid removed to promote active clearance via the asialoglycoprotein receptor, and P10Q/K32E substitutions introduced to its Gla-domain for enhanced phospholipid affinity and activity. Aim We aim to characterize the pro-hemostatic activity of CT-001 in coagulopathic conditions mediated by APC. Results An APTT-APC clotting assay was used to evaluate the procoagulant activity of CT-001 and rFVIIa in plasma rendered coagulopathic by APC. 2.25 nM APC was spiked into the normal plasma to induce a coagulopathic condition and prolong APTT clotting time by 1.5x fold (from the normal of 42 s to 63 s). 31.6 nM CT-001 corrected the APTT time of coagulopathic plasma back to normal (Fig 1). In comparison, a supra-therapeutic concentration of 100 nM rFVIIa was needed. In a thrombomodulin-modified thrombin generation assay (TM-TGA), CT-001 also restored peak thrombin and endogenous thrombin potential (ETP) at a dose lower than rFVIIa. Based on these observations, we next tested the efficacy of CT-001 in mice with coagulopathic bleeding induced by APC. In an APC-mediated coagulopathic hemorrhage mouse model with tails amputated 4 cm from the tip, 3 mg/kg CT-001 reduced blood loss significantly whereas minimal reduction was observed with rFVIIa (Fig 2). The efficacy of CT-001 was further tested in a coagulopathic saphenous vein bleeding model. In this model, mice were treated with activated protein C to induce coagulopathy. The saphenous vein was injured to initiate bleeding. The injury was treated with CT-001 or rFVIIa. After the cessation of the first bleed, the bleeding time was recorded, and the clot was disrupted to initiate a new bleeding episode for a second bleeding time measurement. This process was repeated until 30 mins had elapsed after test articles were administrated. The number of clot formations and duration of bleeding episodes during the 30 min period were collected. In untreated coagulopathic mice, the median number of clot formations within a period of 30 mins was 2.5, and the median clotting time of each bleed was 681s. 3 mg/kg CT-001 provided a significantly higher pro-hemostatic effect and increased the median number of clot formations to 31, while the median clotting time of each bleed was reduced to 25 s. In comparison, 3 mg/kg rFVIIa treated mice had only 6 clots formations and the median clotting time of each bleed was 89s. Conclusions Collectively, these data show that CT-001 has the potential of providing enhanced pro-hemostatic activity to counteract APC pathway-mediated coagulopathic conditions and reduce coagulopathic blood loss in acute bleeds. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Augmenting the Persistence and Efficacy of Engineered Regulatory T Cells to Suppress Anti-Drug Antibody Formation

Background Adoptive cell therapy (ACT) using engineered antigen specific regulatory T cells (Tregs) is a powerful tool for delivering immune tolerance in autoimmune disease, transplantation and in suppressing undesirable anti-drug antibody (ADA) responses against therapeutic proteins. An important example of the latter is hemophilia A (HA), a genetic disorder characterized by deficiency of the blood clotting protein, factor VIII (FVIII). Replacement therapy with recombinant FVIII can be neutralized by ADA development in up to 30% of severe HA patients. We previously showed that ACT with Tregs expressing a chimeric CD3ε based T cell receptor fusion construct (TRuC), encoding extracellular antibody fragments with specificity to human FVIII, suppressed ADA formation to FVIII in a preclinical HA model [Mol Ther. 2021 29(9):2660-2676]. Ensuring adequate in vivo persistence and a suppressive phenotype without risk of plasticity are major concerns that impede translation of ACT with engineered Tregs. Here we evaluate a synthetic single chain antibody fusion IL2 cytokine product (IL2-IC) to selectively expand adoptively transferred FVIII-TRuC Tregs in vivo. We then combine IL2-IC delivery with FVIII-TRuC Tregs expressing the co-inhibitory receptor PD-L1 to test improved suppression to ADA formation. Methods IL2-IC was generated by fusing IL-2 cytokine to the anti-IL-2 monoclonal antibody F5111 via a flexible linker. This complex specifically delivers IL2 to cells that express high levels of IL2 receptor α (IL2Rα) (i.e., Tregs) while avoiding interaction with cells that express low levels (i.e., Tconv, NK and CD8+ T cells). To assess whether IL2-IC could expand TRuC Tregs in vivo, 2x106 FVIII TRuC Tregs were injected intravenously (IV) into HAmice followed 5 hrs later with low dose IL2-IC or IL2 fused to an irrelevant Ab (FITC-IC) as control. 24 hrs later, mice received 2 international units (IU) of FVIII, and spleens were assayed on days 3 and 6. Murine PD-L1 was co-expressed in FVIII TRuC Tregs and in vitro assessed by stimulating TRuC PDL1 Tregs with 2IU FVIII alone or in combination with 2 ug/ml of PD1-Fc to understand the impact of PDL1 signaling on the Treg phenotype. In vivo, 0.5x106 FVIII TRuC PD-L1 Tregs were IV injected in HAmice followed 5 hrs later by mock, IL2-IC or FITC-IC IV injections. 24 hrs later, mice received weekly IV injections of 1IU FVIII. Results IL2-IC treated FVIII TRuC Tregs were preferentially expanded over endogenous Tregs (fold expansion 7.47 vs 4.56 respectively) in spleens on day 3 post ACT. Whereas both IL2-IC and FITC-IC treated FVIII TRuC Tregs had higher division indices over mock treatment (4.047 ± 0.03 and 5.070 ± 0.07 over 2.00 ± 0.00), only IL2-IC treated FVIII TRuC Tregs showed increased frequencies (1.72% ± 0.376% of CD4+ T cells) as compared to FITC-IC (0.28% ± 0.005% of CD4+ T cells) or mock treatment (0.23% ± 0.023% of CD4+ T cells). FITC-IC treatment nonspecifically expanded CD8+ Tconv cells as well as NK cells, whereas IL2-IC selectively expanded Tregs. PD1-Fc triggered PD-L1 signals in FVIII stimulated TRuC Tregs resulted in significant upregulation of Treg associated activation markers FoxP3, CD69, LAP, CTLA4, PD1, ICOS and BCL2 in vitro. In vivo, IL2-IC administration combined with FVIII TRuC PD-L1 ACT led to complete suppression of ADA formation in HA mice. At 4 weeks, mice that only received FVIII infusions developed high functional ADA titers (29.53 ± 19.58 BU/mL), while IL2-IC, TRuC PDL1 Treg and TRuC PDL1 Treg + IL2-IC groups had significantly lower titers of 1.117 ± 0.952 BU/mL, 1.805 ± 0.702 BU/mL and 0.423 ± 0.243 BU/mL, respectively. FITC-IC combined with PDL1 TRuC Tregs resulted in high ADAs of average 109 ± 21.31 BU/mL, confirming the requirement for the IL2Rα targeting antibody to selectively expand Tregs. Conclusion We show that the PD-L1/PD1 axis is an important pathway that can be exploited to improve the suppressive capacity of engineered Tregs. We also demonstrate that exogenous administration of the novel IL2-IC can robustly expand FVIII TRuC Tregs. These 2 strategies can be readily combined to selectively expand and enhance the suppressive capacity of engineered Tregs. The concepts defined here can be extended for improving the persistence and suppressive potential of engineered Tregs for various autoimmune disorders and to prevent transplant rejection. We are currently evaluating long term suppression of ADA responses using this approach.

Guiding Pups with Severe Hemophilia a and Their Parents from Diagnosis to a Full Prophylactic Schedule Mitigating the Risk of Bleed and the Risk for Inhibitor Formation - a Single Centre Approach

Introduction Pediatricians treating newly diagnosed severe hemophilia A patients are confronted with the challenge to protect the children against bleeds that are potentially life-threatening or have long-term damaging effects on joints. A well-established protective effect has early prophylaxis with coagulation factor VIII (FVIII). However, higher doses and "peak treatment moments” have been associated with a rate of inhibitor formation against factor VIII in up to 40%1,2. Another still controversial topic is the impact of the product choice on inhibitor risk. There is some evidence that plasma-derived factor VIII, or certain concentrates, are less immunogenic3,4. We report our experience with an individualized approach to managing PUPs at a single hemophilia treatment center (HTC) in Germany. Methods PUPs with severe hemophilia A (FVIII:C <1%) treated at the Gerinnungszentrum Rhein-Ruhr (GZRR), Germany, between January 2013 and July 2022 were followed prospectively. Our treatment approach includes a thorough training of parents to early detect signs of bleeds and avoid injuries with special focus on the first 50 exposure days (EDs). Patients are treated with a human plasma-derived FVIII (pdFVIII/VWF) concentrate for at least the first 50 EDs with an individually tailored treatment schedule that leads to a full prophylactic schedule. Initial doses are chosen low, given weekly or every 10 days and escalated according to bleeding tendency. In case of more severe bleeds, high doses of 60-80 IU/kg are chosen to avoid treatment for more than 2 days. Minor bleeds or hematomas are not always treated. The parents stay in close contact with the center so that the decision to treat with pdFVIII/VWF can be done on a case-by-case basis. We monitor joint health by prophylactic physiotherapy and regular ultrasound every 3-6 months. Non-urgent surgical procedures are postponed, and venous access system implants are avoided within the first 100 EDs. Inhibitor levels are measured using the modified Bethesda assay every 3-4 EDs until ED 100, and every 3 months thereafter for 2 years. Results Data from 38 consecutive PUPs with severe hemophilia A were collected, of which 35 (92.1%) have been treated with FVIII. Of the 38 patients, 34 started regular treatments within the first 10 EDs. The schedule was tailored individually ranging from 21 IU/kg every 10 days up to 40 IU/kg twice per week. One patient was treated on-demand for an intracranial bleed from ED 1-100. Only 3 spontaneous bleeds in 3 patients were treated; those bleeds had led to diagnosis. All other patients remained free of spontaneous bleeds requiring treatment. One patient developed a high titer inhibitor that was diagnosed at ED 4. He had received 35 IU/kg for treatment of a hematoma and 2 further equal doses in 2 separate weeks. The mother had reported unusual hematoma formation with a dark and more extended middle part following a slow resolution already before the Bethesda assay was positive. Discussion Our approach to managing PUPs includes treatment with human pdFVIII/VWF for at least the first 50 EDs. During this time, we treat bleeds only when considered necessary but then with high doses to minimize further infusions. We report 1 inhibitor development in a prospective cohort of 38 patients. In contrast, between 2003 and 2012, four of nine (44%) PUPs treated at our center developed inhibitors during the first 20 EDs (2 low-, 2 high-titer inhibitors). At that time, we used recombinant FVIII (rFVIII) for prophylaxis in most patients and were less selective about which and how to treat bleeds with FVIII. After at least 50 EDs we usually discuss the option to switch patients from pdFVIII/VWF to rFVIII as the injection volume is generally lower. Conclusion With a rate of 1/30 (3.3%) patients with more than 20 EDs developing an inhibitor, these data support the use of our individualized management approach allowing for a tolerization phase with FVIII on the way to a full prophylactic schedule. We consider the close contact and thorough training of the parents/guardians crucial in order to closely monitor the patients and assure adequate protection from bleeds. 1. Gouw SC et al. J Thromb Haemost 2007; 5(7): 1383-90. 2. Gouw SC et al. Blood 2013; 121(20): 4046-55. 3. Klukowska A, et al. Haemophilia 2018; 24(2): 221-8. 4. Peyvandi F et al. A. New Engl J Med 2016; 374(21): 2054-64. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Eptacog Beta (rFVIIa) Has a Low Incidence of Spontaneous Rebleeding through 24 and 48 Hours in Adult and Adolescent Patients with Hemophilia A or B with Inhibitors

Introduction Bypassing agents (BPAs [ie, recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrate]) are the primary treatment option to control breakthrough bleeding episodes (BEs) in persons with hemophilia A or B with inhibitors (PwHABI). BPAs can have variable efficacy that may lead to rebleeding following initial successful hemostasis; this can result in additional product usage for rebleed treatment or prevention, delayed recovery, and possible re-hospitalization or prolonged hospitalization. Eptacog beta (SEVENFACT®; HEMA Biologics and LFB) is a human rFVIIa that is approved for use in the US for the treatment and control of BEs in PwHABI (aged ≥12 years). In the PERSEPT 1 trial (PwHABI aged ≥12 years), 2 dosing regimens were investigated for the treatment of mild/moderate BEs: the 225µg/kg initial dose regimen (IDR) and the 75µg/kg IDR (Fig 1). Efficacy at 12h was 91% (225µg/kg IDR) and 82% (75µg/kg IDR); efficacy at 24h was 99.5% (225µg/kg IDR) and 96.7% (75µg/kg IDR). The median number of doses to treat a mild/moderate BE were 1 (225µg/kg IDR) and 2 (75µg/kg IDR). No thrombotic events were observed. Aims The aim of this analysis is to examine the incidence of rebleeding in PwHABI ≥12 years when using eptacog beta to treat mild/moderate BEs. Methods PERSEPT 1 (NCT02020369) was a prospective, randomized, crossover trial that investigated eptacog beta (225µg/kg and 75µg/kg IDRs). Informed consent/assent was obtained. Subjects with hemophilia A or B with inhibitors (who were not using prophylaxis) received an initial dose of eptacog beta (225µg/kg or 75µg/kg) for BE treatment followed by 75µg/kg doses at predefined intervals determined by clinical response (Fig. 1). Treatment success for mild/moderate BEs was defined as a patient-reported hemostasis evaluation of excellent or good (using a predefined 4-point scale) with no use of additional eptacog beta, alternative hemostatic agents or blood products, and no increase in pain following the first excellent or good assessment. Rebleeding through the first 24h was reported by the subject and defined as a BE in the same anatomical location after an initial excellent/good assessment. Rebleeding through 48h was an exploratory analysis of all BE data on a subject-by-subject basis: if subsequent bleeds occurred in the same anatomical location, and the initial doses of eptacog beta for each BE were administered within 48h of each other, then the latter BE was considered a rebleed. Results Twenty-seven subjects (aged 12-54) enrolled in PERSEPT 1; 465 mild/moderate bleeds were reported. Subjects used the same IDR to treat both the initial BE and the rebleed. All rebleeds were reported as mild/moderate. During the first 24h of BE treatment a single rebleed was reported (1/465; 0.2% incidence; Table 1). During the first 48h following initial treatment of a BE, this intention-to-treat analysis identified 19 rebleeds at the same anatomical location (incidence of 4.1%) in 11 subjects; the incidence by IDR is shown in Table 1. The majority (91%) of rebleeds through 48h treated with the 225µg/kg IDR were controlled with a single 225µg/kg dose (1 rebleed required an additional 75 µg/kg dose); 50% (4/8) of those treated with the 75µg/kg IDR required a single 75µg/kg dose to achieve bleed control (the remainder required 2 or 3 75µg/kg doses). All rebleeds achieved hemostasis by 12h and none required a hospital/HTC visit. A second analysis that excluded 2 rebleeds in 1 subject who was removed from the trial due to lack of treatment adherence and 1 rebleed in a 2nd subject that was a result of traumatic reinjury, resulted in a 3.4% incidence of rebleeding during the first 48h (Table 1). Conclusion Eptacog beta has a low incidence of rebleeding through 24h (0.2%) and 48h (4.1%) as observed in PERSEPT 1; in the few cases where rebleeding did occur, the majority of rebleeds required a single dose of only eptacog beta to achieve hemostasis and none required a hospital/HTC visit. In hemophilia without inhibitors, it is typical to be able to administer a minimal number of FVIII/FIX doses to control a BE and have a low risk of rebleeding; the data reported in PERSEPT 1 suggest a similar outcome for the control of mild/moderate BEs in inhibitor patients with eptacog beta. These new data may permit providers to make informed treatment decisions for inhibitor patients to reduce or avoid the possibility of rebleeding without the need for additional doses and their associated cost. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Coagulation Factor VIII Enhances the Cleavage of Von Willebrand Factor By ADAMTS13 In Vivo

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nuwiq Dosing and Outcomes in the Management of Women/Girls with Haemophilia a Needing FVIII Treatment for Surgery - an International, Open-Label, Non-Controlled Study (NuDIMENSION)

Background: Historically, hemophilia A has been considered a male disease, leading to a common misconception that carriers are asymptomatic. Hemophilia A carriers have on average half the factor VIII (FVIII) activity (FVIII:C) observed in healthy individuals and they can experience excessive bleeding even with normal FVIII levels. However, bleeding symptoms in women/girls with hemophilia A and in symptomatic carriers are often underrecognized and undertreated. Furthermore, there are limited clinical data available on the management of bleeding in these patients, and improved, evidence-based treatment guidelines on the management of hemophilia A in women/girls are required. Aims: To evaluate the efficacy and safety of the recombinant FVIII simoctocog alfa (Nuwiq®) in women/girls with hemophilia A undergoing major surgery. Methods: NuDIMENSION is a prospective, open-label, single-arm, multicenter study to be conducted at approximately 15 sites worldwide. Women/girls aged ≥12 years of age, diagnosed with moderate or mild hemophilia A (FVIII:C >1% and <40%), scheduled to undergo elective major surgery, and with no current or past FVIII inhibitors are eligible for the study. Simoctocog alfa will be administered once within 3 hours prior to surgery and repeated as necessary every 8-24 hours to achieve a FVIII plasma level of 80-100 international units (IU)/dL until adequate wound healing. Simoctocog alfa dosing will continue for at least another 7 days to maintain a FVIII plasma level of 30-60 IU/dL (Figure 1). The primary endpoint is the overall perioperative hemostatic efficacy ("success" or "failure") of simoctocog alfa in women/girls with hemophilia A undergoing major surgery. Hemostatic efficacy will be assessed at the end of surgery and at the end of the postoperative period (i.e., up to 24 hours after the last injection of simoctocog alfa), both using an objective 4-point ordinal hemostatic efficacy scale, and adjudicated by the Independent Data Monitoring Committee using a pre-defined algorithm. Secondary endpoints include assessment of intra- and postoperative hemostatic efficacy, perioperative FVIII plasma levels, and incidence of adverse events, including inhibitor formation. Target enrollment is 23 patients to document 26 major surgeries. Each subject can have multiple independent surgeries that will be counted as separate procedures. The study is planned to start in Q4 2022. Conclusions: The results from the NuDIMENSION study will contribute to the evidence needed to generate specific treatment guidelines for the management of bleeding in women/girls with hemophilia A undergoing major surgery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Binding to Platelets Differs Among Recombinant Factor VIII Products in Vitro

Introduction: Platelets are activated in response to injury and play a key role in blood clotting. In primary hemostasis, platelets adhere to exposed subendothelial matrix proteins (e.g., collagen) and subsequent downstream signaling pathways induce granule secretion and platelet aggregation. In secondary hemostasis, procoagulant platelets in the periphery activate clotting factors, leading to a more stable fibrin clot. Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient production and localization of thrombin at the site of injury. However, it is less clear how platelet-FVIII interactions spatiotemporally control the initial thrombus formation and pave the way for successful tissue repair. In patients with hemophilia A, replacement FVIII therapy is used to treat and prevent bleeds. A number of recombinant FVIII (rFVIII) products are indicated for use in patients with hemophilia A. Simoctocog alfa (Nuwiq®) is a chemically unmodified rFVIII with no fusion protein produced in a human cell line. Damoctocog alfa pegol (Jivi®) and rurioctocog alfa pegol (Adynovate®) are pegylated rFVIII products, while efmoroctocog alfa (Eloctate®) consists of FVIII fused with a Fc protein. It is not clear whether such modifications impact the platelet binding potential of FVIII. This study aims to examine platelet binding of different rFVIII products in vitro. Methods: Platelets were isolated from healthy donors, activated using thrombin and collagen-related peptide (CRP-XL) and incubated with individual rFVIII concentrates. After 30 min, the reaction was stopped by dilution. Binding was quantified by flow cytometry using one of two approaches: rFVIII immunostaining with a fluorescent (Alexa Fluor 647; AF647) anti-FVIII antibody; or using rFVIII molecules that had been pre-labeled with AF647. For comparison among products, experiments were performed at equimolar concentrations (17.6 nM) or equal activities of rFVIII concentrates. Values were normalized to simoctocog alfa. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc testing. Results: Platelets were obtained from 10 healthy donors. At equimolar concentrations, efmoroctocog alfa and damoctocog alfa pegol bound significantly (p < 0.001) less than simoctocog alfa to stimulated platelets, as visualized via immunostaining (n = 10). Binding to platelets was significantly lower with damoctocog alfa pegol than efmoroctocog alfa (p < 0.001). Similar results were obtained from experiments with pre-labelled rFVIII (n = 6) or when using equal activities of rFVIII concentrates. In experiments with platelets from 2 healthy donors, rurioctocog alfa pegol exhibited lower platelet binding compared with simoctocog alfa, when tested using the immunostaining method and equimolar concentrations of rFVIII. Conclusions: Simoctocog alfa showed increased binding to procoagulant platelets compared with other rFVIII concentrates tested. Further studies, including additional functional platelet assays, will be performed using samples from a larger number of healthy donors and from patients with hemophilia A. The results of this study may provide new insights into the function and efficacy of different rFVIII products in patients with hemophilia A.

Factor VIII and Von Willebrand Factor Inhibit Osteoclastogenesis in a Humanized Cell Culture Model

Background: Patients with hemophilia A suffer from decreased bone mineral density. It is unknown whether the absence of factor VIII (FVIII) might play a direct causative role. Aim: To investigate the effect of recombinant FVIII (rFVIII, simoctocog alfa) and von Willebrand factor (vWF) on osteoclastogenesis in a humanized two-dimensional (2D) cell culture model. Simoctocog alfa is a rFVIII product manufactured using a human cell line without chemical modification or protein fusion. Methods: CD14+ monocytes from male donors were cultured for 14 days with 10% human serum under the influence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), in the presence and absence of osteoprotegerin (OPG). On days 0, 4, 7 and 11 cells were incubated with 10 IU/ml rFVIII, 10 IU/ml vWF or 10 IU/ml rFVIII/vWF. Tartrate-resistant acid phosphatase (TRAP) activity was measured in the supernatant on day 14 using a colorimetric assay with p-nitrophenylphosphate (pNPP) as substrate. Data were normalized to the condition with mCSF/RANKL alone, or mCSF/RANKL/OPG. Quantitative PCR of cathepsin K (CATK) and nuclear factor of activated T cells (NFATc) was performed. Results: In the absence of OPG, osteoclastogenesis was significantly reduced by the addition of vWF (p<0.001) or the combination rFVIII/VWF (p<0.001). In the presence of OPG, addition of vWF (p<0.001), rFVIII (p<0.05), or the combination rFVIII/VWF (p<0.01) significantly reduced osteoclastogenesis. Similar results were obtained for analyses of CATK and NFATc gene expression. Conclusion: We demonstrated an inhibitory effect of rFVIII, vWF and rFVIII/VWF on osteoclastogenesis in an in vitro model using human serum as opposed to the more commonly used fetal calf serum. As osteoclastogenesis has a high donor-to-donor variation, we are currently testing more donors to see if dependencies on age or blood type exist. These data provide a first insight into mechanisms underlying the reduced bone mass observed in patients with hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real-World Safety of Emicizumab: Interim Analysis of the European Haemophilia Safety Surveillance (EUHASS) Database

Background: European Haemophilia Safety Surveillance (EUHASS) is an investigator-led pharmacovigilance program collecting real-world safety data on treatments for inherited bleeding disorders from >90 centers in >25 countries. Emicizumab (HEMLIBRA®) is a bispecific monoclonal antibody approved in Europe for treatment of congenital hemophilia A (HA) of all severities with factor VIII (FVIII) inhibitors and severe HA without FVIII inhibitors. Adverse events (AEs) of interest for emicizumab that impact morbidity, mortality, and treatment options include: FVIII inhibitor occurrence/recurrence, which has been assessed by modelling but is lacking real-world evidence; thrombotic events (TEs); and thrombotic microangiopathies (TMAs). There is a known risk of TMAs when using emicizumab concomitantly with activated prothrombin complex concentrate (aPCC, FEIBA®) at a dose of >100 U/kg/24 hours for ≥24 hours. This analysis will share EUHASS data from 2020 and a 2017-2020 overview in people receiving emicizumab. Methods: AE data were collected from participating centers from people who received emicizumab alone or in addition to other hemophilia treatments. EUHASS data are not collected according to Medical Dictionary for Regulatory Affairs (MedDRA) classifications; however, AEs were aligned to MedDRA preferred terms for this analysis. Most people receiving emicizumab in 2017 (n=25), 2018 (n=181), and 2019 (n=414) will be included in the 2020 data, as most people continue emicizumab treatment over time (Khairnar et al. Haemophilia 2022). The number who received emicizumab between Jan 1, 2017−Dec 31, 2020 is unknown, as records are not matched across the years. Results: From Jan 1, 2020−Dec 31, 2020, data from 985 people who received emicizumab were reported to EUHASS: emicizumab alone (n=832); emicizumab and FVIII products indicated for HA (n=96); emicizumab and activated recombinant FVII (rFVIIa, NovoSeven®; n=56); emicizumab and aPCC (n=1) (Table). A further 22 people received emicizumab and tranexamic acid, but AE data are unavailable. In 2020, a 26-year-old male with severe HA who received emicizumab alone had FVIII inhibitor recurrence after >50 emicizumab exposures (the date of the last FVIII inhibitor test prior to this positive test is unknown). No TEs or TMAs occurred. Two males with HA (40 years and 55 years of age) who received emicizumab alone had an allergic or other acute reaction (rash) 1 hour and 12 hours after dosing, respectively, which were considered definitely/probably related to emicizumab. Both events resolved. Across ≥985 people spanning 4 years from 2017-2020, four TEs occurred within 30 days of emicizumab dosing (relatedness to emicizumab unknown). Two occurred in 78-year-old males with HA who received emicizumab and another hemophilia treatment: one received emicizumab, rFVIIa, and a FVIII product and had a thrombosis in his port-a-cath 1 day after emicizumab dosing; the other received emicizumab and aPCC and had a myocardial infarction (MI) 10 hours after emicizumab dosing (Shang et al. Blood 2020). Two TEs occurred in males with HA (32 years and 53 years of age) who received emicizumab alone: an MI 24 hours after dosing and a superior mesenteric artery thrombus 6 days after dosing, respectively. Other AEs (both allergic or other acute reactions) reported in 2017-2020 not previously mentioned include a rash (Shang et al. Blood 2020) and headache, visual impairment, and dizziness 24 hours after emicizumab dosing in a 22-year-old male, which were reported as probably related to emicizumab. This analysis has some limitations. Firstly, off-label use of emicizumab in people with other inherited blood disorders may be recorded, although it has been confirmed that the AEs reported here occurred in people with HA. Secondly, people receiving emicizumab may not be routinely tested for FVIII inhibitors. Finally, comorbidities or other risk factors, AE severity, and whether hemophilia treatments were received concurrently or sequentially or were discontinued, are unknown. Conclusions: Across 2017-2020 in ≥985 people receiving emicizumab, one FVIII inhibitor recurrence, four TEs, and four allergic or other acute reactions were reported to EUHASS. Based on the EUHASS data to date, the safety profile of emicizumab in people with HA is favorable and consistent with clinical trial data. Roche will continue to monitor real-world safety of emicizumab and share data with the community. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Two Part Case Series on Acquired Hemophilia A

Abstract Introduction/Objective Acquired Hemophilia A is a rare condition with an incidence of 1 per 1.5 million per year. The average age at presentation is 74 years. It is due to spontaneous production of IgG autoantibodies that target endogenous factor viii. This disease should be considered when a patient presents with bleeding, an abnormal prolongation of activated partial thromboplastin time and /or no prior personal or family history of bleeding. Autoimmune disorders and malignancy are common associations. Pregnancy and surgery are common presenting scenarios. Methods/Case Report Case 1: 71 year old female with a history of deep venous thrombosis, and rheumatoid arthritis. Patient presented to the emergency department with a hemoglobin of 4.7 and coagulation studies showed a prolonged partial thromboplastin time of 119 seconds. Imaging revealed retroperitoneal bleeding. A mixing study showed no correction of partial thromboplastin time. The patient was treated with packed red blood cell transfusions until hemodynamically stable. Further coagulation studies revealed a factor VIII activity of 3L and factor VIII inhibitor level of 103 bethesda units. This confirmed suspicion of an acquired factor VIII inhibitor associated with the patient’s rheumatoid arthritis as the source of abnormal lab values and bleeding. Patient was treated with recombinant factor VIIa, novoseven, along with continuous prednisone and cyclophosphamide. Case 2: 82 year old male with history of IgG Kappa Monoclonal Gammopathy of Undetermined Significance. Patient presented with gastrointestinal bleeding and 4g/Dl hemoglobin. He received 5 units of packed red blood cells. Sigmoidoscopy revealed a rectal ulcer causing lower gastrointestinal bleeding. An advanced coagulation study revealed factor VIII activity of 1%, and inhibitor levels of 17 bethesda units. Patient was started on prednisone initially, followed by cyclophosphamide. However, bleeding continued. He was treated with 6 units of recombinant factor VIIa and packed red blood cell transfusions as required to keep hemoglobin levels at 7g/Dl. He was continued on prednisone at an increased dosage and discharged. Results (if a Case Study enter NA) NA. Conclusion Acquired Hemophilia A is a rare disease. It should be suspected anytime there is unprecedented bleeding and/or unexplained prolongation of partial thromboplastin time. It is diagnosed with factor VIII panel, including inhibitor. Treatment includes immunosuppresants, intravenous immunoglobulins, and recombinant factor VIIa.

Comparison of European recommendations about patient blood management in postpartum haemorrhage.

Postpartum haemorrhage (PPH) is the leading cause of maternal mortality and morbidity worldwide. Some documents with practical recommendations for the management of PPH do not include the most updated directives. This review offers a quality comparison of the recommendations stated in Europe since 2015. A literature search was conducted to identify the documents published in Europe from 2015 to 2020 containing recommendations about management of PPH. The search returned 10 publications. A narrative synthesis and a summary of the information about PPH definition and its management were performed. Differences in the definition of PPH were identified: some documents considered the delivery procedure, and many publications included severity criteria. The therapeutic goal for red blood cells transfusion ranged from 6 to 9g/dl. There were divergences in the need for considering haemostatic results before fresh frozen plasma transfusion. The therapeutic goal of platelet transfusion ranged from 50 x 109 to 100 x 109 μ/L. There was a wide consensus about the therapeutic goal of fibrinogen replacement (>2g/L), but not about its use in an unmonitored or pre-emptive manner. Most publications included therapeutic approaches such as tranexamic acid and recombinant factor VII activated, but not prothrombin complex concentrate or coagulation factor XIII. The recommendations about PPH management offered in European documents are heterogeneous and have changed over time. The standardisation of all them could be useful to make progress in PPH clinical management and research which, in turn, could strongly impact in patient outcomes.

Phase III dose selection of marzeptacog alfa (activated) informed by population pharmacokinetic modeling: Anovel hemostatic drug.

Marzeptacog alfa (activated) (MarzAA) is an activated recombinant human FVII (rFVIIa) variant developed as subcutaneous (s.c.) administration for the treatment or prevention of bleeding episodes in patients with hemophilia A (HA) or hemophilia B (HB) with inhibitors and other rare bleeding disorders. Population pharmacokinetic (PK) modeling was applied for dose selection for a pivotal phase III clinical trial evaluating s.c. MarzAA for episodic treatment of spontaneous or traumatic bleeding episodes. The population PK model used MarzAA intravenous and s.c. data from previously completed clinical trials in patients with HA/HB with or without inhibitors. Based on the model, clinical trial simulations were performed to predict MarzAA exposure after different dosing regimens. The exposure target was identified using an exposure-matching strategy with a wild-type rFVIIa but adjusting for the difference in potency between the two compounds. Simulations demonstrated a sufficient absorption rate and prolonged exposure following a single 60 μg/kg dose leading to 51% and 70% of the population reaching levels above the target after 3 and 6 h, respectively. According to the phase III protocol, if a second dose was required after 3 h because of a lack of efficacy, 90% of the population was observed to be above target 6 h after the initial dose. The model-informed drug development approach integrated information from several trials and guided dose selection in the pivotal phase III clinical trial for episodic treatment of an acute bleeding event in individuals with HA or HB with inhibitors without the execution of a phase II trial for that indication.

Activated prothrombin complex concentrate to treat bleeding events in acquired hemophilia A: BAHAS study

Activated prothrombin complex concentrate (aPCC) is a bypassing agent indicated to treat bleeds in patients with acquired hemophilia A (AHA). Nevertheless, its efficacy and safety in the real-world setting have not often been addressed. We report the experience of Spanish reference centers for coagulation disorders and from acquired hemophilia Spanish Registry (AHASR) from August 2012 to February 2021. Follow-up period of 30 days after aPCC withdrawal. Thirty patients with a median age of 70 years old, suffering from 51 bleeds treated with aPCC were finally evaluated. As first-line treatment, aPCC stopped bleeding in 13 of 14 (92.9%) cases. aPCC as the second line after recombinant factor VIIa failure, stopped bleeding in all cases. In 17 patients, aPCC was used far from initial bleed control as prophylaxis of rebleeding with 94% effectiveness. No thromboembolic episodes were communicated. One patient developed hypofibrinogenemia, which did not prevent aPCC from halting bleeding. No other serious adverse events possibly or probably associated with aPCC were reported. This data support aPCC as hemostatic treatment in AHA with high effectiveness and excellent safety profile in acute bleeds and as extended use to prevent rebleedings, even in aging people with high cardiovascular risk.

Life-threatening chlorpromazine-induced acquired haemophilia A in a patient with a cavernous malformation involving the medulla oblongata

IntroductionChlorpromazine is a commonly used drug in several medical conditions associated with a wide range of side effects. Few cases of hemostatic disorder have been reported in the literature. Case reportA 39-year-old man had previously been diagnosed with a cavernous malformation of the medulla oblongata. Chlorpromazine was started to treat persistent hiccups. Twenty days later, the patient presented hepatitis and a pruritic rash. Haemostasis tests revealed a prolonged partial thromboplastin time associated with isolated decrease of factor VIII level and anti-factor VIII antibodies. Magnetic resonance imaging revealed recent asymptomatic bleeding. Introduction of eptacog alfa and prednisone allowed clinical and biological improvement as well as a prolonged remission after 12 months of follow-up.

Emicizumab in hemophilia A with inhibitors: clinical and economic impact of its use in a Cuban patient

Introduction: The management of a patient with severe hemophilia A with inhibitors is a challenge for any healthcare professional. The present analysis shows the clinical and economic impact of the therapeutic approach in the most critical patient of the Cuban Hemophilia Cohort. Objective: To evaluate the economic and clinical impact of the implementation of Emicizumab therapy in the first Cuban patient. Case presentation: Adult patient who started to use recombinant activated factor VII episodically during adolescence when he was diagnosed with the presence of high-responding antibodies against factor VIII. During the years that he used this medication, he had recorded between 95 and 105 bleeds annually. In 2018, he presented with severe hemoperitoneum and was admitted to the intensive care unit with high doses of recombinant factor VII activated, multiple transfusions of packed red blood cells, and other care typical of a critically ill patient; his evolution was satisfactory. He started emicizumab prophylaxis 7 months after this event and is currently on Week 133 of treatment. He has not presented with further bleeding, nor has he reported adverse reactions to this treatment. The biannual cost savings for on-demand treatment and prophylaxis have been US $792,509.24. Conclusions: Emicizumab prophylaxis improved the patient’s quality of life and that of his family. From the perspective of the Cuban health system, the use of emicizumab in this clinical case was satisfactory and its experience will be analyzed in new 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.