Clinical Therapeutics/Volume ], Number ], 2015
Therapeutic Development in Amyotrophic Lateral Sclerosis Monica Bucchia, MS; Agnese Ramirez, MS; Valeria Parente, PhD; Chiara Simone, PhD; Monica Nizzardo, PhD; Francesca Magri, MD; Sara Dametti, MS; and Stefania Corti, MD, PhD Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ca’ Granda Maggiore Hospital Policlinico, Milan, Italy ABSTRACT Purpose: Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults. It is almost invariably lethal within a few years after the onset of symptoms. No effective treatment is currently available beyond supportive care and riluzole, a putative glutamate release blocker linked to modestly prolonged survival. This review provides a general overview of preclinical and clinical advances during recent years and summarizes the literature regarding emerging therapeutic approaches, focusing on their molecular targets. Methods: A systematic literature review of PubMed was performed, identifying key clinical trials involving molecular therapies for ALS. In addition, the ALS Therapy Development Institute website was carefully analyzed, and a selection of ALS clinical trials registered at ClinicalTrials.gov has been included. Findings: In the last several years, strategies have been developed to understand both the genetic and molecular mechanisms of ALS. Several therapeutic targets have been actively pursued, including kinases, inflammation inhibitors, silencing of key genes, and modulation or replacement of specific cell populations. The majority of ongoing clinical trials are investigating the safety profiles and tolerability of pharmacologic, gene, and cellular therapies, and have begun to assess their effects on ALS progression. Implications: Currently, no therapeutic effort seems to be efficient, but recent findings in ALS could help accelerate the discovery of an effective treatment for this disease. (Clin Ther. 2015;]:]]]–]]]) & 2015 Elsevier HS Journals, Inc. All rights reserved. Key words: amyotrophic lateral sclerosis, clinical trials, molecular targets, motor neuron disease, small molecules.
INTRODUCTION The most common adult form of motor neuron disease is amyotrophic lateral sclerosis (ALS), which leads to paralysis and death caused by respiratory failure 3 to 5 years after the onset of symptoms due to the progressive degeneration of motor neurons in the spinal cord, brainstem, and cortex.1 Although the majority of ALS cases are sporadic (sALS), with no family history, 10% are familial and are caused by mutations in the superoxide dismutase 1 (SOD1), TARDBP, and FUS genes. Recent studies have identified expanded repeats in a noncoding region of chromosome 9 open reading frame 72 (C9orf72) as the most frequent genetic cause of ALS.2 Rare mutations in other genes (eg, ANG, VAPB, DAO, OPTN, VCP, and UBQLN2) have also been reported. It has been observed that the pathologic mechanisms of this pathology involve mitochondrial dysfunction, protein aggregation, excitotoxicity, and oxidative stress,3 with consequent loss of neuromuscular junction (NMJ) integrity, retrograde axonal degeneration, and motor neuronal cell death. These discoveries support the idea that complex and multiple mechanisms can induce motor neuron degeneration, with significant implications for the development of new therapeutic strategies. In fact, no effective treatments are currently available. Riluzole is the only approved compound for ALS; it has been linked to increased survival, but has no effect on the degradation of muscular function. The understanding of mechanisms of motor neuron degeneration in ALS is in an early stage of knowledge. The study of multiple targets for therapeutic treatments in neurons and other cell types can contribute Accepted for publication December 29, 2014. http://dx.doi.org/10.1016/j.clinthera.2014.12.020 0149-2918/$ - see front matter & 2015 Elsevier HS Journals, Inc. All rights reserved.
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Clinical Therapeutics to identification of adequate approaches for ALS pathogenesis and progression. The aim of this review is to summarize preclinical and clinical studies, drawing attention to the main molecular targets that are being investigated or for which testing in ALS patients is planned (Figure).
METHODS A systematic literature review until September 2014 was carried out on PubMed using the following keywords: clinical trials, small molecules, molecular targets, motor neuron disease, and amyotrophic lateral sclerosis. The search criteria are restricted to English-language articles and are based on clinical and preclinical studies divided according to their molecular targets. All articles found were systematically analyzed and taken into consideration when preparing the review. In addition, the ALS Therapy Development Institute website was carefully analyzed and a selection of ALS clinical studies registered at ClinicalTrials.gov—an official platform and catalog for registering a clinical trial—have been consulted and selected studies have been included in the article.
RESULTS Protection of Mitochondria: Dexpramipexole and Rasagiline Increasing evidence indicates that mitochondrial dysfunction and oxidative stress play a central role in the etiopathogenesis of many neurodegenerative disorders, including ALS.4 The energy requirements of neurons are very high, so inefficient energy production caused by mitochondria perturbations can trigger neurons to die because these cells have an elevated susceptibility to aging and stress. Two drugs, dexpramipexole and rasagiline, have been proposed to protect motor neurons in ALS patients by reducing levels of oxidative stress. Dexpramipexole, like riluzole, belongs to the benzothiazanate family and it represents an optimized development of pramipexole, which also had neuroprotective properties, but strong dopaminergic agonist effects. Preclinical studies showed a reduction of neuronal death after dexpramipexole (KNS-760704) administration when energy demand exceeds supply by inhibiting aberrant mitochondrial leak conductance. It has been demonstrated the maintenance or increase in the production of adenosine triphosphate, the decrease of oxygen consumption and the stabilization of the
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metabolic profile of damaged cells. In addition, it has shown protective effects against the toxicity of proteasome inhibition.5 Dexpramipexole is currently used for Parkinson’s disease. To test the efficacy of this putative mitochondrial modulator in individuals affected by ALS, Biogen Idec proceeded with clinical trials in partnership with academic investigators. Dexpramipexole was well tolerated in a Phase II safety study (ClinicalTrials.gov identifier: NCT00647296), and it showed a dosedependent trend toward slowing functional decline and improving survival. Based on these promising data, tolerability and efficacy of dexpramipexole were assessed in a doubleblind and placebo-controlled multicenter Phase III clinical trial (EMPOWER, ClinicalTrials.gov identifier: NCT01281189). Investigators enrolled approximately 1000 patients who were randomly assigned to be treated with 150 mg twice daily of dexpramipexole or placebo and were followed for a period of at least 12 months. On the basis of changes in time to death and ALS Functional Rating Scale-Revised (ALSFRS-R) total scores and combined assessment of function and survival scores, the main goal was to measure the joint rank of functional outcomes adjusted for mortality up to 12 months. After this period, there was no difference in combined assessment of function and survival score, ALSFRS-R total score, and time to death between dexpramipexoletreated and placebo-treated patients. However, 8% of dexpramipexole-treated patients developed neutropenia, compared with only 2% of placebo-treated participants.6 Although dexpramipexole performed better than many of its predecessors with regard to tolerability in the Phase II trial, the Phase III study did not meet its primary end point, no efficacy was seen in the individual components of function or survival and it did not improve symptoms or disease progression in ALS patients, so Biogen Idec discontinued development of dexpramipexole for ALS. Phase II trials should have dose selection as a goal, not efficacy, and the EMPOWER investigators based their choice of dose on results obtained from the small number of patients enrolled in Phase II. The negative Phase III results suggest that the Phase II clinical trials for ALS might need to be redesigned. However, EMPOWER investigators intend to move forward in the development of dexpramipexole for ALS.7
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M. Bucchia et al.
Target
Drug
Preclinical
Dexpramipexole Mitochondria
Immunomodulation (T-cell modulators) Immunomodulation (Macrophage modulators)
Rasagiline (Azilect) Fingolimod (Gilenya) CDP7657 Tocilizumab NP001
Clinical Phase I
Clinical Phase II
Clinical Phase III
Reduces neuronal death, Inhibiting aberrant mitochondrial leak conductance, Maintains/increases ATP production, Decreases oxygen consumption
failed end point
Inhibits MAO-B, enhancer of mitochondrial viability and stabilizer of permeability transition
Safety profile and efficacy evaluation
Sphingosine 1-phosphate receptor agonist that blocks T cells in lymph nodes
Safety profile and tolerability evaluation
Anti-CD40Lthat reduces activation of T cells
Immunomodulation Safety profile and tolerability evaluation
IL-6 receptor antibody that reduces activation of macrophages (monocytes and T cells)
Well tolerated and tolerability evaluation
It reduces macrophage activation
Safety profile and efficacy evaluation
Autophagy
Lithium Carbonate
It may boost autophagy-clearing misfolded proteins
Mast cells
Masitinib (AB1010)
Kinase inhibitor that targets the stem cell factor receptor, KIT. It blocks mast cell–mediated degranulation, cytokine production, and mast cell migration
Astroglial and microglial cells
Motor neurons
Fasudil
Reducing accumulation of TDP-43
PERK inhibitor that reduces motor neuron loss
Kenpaullone
Reducing SOD1 protein aggregates
GSK-3 inhibitor that reduces neuronal apoptosis 1
Neurimmune NI-204
Targeting of misfolded SOD1
Antibody directed to misfolded SOD1 reduces its accumulation within motor neurons Safety profile and tolerability evaluation
SOD1RX
Nuedexta
Astrocytes
Well tolerated in mice
Neural stem cell therapy
Neuromuscolar junctions
Ozanezumab Mexiletine
Antisense RNA directed to the SOD1 gene to reduce the accumulation of misfolded SOD1
Antisense oligonucleotides against C9orf72 inclusions
Against sigma1 receptor of neuronal cells, it can help to reduce PBA
Astrocytes replacement therapy
Motor neurons and astrocytes
Increasing survival in mouse model
APY, KYL, and VTM Tirasemvit
Safety profile and efficacy evaluation
Cell therapy to replace toxic astrocytes
Cell therapy as a tool for cell replacement
Well tolerated in patients, Phase II planned
Against NOGO-A protein, it can repair and reconnect motor nerves and muscle fibers
Safety profile and efficacy evaluation
It reduces hyperexcitability through sodium channel inhibition
Safety profile and efficacy evaluation
Neurons
Fast skeletal muscles
Safety profile and efficacy evaluation
ROCK inhibitor that reduces astroglial and microglial cell infiltration of the spinal cord
GSK2606414
Antisense C9orf72
MN survival increases
Safety profile and efficacy evaluation
Regulator of the regeneration of the central nervous system acting against EphA4 receptor
Troponin activator that modulates muscle contractility
Safe and tolerable. Phase IIb started
Figure. Amyotrophic lateral sclerosis clinical trials (Azilects Teva Pharmaceutical Industries, Petah Tikva, Israel]; Gilenyas [Novartis Pharmaceuticals Corporation, East Hanover, New Jersey]; Nuedextas [Aventir, Alisa Viejo, California]). ATP ¼ adenosine triphosphate; C9orf72 ¼ chromosome 9 open reading frame 72; EphA4 ¼ Ephrin receptor A4; GSK-3 ¼ glycogen synthase kinase 3; IL ¼ interleukin; MAO-B ¼ monoamine oxidase B; MN ¼ motor neuron; PBA ¼ pseudobulbar affect; PERK ¼ protein kinase Rlike kinase; ROCK ¼ Rho-associated protein kinase; SOD1 ¼ superoxide dismutase 1; TDP-43 ¼ TAR DNA-binding protein 43. ] 2015
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Clinical Therapeutics Rasagiline* (or N-propargyl-1-R-aminoindan) is an antiapoptotic drug that acts as a highly potent irreversible inhibitor of monoamine oxidase B and is already approved by the US Food and Drug Administration for the treatment of Parkinson’s disease. Rasagiline may help slow motor neuron loss by regulation of the mitochondrial permeability transition and increase mitochondrial survival.8 Its neuroprotective potential has been reported in in vitro studies and in the G93A model of familial ALS; survival and motor activity were examined after administration of rasagiline alone or in combination with riluzole. The drug had a significant dose-dependent therapeutic effect on both preclinical and clinical motor function and survival of the animals. Motor function increased 4% in mice treated with rasagiline 0.5 mg and 13.9% in those treated with rasagiline 2 mg during weeks 9 to 12. This increased activity is significant during 13 to 16 weeks (between 38% and 52.7%). Then, only the group treated with rasagiline 2 mg had motor performance that was still significantly more active. Survival rate increased to 6.2% and 13.8% in mice treated with rasagiline 0.5 mg and 2 mg only, with both riluzole and rasagiline 2 mg it increased to 20%, and with riluzole and rasagiline 0.5 mg it increased to 17.6%.9 Currently, rasagiline is undergoing a Phase II placebo-controlled clinical trial (Western Amyotrophic Lateral Sclerosis study group) in which the safety profile and efficacy of this drug in ALS patients will be evaluated (ClinicalTrials.gov identifier: NCT0123273). In this study, patients are treated with 2 mg rasagiline or placebo for 12 months. The primary outcome evaluates the number of adverse events and the change in ALSFRS-R score. Rasagiline appears to be well tolerated in this ALS population, and the change in mitochondrial membrane potentials indicates improved mitochondrial function and target engagement of the drug.
Immune Modulation: T-Cell Modulators and Macrophage Modulators Despite the metabolic dysfunctions found in ALS motor neurons, neuroinflammation seems to have as relevant a role in the etiopathogenesis of this pathology as in other neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease.10,11 Astrocytes, microglia, T lymphocytes, and oligodendroglial cells all Tradename: Azilects (Teva Pharmaceutical Industries, Petah Tikva, Israel).
*
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seem to be involved in ALS pathogenesis. Sustained inflammation in the brain and spinal cord amplifies the neurodegenerative process involving resident microglia and blood-derived immune cells.12 Glia is induced to release neuronal cytotoxic substances in the presence of mutated motor neurons, causing their death by apoptosis.13 Microglia was reported to be crucial in inducing motor neuron degeneration through increased M1 cytotoxic response, to the detriment of the classic M2 neuroprotective state.14 Macrophages can also damage motor neurons by entering the central nervous system and releasing toxic cytokines. In addition, activated resident macrophages and phagocytes contribute to the enrollment of CD4 T lymphocytes, triggering a CD8and B lymphocytemediated cytotoxic antineuron immune response. On the basis of this knowledge, different compounds that act on T lymphocytes and macrophages have been proposed to reduce neuroinflammation, and autophagic stimulation has been hypothesized to have a beneficial effect on ALS.15,16 Based on recent studies showing that, in ALS patients, T lymphocytes contribute to disease progression by penetrating brain and spinal cord parenchyma and interacting with resident microglia,12 researchers plan to develop interventions to protect motor neurons by keeping T cells out of the nervous system by potentially using the T-cell modulators fingolimod† and CDP7657. Fingolimod is a sphingosine 1 phosphate receptor agonist that blocks T cells in the secondary lymphoid tissue17 and is approved by the US Food and Drug Administration for multiple sclerosis treatment. It is being tested in an ongoing Phase IIa double-blind, placebo-controlled study, with the purpose of assessing the tolerability and safety profile of 0.5 mg daily fingolimod administration in ALS patients (ClinicalTrials.gov identifier: NCT01786174). Patients are currently being recruited; final data collection for primary outcomes measures is expected in June 2014. This strategy aims to slow ALS by reducing inflammation, but it might be considered risky because multiple sclerosis compounds have never shown efficacy in patients with ALS in the past. UCB Pharma and Biogen Idec have investigated the use of UCB’s CDP7657 as a prospective treatment for †
Trademark: Gilenyas (Novartis Pharmaceuticals Corporation, East Hanover, New Jersey).
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M. Bucchia et al. ALS. CDP7657 is a humanized antibody against CD40L that interferes with the interaction between CD40L, expressed on an activated T cell, and its ligand on B cell and other antigen-presenting cells, targeting a key regulatory step of the T-celldependent activation of accessory cells. A Phase I trial in patients with systemic lupus erythematosus, which produces an anti-CD40L monovalent pegylated Fab antibody fragment (CDP7657), is currently ongoing (ClinicalTrials.gov identifier: NCT01764594). Researchers believe that CD40L inhibition represents a potential therapeutic approach for ALS by reducing immune response in ALS patients. A preclinical study showed that targeting CD40L with a specific monoclonal antibody in SOD1 ALS mouse model results in delay of paralysis (7.1%) and extended survival (7.26%).18 These promising results in a preclinical model have triggered researcher interest in immunomodulation in ALS, and recruitment of ALS patients for a Phase I clinical study targeting the CD40L pathway has begun. To reduce macrophage activation, 2 different compounds have been proposed: NP001 and tocilizumab. Neuraltus Pharmaceuticals’ NP001 is a small molecule designed to reestablish normal macrophage function in the central nervous system. After preclinical reports that NP001 is able to lower markers of abnormal inflammatory macrophages in in vitro and in vivo studies, and an earlier-stage clinical work reported that this effect is dose dependent. Based on these promising observations, a Phase II study was started that evaluated the effects of NP001 on measures of clinical function for 9 months as the primary outcomes. Secondary outcomes measured were the safety profile and tolerability in ALS for the duration of the study and pulmonary function and biomarkers associated with inflammation during 9 months. The results of a Phase II clinical trial indicate that NP001 well tolerated (ClinicalTrials.gov identifier: NCT0 1281631). In addition, the halting of disease progression in 27% of the highest-dose treated (2 mg/kg) patients was observed approximately 2.5 times that seen in the concurrent placebo group. These promising results justified further development of NP001 and a Phase III clinical trial has been planned. Enrollment has begun in the second half of 2014. The positive trend in the ability of NP001 to slow the rate of disease progression suggests that regulatory proteins controlling activation of macrophages may be an
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important new therapeutic target in ALS, confirming the hypothesis that dysregulated macrophage activation and subsequent inflammation are linked to ALS pathology. Treatment with another immunomodulator aims to reduce neuroinflammation; tocilizumab,‡ an antiinflammatory drug currently approved to treat rheumatoid arthritis. Specifically, it is an antiinterleukin 6 receptor antibody with the function to reduce the activation of key immune cells (eg, macrophages, monocytes, and T cells) that infiltrate the nervous system in ALS. The blockage of interleukin 6 receptor signaling by tocilizumab may protect motor neurons from damage by decreasing production of proinflammatory cytokines, thereby reducing disease progression. A pilot study reported that in vitro tocilizumab suppresses many factors that drive inflammation in sALS patients.19 Another study supported the hypothesis that tocilizumab infusions may benefit sALS patients by normalizing interleukin 1 and interleukin 6 expression, but the effects are individual dependent: in fact, tocilizumab infusion attenuated strong inflammation in an sALS patients group, but actually increased weak inflammation in a second group of sALS patients.20 A Phase II clinical trial is ongoing to define the safety profile and tolerability of tocilizumab in ALS patients; participants are treated with tocilizumab or placebo for 6 months. Other outcomes include analysis of peripheral blood mononucleated cells to determine inflammation levels.
Stimulation of Autophagy: Lithium Carbonate Neurodegenerative diseases share a common cellular and molecular pathogenic mechanism that requires misfolded protein or peptide aggregation and deposition. The degradation of aggregated cellular proteins and dysfunctional organelles is possible through autophagy. Recent studies have reported that upregulation of autophagy can decrease levels of toxic protein aggregates and is helpful in the context of aging and in various models of neurodegenerative disease.21 Lithium carbonate is an inorganic compound originally developed to treat bipolar disorder, but is frequently prescribed to treat depression; it has antiapoptotic and antiglutamatergic activity and, in the context of ALS, may stimulate autophagy of misfolded ‡
Trademark: Actemras (Genentech, South San Francisco, California).
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Clinical Therapeutics proteins. Preclinical studies suggest that lithium carbonate decreases the proliferation of glial cells and the accumulation of proteins such as Alpha-Synuclein, Ubiquitin and SOD1. It is also demonstrated an induction of neurogenesis and neurodifferentiation at spinal cord level after treatment.22 In addition, reduced motor neuron loss in SOD1-G93A mice was observed. Preclinical data showed 16% delay in onset of decreased motor function and 9.2% extension in life expectancy in treated animals versus vehicletreated mice.23 A pilot study of ALS patients has provided encouraging results for lithium.24 This promising evidence has drawn the attention of the scientific community to lithium. The UKMND-LiCALS Study Group conducted a multicenter, randomized, double-blind, placebocontrolled trial in which ALS patients were administered oral lithium (n ¼ 107) or a placebo (n ¼ 107) for 18 months. No significant changes in survival functions were observed; however, there were no significant differences in serious adverse events between treated and control individuals, so there were no safety profile concerns. Five other published papers reported negative trial results for lithium in ALS,25–29 independent of the different trial designs; in none of them was the possibility of a small biologic effect of lithium on ALS survival and progression definitely excluded or confirmed. The effect of lithium in this pathology remains unknown, and the role of autophagy in ALS remains controversial, but experimental evidence suggests an impaired autophagic process in ALS, so more specific autophagic regulators should be investigated to better understand this mechanism and evaluate treatments in this direction to be tested in the clinic.
Kinase Inhibition: Masitinib, Fasudil Hydrochloride, GSK2606414, and Kenpaullone Kinases are enzymes that transfer a phosphate group to specific substrates. They play an important role in signal transduction and coordinate complex functions, such as the cell cycle, apoptosis, and inflammation. The inhibition of kinases involved in apoptosis and inflammation has been proposed as ALS therapy. Masitinib (AB1010) is a new tyrosine kinase inhibitor, already evaluated to treat a wide range of inflammatory diseases, including multiple sclerosis. Masitinib selectively inhibits c-kit tyrosine kinase, a stem cell factor receptor, blocking stem cell factorinduced proliferation. It might slow ALS progression by
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blocking mast cellmediated degranulation, bone marrow mast cell migration, and cytokine production. Masitinib might have a lower toxicity profile than other tyrosine kinase inhibitors,30 and preclinical studies confirm the absence of cardiotoxicity and genotoxicity. A Phase III clinical trial is currently ongoing, recruiting 210 patients, and will evaluate the safety profile and efficacy of masitinib for the treatment of ALS. The results of the Phase III study are expected in 2015 (EudraCT Number: 2010-024423-24). Other preclinical studies considered fasudil hydrochloride, a Rho kinase inhibitor, as a therapeutic approach in ALS. Rho-associated protein kinase has been identified as a target to modulate neurodegeneration and its inhibition resulted in slower disease progression. It combines both neuroprotection and immunomodulation.31 Fasudil hydrochloride has a strong survival effect on damaged motor neurons in vitro because it has been shown to reduce the release of cytokines and chemokines, such as tumor necrosis factorα, interleukin 6, CCL2, CCL3, CCL5, which stimulate the proinflammatory microglial phenotype. In addition, SOD1G93A mice treated with fasudil hydrochloride show prolonged survival (with delayed onset of 9.2% with low dosage and 9.4% at high dosage; and prolonged survival of 6% and 7% with low and high dosage, respectively, compared with vehicle-treated littermates) and improved motor functions32; in these mice, reduced infiltration of astroglia and microglia into the spinal cord; and an increase of remodeling processes in sciatic nerve motor axons and neuromuscular junction occur. An ongoing Phase II clinical trial for ALS is examining whether fasudil is effective and well tolerated in ALS patients (ClinicalTrials.gov identifier: NCT01935518). GSK2606414 is known to inhibit protein kinase Rlike kinase, a kinase that is upregulated during stress. When activated by phosphorylation, protein kinase Rlike kinase turns off eukaryotic initiation factor 2a and triggers the formation of stress granules, proteins, and RNA aggregations in the cytosol, which are the typical features of neurodegenerative disorders, such as ALS, Parkinson’s and Alzheimer’s diseases, and prion diseases. The therapeutic effect of GSK2606414 was reported in prion-infected mice33 and, on the basis of these results, preclinical studies are ongoing to assess if GSK2606414 can help reduce motor neuron loss in ALS.
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M. Bucchia et al. In particular, Kim et al34 reported that protein kinase Rlike kinase inhibition with GSK2606414 reduced TAR DNA-binding protein 43 (TDP-43) toxicity in primary rat neurons and in Drosophila (TDP-43expressing flies had only a mean [SD] of 18% [5%] climbing ability and with GSK2606414 treatment they retained 42% [7%]). The beneficial activity of GSK2606414 is obtained by preventing eukaryotic initiation factor 2a phosphorylation, blocking stress granule formation, and reducing accumulation of TDP-43, a protein usually localized to the nucleus that accumulates in the cytoplasm of ALS motor neurons, where it associates with stress granules and forms aggregates.34 Kenpaullone is an adenosine triphosphate competitive inhibitor of several cyclin-dependent kinases, as well as glycogen synthase kinase 3β. In neurons and other cells, glycogen synthase kinase 3 activation occurs during starvation; its inhibition reduces neuronal apoptosis. It has been proposed as a new therapeutic strategy after a report of a correlation between glycogen synthase kinase 3 and ALS.35 A previous study reported that after the 2- to 3-week period during which ALS-characteristic protein aggregates appear, the enhanced death of SOD1G93A motor neurons takes place in culture; kenpaullone protects against this delayed form of death (motor neuron survival rate is 35%).36 Motor neurons treated with kenpaullone exhibit lower SOD1 protein; its ability to enhance mutant motor neurons survival may be due to SOD1 aggregates suppression. The potential effect of kenpaullone is likely due to an additional target, HGK, a kinase that is selectively expressed in motor neurons; however, there are no studies in the context of ALS or other motor neuron diseases. The inhibition of HGK blocks the activation of an apoptosis pathway37; this is why HGK is emerging as a potentially promising therapeutic target for ALS. In addition, kenpaullone has the ability to inhibit mitogen-activated protein kinase kinase kinase kinase 4, an enzyme that promotes cellular death.38 Kenpaullone may represent a future therapeutic option for ALS patients. In conclusion, prolonged stress in motor neurons can lead to or contribute to ALS, so targeting stress-induced kinases might be a successful treatment for neurodegenerative diseases; in particular, finding small druglike molecules able to reduce levels of mutant SOD1 protein in ALS patients with SOD1 mutations might be a promising strategy for developing therapeutics.
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Gene Targeting: Anti-SOD1 and Antisense Oligonucleotides Misfolding of the antioxidant enzyme SOD1 in motor neurons may contribute to ALS, and mutations in the SOD1 gene cause 13% of familial ALS. The use of antibodies against misfolded antioxidant enzyme SOD1 might be a therapeutic option for ALS patients: one study found that anti-SOD antibodies are associated with prolonged survival in sALS patients.39 These results suggest that reducing SOD1 levels could have the potential to slow progression of the disease and decrease the accumulation of misfolded SOD1 in motor neurons. Another similar approach considers neuroimmunization by NI-204, a human-derived monoclonal antibody that targets misfolded SOD1 to reduce neurodegeneration. According to a growing number of studies, it could be important to the targeting of misfolded SOD1 for the treatment of both sALS and familial ALS. Another possible therapeutic strategy is represented by the use of antisense oligonucleotides to reduce the expression of toxic or misfolded proteins, such as SOD1 and C9orf72. Reduction of accumulation of misfolded SOD1 using an antisense RNA that specifically targets the SOD1 gene has already been tested in the rat model of ALS SOD1 Gly93Ala: the antisense oligonucleotide ISIS333611 reduced SOD1 mRNA (25%40%) and protein concentrations in spinal cord (50%) and also increased survival (9.83%).40 A Phase I clinical trial with ISIS-SOD1RX in patients with SOD1-related familial ALS is ongoing. In this study, ISIS-SOD1RX is administered intrathecally into the spinal canal; an external pump is used to deliver the drug directly into the spinal fluid.41 SOD1 levels in the cerebrospinal fluid of patients given this therapy were reduced, and the therapy also appears to be well tolerated. Finally, recent studies have identified expanded GGGGCC repeats in the C9orf72 gene as the most common genetic cause of familial ALS and sALS in Europe and the United States. This mutation leads to reduced expression of a C9orf72 transcript that encodes a protein with unclear function, suggesting a possible loss of function as a pathogenic mechanism.2 However, recent studies reveal a gain of function of RNA with the presence of RNA nuclear foci in various tissues from C9orf72-linked patients.42–44 Repeat-rich RNAs, particularly 6-dipeptides repeats, appear to be stored in affected neurons.45 These nuclear foci are toxic, and the sequestration of RNA-binding proteins and
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Clinical Therapeutics repeat-associated proteins called C9RANTs appears to increase their concentration in affected motor neurons. More than 7 regulatory proteins appear to be inactivated by C9orf72 expanded RNAs in the brain and spinal cord.42 The role of C9RANTs in ALS has not yet been clarified. Researchers at the University of Massachusetts are working to create a model of C9orf72-linked ALS, developing transgenic mice with 580 repeats in the first intron of the C9orf72 gene; these mice do not appear to develop paralysis, but might have other emerging characteristics of the disease. To treat this mutation, ISIS Pharmaceuticals has developed a potential strategy with antisense oligonucleotides, known as gapmers, against expanded C9orf72 RNAs. This strategy appears to be well tolerated in mice,42 and RNA foci were reduced by nearly 50%.43–45 However, scientists do not know whether antisense is the best approach to treat C9orf72-linked ALS because some existing dipeptide repeat proteins remain untouched. This happens because expanded RNAs fold tightly into stable quadruplexes, rendering them potentially less vulnerable to attack by oligonucleotides. In addition, many antisense oligonucleotides do not appear to reduce C9orf72 gene expression, and so this strategy may be helpful in some patients with C9orf72-linked ALS. An alternative approach is the use of small molecules that target RNAs to keep key regulatory proteins in action.46 Their design is quite tricky because small molecule libraries contain few RNA-binding compounds. Designing new molecules that target expanded RNAs to treat disease is one possible way to overcome these challenges. The library versus library approach may identify key small molecules that target RNA. It is possible to use computational methods to optimize their RNA-binding and detoxification abilities. The resulting small molecules could then be exposed on peptide-like scaffolds that would allow them to bind cooperatively to the repeat-rich RNAs, maximizing their efficacy. This approach is currently being used for myotonic dystrophies treatment. Now scientists are focused on the creation of expanded C9orf72 repeat-rich RNA-targeted small molecules.
Cell Therapy: Astrocyte Replacement, Neural Stem Cell Therapy, Neural Stem Cell Therapy, and Immunosuppressants Possible cell therapies for ALS include astrocyte transplantation and neural stem cell (NSC)
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transplantation. This strategy can be therapeutic through multiple mechanisms in addition to cell replacement, including reduced inflammation and reduced neuronal protein tangles or aggregates.47 In the mid-2000s, researchers looked to astrocytes as a possible treatment strategy. They suspected that the introduction of healthy astrocytes might provide “life support” to existing motor neurons and protect them from degeneration. This treatment, known as astrocyte replacement, comprises the injection of astrocyte precursors into the spinal cord to protect motor neurons and restore the balance of key neuronal substances, including glutamate. One such approach, termed Q cells in preclinical studies, appears to delay the onset of symptoms (2%) and increase survival (2%) in a mouse model.48 Astrocytes regenerated from the skin fibroblasts of SOD1-linked ALS patients appear to contribute to motor neuron death. However, motor neurons appeared undamaged when cocultured with astrocytes harboring a mutation in TDP-43.48,49 It is currently thought that astrocytes in C9orf72-linked ALS patients may be centrally involved in the disease. The “induced” astrocytes, differentiated from skin fibroblasts of patients with C9orf72-linked ALS, reduced the survival of mouse motor neurons by at least 50%. Preliminary coculture findings suggest that astrocytes may contribute to motor neuron death in certain forms of sALS,50 and astrocyte replacement may benefit patients with ALS. Suzuki et al51 have developed a different strategy to deliver potential therapeutic drugs into the spinal cord: using human embryo-derived astrocytes. This approach aims to reduce motor neurons degeneration in ALS patients by feeding them with neuroprotective factors. The stem cellbased therapy is currently at the investigational new drug application stage. NSCs are self-renewing and multipotent cells that primarily differentiate into neurons, astrocytes, and oligodendrocytes. They are a life-long source of neuronal cells and glia. Thanks to plasticity, they are able to generate and repair the nervous system. Human somatic cellderived NSCs and their progeny may represent a promising tool with which to model neurologic diseases.52 NSCs also represent a tool for cell replacement. Their transplantation could be a promising therapeutic strategy for several diseases,
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M. Bucchia et al. including ALS. Several stem cell strategies are currently being developed for ALS across the globe. Stem cell transplantation may be suitable for a variety of therapeutic uses, such as the replacement of microenvironmental cells (astrocytes and neuronal precursors), reduction of inflammation and tangles or aggregates, and the protection and replacement of motor neurons and neuronal circuitry.47 Different groups have reported the positive effects of the transplantation of neural and non-neural stem cells in vitro and in animal models of ALS.47,53,54 It was recently reported that ALS was effectively slowed and survival prolonged in mice treated with transplanted NSCs; in 25% of treated ALS mice, the pathologic phenotype is completely rescued.54 These data represent unprecedented success in motor neuron disease models. However, 40% of mice injected with NSCs exhibit a very mild increase in survival. The reason for this variability is unclear, and is probably related to the type of cells transplanted. Human NSCs are derived from primary central nervous system tissue (fetal brain), which is a limited source of cells. In addition, if this strategy is translated to humans, multiple direct injections of stem cells into the spinal cord will require major invasive surgery. Human-induced pluripotent stem cells represent an alternative source of NSCs derived from the reprogramming of adult somatic cells. Nizzardo et al53 isolated a specific NSC subpopulation from humaninduced pluripotent stem cells that feature high pluripotency and are able to migrate, and then reported the beneficial effects of these cells on the pathologic phenotype of ALS mice. Treated mice exhibited improved neuromuscular function and significantly increased lifespan, particularly when treated with systemic injections of NSCs. These results show that minimally invasive injections of induced pluripotent stem cellderived NSCs can exert a therapeutic effect in ALS.53 On the basis of the positive results of NSC transplantation in preclinical studies, the US Food and Drug Administration approved a Phase I trial of direct intraspinal transplantation of NSCs into patients with ALS. In this trial, 200,000 NSCs derived from a human fetus were injected into the C3 to C5 region on one or both sides of the spinal cords of 15 patients with ALS in hopes of protecting the motor neurons needed for breathing. The results indicate that NSC transplantation appears to be well tolerated, and no
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significant changes were detected with respect to the ALSFRS-R. A Phase II trial is being planned.55 In ongoing clinical trials of NSC therapy, antirejection drugs, including mycophenolate mofetil§ and tacrolimusJ are prescribed to patients with ALS. One participant, known as “Patient 11,” exhibited improvement after NSC transplantation; however, the improvement was too rapid to be explained by NSC transplantation, and this benefit might actually be due to the antirejection medications. Therefore, researchers are taking another look at immunosuppressants as a potential treatment for ALS. Thirty patients with ALS are expected to participate in a current Phase II clinical trial. The multidrug regimen includes intravenous injections of basiliximab¶ and methylprednisolone during the first week, as well as mycophenolate mofetil and tacrolimus for 6 months. The study aims to identify whether individuals with ALS might benefit from these medicines. However, Phase I results indicate that these antirejection medicines are not tolerated by some ALS patients (ClinicalTrials.gov identifier: NCT01884571).
OTHER MECHANISMS Other mechanisms involved in ALS pathogenesis have been investigated as possible therapeutic targets and are currently in clinical and preclinical trials. Several are aimed at reducing symptoms such as cramps and uncontrollable laughing or crying (pseudobulbar affect [PBA]). PBA affects approximately 20% to 50% of patients with ALS, possibly due to structural damage in certain parts of the brain that control emotions; reduction of these symptoms might improve quality of life and survival of patients. In the 1990s, scientists hypothesized that dextromethorphan, a cough suppressant, later combined with quinidine (dextromethorphan hydrobromide and quinidine sulfate#), generally used to treat certain rhythm disorders, might help to slow ALS progression. Clinical trial results indicated that patients given dextromethorphan hydrobromide and quinidine sulfate were Trademark: CellCepts (Genentech, South San Francisco, California). J Trademark: Prografs (Astella Pharma US, Northbrook, Illinois). ¶ Trademark: Simulect (Novartis Pharmaceuticals Corporation, East Hanover, New Jersey). # Trademark: Nuedextas (Aventir, Alisa Viejo, California). §
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Clinical Therapeutics better able to control their emotions; the number of episodes of PBA decreased by approximately 50%. In 2010, the FDA approved dextromethorphan hydrobromide and quinidine sulfate for the treatment of PBA; PBA symptoms are reduced when dextromethorphan hydrobromide and quinidine sulfate act on SIGMA1 receptors localized in motor neurons in the brainstem. The SIGMA1 receptor is a transmembrane protein found in the endoplasmic reticulum, plasma membrane, and mitochondria-associated membranes. The SIGMA1 receptor is centrally involved in the membrane excitability of neuronal cells; it regulates the activity of ionic channels.56 Increasing evidence suggests that the degeneration of NMJs might precede and actively cause motor neuron death; however, the role of the NMJ receives little attention in ALS, likely because compensatory mechanisms mask NMJ loss for prolonged periods. The mechanisms involved in NMJ degeneration include irregular cellular metabolism, changes in muscle gene expression, and disruption of axonal transport. Research should be undertaken to preserve NMJs to delay or prevent ALS. One compound used to preserve NMJs is ozanezumab (GSK1223249), a monoclonal antibody that targets NogoA, a protein that inhibits neurite outgrowth. NogoA level is increased in the muscle tissue of ALS patients.57 Ozanezumab can both repair damage to the motor neuronal axons and reconnect them to muscle fibers.58 Ozanezumab is currently being tested in a 48-week Phase II trial (ClinicalTrials.gov identifier: NCT01753076). Hyperexcitability due to overactivity of sodium channels is reduced in ALS patients given mexiletine, a local anesthetic antiarrhythmic drug, which protects motor neurons from additional damage and slows disease progression. According to a small study of subjects that were at high risk for developing familial ALS, these changes might happen before the initial onset of symptoms. In the early 1990s, clinicians used mexiletine to reduce muscle stiffness in patients with myotonias. This drug inhibits the inward sodium current59 and is considered a potential treatment for muscle cramps in many disorders, including ALS. In vitro exposure of primary wild-type spinal cord cultures to conditioned medium derived from SOD1 astrocytes increases persistent sodium inward currents, repetitive firing, and intracellular calcium transients, inducing specific and extensive (40%) death of
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motor neurons.60 However, chronic treatment with 25 nM mexiletine showed a reduction of motor neurons death (mean [SD] survival rate, 88% [2%]).61 A Phase II clinical trial is ongoing (ClinicalTrials.gov identifier: NCT01849770). Tirasemtiv (CK-357) is a selective troponin activator that specifically modulates the contractility of fast skeletal muscle, increasing the sensitivity of muscle to calcium.62 Direct modulation of contractility might represent an innovative therapeutic strategy for improving physical activity in ALS patients. Tirasemtiv could potentially help increase the strength of different skeletal muscles, particularly those crucial for breathing. Tirasemtiv was reported to be well tolerated in a Phase IIa clinical trial. A Phase IIb clinical trial is now recruiting 400 participants.63 Finally, Ephrin receptor A4 (EphA4) is an emerging key regulator of brain and spinal cord regeneration upon injury or disease. In ALS patients, EphA4 is upregulated and its expression is inversely correlated with disease start and survival. In addition, loss-of-function mutations in EphA4 are linked to long survival in ALS and its knockdown rescues the axonopathy induced by expression of mutant TDP-43.64 This suggests that EphA4 regulates the susceptibility of motor neurons to axonal degeneration and could symbolize a new target for ALS. Unfortunately, the exact reason why levels of EphA4mediated signaling affect the outcomes of patients with ALS remains unclear. In preclinical studies, the reduction of EphA4 levels appears to slow the decline in motor performance by 40% and survival appears to increase >50%. Van Hoecke et al64 reported that ephrin B2, a protein that binds and activates EphA4, may explain its role in modifying the disease. In a mouse model of ALS, ephrin B2 appears to relocate to astrocytes after the onset of symptoms and fuel disease progression. In addition, astrocytes lacking ephrin B2 appear to progress more slowly. This result suggests that ephrin B2 might prevent the repair of motor neurons damaged by ALS, accelerating disease progression.64 Researchers hope to reduce EphA4 signaling by using antagonists as a potential compensatory mechanism that can help protect motor neurons in some ALS patients. Lamberto et al65 found 3 peptides (APY, KYL, and VTM) bound to EphA4 that block its ability to bind ephrins (including, potentially, ephrin B2).65
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M. Bucchia et al.
CONCLUSIONS ALS is a fatal neuromuscular disease with a significant impact on society and public health. Finding an effective therapeutic strategy is crucial for ALS patients. At this time, ALS treatment is largely limited to palliative care and, after a half century of studies, riluzole is the only approved compound that modestly modifies ALS survival time, but it does not have any effect on muscle strength or function. There is an urgent medical need to identify and comprehend the molecular basis of ALS and find an effective cure. Consequently, the development of therapies to treat ALS now has strong academic, government, and industry involvement, in addition to the interest of several patients’ organizations and foundations. Although a study of a third interventional compound, dexpramipexole, was substantially disappointing, therapeutic strategies are now entering clinical phases. Researchers aim to slow disease progression by targeting etiopathophysiologic pathways or genetic defects. Current strategies are directed against inflammation factors, kinases, and key genes, such as SOD1 and C9orf72. Stem cell transplantation is a promising therapeutic strategy for a number of disease processes, including ALS, because of its ability to reduce inflammation and replace microenvironmental cells, such as astrocytes and NSCs. Recent discoveries of the pathogenic mechanisms that cause ALS give rise to the opportunity to develop new efficacious treatment for this incurable disorder. The relative importance of different pathways will vary according to the subgroup of the patient, and a crucial task for the future is to subclassify ALS more accurately, which will improve prognostic prediction and therapeutic targeting. Much of our current understanding of disease biology has come from studying the subtype of ALS associated with mutations in SOD1, in which a complex interplay between multiple pathophysiologic mechanisms culminates in motor neuron injury.
ACKNOWLEDGMENTS Stefania Corti was supported by an MIUR grant, RBFR08RV86 (“Development of a Stem Cell Approach for Motor Neuron Diseases”). The authors wish to thank the Associazione Amici del Centro Dino Ferrari for their support. Finally, the authors wish to thank Dr. Dario Ronchi for critical reading of the work.
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Monica Bucchia, Agnese Ramirez and Valeria Parente wrote the manuscript; Chiara Simone and Monica Nizzardo did literature search and correction of the manuscript; Francesca Magri and Sara Dametti did figure creation; Stefania Corti supervised the work and manuscript writing.
CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.
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Address correspondence to: Stefania Corti, MD, PhD, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ca’ Granda Policlinico, Via F. Sforza 35, 20122 Milan, Italy. E-mail:
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