design of the HELP study extension

Background Hereditary angioedema (HAE) is characterized by recurrent attacks of subcutaneous or submucosal edema. Attacks are unpredictable, debilitating, and have a significant impact on quality of life. Patients may be prescribed prophylactic therapy to prevent angioedema attacks. Current prophylactic treatments may be difficult to administer (i.e., intravenously), require frequent administrations or are not well tolerated, and breakthrough attacks may still occur frequently. Lanadelumab is a subcutaneously-administered monoclonal antibody inhibitor of plasma kallikrein in clinical development for prophylaxis of hereditary angioedema attacks. A Phase 1b study supported its efficacy in preventing attacks. A Phase 3, randomized, double-blind, placebo- controlled, parallel-arm study has been completed and an open-label extension is currently ongoing. Methods/design The primary objective of the open-label extension is to evaluate the long-term safety of repeated subcutaneous administrations of lanadelumab in patients with type I/II HAE. Secondary objectives include evaluation of efficacy and time to first angioedema attack to determine outer bounds of the dosing interval. The study will also evaluate immunogenicity, pharmacokinetics/pharmacodynamics, quality of life, characteristics of breakthrough attacks, ease of self-administration, and safety/efficacy in patients who switch to lanadelumab from another prophylactic therapy. The open-label extension will enroll patients who completed the double-blind study (“rollover patients”) and those who did not participate in the double-blind study (“non-rollover patients”), which includes patients who may or may not be currently using another prophylactic therapy. Rollover patients will receive a single 300 mg dose of lanadelumab on Day 0 and the second dose after the patient’s first confirmed angioedema attack. Thereafter, lanadelumab will be administered every 2 weeks. Non- rollover patients will receive 300 mg lanadelumab every 2 weeks regardless of the first attack. All patients will receive their last dose on Day 350 (maximum of 26 doses), and will then undergo a 4-week follow-up. Discussion Prevention of attacks can reduce the burden of illness associated with HAE. Prophylactic therapy requires extended, repeated dosing and the results of this study will provide important data on the long-term safety and efficacy of lanadelumab, a monoclonal antibody inhibitor of plasma kallikrein for subcutaneous administration for the treatment of HAE. Trial registration NCT0274159

The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity

Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies

Recombinant human C1 esterase inhibitor (Conestat alfa) for prophylaxis to prevent attacks in adult and adolescent patients with hereditary angioedema

Introduction: Hereditary angioedema (HAE) due to C1 inhibitor (C1-INH) deficiency is a debilitating and potentially lethal disease. Management includes on-demand treatment of angioedema and their prophylaxis. Plasma derived C1-INH is an established treatment for both on demand and prophylaxis of HAE. Conestat alfa is a recombinant form of human C1-INH (rhC1-INH) produced in transgenic rabbits. It has granted drug\u2019s registration as treatment option for acute HAE attacks in adults and adolescents in Europe, America, and other countries. Long-term prophylaxis with rhC1-INH received recent consideration in clinical trials. Areas covered: This review will critically appraise available information about rhC1-INH (conestat alfa) prophylactic treatment in adult and adolescent patients with congenital C1-INH deficiency. Results from a phase II randomized placebo-controlled trial for prophylaxis of severe HAE evidenced positive treatment outcomes for its application, both twice or once weekly. Expert commentary: Phase II clinical studies suggest that rhC1-INH is a viable option for prophylaxis of HAE. Safety and tolerability data are comparable to other available HAE specific drugs, zeroing the possibility for blood-born viral transmission. Sustainability of modern technologies is granting a practically stable and continuous recombinant production process. With other available options, rhC1-INH facilitates tailoring HAE treatment to patients\u2019 needs

The central role of endothelium in hereditary angioedema due to C1 inhibitor deficiency

An impairment of the endothelial barrier function underlies a wide spectrum of pathological conditions. Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) can be considered the \u201cpathophysiological and clinical paradigm\u201d of Paroxysmal Permeability Diseases (PPDs), conditions characterized by recurrent transient primitively functional alteration of the endothelial sieving properties, not due to inflammatory-ischemic-degenerative injury and completely reversible after the acute flare. It is a rare yet probably still underdiagnosed disease which presents with localized, non-pitting swelling of the skin and submucosal tissues of the upper respiratory and gastrointestinal tracts, without significant wheals or pruritus. The present review addresses the pathophysiology of C1-INH-HAE with a focus on the crucial role of the endothelium during contact and kallikrein/kinin system (CAS and KKS) activation, currently available and emerging biomarkers, methods applied to get new insights into the mechanisms underlying the disease (2D, 3D and in vivo systems), new promising investigation techniques (autonomic nervous system analysis, capillaroscopy, flow-mediated dilation method, non-invasive finger plethysmography). Hints are given to the binding of C1-INH to endothelial cells. Finally, crucial issues as the local vs systemic nature of CAS/KKS activation, the episodic nature of attacks vs constant C1-INH deficiency, pros and cons as well as future perspectives of available methodologies are briefly discussed

Trehalose prodegradative role on AR aggregates in a muscle model of Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is a motor neuronal disease whose onset and progression have been recently linked also to a muscular defect. SBMA is caused by a polyglutammine tract in the exon 1 of the androgen receptor protein (ARpolyQ). When AR is activated by testosterone a fraction of the protein misfolds and become toxic to cells. Moreover if ARpolyQ is not correctly removed from cellular environment it also forms aggregates that could damage many cellular process. In this work we have studied the protein quality control system (composed of a chaperone network and two main degradative pathways: proteasome and autophagy) in a cellular muscular model of SBMA. We use C2C12 stably transfected with ARwt or ARpolyQ bearing an elongation of 100 glutammine. Initially we performed a filter trap assay (FTA) on both cell line treated with testosterone. We observed that testosterone triggers the aggregation of ARpolyQ but not of ARwt. We also observed that testosterone treatment caused mortality in C2C12 with ARpolyQ. By real time PCR we found that there was not activation of the PQC system in presence of ARpolyQ but that the expression of AchR was significantly lower than in control cell. These data suggest that ARpolyQ led to muscular atrophy. We investigate degradative systems that degrade AR and found that autophagy is highly involved in AR degradation. We facilitate autophagy towards the overexpression of HspB8. We observed that HspB8 counteracted testosterone dependent aggregation of ARpolyQ. We know that trehalose in motorneuronal model of SBMA induce the expression of HspB8. We then enhanced autophagy with trehalose and found that ARpolyQ aggregation was almost completely reverted. We co-treated cells with trehalose and bafilomycin and found that this condition abolished trehalose effect. We demonstrated that trehalose effect depend upon an efficient autophagic flux. By rtPCR we observed that trehalose enhance the expression of a wide range of genes related to autophagy. Interestingly we also found VCP overexpression in presence of trehalose. The valosin containing protein VCP is a multi-functional protein involved also in the ERAD pathway. So we inhibit VCP with DBEQ a specific inhibitor of the ATPase activity of VCP and found that testosterone dependent aggregation was significantly increased. Interestingly we found that trehalose treatment counteracted this DBEQ associated aggregation. In conclusion we characterized C2C12 as a reliable muscle model of SBMA. we also found that autophagy is highly involved in ARpolyQ degradation and consequently we demonstrated that autophagy activation rescues ARpolyQ aggregation in muscle cells. We finally observed that also the ERAD pathway plays an important role in ARpolyQ degradation. AFM-TELETHON; FONDAZIONE TELETHON; FONDAZIONE CARIPLO; FONDAZIONE ARISLA; Ministero della Sanità; Joint Programme Neurodegenerative Disease (JPND)

Costs and effects of on-demand treatment of hereditary angioedema in Italy : a prospective cohort study of 167 patients

Objectives To explore treatment behaviours in a cohort of Italian patients with hereditary angioedema due to complement C1-inhibitor deficiency (C1-INH-HAE), and to estimate how effects and costs of treating attacks in routine practice differed across available on-demand treatments. Design Cost analyses and survival analyses using attack-level data collected prospectively for 1 year. Setting National reference centre for C1-INH-HAE. Participants 167 patients with proved diagnosis of C1-INH-HAE, who reported data on angioedema attacks, including severity, localisation and duration, treatment received, and use of other healthcare services. Interventions Attacks were treated with either icatibant, plasma-derived C1-INH (pdC1-INH) or just supportive care. Main outcome measures Treatment efficacy in reducing attack duration and the direct costs of acute attacks. Results Overall, 133 of 167 patients (79.6%) reported 1508 attacks during the study period, with mean incidence of 11 attacks per patient per year. Only 78.9% of attacks were treated in contrast to current guidelines. Both icatibant and pdC1-INH significantly reduced attack duration compared with no treatment (median times from onset 7, 10 and 47 hours, respectively), but remission rates with icatibant were 31% faster compared with pdC1-INH (HR 1.31, 95% CI 1.14 to 1.51). However, observed treatment behaviours suggest patterns of suboptimal dosing for pdC1-INH. The average cost per attack was \u20ac1183 (SD \u20ac789) resulting in \u20ac1.58 million healthcare costs during the observation period (\u20ac11 912 per patient per year). Icatibant was 54% more expensive than pdC1-INH, whereas age, sex and prophylactic treatment were not associated to higher or lower costs. Conclusions Both icatibant and pdC1-INH significantly reduced attack duration compared with no treatment, however, icatibant was more effective but also more expensive. Treatment behaviours and suboptimal dosing of pdC1-INH may account for the differences, but further research is needed to define their role

Mutations in VCP induce lysosomal alterations and autophagy activation in ALS neuronal models

Valosin Containing Protein (VCP) is an ATPase protein member of the AAA+ protein family. VCP is involved in various pathways that concur in maintaining cellular homeostasis. VCP mutations have been correlated different proteinopathies including neurodegenerative diseases as ALS. VCP-mutants are associated associated with the presence of alteration of the Protein Quality Control System: ubiquitin inclusions, TDP- 43 mis-localization and aggregation, and abnormal vacuoles. To date, the mechanisms correlated to VCP- mutants that lead to cell toxicity and death are still not defined. In this study, we identify VCP-mutants pathological mechanisms in an ALS-model. We overexpress VCP WT, VCP R155H and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. In first instance, we found that both VCP mutants from insoluble aggregates and induce lysosomal alteration in size, morphology, activity and membrane breakage. Lysosomal damage is known to lead to cell toxicity and death, so cells activate different mechanisms to remove damaged lysosome as the activation of autophagy. Therefore, we studied variance in the autophagic flux in presence of VCP-mutants by analysing LC3 conversion and p62 accumulation. We could determine that VCP-mutants are correlated with an activation of the autophagic flux. Moreover, by analysing transcription factors that regulate autophagy we determined that VCP-mutants positively regulates autophagic flux by specifically activating the transcription factor TFE3. Results also determined that TFE3 activation triggered by VCP-mutants presence is mediated by calcineurin, a Ca2+ dependent phosphatase. In parallel, we excluded the involvement of TFEB in this pathway. Overall, these data suggest that lysosomal damage and leakage induced by VCP-mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support to this we found that VCP mutants enhances insoluble protein-aggregates with a specific dependency from the autophagic pathway

VCP mutants cause lysosomal alterations and autophagy induction in ALS-neuronal model

Valosin Containing Protein (VCP) is an ATPase protein that has a key role in various pathways critical for the maintenance of cellular homeostasis and vitality. In particular, VCP is involved in the Protein Quality Control System. Indeed, VCP- mutants have been correlated to different proteinopathies as IBMPFD and ALS. The presence of VCP mutations has been associated with ubiquitin inclusions, TDP-43 mislocalization and aggregation, and abnormal vacuoles. To date VCP-mutants pathological mechanisms are still controversial. Thus, we decided to better define VPC-mutants pathological mechanisms in an ALS-model overexpressing VCP WT, VCP R155H, and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. Firstly, we determined that VCP-mutants form insoluble aggregates in this neuronal model. In addition, we observed that the presence of VCP-mutants triggers significant lysosomal alterations in morphology, size, activity, and membrane breakage. Lysosomal alterations have been described to induce cell toxicity and death. To remove damaged lysosomes and therefore to maintain cell vitality, cells activate different mechanisms like autophagy induction. Thus, we analysed LC3 conversion and p62 accumulation, markers of autophagic flux, to determine if the presence of VCP-mutants triggered activation of the autophagic flux. Data showed that VCP- mutants were correlated with an activation of the autophagic flux. Moreover, we determined that the activation of the autophagic flux was specifically regulated by TFE3 calcineurin-dependent dephosphorylation and activation. Calcineurin is a calcium-dependent phosphatase that could be activated by lysosomal leakage supporting a correlation between VCP-mutants lysosomal damage and autophagy activation. In addition, we excluded the involvement of TFEB in this pathway. Together these data suggest that lysosomal damage and leakage induced by VCP- mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support of this, we found that VCP- mutants enhanced insoluble protein-aggregates with a specific dependency on the autophagic pathway