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A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

ABSTRACT: Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

SUBMITTER: Stennicke HR

PROVIDER: S-EPMC3596971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundProphylactic treatment regimens lead to improvements in health-related quality-of-life (HRQoL) among individuals with hemophilia. Turoctocog alfa pegol (N8-GP) provides the benefit of extending the duration of protection from bleeding and reducing the number of injections, which is expected to impact HRQoL and treatment satisfaction (TS).AimTo investigate the HRQoL and TS of patients with severe hemophilia A from two phase III trials evaluating the safety and efficacy of N8-GP.MethodsHRQoL was assessed using the Haemo-QoL (reported by children and their parents) and Haem-A-QoL (reported by adults). TS was assessed using Hemo-Sat. Domain and total scores for all questionnaires ranged from 0 to 100, with lower scores indicating a better HRQoL or TS. A negative change in score indicates an improvement in HRQoL/TS.ResultsMean changes in HRQoL scores were reported for 14 children aged 4-7 years, 21 children aged 8-11 years, 10 adolescents aged 13-16 years, and 163 adults (17 years and above). Mean changes in children/adolescents-reported Haemo-QoL total score were -14.0 for ages 4-7 years, -3.6 for ages 8-11 years, and -0.1 for ages 13-16 years. Mean changes in parent-reported Haemo-QoL total scores were -11.5 for 4-7 years, -8.6 for ages 8-11 years, and -4.0 for 13-16 years. Adults' mean change in Haem-A-QoL total score was -3.1 for those receiving on-demand treatment and -2.3 for those receiving prophylaxis treatment. High levels of TS with N8-GP were reported by parents of children/adolescents and the adults at the end of the trial.ConclusionWhile most patients reported a relatively good baseline HRQoL when entering the respective trials, the HRQoL of patients was either maintained or further improved when treated with N8-GP. Adults and parents of children and adolescents reported a high level of treatment satisfaction with N8-GP.

| S-EPMC6460998 | biostudies-literature

Long-term safety and efficacy of N8-GP in previously treated pediatric patients with hemophilia A: Final results from pathfinder5.

Project description:BACKGROUND:N8-GP (turoctocog alfa pegol; Esperoct® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated, extended half-life human recombinant factor VIII (FVIII). OBJECTIVE:Here, we report end-of-trial safety and efficacy results from the completed N8-GP pathfinder5 trial. METHODS:pathfinder5 (NCT01731600) was a multi-national, open-label, single-arm, non-randomized, non-controlled trial in previously treated male patients aged <12 years old with severe hemophilia A that comprised a main and an extension phase. During the main phase, patients received twice-weekly N8-GP 60 IU/kg for 50 exposure days (~26 weeks). During the extension phase, patients received the same regimen until the end of trial (first patient in main phase, 20 February 2013; trial end, 28 September 2018). RESULTS:Sixty-eight patients were exposed to N8-GP for a median time of ~4.9 years on regimen. Of the 63 patients who started in the extension phase, 62 completed the trial. No FVIII inhibitors (?0.6 BU) or other safety concerns were detected. The overall estimated annualized bleeding rate was 1.08 (median 0.81), and nearly 20% of patients had no bleeds during the entire trial. The proportion of patients with no annual bleeds increased with time, with 56% of patients experiencing no bleeds and 86% experiencing no spontaneous bleeds during the fourth year of exposure. All baseline target joints of patients who participated in both phases of this trial were resolved in slightly over 2 years. CONCLUSION:Overall, data from the completed pathfinder5 trial show that long-term (median 4.9 years) N8-GP treatment was efficacious and well tolerated in previously treated pediatric patients with severe hemophilia A.

| S-EPMC7540298 | biostudies-literature

Long-term safety and efficacy of N8-GP in previously treated adults and adolescents with hemophilia A: Final results from pathfinder2.

Project description:BACKGROUND:N8-GP (turoctocog alfa pegol; Esperoct® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated human recombinant factor VIII with a half-life of ~1.6-fold of standard FVIII products. pathfinder2 (NCT01480180) was a multi-national, open-label trial of N8-GP in previously treated adolescent and adult patients with severe hemophilia A. OBJECTIVE:We report end-of-trial efficacy and safety of N8-GP from pathfinder2. METHODS:pathfinder2 main phase and extension phase part 1 results have been previously reported. During extension phase part 2, patients could switch from N8-GP prophylaxis 50 IU/kg every fourth day (Q4D) or 75 IU/kg once weekly (Q7D), depending on bleeding status. Extension phase part 2 collected long-term safety and efficacy data for all regimens until trial end (first patient in main phase, 30 January 2012; trial end, 10 December 2018). RESULTS:Overall, 186 patients were exposed to N8-GP for up to 6.6 years (median 5.4 years). The estimated annualized bleeding rate (ABR) was 2.14 (median 0.84) for the Q4D prophylaxis arm and 1.31 (median 1.67) for the Q7D prophylaxis arm. Nearly 30% of patients experienced zero bleeds throughout the entire duration of the trial, the hemostatic response was 83.2% across all treatment arms, and patient-reported outcomes were maintained or slightly improved. No safety concerns were detected. CONCLUSION:Data from the completed pathfinder2 trial, one of the largest and longest-running clinical trials to investigate treatment of severe hemophilia A, demonstrate the efficacy and safety of N8-GP in previously treated adolescent and adult patients.

| S-EPMC7540590 | biostudies-literature

An overview of the pathfinder clinical trials program: Long-term efficacy and safety of N8-GP in patients with hemophilia A.

Project description:N8-GP (turoctocog alfa pegol, Esperoct® ; Novo Nordisk A/S, Bagsvaerd, Denmark) is a state-of-the-art, extended half-life factor VIII (FVIII) molecule used for prophylactic and on-demand treatment of patients with hemophilia A. The pathfinder clinical trial program, which began with the pathfinder1 trial in 2010, was developed to assess the long-term efficacy and safety of N8-GP in children, adolescents, and adults. The pivotal pathfinder2 (adolescents and adults) and pathfinder5 (children) trials were completed in late 2018, and comprehensive analyses of the end-of-trial results are published together with this article as part of an N8-GP Supplement. Furthermore, results from the pathfinder3 trial, which was designed to evaluate the safety and efficacy of N8-GP during major surgery, have also recently been finalized. Here, we provide an overview of the pathfinder clinical development program and summarize key data from the completed pathfinder trials. We also provide perspectives on the future of extended half-life FVIII molecules in the treatment of patients with hemophilia A and describe currently ongoing pathfinder trials.

| S-EPMC7540506 | biostudies-literature

Suppression of FVIII inhibitor formation in hemophilic mice by delivery of transgene modified apoptotic fibroblasts.

Project description:The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20-40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.

| S-EPMC2839230 | biostudies-literature

How Full-Length FVIII Benefits from Its Heterogeneity - Insights into the Role of the B-Domain.

Project description:PURPOSE:To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS:Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS:FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-? sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS:These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

| S-EPMC6443606 | biostudies-literature

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice.

Project description:Here we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)-secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

| S-EPMC6418497 | biostudies-literature

Once-weekly prophylaxis with glycoPEGylated recombinant factor VIII (N8-GP) in severe haemophilia A: Safety and efficacy results from pathfinder 2 (randomized phase III trial).

Project description:INTRODUCTION:Turoctocog alfa pegol (N8-GP) is a site-specific, 40 kDa glycoPEGylated recombinant factor VIII (FVIII) product with an extended half-life. The comprehensive main phase of the pivotal pathfinder 2 trial showed N8-GP dosed every 4 days (Q4D) provided favourable safety and efficacy for preventing bleeds in 175 patients with haemophilia A. AIM AND METHODS:We investigated the safety and efficacy of N8-GP prophylaxis when administered weekly (Q7D) for 24 weeks to patients with low bleeding rates in the pathfinder 2 extension trial. Patients (?12 years) with ?2 bleeds during the preceding 6 months of the pathfinder 2 main phase were eligible for randomization to receive N8-GP 50 IU/kg Q4D or 75 IU/kg Q7D. Safety and efficacy endpoints were incidence of FVIII inhibitors and annualized bleeding rate (ABR), respectively. RESULTS:Fifty-five of 143 (38.5%) patients on prophylaxis who continued into the extension phase were randomized to receive 50 IU/kg Q4D (n = 17) or 75 IU/kg Q7D (n = 38). Nine patients in the Q7D cohort reverted to 50 IU/kg Q4D. No inhibitors were detected. In both cohorts, >50% of patients experienced no bleeds. Median ABR for overall, joint, spontaneous, traumatic and muscle was 0.00 for both cohorts. Overall estimated success rate for treating bleeding episodes was 87.5%; 94.7% of bleeds were controlled with ?2 injections. CONCLUSIONS:Weekly N8-GP was well tolerated and efficacious and may benefit selected "low bleeder" patients with haemophilia A.

| S-EPMC6850405 | biostudies-literature

A field study evaluating the activity of N8-GP in spiked plasma samples at clinical haemostasis laboratories.

Project description:AimN8-GP (turoctocog alfa pegol) is a glycoPEGylated, extended half-life human recombinant factor VIII (FVIII) shown to be an efficacious treatment for patients with haemophilia A. Accurate monitoring of replacement therapy helps ensure proper dosing, leading to better patient care. The objective of this field study was to evaluate the accuracy and intra- and inter-laboratory variabilities of N8-GP and rAHF (Advate® ) FVIII activity (FVIII:C) measurements in clinical laboratories using their routine methods and reagents.MethodsLaboratories measured plasma samples spiked with 0.03, 0.2, 0.6 and 0.9 IU/mL N8-GP or rAHF. Samples were blinded, and laboratories were instructed to perform evaluations using their routine FVIII activity assays and calibrators.ResultsOf the 67 participating laboratories from 25 countries, 60 used a one-stage assay, 36 used a chromogenic assay, and 29 used both one-stage and chromogenic assays. Participating laboratories used nine different activated partial thromboplastin time (aPTT) reagents, the most common being SynthASil® and Actin® FS. Most aPTT reagents recovered N8-GP close to target. Three silica-based aPTT reagents (APTT-SP, TriniCLOT™ and STA® PTT-Automate) underestimated N8-GP, recovering 40%-83% of target concentration. For chromogenic assays, N8-GP and rAHF recoveries were comparable at all concentrations, with overall mean recoveries for both products close to 130%. Assay variability was similar for both assay types and both products; inter-laboratory variability was greater than intra-laboratory variability and highest at 0.03 IU/mL.ConclusionsMost clinical laboratories accurately measured N8-GP and rAHF when using their in-house one-stage or chromogenic FVIII:C assays. However, three silica-based aPTT reagents underestimated N8-GP recovery.

| S-EPMC6852407 | biostudies-literature

Predictive Modeling Identifies Total Bleeds at 12-Weeks Postswitch to N8-GP Prophylaxis as a Predictor of Treatment Response.

Project description:Background Predicting annualized bleeding rate (ABR) during factor VIII (FVIII) prophylaxis for severe hemophilia A (SHA) is important for long-term outcomes. This study used supervised machine learning-based predictive modeling to identify predictors of long-term ABR during prophylaxis with an extended half-life FVIII.Methods Data were from 166 SHA patients who received N8-GP prophylaxis (50 IU/kg every 4 days) in the pathfinder 2 study. Predictive models were developed to identify variables associated with an ABR of ≤1 versus >1 during the trial's main phase (median follow-up of 469 days). Model performance was assessed using area under the receiver operator characteristic curve (AUROC). Pre-N8-GP prophylaxis models learned from data collected at baseline; post-N8-GP prophylaxis models learned from data collected up to 12-weeks postswitch to N8-GP, and predicted ABR at the end of the outcome period (final year of treatment in the main phase).Results The predictive model using baseline variables had moderate performance (AUROC = 0.64) for predicting observed ABR. The most performant model used data collected at 12-weeks postswitch (AUROC = 0.79) with cumulative bleed count up to 12 weeks as the most informative variable, followed by baseline von Willebrand factor and mean FVIII at 30 minutes postdose. Univariate cumulative bleed count at 12 weeks performed equally well to the 12-weeks postswitch model (AUROC = 0.75). Pharmacokinetic measures were indicative, but not essential, to predict ABR.Conclusion Cumulative bleed count up to 12-weeks postswitch was as informative as the 12-week post-switch predictive model for predicting long-term ABR, supporting alterations in prophylaxis based on treatment response.

| S-EPMC9251711 | biostudies-literature

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