Genetic and Mechanistic Insights Inform Amyotrophic Lateral Sclerosis Treatment and Symptomatic Management: Current and Emerging Therapeutics and Clinical Trial Design Considerations

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting both upper and lower motor neurons. ALS is classically characterized by painless progressive weakness, causing impaired function of limbs, speech, swallowing, and respiratory function. The disease is fatal within 2–4 years, often the result of respiratory failure. The pathologic hallmark for a majority of ALS cases is aberrant cytoplasmic accumulations of the nuclear protein TAR-DNA binding protein (TDP-43). A total of 10–15% of ALS can be attributed to a single gene mutation, known as genetic or “familial” ALS, while the remainder of cases are termed nongenetic or “sporadic” although heritability has been measured in up to 37% in this population. Complex interactions between genetics, environment, and physiologic susceptibility are thought to contribute to disease. Management is primarily supportive in nature, though there are several approved treatments worldwide. This review details the mechanisms and evidence of approved disease-modifying treatments, relevant measures to track disease burden and progression used in clinical trials, and approaches to pharmacologic management of common symptoms in ALS. As there is not currently a cure for ALS, research into the complex pathophysiologic and genetic alterations contributing to disease is of great interest. This review further discusses the current understanding of genetic etiologies and altered physiology leading to disease, such as neuroinflammation, integrated stress response, aberrant proteostasis and mitochondrial dysfunction, among others. The translation of preclinical discoveries into current investigational therapeutics, novel therapeutic categories such as antisense oligonucleotides and stem cell transplantation, as well as future horizons harnessing the power of artificial intelligence in drug development and clinical trials are discussed.

Key Points

Management of amyotrophic lateral sclerosis is best delivered in a multidisciplinary clinic and utilizes pharmacologic and non-pharmacologic approaches.

An improved understanding of amyotrophic lateral sclerosis pathomechanisms informs new therapeutic targets.

The therapeutic landscape of amyotrophic lateral sclerosis is very active, with numerous clinical trials.

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting, most notably, both upper and lower motor neurons in the brain, brainstem, and spinal cord, leading to progressive weakness of limbs and bulbar muscles as well as neuromuscular respiratory failure [1]. Up to 40–50% of patients with ALS demonstrate mild-to-moderate cognitive impairment, primarily involving executive dysfunction and impaired verbal fluency, with 15% of patients meeting criteria for frontotemporal dementia (FTD) [2]. Most patients who forego invasive mechanical ventilation via tracheostomy die within 2–4 years from diagnosis, primarily from respiratory failure and complications of bulbar and diaphragmatic weakness [3]. The observed prevalence of ALS in the USA is 6.6/100,000 with a predominance in male sex, white race, and age > 66 years [4]. Presentation is often insidious and motor and cognitive phenotypes vary greatly between patients, contributing to diagnostic delays of on average 12 months from symptom onset, a timeline that has unfortunately not declined over the last decade [5]. Shortening the time from the onset of ALS symptoms to diagnosis is essential to evaluate and initiate treatments and therapeutics sooner, especially if they exert a meaningful disease-modifying effect. In total, 85–90% of ALS cases are “sporadic,” occurring without known genetic cause or family history. Even in these cases, heritability has been estimated to be up to 36.9% [6]. Alternatively, 10–15% of ALS cases can be attributed to a single gene mutation, and these mutations can be found in patients without a known family history. A total of 5–10% of cases are termed genetic, or “familial,” ALS and have Mendelian inheritance; yet penetrance is incomplete [6], perhaps resulting from environmental exposures. To date, at least 40 genes have been linked to ALS [7], with only a subset felt to be directly causative. Among diverse and complex genetic mechanisms, many mutations lead to protein aggregates with toxic gain of function, though loss of protein function, particularly in the nucleus, is important as well [8].

In > 97% of patients with ALS (and 50% with FTD), a hallmark pathologic finding is accumulation of TAR-DNA binding protein (TDP-43) [9], a nuclear protein normally involved in RNA and cryptic exon splicing [8]. When TDP-43 is mislocalized from the nucleus to the cytoplasm, there is both loss of nuclear function and toxic sequestration of proteins and ribonucleic acids (RNAs) within the aggregates. The superoxide dismutase 1 (SOD1) and fused in sarcoma (FUS) genetic forms of ALS are exceptions as they do not develop TDP-43 aggregates, but instead show cytoplasmic SOD1 and FUS aggregates, respectively [8, 10, 11].

Despite the prominence of aberrant TDP-43 accumulation in most forms of ALS, it is a heterogenous disease with multiple aberrant pathogenic mechanisms implicated in progression, which contributes to challenges in disease modeling and developing a single disease-modifying therapy. The initiating factor of ALS is hypothesized to be a combination of multigenic and environmental factors [7] acting as “multiple hits” toward a threshold at which genetic and epigenetic changes converge into a pathologic cycle of degeneration. Some mechanisms of motor neuron injury include cytoplasmic protein aggregation [12], pathologic RNA processing, dysregulated microglia with associated neuroinflammation, and oxidative stress, among others [8]. Drugs approved to date have focused on glutamate excitotoxicity and oxidative stress, discussed in detail below. Current trials have aimed to target the early state of disease to intervene prior to development of heterogenous mechanisms of neurodegeneration [5, 13]. An improved understanding of genetic forms of disease has more recently facilitated the identification of amenable targets for novel gene therapies in subsets of patients with ALS. Notwithstanding, environmental and epigenetic contributions to disease are still under intense investigation as they may influence disease penetrance and therapeutic responses.

Pharmacologic successes have unfortunately not kept pace with the increasing pathophysiologic insights into ALS, leading to only a few medications approved for use globally, none of which stop or reverse disease progression. Therefore, there is an urgent need for edifying understanding of ALS pathophysiology and genetic underpinnings and developing novel therapeutics. This lack of therapeutic success does result in some patients seeking off-label therapies, supplements, and other “treatments” that lack high-quality levels of evidence but provide hope of altering disease trajectory. These therapies are often not covered by insurance and can be quite costly, leading to inequitable access and variability of formulations and dosing. Patients with ALS may not report these complementary therapies, leading to risks of adverse effects and possible interference with clinical trial eligibility [13].

ALS Genetics

While only a minority of patients with ALS harbor a monogenic cause, the downstream consequences of genetic mutations in patients with ALS have improved mechanistic insights. In turn, the genetic mutations or resulting dysregulated mechanisms provide therapeutic targets. Of the dozens of known mutations, four—SOD1, chromosome 9 open reading frame 72 (C9orf72) hexanucleotide repeat expansion (HRE), transactive response DNA binding protein (TARDBP), and FUS—are most prevalent [14, 15].

SOD1 was the first identified genetic cause of ALS. Discovered in 1993, it is the second most common cause of familial/genetic ALS in those of European ancestry, affecting 12% of patients with familial ALS [7]. Normal SOD1 protein functions as an antioxidant enzyme. In ALS, mutations lead to misfolding or aberrant conformational changes via oxidation leading to toxic gain of function and anomalous protein aggregation [16].The aggregates, in turn, lead to oxidative stress and mitochondrial dysfunction [17]. Therapeutic approaches have focused on reducing SOD1 protein and mRNA levels [18]. Despite the eventual recognition that most forms of ALS have different pathologic hallmarks, the discovery of SOD1 led to the development of SOD1 transgenic mouse models, which continue to be the foundation for studying potential ALS therapies. However, by lacking TDP-43 pathology, they may not accurately predict therapeutic success for all forms of ALS [19]. Please see recent reviews on SOD1 ALS for more details [10].

The most common genetic mutation in both sporadic and familial forms of ALS is the C9orf72 HRE, representing 40% of familial cases [7]. C9orf72 HRE is also the most common genetic form of FTD, thereby linking the two diseases. C9orf72 plays a role in RNA processing and autophagy. In ALS, pathological intronic hexanucleotide GGGGCC expansions in the C9orf72 gene are thought to contribute to neurodegeneration by multiple mechanisms, including toxic cytoplasmic dipeptide repeat protein aggregates, toxic RNA gain-of-function, or loss of function due to haploinsufficiency [20, 21]. The reader is directed to other recent reviews [22] for more detailed information regarding C9orf72 pathophysiology.

Other notable causes of genetic ALS currently undergoing dedicated therapeutic study include FUS and TARDBP [7]. FUS, similar to SOD1, is not a TDP-43 proteinopathy [8]. It is a nuclear RNA binding protein that mediates transport of mRNA to the cytoplasm. Mutated FUS leads to cytoplasmic protein aggregates [12, 23]. Mutations in TANK-binding kinase (TBK1) [24] and NIMA-related kinase 1 (NEK1) [25, 26], though more prevalent than FUS, currently do not have any specific targeted therapeutics in trial. TARDBP is the gene encoding TDP-43 and leads to TDP-43 aggregates, similar to findings in nongenetic ALS, thus making it an intriguing target for intervention.

Clinical Outcomes in ALS

The US Food and Drug Administration (FDA) provides guidance on important outcomes in ALS clinical trials, such as mortality, functional status, respiratory and strength measures, and patient-reported outcomes [27]. Survival and other time-to-event (such as time to death or permanent assisted ventilation) outcomes are standard and discussed elsewhere [28, 29]. Here, we review the most used non-survival outcome measures in ALS clinical trials.

The ALS Functional Rating Scale-Revised (ALSFRS-R) is a 12-point ordinal scale measuring domains of bulbar, upper limb, lower limb, and respiratory function. Scores range from 0 to 48, with lower scores indicating greater functional impairment [30]. The average rate of decline in ALSFRS-R is 1 point per month [31], with more negative slopes correlating with poorer survival [32, 33]. Among experts, a clinically meaningful change in ALSFRS-R in clinical trials would be a reduction of about 20% in the slope of rate of decline [34]. Ordinal scales, such as ALSFRS-R, where total sum scores are compared within participants, sometimes under- or over-value changes (i.e., the difference in scores in patients who go from walking up stairs to using a handrail is considered equivalent to the difference between feeding oneself and relying on others for all feeds). Thus, the Rasch-based method for developing weighted scales was applied to questions related to activities of daily living in patients with ALS to create the 28-question Rasch-built Overall ALS Disability Scale (ROADS) [35, 36]. Nonetheless, the selection of the optimal scale for use in ALS research remains of considerable debate and was the topic of a recent global summit, as the ALSFRS-R exhibits multidimensionality and revision is suggested [37–39].

Measures of respiratory function are important markers of disease progression and clinical trial outcomes. Forced vital capacity (FVC), the vital capacity when exhalation is performed with a rapid maximum exhalation, is the most common respiratory measurement in ALS trials and correlates with survival [40, 41]. Worsening rates of decline of predicted vital capacity over time increases the risk of mortality up to 17% [40]. Other measures, such as slow vital capacity (SVC), where the patient exhales slowly into a spirometer, and mean inspiratory pressure, where the patient inhales against an occluded airway, have also been used [42, 43]. SVC is sometimes favored because it is more accurate for patients with concurrent obstructive lung pathology [44]. Unfortunately, most measurements of pulmonary function can be affected by cognition, or more commonly, orofacial weakness, impairing the ability to create a seal with the respiratory measurement interface [45].

Objective measures of strength are clear target outcomes in a disease of progressive weakness. Handheld dynamometry (HHD) has been studied and consists of the evaluator applying confrontational resistance to prespecified muscle groups, measuring outputs against healthy population Z-scores and combining Z-scores into cumulative scores for an extremity. This can be monitored over time more reliably than manual muscle testing [46]. In ALS populations, change in HHD scores was correlated with ALSFRS-R and vital capacity (VC), with coefficient of variation performing better than VC, but not as well as ALSFRS-R [47]. A tool for isometric measurement of limb strength, Accurate Test of Limb Isometric Strength (ATLIS), was developed to eliminate user variability with handheld dynamometers and was found to be more sensitive to change than ALSFRS-R and VC [48]; however, training and accessibility to the ATLIS device has limited widespread use.

The FDA encourages outcomes that combine both survival and function as efforts to identify meaningful interventions persists [27]. The most commonly used combined outcome is the Combined Assessment of Function and Survival (CAFS), which is a combined, nonparametric test assessing change of ALSFRS-R scores at multiple timepoints while accounting for survival, patient drop-out (censorship), or other prespecified “time-to-event” endpoints, similar to a rank sum model [49, 50]. Though useful for accounting for study endpoint events in a rapidly fatal disease (i.e., death versus study completion versus trial withdrawal), combined outcomes suffer from issues with cross-trial validity and weighting the importance of relative contributions to outcome (death versus disease progression versus loss of follow-up) in creating the summed rank model [51]. More recently, other techniques used to combine function and survival include progression-free survival [52] and Bayesian shared parameter analysis [53].

Influenced by the 21st Century Cures Act [54], patient-reported outcome (PRO) measures are playing a more prominent role in clinical trials. Though the number of questionnaires and PROs used across ALS trials is too extensive to discuss here, we highlight a few of the most frequently used scales often included as secondary or exploratory outcomes in recent clinical trials. The Patient-Reported Outcome Measurement Information System (PROMIS-10), which measures a combination of physical, mental, and social determinants of health [55], as well as the NeuroQoL-fatigue, an 8–9 question survey about fatigue [56], correlate with ALSFRS-R scores [57] and are being incorporated more consistently across neurology trials, but are not specific to ALS. The ALS Specific Quality of Life Scale (ALSSQOL) and the ALSSQOL-Short Form (ALSSQOL-SF) were developed to measure quality of life beyond limitations in physical function and instead measure across six domains: negative emotion, interaction with people and the environment, intimacy, religiosity, physical symptoms, and bulbar function. The ALSSQOL is validated in patients with ALS and correlates with ALSFRS-R scores, as well as other measures of quality of life [58, 59]. The 40-item ALS Assessment Questionnaire (ALSAQ-40) has also been used frequently in trials, measuring five domains (eating and drinking, communication, activities of daily living/independence, physical mobility, and emotional reactions) with strong internal reliability, construct validity, and correlations with ALSFRS-R [60]. Lastly, the Center for Neurologic Study Bulbar Function Scale (CNS-BFS) is a 21-question self-administered questionnaire regarding bulbar function, including specific speech, saliva, and swallowing domains that correlates well with clinical findings and other objective measures of speech and swallow [61]. Additional details on qualitative outcomes in ALS can be found elsewhere [62].

ALS clinical trials have also incorporated advanced electrophysiologic studies as exploratory outcomes measures for quantifying lower and upper motor neuron function, although they are limited by generalizability and interrater reliability [63, 64]. For lower motor neuron function, motor unit number estimation (MUNE) and motor unit number index (MUNIX) are strategies that attempt to estimate the number of remaining motor units or lower motor neurons. Both are based on the ratio of compound muscle action potential to an average single motor unit to establish a global index, relating single motor unit function relative to muscle activation, findings of which correlate with disease progression [63, 65–67]. MUNIX is generally more reproducible than MUNE, correlates with decreasing ALSFRS-R scores better than standard surface compound muscle action potential (CMAP) measurements, and performs with exceptionally high specificity [68]. Electrical impedance myography (EIM) is a noninvasive measure of muscle via low-intensity current to detect changes in impedance over time [69]. In essence, EIM quantifies the “health” of the muscle tissue and correlates with survival in ALS as well as ALSFRS-R and HHD [70]. Given the difficulty in quantifying upper motor neuron dysfunction on clinical exam alone, some studies have used transcranial magnetic stimulation (TMS) to indirectly measure cortical hyperexcitability as a surrogate for pyramidal cell dysfunction. Though techniques vary, threshold tracking TMS has demonstrated abnormal cortical hyperexcitability as an early feature of sporadic ALS [71]. Other TMS measures, such as short-interval intracortical inhibition (SICI) and resting motor threshold (RMT), have been used in trials with variable success, though issues with reproducibility, reliability, and inconsistency in specific measurement protocols have limited the utility of these in further studies [72, 73]. Detailed review of MUNIX, TMS, and other advanced neurophysiologic studies, such as peripheral threshold tracking nerve conduction studies to measure spinal motor neuron excitability, are reviewed elsewhere [74–77].

One of the fundamental principles of therapeutic drug development is confirmation of end-target engagement, measured most readily and ideally via serum biomarkers. Though there is not a single specific serum or plasma biomarker for ALS, higher levels of serum neurofilament light chain (NfL), a neuron-specific marker of axonal degeneration, have been found to differentiate ALS from disease mimics, such as inflammatory demyelinating polyneuropathy or structural polyradiculopathy [78], and correlates with rate of progression in ALS as well as disease severity [79]. NfL is emerging as an important biomarker in ALS clinical trials [80] and has been considered by the FDA in approval decisions as an outcome to measure the effect of an intervention [81]. Further details on NfL in ALS, as well as other promising biomarkers, including markers of oxidative stress and alterations in stress and energy metabolism, are reviewed separately [82–85].

Clinical trial design in ALS continues to advance, allowing for better measurement of changes in outcomes via patient stratification, identification of patients most likely to benefit from specific drug mechanisms, and methods for faster drug development and availability to patients. Further details on trial design development in ALS are referenced [53, 86–89].

Current ALS Treatment Approaches

The overall treatment approach to the patient with ALS incorporates disease-slowing therapeutics, symptomatic management, and respiratory and nutritional support. Given the absence of disease-modifying therapies that meaningfully slow disease progression, supportive care remains the mainstay of treatment for patients with ALS. Multidisciplinary clinics substantially improve survival and quality of life in patients with ALS and are the “gold standard” of care [90, 91]. This survival benefit likely results from increased use of noninvasive ventilation and feeding tube placement [92], although it should be noted that a recent review did show both negative and positive outcomes with respect to feeding tubes, indicating future research is needed [93]. Though patients often report good quality of life, symptoms such as fatigue, pain, pseudobulbar affect, depression, anxiety, thick secretions, and sialorrhea are reported frequently and contribute to worse quality of life [94–96]. Though there is not a standardized approach to multidisciplinary care of patients with ALS, expert consensus statements and society guidelines such as those from the European Academy of Neurology [97], United Kingdom National Institute for Health and Care Excellence (NICE), American Academy of Neurology [98, 99], and Japanese Society of Neurology [100], as well as national guidelines from Canada [100] and Iran [101], provide valuable frameworks for care.

Disease-Modifying Treatments

Only a few medications are approved globally for the treatment of ALS (Fig. 1). A combination of PubMed searches and guideline reviews were used to generate lists of these treatments. A PubMed literature search used keywords, such as “amyotrophic lateral sclerosis” or “ALS,” as well as combinations of words using the controlled vocabulary thesaurus feature, Medical Subject Headings (MeSH). The final PubMed search was completed in February 2025. International society guidelines published prior to this date were reviewed from the American Academy of Neurology (AAN), European Academy of Neurology (EAN), and Japanese Society of Neurology, as well as national guidelines from Canada and Iran. Specific note is made of therapies approved internationally but not currently used in the USA. The primary focus of this review is on current FDA-approved therapies; however, given patient interest in supplements, data on safety and efficacy of supplements were obtained from the Table of Evidence provided by the international research consortium of ALS Untangled [102]. ALS Untangled is a body of international ALS experts who rigorously review alternative therapies and off-label treatments to provide objective and informative information for patients and providers [103]. Filters for “grade A” mechanism, indicating peer-reviewed publications to target relevant mechanisms in humans, and “grade A” trial or case data, indicated by two or more peer-reviewed publications describing benefit in well-designed placebo-controlled human trials, or one or more peer-reviewed publications reporting benefits in validated diagnoses were applied for inclusion in our review. References were thoroughly reviewed for quality and additional primary data. This criteria identified methylcobalamin, acetyl-L-carnitine, and vitamin E, though upon review of the literature, randomized trials of vitamin E in high and “regular” dosing did not meet primary endpoints [104–106], and is associated with increased risk of hemorrhage [107] and slight increased risk of prostate cancer [108], and is not recommended by consensus guidelines [98]. It is thus not reviewed in further detail here.

Fig. 1.

Mechanisms linked to current ALS therapeutics. Sites of action for medications and selected supplements used for the treatment of ALS. Tofersen is limited to patients with SOD1 mutations. ALCAR: acetyl-L-Carnitine; ATP: adenosine triphosphate; EEAT: Excitatory amino acid transporters; HCY: homocysteine; HSP: heat shock protein; MethylCo: methylcobalamin; mRNA: messenger RNA; Mt SOD1: mutant superoxide dismutase 1; ROS: reactive oxidative species; SAM: S-adenosylmethionine. Created in BioRender.

Source: Goutman, S. (2025) https://BioRender.com/h74e601

Riluzole

Riluzole (2-amino-6-(trifluoromethoxy)benzothiazole) was developed to modulate glutaminergic transmission to target excess of the excitatory neurotransmitter, glutamate, at synapses in the central nervous system [109]. The mechanism by which it does this is not fully understood [110], though proposed mechanisms include: (1) antagonism of the persistent sodium (Na+) current, thereby reducing hyperexcitability; (2) blockage of voltage gated calcium cannels to prevent glutamine release [111]; and (3) inhibition of inhibitory gamma-aminobutyric acid (GABA) reuptake [112]. A randomized, double-blind, placebo-controlled, stratified trial of riluzole 100 mg daily, given as 50 mg twice daily, demonstrated a statistically significant survival benefit at 12 and 21 months, with median survival increasing by 83 days [113], with more robust survival effect seen in patients with bulbar onset disease. Primary indications for withdrawal of treatment were asthenia, worsening spasticity, and increases in serum aminotransferase levels and blood pressure. Riluzole (Rilutek) was approved for marketing by the FDA in 1995 [114] and is readily available worldwide [115]. A 2012 Cochrane Review of pooled data from three trials examining tracheostomy-free survival in 974 patients treated with riluzole and 503 placebo-treated patients demonstrated a statistically significant increase in survival from 11.8 months to 14.8 months, but without any benefit on measures of muscle strength. Hazard ratios decreased while using riluzole for the first 6 months of assessment but increased when measured out to 18 months, at which point the survival benefit was no longer statistically significant [116]. Included in the Cochrane Review was a trial evaluating riluzole in patients > 75 years old with disease duration longer than 5 years and vital capacity < 60% of predicted normal, but could not recruit enough to meet their prespecified power requirements and could not comment on survival, though the medication was well tolerated in this population [117].

“Real-world” riluzole data complements clinical trial data, as retrospective studies and ALS registry data also support a riluzole survival benefit ranging from 4 months [118] to 21 months [119, 120]. Some studies suggest that earlier initiation of riluzole (within 1 year of symptom onset) can provide an additional survival benefit of up to 1.9 months [121], especially in patients with an older age at disease onset [122]. Other studies suggest riluzole prolongs time at milder disease stages [123]; however, others indicate greater benefit of riluzole later in disease, except for bulbar-onset patients, when adjusted for age and slope of ALSFRS-R decline at time of diagnosis [124]. Meta-analyses and population-based studies suggest a small benefit in bulbar and limb function, but not muscle strength [116], and contradictory to other data, greater benefit in those with bulbar onset compared with limb onset [122].

Dysphagia is an expected symptom in the progression of ALS and crushing riluzole tablets or ingesting riluzole with high-fat foods results in loss of efficacy [125]. An oral suspension was developed to obviate the need for crushing and mixing with foods, such as yogurts and puddings, and was specifically developed to avoid the side effect of mouth anesthesia that is reported with oral riluzole tablets [126]. The FDA approved the liquid version (marketed as Tiglutik [127]) in 2018 and an oral film (marketed as Exservan [128]) in 2019, though the latter was taken off the market in fall 2024 [129].

Tofersen

Tofersen was developed as an antisense oligonucleotide (ASO) targeting SOD1 mRNA to reduce synthesis of SOD1 proteins in patients with ALS resulting from mutations encoding this gene. SOD1 mutations classically have accounted for about 12% of familial ALS in European countries [7], though recent data suggest this may be an underestimate and that SOD1 mutations are also common in sporadic ALS [130]. The phase 1–2 trial of tofersen [131] demonstrated target efficacy, as measured by reduction of SOD1 in cerebrospinal fluid (CSF), without treatment-related serious adverse events (SAEs) or participant discontinuation in the trial. The phase 3 study was constructed in a three-part sequence, first comparing eight intrathecal doses of 100 mg tofersen versus placebo over 28 weeks, then proceeding into a 52-week open label extension with ongoing data collection planned through 236 weeks [132]. A total of 108 participants were enrolled, with 72 participants assigned to receive tofersen. Endpoints were designed specifically to assess changes among a prespecified “faster progression group,” though others were not excluded from the trial. Final trial results were reported for 60 of the 108 enrolled participants who met criteria for the faster progression group. Faster progression was defined by SOD1 genotype and rate of decline on ALSFRS-R calculated using the deltaFS method [33]. The primary outcome in the placebo-controlled period was change in ALSFRS-R from baseline to week 28 based on a joint rank test, which allows for measurement of treatment effects while accounting for deaths during the trial, though interestingly, there were not many deaths during either the 28- or 52-week period. The primary endpoint for the 52-week open label portion was change in neurofilament light chain (NfL). This portion of the study was not powered to assess clinical outcomes. The placebo-controlled portion of the trial did not meet its primary endpoint, but there was a statistically significant decline in CSF measurement of SOD1 and NfL at week 28 but not at week 52. There was a trend toward reduction in clinical progression at week 40 and beyond in the open-label extension. Serious neurologic adverse events occurred in 6% and 7% of the tofersen trial group and open-label group, respectively, in the form of myelitis, chemical or aseptic meningitis, lumbar radiculopathy, and papilledema, each requiring discontinuation of treatment. The mechanism of myelitis is unclear. Otherwise, headache and procedural pain from the lumbar puncture were the most common adverse events. Tofersen (marketed as Qalsody) was approved under the FDA’s accelerated approval pathway on the basis of the target engagement of reduced NfL in April 2023 [81]. It is also approved in the European Union, Norway, China, Japan, and the UAE, among others [133, 134].

Edaravone

Edaravone (MCI-186) is a free-radical scavenger, originally approved for stroke [135], developed to reduce oxidative stress in and around ischemic tissue in motor neurons. In the ischemic stroke literature, the use of intravenous (IV) edaravone is associated with rash, abnormal liver and abnormal renal function [136], fever, and dizziness; although pooled meta-analyses have not found significant differences as compared with placebo in the acute stroke population [137]. A phase 2 trial of edaravone in patients with ALS demonstrated tolerability of 60 mg, with the most common side effect reported as soft stools and diarrhea [138]. The study found a significant decline in 3-nitrotyrosine, a marker of oxidative stress, but not other inflammatory markers, such as coenzyme Q10, 8-hydroxydeoxyguanosine, or 4-hyroxy-2,3,nonenal [138]. Two subsequent phase 3 trials were completed, which did not meet their endpoints, but post hoc data revealed that a specific subset of the early stage patients may have had a greater response [139]. This led to the MCI186-19 trial, which enrolled patients with early stage disease, defined as symptom onset within 2 years and score of at least 2 out of 4 on each question of the ALSFRS-R, slow progression, defined as < 4 point decline over a 12-week observation period, and forced vital capacity (FVC) > 80%. This study compared 60 mg IV edaravone for 6 cycles (24 weeks) with placebo in 137 patients with ALS and successfully met the primary endpoint of adjusted mean difference of ALSFRS-R [140]. Biomarkers were not part of the analysis in MCI186-19 trial. The study allowed for an open-label extension, and post hoc data of patients who had FVC < 80% during the open-label extension demonstrated an overall reduction in ALSFRS-R decline from baseline to week 48. However, a propensity matched study of 194 patients with motor neuron disease in Germany failed to demonstrate any difference in disease progression as compared with riluzole alone over 1 year [138].

IV formulations are felt to add undue burden on patients with ALS, and thus an oral bioequivalent liquid form of edaravone was developed [141]. In an early phase study of oral edaravone in ALS, the drug demonstrated predictable kinetics, though with variable metabolism of drug metabolites, and was tolerated orally and via gastrostomy tube [142]. An open-label, phase 3 study of oral edaravone over 48 weeks with dosing mimicking the cycle dosing of IV edaravone was completed in 2020, with fatigue, dizziness, headache, nausea, and constipation the most frequently reported treatment-related adverse event [143]. There were no changes in serum lab monitoring or vital signs. The FDA approved oral edaravone in cycle dosing in May 2022 [144]. A phase 3 trial of oral daily dosing of edaravone failed to demonstrate superiority to cycle dosing of oral edaravone (NCT04569084) [145] but full trial results have not yet been published. Similarly, a phase 3 randomized trial of once daily oral edaravone did not demonstrate benefit as measured by change in ALSFRS-R and CAFS, though full results have not been published (NCT05178810) [146].

Sodium Phenylbutyrate-Taurursodiol (AMX0035)

The combination of sodium phenylbutyrate and tauruorsodeoxycholic acid (TUDCA) had brief approval following an encouraging phase 2 study but was removed from the market following negative phase 3 results. Sodium phenylbutyrate is a histone deacetylase inhibitor, acting on histone deacetylases, which inhibit transcription leading to disruption of cellular function and neuronal death. This is thought to be a contributing mechanism of pathogenesis in numerous neurodegenerative disorders, including ALS. Data in Alzheimer’s disease had suggested an anti-inflammatory effect, including reduction of glial activation, using TUDCA [147]. A phase 2 tolerability and dose-finding study of sodium phenylbutyrate alone in ALS demonstrated biomarker engagement and overall tolerability at a dose of 9 g/day [148]. The combination of sodium phenylbutyrate and TUDCA had been shown to reduce neuronal death in experimental models of ALS [149], thus leading to a phase 2 randomized, double-blind, placebo controlled trial in 137 patients of the combination drug, which demonstrated a statistically significant difference of 0.42 points per month decline in ALSFRS-R between treatment and placebo groups (−1.24 and −1.66 points per month, respectively) [149]. The most common side effects requiring discontinuation or dose adjustments were gastrointestinal side effects, primarily diarrhea. In a controversial decision [150]), the FDA approved AMX0035 for treatment of ALS, with the condition that it would be removed if an ongoing phase 3 trial, due to complete in 2024, was negative [151]. Two post hoc analyses of the long-term trial data showed increased overall median survival in the treatment group (25.0 months versus 18.5 months) [152] and reduced risk of death, tracheostomy, permanent assisted ventilation, and hospitalization [153]. Despite optimism from the phase 2 trial, the phase 3 trial did not meet primary or secondary endpoints and thus was voluntarily removed from the market in April 2024 [154]. Full study results have not yet been published as of March 2025.

Methylcobalamin

Methylcobalamin is the active form of cobalamin, or vitamin B12, and reduces oxidative stress [155], glutamate toxicity [156], and apoptosis, and lowers plasma homocysteine levels [156], all of which are demonstrated pathologies in patients with ALS. Given relatively low risk of administration, it has long been used by patients with ALS but has lacked convincing trial evidence to support disease-modifying effects [157]. A phase 2/3 study of ultra-high dose methylcobalamin failed to meet primary survival and functional outcomes [158]. Driven by a subgroup of these results, a phase 3 trial of ultrahigh-dose methylcobalamin was repeated in Japanese patients with symptom onset within 1 year of enrollment, slow decline in ALSFRS-R pretreatment, and limited respiratory involvement (FVC > 60%, no history of NIV or mechanical ventilation, and ambulatory). Results demonstrated a statistically significant difference in decline in ALSFRS-R over 16 weeks [159]. Methylcobalamin was approved in Japan for the treatment of ALS, marketed under the brand name Rozebalamin in 2024 [160]. The long-term open label extension of the Japanese Early-Stage Trial of High Dose Methylcobalamin for Amyotrophic Lateral Sclerosis (JETALS) completed enrollment in March 2025 (NCT03548311), with the medication showing good tolerability at an interim analysis performed in early 2024 [161].

Lenzumestrocel

Lenzumestrocel (marketed as Neuronata-R) is an intrathecal autologous bone-marrow-derived mesenchymal stem cell (MSC) therapy currently approved in South Korea for the treatment of ALS. Early preclinical data showed a prolonged life span and delay in decline in motor performance with preservation of motor neurons in the lumbar spinal cord of SOD1 mouse models [162]. In a phase 1 study, two monthly injections of intrathecal MSC were given to seven patients with ALS. There were no treatment-related serious adverse events, and all adverse events were transient in nature, such as flu-like illnesses, fevers, headache, and back pain. At 6 months, the rate of decline in ALSFRS-R was less than what had been measured in a 3-month lead in period, though this was not statistically significant [163]. The phase 2 randomized, parallel trial similarly followed the protocol of a 3-month lead-in period, followed by two intrathecal injections of MSCs in consecutive months followed by 6 months of clinical outcomes and follow-up. The control group did not undergo sham lumbar punctures or injection, but outcome evaluators were blinded to treatment assignments. A total of 33 patients underwent treatment. Adverse events were nearly identical to the phase 1 study and there were no treatment-related SAEs reported. There was a statistically significant difference in the mean difference in ALSFRS-R scores between groups at both 4 months and 6 months after injection. The mean difference in the slope of decline between lead-in and 6 months after injection was also significantly lower in the treated group. There was no change between groups in FVC or slope of FVC decline [164]. The results from these studies led to conditional approval of the drug in South Korea by the Korean Ministry of Food and Drug Safety (KMFDS) in 2013. Post-marketing surveillance data were published from 170 patients who underwent treatment with lenzumestrocel, with symptom duration at time of injection < 2 years and documented follow-up, though only 157 were included in the final survival analysis. Most patients received two injections (one cycle), but 21 underwent repeated cycle dosing. Treated patients were propensity score matched to the external controls from the PRO-ACT database. Median survival probability was significantly higher in treated patients (single and multiple cycles) compared with controls. In total, 17% of patients experienced adverse drug reactions, again mostly fever, headache, pain, back pain, and nausea, though 5% had persistent pain and allodynia lasting greater than 2 weeks [165]. The FDA and KMFDS approved a phase III, multicenter, randomized, double-blind, sham-procedure-controlled trial, conducted exclusively in South Korea [166] NCT 04745299, KCT0005954, which did not meet primarily efficacy outcomes [167], though long-term follow-up and safety reporting continues through 2026. Detailed results of the primary analysis have not been published.

Disease-Modifying Treatments Pending Regulatory Approval

Three additional agents, masitinib, co-formulated ciprofloxacin and celecoxib (marketed as PrimeC), and debamestrocel-MSC-NTF (marketed as NurOwn), are notable due to requests for regulatory opinions [168–170]. Masitinib is an oral tyrosine kinase inhibitor targeting central and peripheral inflammation [171]. Co-formulated ciprofloxacin and celecoxib is a combination of ciprofloxacin and celecoxib, thought to act synergistically to regulate inappropriate microRNA processing and reduce inflammation [172]. NurOwn is an autologous bone-marrow-derived mesenchymal stem cell designed to induce neurotrophic factors [173]. Originally, the FDA did not support the biologics license application submission for NeurOwn [174]; however, it has provided clearance for initiation of a phase 3b trial (NCT06973629) estimated to start in June 2025.

Use of Supplements in Management of ALS

Acetyl-L-Carnitine (ALCAR)

Carnitine is a human amino acid that contributes to mitochondrial fatty acid oxidation. There are multiple proposed mechanisms for its use in ALS, including mitigating oxidative stress by up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione, and heme oxygenase 1 (HO-1) [175], promoting mitochondrial function by increasing serum levels of ATP [176], and protecting against glutamate excitotoxicity [177]. It has been demonstrated that baseline fatty acid peroxidation markers are elevated in patients with ALS, whereas antioxidant markers are lower. This finding was reversed after 6 months of treatment with acetyl-L-carnitine, though the same pattern was not seen in nitric oxide measurements [178]. A phase 2 randomized, placebo-controlled trial compared acetyl-L-carnitine with placebo over 48 weeks and did not find a significant difference in primary endpoint of time to non-self-sufficiency [179]. Unfortunately, the study had numerous pitfalls, including protocol deviations during randomization and slow recruitment. Post hoc analysis did show a trend toward prolonged time to non-self-sufficiency but was not statistically significant. A more recent retrospective, case-controlled study found a survival benefit in patients with ALS taking 1.5 g/day of acetyl-L-carnitine, but not for patients taking 3 g/day. Functional outcomes were not significantly different from controls [180]. A phase 2/3 multicenter randomized trial of ALCAR versus placebo is expected to begin enrollment in 2025 (NCT06126315).

Symptomatic Treatment

Symptomatic management of ALS is critical and many of the medications utilized for symptomatic management (Fig. 2) are based on expert guidelines, as randomized clinical trials are lacking [181]. Medications consistently recommended in consensus guidelines—along with their mechanisms of action, important safety monitoring parameters, common adverse effects, and available clinical trial data—are listed in Table 1. In-depth reviews for ALS symptomatic management are published [182, 183].

Fig. 2.

Therapeutics for the management of ALS symptoms. The mainstay of ALS clinical management is addressing and controlling symptoms. While non-pharmacologic and secondary sources of symptoms should be evaluated at every visit, pharmacologic options are frequently utilized. A range of medications are available to target the diverse symptoms experienced by patients with ALS. Created in BioRender.

Source: Goutman, S. (2025) https://BioRender.com/w04b047

Table 1.

Selected medications used in the treatment of symptoms in ALS

Symptom	Medication	Mechanism of action	Other uses	Common side effects	Monitoring	Special considerations
Sleep/fatigue	Modafinil1	Thought to block dopamine transporters, increasing the amount of dopamine in the brain		-Headache -Nausea -Gastrointestinal distress	-Regularly monitor heart rate, blood pressure, and weight	-Can exacerbate anxiety and depression -Increased cardiac monitoring in patients with unstable angina -Fatigue is a common side effect of riluzole
	SNRI* (duloxetine, venlafaxine)	Inhibits neuronal serotonin and norepinephrine reuptake	Depression/anxiety Neuropathic pain	-Nausea -Headache -Dose-related drowsiness -Constipation -Abnormal dreams	-Blood pressure -Baseline kidney and liver function	-Can precipitate manic attacks if used as monotherapy in patients with bipolar disorder -Serotonin syndrome can occur if used with multiple other serotonergic medications
	Melatonin*	Melatonin agonist at the suprachiasmatic nucleus to reduced evening arousal and promote sleep onset		-Vivid dreams and nightmares -Dizziness -Irritability -Stomach cramps	-Daytime sleepiness may indicate need for dose adjustment or discontinuation	-Dietary supplement, thus quantities and properties may vary between formulations
Depression/anxiety	Tricyclic antidepressants* (amitriptyline, nortriptyline, imipramine)	Prevents reuptake of serotonin and norepinephrine	Neuropathic pain Pseudobulbar affect Sialorrhea (amitriptyline) Insomnia	-Anticholinergic effects (constipation, dry mouth, urinary retention, tachycardia) -Sedation -Weight gain -Cardiac rhythm abnormalities at high doses and those at risk of cardiac arrhythmia	-Heart rate -Serum sodium in those with poor oral intake	-Side effects can be exacerbated and can increase risk of falls in older adults -Nortriptyline is thought to have a more modest anticholinergic effect than amitriptyline
	Benzodiazepine*	Increases GABA-ergic post-synaptic membrane permeability to chloride, enhancing inhibitory GABA effects	Short-acting or end-of-life anxiety Refractory insomnia Refractory spasticity	-Sedation -Dizziness -Respiratory depression	-Respiratory status -Heart rate, blood pressure -Mental status	-Risk of paradoxical reaction in those with preexisting aggression or alcoholism -Consider implementing benzodiazepines as first-line therapy in patients with advanced disease in whom non-pharmacologic options for therapy are not possible
	Gabapentinoids* (gabapentin, pregabalin)	Structurally related to GABA, but work on pre-synaptic voltage gated calcium channels to inhibit excitatory neurotransmitter release	Pain Cramps Spasticity	-Dizziness -Drowsiness -Ataxia -Fatigue -Peripheral edema	-Renal function -Sedation -Respiratory function	-Initiate at the lowest dose for those with respiratory involvement
Appetite stimulation	Cannabinoids2 (dronabinol [THC])	Acts on CB1 and CB2 receptors in the brain	Pain Insomnia Spasticity	-Dizziness -Drowsiness -Nausea/vomiting -Paranoia/changes in thinking -Euphoria	-Heart rate, blood pressure -Mood	-May exacerbate depression -May cause syncope with dose initiation -Cost may be prohibitive to patients -Nonregulated forms of THC face legal, safety, and dose-reliability issues
	Megestrol acetate*	Synthetic progestin with antiestrogenic properties and thought to antagonize metabolic effect of catabolic cytokines		-Dose and duration-dependent hypothalamic-pituitary axis suppression -Rash -Impotence/decreased libido -Flatulence -Insomnia	-Serum glucose -Signs of adrenal insufficiency with chronic use -Blood pressure	-May increase risk of venous thromboembolism

CB1, cannabinoids 1; CB2, cannabinoids 2; GABA, gamma-aminobutyric acid; THC, tetrahydrocannabinol

^*Expert/consensus recommendations

¹Modafanil was well tolerated in doses up to 300 mg/day and demonstrated improvement in patient-reported measures of fatigue after 8 weeks of use in patients with ALS as compared with placebo [326]

²Moderate evidence supports the use of cannabinoids as an appetite stimulant [327, 328] but it has not been specifically studied in ALS, thus guidelines are variable in recommendations for use in this patient population

Pseudobulbar Affect

Pseudobulbar affect (PBA) represents unintentional or uncontrollable laughing and crying related to impaired corticobulbar function and is a common symptom in ALS. Currently there is one medication, dextromethorphan/quinidine (DMQ) (marketed as Nuedexta), specifically FDA-approved for PBA. Dextromethorphan is a weak non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist resulting in glutamate inhibition and protected from first-pass metabolism by its co-drug, quinidine, which was shown to be well tolerated in patients with ALS [184]. Patient-reported episodes of PBA were reduced in patients taking DMQ compared with quinidine alone with significant improvement in quality-of-life measures over 29 days. The most common side effects were nausea, dizziness, and loose stools. There was a significant difference in QT interval prolongation on electrocardiogram in those receiving DMQ; however, it was not judged to be clinically relevant. Similar findings were reproduced over 12 weeks in a combined study of DMQ for ALS and multiple sclerosis [185]. A small, phase 2 crossover trial of DMQ versus placebo found significantly improved bulbar function as measured by the patient-reported CNS-BFS, as well as the bulbar subdomain of the ALSFRS-R [186]; however, these findings have not been replicated in larger cohorts and DMQ is not currently FDA-approved as a disease-modifying therapy in ALS. Other considerations based on expert guidelines include selective serotonin reuptake inhibitors (SSRI) or tricyclic antidepressants (TCA) [97]. Treatment of PBA should always include an evaluation for underlying mood disorders, such as depression, which may be contributing to emotionality.

Disorders of Saliva (Sialorrhea and Thick Mucus)

Treatment for sialorrhea (excessive saliva) includes anticholinergics, such as amitriptyline, glycopyrrolate, atropine, or scopolamine, with the latter two providing non-tablet administration options [97, 187]. As detailed above, there is patient-reported improvement in overall bulbar function with DMQ [186], and thus, on the basis of EAN guidelines, DMQ is considered for treatment of sialorrhea in those patients with concurrent pseudobulbar affect [97]. A recent Cochrane review identified low-certainty and moderate-certainty evidence for the use of botulinum toxin B injections for patients with “refractory sialorrhea” or “bulbar dysfunction” [187].

The aforementioned medications are helpful for thin, pooled secretions and drooling, but may exacerbate thicker mucus that can arise from poor cough or sinus drainage. Treatment of thick secretions often includes non-pharmacologic therapies, such as insufflation–exsufflation devices or saline nebulizers; however, mucolytics (acetylcysteine, guaifenesin) or beta blockers [188] can be used if these are insufficient.

Carbonated thin liquids reduce penetration and aspiration in patients with neurogenic dysphagia [189], and sour taste has been shown to increase swallowing frequency and prolong cortical response to the initiation of swallow [190]. A variety of foods have been tested to promote intake in neurogenic dysphagia [191], though rigorous trial data are lacking. Anecdotally, grapefruit juice helps relieve symptoms of excessive saliva, whereas papaya and pineapple juice are more helpful for thicker secretions.

Cramps

Up to 95% of patients with ALS experience muscle cramps [192]. A phase 2 trial of mexiletine, a sodium channel blocker, demonstrated dose-dependent reduction in muscle cramps, though 300 mg doses were associated with fewer adverse events. Other medications with effects on sodium channels, such as gabapentinoids and baclofen, are also commonly used, though there is inconsistent evidence on the efficacy of gabapentin for cramps in ALS [181]. The antimalarial medication quinine has long been used for cramp relief, but after increasing reports of side effects, most severely disseminated intravascular coagulopathy [193], the FDA limited and provided guidelines for the amount of quinine readily available in common formulations [194].

Spasticity

Spasticity related to upper motor neuron dysfunction can be treated with classical muscle relaxants, such as baclofen, tizanidine, and cyclobenzaprine. Surveys of patients with ALS report reduction in spasticity with use of cannabis [195]. Focal injections of botulinum toxin are frequently used as well. For intractable spasticity, intrathecal baclofen pumps can be considered [196].

Preclinical Drug Development Disease Models

The early discovery of SOD1 genetic ALS led to the development of SOD1 transgenic mouse models. These mice display motor neuron degeneration and resultant muscle atrophy and weakness similar to human ALS phenotypes. As such, they continue to be the foundation for the study of potential ALS therapeutics [19]. Alternative models for drug development are used less frequently. In silico screens utilize computational biology to predict interactions between drug compounds and target proteins. In vitro drug screens are performed on either cell-free protein aggregates, animal-derived cell lines, or patient-derived motor neurons [7, 197]. Finally, in addition to murine models, in vivo drug screens are performed with transgenic Caenorhabditis elegans, Drosophila melanogaster, and zebrafish (please see reference for review of animal models used in ALS drug discovery [198]).

Investigational Agents in Clinical Trials

A large number of investigational agents are currently in various phases of clinical trials for the treatment of ALS. These investigational agents target a range of mechanisms implicated in ALS. The below is not intended as a comprehensive review of ALS pathophysiology, but instead to highlight mechanisms targeted by drugs currently in phase 1, 2, and 3 studies.

ALS Mechanisms Targeted by Current Investigational Agents

To identify current ALS investigational agents, we accessed the ClinicalTrials.gov database on 1 October 2024. We searched for “amyotrophic lateral sclerosis” but excluded other motor neuron disorders including primary lateral sclerosis, spinal muscular atrophy, and spinal bulbar muscular atrophy. To focus on contemporaneous studies, trials were further filtered for interventional studies with a posted date from 1 January 2020 through 1 October 2024. We restricted the analysis to phase 1, 2, and 3 studies. A total of 210 results were independently reviewed and 150 of these were excluded on the basis of author consensus to scope the review around active trials (Fig. 3). This identified 60 active or recently completed drug trials included in the final review. Additional investigational agents were included from independent sources known to the authors.

Fig. 3.

Screening criteria for ALS clinical trials included in final review. ClinicalTrials.gov search strategy to identify current investigational therapeutics in phase 1, 2, and 3 trials for ALS; 211 trials were evaluated and ultimately 60 trials were included in the final review

Broad categories of drugs are under investigation as new ALS therapeutics. These include novel small molecules, repurposed drugs, biologics (including antibodies and immune cells), gene therapies, and stem cells. Further, these investigational agents are targeting a range of pathological mechanisms including protein homeostasis and neuroinflammation. Other targeted ALS pathways include neurotrophic factors, oxidative stress, neuromuscular junction and axonal integrity, mitochondrial function and energy expenditure, RNA metabolism, and the gut–brain axis (Fig. 4).

Fig. 4.

ALS therapeutic targets of drugs in phase 1, 2, and 3 clinical trials. ALS drugs in clinical trials currently target multiple pathophysiologic mechanisms implicated in ALS pathogenesis. These mechanisms include impaired proteostasis and integrated stress response within motor neurons; neuroinflammation mediated by microglia, astrocytes, and blood–brain barrier infiltration of peripheral immune cells, such as regulatory T cells (T_regs); oxidative stress mediated by reactive oxygen species (ROS); impaired neuromuscular junction and axonal transport; mitochondrial dysfunction; alterations in RNA metabolism; excitotoxicity due to glutamate-mediated calcium influx into motor neurons; and changes to the gut microbiome. Additionally, use of stem cells and isolated neurotrophic factors are used to stimulate motor neuron growth and survival. Gene-specific therapies, such as antisense oligonucleotides, are also employed to target various ALS pathophysiologic mechanisms, particularly in genetic ALS. Created in BioRender.

Source: Goutman, S. (2025) j37l770

Gene-Specific Therapies

Genetic forms of ALS offer hope that treatments can be personalized to the specific patients harboring a monogenic form of disease. While this potential shows promise for some genetic forms, such as SOD1 [131, 132] and FUS [199], similar efficacy has unfortunately not been realized to date with others, including C9orf72 HRE [200] and ATXN2 [201]. In cases of genetic ALS, mutations lead to motor neuron pathology via multiple mechanisms, such as oxidative stress with SOD1 and RNA processing with C9orf72. However, even in sporadic ALS, genetic therapies may have amenable targets. An example is the microtubule binding protein stathmin-2 (STMN2), which exhibits reduced expression due to TDP-43 mislocalization [202] and is currently being targeted by an antisense oligonucleotide in clinical trial.

There are multiple approaches under development to target ALS genes. Strategies include antisense oligonucleotides (ASOs), ribonucleic acid (RNA) interference, and viral vectors [203]. ASOs are currently in clinical trials for C9orf72, SOD1, FUS, and coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) ALS [204]. ASOs are single-stranded RNA or DNA sequences of 13–25 nucleotides. They can deliver a normal copy of a mutated (target) gene or knock out a causative gene by targeting its RNA [203]. ASO mechanisms vary and include messenger RNA degradation, splicing modulation, or inhibition of RNA-binding protein-mediated protein translation. Limitations of ASOs include rapid degradation and inability to cross the blood–brain barrier, requiring treatment with repeated intrathecal injections [205]. As a result, the drug is usually administered via repeated lumbar punctures, but other strategies are contemplated [206].

RNA interference molecules, namely small interfering RNAs (siRNA), are also being studied in ALS clinical trials [204]. siRNA are double-stranded, 19–23 nucleotide RNA sequences, which are assembled within the cell to form RNA-induced silencing complex (RISC). The RISC is then used similarly to an ASO to degrade, silence, or alter splicing of messenger RNA to inactivate gene expression [207]. Exogenously delivered double-stranded RNA interference molecules are also rapidly degraded, necessitating high doses. Strategies can be employed to mitigate this. One such strategy is the use of precursor RNAs including short hairpin RNA and microRNAs, which are processed in the cytoplasm to siRNA and are continuously produced within cells. Another strategy for both siRNAs and ASOs is conjugating them to carrier molecules such as peptides or nanoparticles. Finally, viral vectors can be employed to more stably deliver and express these transgenes [203].

Viral vectors, most commonly adeno-associated viral (AAV) vectors, are non-enveloped, single-stranded viruses that have DNA modifications to deliver the desired therapeutic gene. The AAV can either deliver a normal copy of a diseased gene or deliver ASOs or RNA interference molecules, as mentioned previously [205]. One benefit of viral vectors is their ability to target specific cell types allowing for more targeted delivery, known as viral tropism. Specifically, AAV9 has tropism for neurons and is frequently used in ALS drug development [205]. Additional benefits of viral vectors include their persistence in cells as extra-chromosomal plasmids, limiting risk of insertional DNA mutations in the host [203, 208], and their ability to cross the blood–brain barrier, allowing for intravenous delivery [205]. AAV are not without limitations, which include small packaging capacity (5 kilobases), limiting delivery of large genes, and development of antibodies (while AAV is poorly immunogenic, both the capsid and transgene can elicit an immune response). Mechanisms to overcome these limitations are discussed elsewhere [209, 210].

Proteostasis

Protein homeostasis, or proteostasis, is essential for normal cellular function and involves maintaining proteins in appropriate conformation, concentration, and location within the cell [211]. A delicate balance of protein synthesis, trafficking, and degradation requires the interplay of complex cellular factors to achieve proteostasis [212]. A functioning proteome allows a cell to adapt to environmental conditions and cellular stress; without it, a cell becomes dysfunctional and eventually undergoes apoptosis if the stressor is not ameliorated [211].

Prolonged cellular stress conditions within motor neurons dysregulate normal protein homeostasis [212]. Cellular stress leads to both impaired protein production and reduced protein degradation. Affected pathways involved in protein production include messenger RNA dysregulation, protein misfolding, and protein mislocalization. Changes to the unfolded protein response (UPR) and autophagy pathways reduce clearance of misfolded proteins [211]. Overall, downstream effects of an abnormal proteome lead to a vicious cycle of disruption in proteostasis, including endoplasmic reticulum stress, mitochondrial dysfunction, and synapse destruction within motor neurons [213].

The hallmark of defective proteostasis in ALS is cytoplasmic ubiquitinated protein inclusions (such as TDP-43 or FUS) [211, 214]. Some of these proteins, including TDP-43, have prion-like domains that are particularly prone to aggregation [12]. It remains unclear whether these inclusions are the inciting factor or downstream consequence of motor neuron pathology [211]. Motor neurons are perhaps more susceptible to dysregulated proteostasis given their large size [215]. However, protein aggregates are also found in other neurodegenerative disorders, supporting a causal role in neuronal degeneration [12].

Protein mislocalization and cytoplasmic aggregation are prominent features in both genetic and nongenetic ALS, leading to loss of essential cellular functions. Many genes implicated in ALS involve pathways of protein production [211]. As a primary example, TDP-43, a product of the TARDBP gene, is a DNA- and RNA-binding protein that plays an essential role in RNA metabolism including splicing, repression, and messenger RNA nucleocytoplasmic shuttling [12, 216]. Loss of nucleolar function leads to altered levels of more than 600 messenger RNAs [217]. Similarly, FUS is an RNA-binding protein whose loss of function may reduce multiple protein activities [214].

Perhaps more importantly, protein aggregates lead to harmful downstream effects due to toxic gain-of-function. Misfolded or aggregated proteins in the endoplasmic reticulum (ER) lumen trigger the UPR. The UPR is a signaling pathway activated by ER stress that tries to promote protein refolding or clearance, and if unsuccessful, activates apoptotic pathways [218]. Separately, misfolded or aggregated proteins in the cytosol participate in stress granule formation. Stress granules are dynamic, membrane-less clusters of messenger RNAs and RNA-binding proteins (including TDP-43) that assemble in response to cell stress. They redirect translation toward stress-response proteins to restore cellular homeostasis [8]. When stress is resolved, these granules are normally disassembled. However, when mutant FUS, TDP-43, and ATXN2 aggregate within stress granules, they form insoluble aggregates, impacting appropriate transcription [12, 219].

Finally, autophagy, the process of degrading misfolded proteins and damaged organelles, is required for cell survival but becomes aberrant under stress conditions. Normally, autophagosomes deliver cellular constituents (such as TDP-43 aggregates and stress granules) to lysosomes, where they are degraded and recycled [8, 220]. This lysosomal stress response is regulated by mammalian target of rapamycin (mTOR) and transcription factor EB [221]. However, many ALS-implicated genes, including C9orf72, are also involved [8, 220]. For example, C9orf72 is part of a protein complex that controls autophagosome formation and lysosomal fusion, and when impaired, leads to accumulation of TDP-43 aggregates [222]. Similar to autophagy, which is mediated within the cell, neurons can also exocytose protein aggregates, which can be influenced by PIKfyve kinase inhibition [223].

A total of 14 current investigational agents target proteostasis (Table 2) by attempting to reduce mutated proteins prone to aggregation or enhancing cellular mechanisms to clear aggregates [212]. Various mechanisms include targeting protein aggregates (e.g., with ASOs, antibodies, or other methods), enhancing autophagy or exocytosis, or reducing apoptosis in response to ER stress.

Table 2.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting proteostasis

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
Trehalose NCT05136885 Completed	2/3	Small molecule	Activates transcription factor EB and inhibits mTOR	Proteostasis: promotes lyososomal biogenesis and induces autophagy.	Oxidative stress: free radical scavenger. Neuroinflammation: inhibits proinflammatory cytokines and NF-kB.	All	24 weeks	Randomized, blinded	161	Failed to meet primary or secondary endpoints. Some signal of slowed ALSFRS-R decline in the TUDCA-sodium phenylbutyrate free group. Ongoing post-hoc analyses.	[329] [330]
ION363 (ulefnersen) NCT04768972 Recruiting	3	Gene therapy	ASO versus FUS transcript	Proteostasis: reduces FUS aggregates.		FUS	60 weeks	Randomized, blinded	95 (est)		[214]
Lithium carbonate NCT06008249 Recruiting	3	Repurpose (bipolar disorder)	Interacts with multiple neurotransmitters and attenuates calcium influx	Proteostasis: increases autophagic vacuoles in mice.	Mitochondrial dysfunction: promotes biogenesis of mitochondria.	All, with specific UNC13A genotype	24 months	Randomized, blinded	171 (est)	Prior lithium study in ALS terminated for futility.	[331] [332]
3K3A-APC NCT05039268 Completed	2	Small molecule	Recombinant variant of activated protein C with reduced anticoagulant activity	Proteostasis: in C9orf72 mouse models, lowers C9orf72 dipeptide repeat protein levels and restores nuclear TDP-43 localization. Rescues the survival of sporadic ALS induced motor neurons.	Excitotoxicity: lowers glutamate receptor levels.	All	7 days	Open label	16		[333]
LAM-002A NCT05163886 Active, not recruiting	2	Small molecule	Inhibits PIKfyve	Proteostasis: inhibits transcription factor EB pathway to increase lysosome production and induce autophagy.		C9orf72	12 weeks	Randomized, blinded	14	Demonstrated CNS penetrance and target engagement. Resulted in the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.	[334] [335]
AP-101 NCT05039099 Active, not recruiting	2	Biologic	Monoclonal antibody (IgG1) versus SOD1 protein	Proteoastasis: targets misfolded SOD1		Sporadic or SOD1	48 weeks	Randomized, blinded	63		[336]
Tideglusib NCT05105958 Not yet recruiting	2	Small molecule	Inhibits GSK-3	Proteostasis: Reduces TDP-43 phosphorylation to lower cytoplasmic aggregation and recover nuclear localization.		All (excluding SOD1 or FUS mutations)	14 weeks	Randomized, blinded	98 (est)		[337]
Bosutinib NCT04744532 Unknown	1/2	Repurpose (chronic myeloid leukemia)	Inhibits Src/c-Abl tyrosine kinase	Proteostasis: promotes autophagy to reduced protein aggregates and inhibit motor neuron death.		Sporadic or SOD1	12 weeks phase 1, 24 weeks phase 2	Open label	25 (est, phase 2 arm)	In phase 1 trial, dose-limiting toxicity observed at 400mg dose. ALSFRS-R remained stable or declined in 7 of 9 patients on 100-300mg dosing.	[338]
AMT-162 NCT06100276 Recruiting	1/2	Gene therapy	AAV vector with microRNA versus SOD1 transcript	Proteostasis: microRNA silences SOD1 in lower motor neurons.		SOD1	5 years	Open label (phase 1)	6–8 (est, phase 2 arm)		[339]
Monepantel NCT04894240 Completed	1	Repurpose (livestock wormicide)	Inhibits mTOR	Proteostasis: inhibits mTOR, reducing protein accumulation. mTOR inhibition slows progression in preclinical ALS models.		All (excluding SOD1 or VCP mutations)	6 months	Open label	12	43-58% slowing in decline of ALSFRS-R (secondary endpoint) in high dose cohort in 2-year open label extension. Enrolling in HEALEY trial.	[340]
RAG-17 NCT06556394 Recruiting	1	Gene therapy	Small interfering RNA bound to accessory oligonucleotide versus SOD1 transcript	Proteostasis: reduces mutant SOD1 protein by binding mRNA.		SOD1	Unknown	Randomized, blinded	32 (est)	Well tolerated at all doses.	[341]
VRG50635 NCT06215755 Recruiting	1	Small molecule	Inhibits PIKfyve	Proteostasis: stimulates exocytosis of protein aggregates from motor neurons.		All	28 days	Randomized, blinded	50 (est)		[223]
ALN-SOD NCT06351592 Recruiting	1	Gene therapy	Small interfering RNA versus SOD1 transcript	Proteostasis: reduces mutant SOD1 protein by binding mRNA.		SOD1	4 weeks	Randomized, blinded	42 (est)
Prosetin NCT05279755 Unknown	1	Small molecule	Inhibits MAP4K	Proteostasis: reduces apoptotic response to misfolded proteins, which trigger ER stress and the unfolded protein response.		All	7 days	Randomized, blinded	32 (est)		[342]

Abbreviations: AAV, adeno-associated virus; ALSFRS-R, ALS functional rating scale revised; ASO, antisense oligonucleotides; CNS, central nervous system; CSF, cerebral spinal fluid; ER, endoplasmic reticulum; est, estimated; FUS, fused in the sarcoma; GP, glycine-proline; GSK-3, glycogen synthase kinase-3; MAP4K, MAP kinase kinase kinase kinase; mTOR; mechanistic target of rapamycin; NF-kB, nuclear factor kappa-beta; TDP-43, transactive response DNA binding protein; TUDCA, tauroursodeoxycholic acid

Integrated Stress Response

Integrated stress response (ISR) is an integral part of protein homeostasis that is directly targeted by several drugs in clinical trials. Under conditions of cellular stress, including ER stress (via the UPR), cytosolic protein aggregation, starvation, and infection, the ISR is activated to restore cellular homeostasis [224]. It does this by reducing global protein synthesis and enhancing gene expression targeted toward cellular recovery (chaperones, protein folding, etc.) [225, 226].

Specifically, under normal conditions, the eukaryotic initiation factor-2 (eIF2) complex (composed of alpha, beta, and gamma subunits) assists with RNA translation via ribosomal recognition of the AUG start codon [227]. However, during the integrated stress response, this process is halted and instead open reading frame messenger RNAs (mRNAs) are preferentially translated [224]. Of these mRNAs, perhaps most important is ATF4, a transcription factor that binds DNA targets to regulate gene expression, essential for restoring mitochondrial homeostasis [228]. When cellular homeostasis has been restored, the ISR is turned off via protein phosphatase 1 complex (PP1R15A, also known as growth arrest and DNA damage-inducible protein GADD34, and PPP1R15B). This negative feedback loop is important to restore normal protein synthesis [225, 229].

In ALS, the ISR can lead to increased translation of mutant genes. For example, C9orf72 GGGGCC repeat expansions can be translated into toxic dipeptide repeats via pathogenic non-AUG repeat-associated (RAN) translation. RAN translation is enhanced by the preferential translation of open reading frame mRNAs during the ISR [230]. Additionally, as cellular homeostasis cannot be restored in ALS, a prolonged integrated stress response eventually leads to cell death. This is in part mediated by PPR15A/B dephosphorylation of eIF2a, allowing for synthesis of death-inducing proteins [225, 231]. Elevated markers of the integrated stress response and unfolded protein response have been identified in postmortem samples from patients with nongenetic ALS [224, 232].

Investigational agents involving the ISR in current ALS clinical trials target the eIF2 complex, PP1R15A, and pathological RAN translation (Table 3).

Table 3.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting the integrated stress response

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
ABBV-CLS-7262 (fosigotifator) NCT05740813 Enrollment complete	2/3	Small molecule	Activates eIFB2	ISR: inhibits the ISR to restore protein synthesis and dissolve TDP-43 containing stress granules		All	24 weeks	Randomized, blinded	310	No significant difference in combined analysis of ALSFRS-R and mortality (primary endpoint) or in respiratory function (secondary endpoint). Slower deterioration of handheld dynamometry (secondary endpoint) in high dose group.	[343]
DNL 343 NCT05842941 Enrollment complete	2/3	Small molecule	Activates eIFB2	ISR: inhibits the ISR to restore protein synthesis and dissolve TDP-43 containing stress granules		All	24 weeks	Randomized, blinded	240 (est)	Failed to meet primary endpoint in decline of ALSFRS-R as well as secondary survival endpoints. Detailed results pending publication.	[344]
Metformin NCT04220021 Active, not recruiting	2	Repurpose (diabetes)	Glucose metabolism	ISR: reduces repeat-associated non-AUG translation of C9orf72 dipeptide repeats, which occurs more frequently during the ISR.		C9orf72	24 weeks	Open label	18		[345]
IFB-088 (icerguastat) NCT05508074 Active, not recruiting	2	Small molecule	Inhibits PPP1R15A and NMDA subtype 2B receptors	ISR: prolongs ISR to adjust protein production rate and encourage protein folding.	Excitotoxicity: inhibits NMDA receptors to reduce glutamate excitotoxicity.	All (bulbar-onset only)	26 weeks	Randomized, blinded	51 (est)		[231, 346]

Abbreviations: ALSFRS-R, ALS functional rating scale revised; eIFB2, eukaryotic initiation factor 2; est, estimated; ISR, integrated stress response; PPP1R15A, protein phosphatase 1, regulatory subunit 15A; TDP-43, transactive response DNA binding protein; NMDA, N-methyl-D-aspartate

Neuroinflammation

Central and systemic inflammation play a role in ALS pathogenesis, with some immune cell targets [8, 233] leading to investigational considerations (Table 4). Astrocytes in the central nervous system (CNS) help to maintain the integrity of the blood–brain barrier and mediate release of antiinflammatory molecules, such as interleukins (IL) and tumor necrosis factor (TNF) [234]. Overproduction of these signals drives necrotic cell death, or necroptosis [235]. Higher levels of these inflammatory cytokines have been found in CSF and serum of patients with ALS and correlate negatively with survival, specifically TNF-alpha, interleukin-10 and interleukin-1 beta [236]. These molecules further activate the nuclear factor-kappa B (NF-kB) pathway, which perpetuates proinflammatory destruction in the CNS, muscle, and neuromuscular junction [237]. Activation of NF-kB has also been linked to both SOD1 gene function [238] and C9orf72 [239]. Of further support, cultured astrocytes from postmortem tissue of patients with ALS are toxic to motor neurons [240].

Table 4.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting neuroinflammation

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
Zilucoplan NCT04436497 Completed	2/3	Small molecule	Inhibits C5 complement	Neuroinflammation: inhibits terminal complement cascade (active in SOD1 mouse models) to reduce neuroinflammation.	NMJ: reduces NMJ destruction by inhibiting complement activation at motor neuron terminals and Schwann cells.	All	24 weeks	Randomized, blinded	162	Failed to meet primary endpoint in ALSFRS-R at 24 weeks, therefore terminated for futility.	[347–349]
Dolutegravir, Abacavir and Lamivudine (Triumeq) NCT05193994 Active, not recruiting	3	Repurposed (HIV)	Combined nucleoside reverse transcriptase inhibitor and integrase strand transfer inhibitor	Neuroinflammation: reduces HERV-K, which has increased transcripts in ALS that may drive inflammation.		All	24 months	Randomized, blinded	390 (est)	Previous antiretroviral clinical trials were ineffective (zidovudine monotherapy in 12 patients, indinavir monotherapy in 46 patients).	[350]
RAPA-501 NCT04220190 Recruiting	2/3	Biologic	T regulatory/Th2 cells	Neuroinflammation: ex vivo manufactured cells with epigenetic reprogramming to enrich an anti-inflammatory phenotype. Regulatory T cell expansion prolonged survival in mouse models.		All	24 weeks	Open label	41 (est)		[351]
NP001 NCT02794857 Completed	2b	Small molecule	Sodium chlorite	Neuroinflammation: Downregulates NF-kB expression in macrophages to inhibit production of pro-inflammatory cytokines.		All	24 weeks	Randomized, blinded	138	Seeking FDA approval as of 10/2024. No difference in ALSFRS-R score (primary endpoint), SVC (secondary endpoint), or percentage of non-progressors overall. Subpopulation of patients 40–65 years old had 36% slower decline in ALSFRS-R and 51% slower decline in SVC.	[352, 353]
AT-1501 (tegoprubart) NCT04322149 Completed	2	Biologic	Antibody versus CD40 ligand	Neuroinflammation: blocks CD40 ligand on T cells to suppress the adaptive immune system that is overactive in ALS.		All	11 weeks	Open label	54	Dose-dependent reduction in proinflammatory biomarkers.	[354, 355]
ANX005 NCT04569435 Completed	2	Biologic	Antibody versus C1q complement	Neuroinflammation: inhibits classical complement cascade (elevated in spinal cord and motor cortex in ALS) to reduce inflammation and subsequent neurodegeneration.		All	22 weeks	Open label	17	Prior study showed Cq1b inhibition had no effect.	[356, 357]
ZYIL1 (usnoflast) NCT05981040 Completed	2	Small molecule	Inhibits NLRP3 inflammasome	Neuroinflammation: reduces inflammation triggered by pathogen or damage-associated molecular patterns. Validated in inflammatory preclinical animal models.		All	12 weeks	Randomized, blinded	24	FDA approved US phase IIb study of 210 patients.	[358, 359]
TPN-101 NCT04993755 Active, not recruiting	2	Repurposed (HIV)	Nucleoside reverse transcriptase inhibitor (derivative of stavudine)	Neuroinflammation: Loss of nuclear TDP-43 causes reactivation of virus-derived retrotransposable DNA including LINE-1. Inhibiting LINE-1 reverse transcriptase reduces the antiviral immune response that otherwise drives neurodegeneration.		C9orf72	24 weeks	Randomized, blinded	42	At 48 weeks, 40% less decline in ALSFRS-R, 50% slower decline in vital capacity, and reduced neurofilament level (secondary endpoints).	[360, 361]
WP-0512 (fasudil) NCT05218668 Active, not recruiting	2	Small molecule	Inhibits rho kinase	Neuroinflammation: inhibits proinflammatory and apoptotic signaling pathways. Reduces microglial activation.	Axonal transport: regulates cytoskeleton including actin and microtubules, involved in vesicle transport among many other functions.	All	24 weeks	Open label	40 (est)	Approved in China and Japan for treating subarachnoid hemorrhage. Prior phase 2 trial on intravenous fasudil showed reduced decline in MUNIX scores versus placebo.	[362–366]
Dazucorilant NCT05407324 Active, not recruiting	2	Small molecule	Modulates glucocorticoid receptors	Neuroinflammation: counteracts high cortisol levels found in ALS and reduces microglial proinflammatory response.		All	24 weeks	Randomized, blinded	249	No slowing in ALSFRS-R decline (primary endpoint) but did improve survival. Gastrointestinal side effects more common. Ongoing open-label extension.	[367, 369]
TCD601 (siplizumab) NCT06453668 Active, not recruiting	1	Biologic	Monoclonal antibody vs CD2	Neuroinflammation: targets CD2 receptor on T cells to reduce mature T cells and increase regulatory T cells.		All	52 weeks	Open label	48 (est)		[368]
Abatacept and Proleukin (IL-2) NCT06307301 Active, not recruiting	1	Repurposed, biologic (rheumatoid arthritis)	CTLA-4 analog and interleukin-2	Neuroinflammation: prevents interaction of antigen presenting cells (CD80/CD86 ligands) with T cells (CD28/CTLA4 receptors) to inhibit T cell activation.		All	59 weeks	Open label	5		[369]
FB418 NCT05995782 Recruiting	1	Small molecule	Inhibits LRRK2 and c-Abl	Neuroinflammation: inhibits neuroinflammatory response in microglia and modulates lysosome pathway and autophagy in immune cells.		All	Unknown	Randomized, blinded	64 (est)		[370]
CK0803 NCT05695521 Recruiting	1	Biologic	Umbilical cord blood derived regulatory T cells	Neuroinflammation: regulatory T cells inhibit proinflammatory immune cells and higher cell counts prolong survival in ALS.		All	24 weeks	Randomized, blinded	66 (est)		[371]

Abbreviations: ALSFRS-R, ALS functional rating scale revised; CLTA-4, cytotoxic T-lymphocyte-associated protein 4; est, estimated; HERV-K, human endogenous retrovirus-K; HIV, human immunodeficiency virus; LRRK2, leucine-rich repeat kinase 2; LINE-1, long interspersed nuclear elements 1; MUNIX, motor unit number index; NF-kB, nuclear factor kappa-beta; NLRP3, NOD-like receptor protein 3; NMJ, neuromuscular junction; TDP-43, transactive response DNA binding protein; TH2, T helper cell type 2; SVC, slow vital capacity

Microglia are felt to have a more direct role in pathologic inflammatory changes in ALS. In the M1 state, microglia are thought to be more proinflammatory and toxic, while in the M2 state microglia are anti-inflammatory and more restorative [241]. In SOD1 mouse models, end-stage disease is associated with higher markers of M1 activation (such as NOX2 and other reactive oxidative species) compared with higher levels of M2 activation (such as CD163 and brain-derived neurotrophic factor (BDNF)) at disease onset [242]. M1 macrophages in patients with ALS are found to release more toxic cytokines, such as IL-6 and TNFα, as compared with healthy controls [243]. Positron emission tomography (PET) studies have also demonstrated higher levels of microglial activation in the motor cortices of patients with ALS [244].

There is increasing evidence of peripheral and central immune system cross connectivity contributing to acceleration of a proinflammatory state in ALS [245]. Mechanisms by which the barrier is disrupted include direct changes to the integrity of the blood–brain barrier via impaired tight junctions and adhesion proteins, leading to increased permeability [246, 247] and changes in barrier cell concentration and increased localized secretion of inflammatory proteins [248, 249]. Chronic activation of the peripheral immune system exacerbates disruption of the central and peripheral immune barrier, and more specifically to ALS, levels of specific pro- or anti-inflammatory cytokines and chemokines are inappropriately altered [250]. As a result of the barrier disruption, additional pathologic changes in the peripheral immune system are capable of affecting the central immune response [251]. For example, dendritic cells, which prime T cells as part of the innate immune response, are found in abundance in the ventral horn and corticospinal tracts, specifically, in postmortem spinal cord tissue [252]. Regulatory T cells (T_regs), which act to prevent an excessive immune response or autoimmunity, are found in greater numbers during a slow-progression phase of SOD1 mouse models but increase during rapid disease progression, suggesting that control of T_regs may influence disease progression [253]. Ratios of “classical” monocytes (CD14-/CD16-), which are more responsible for phagocytosis and initiation/priming of the immune system [254], are higher in patients with ALS and pre-symptomatic SOD1 carriers as compared with healthy controls, but function at a decreased efficacy and invade the CNS, though the difference between the roles of monocytes in the CNS versus systemic circulation are still unknown [255]. Evaluation of the complement cascade, the initiation of the innate immune response, is a natural investigation. The findings in mouse models of SOD1 ALS show an increase of complement activators (C1qB, C4, C3, C5, and CD88) and decrease in complement regulators (CD55, CD59a) during disease progression in spinal-cord-tissue supported investigations of complement inhibitors in ALS [256]. Higher levels of circulating serum cortisol are found in patients with nongenetic ALS, though correlation with symptom onset and progression are variable [257, 258]. Glucocorticoid receptor (GR) modulation has been shown to reduce inflammation and glial reactivity in mouse models [259]. Other peripheral immune cells, such as neutrophils [260] and natural killer cells [52, 261–263], show associations with ALS and may influence future therapeutic targets [264].

The rapidity of clinical decline and death in ALS has led many to investigate whether there could be an infectious etiology of the disease, primarily leading to research into transposable elements of the genome, meaning parts of genes that can “jump” from one chromosome to another and are present and essential in the healthy human genome [265]. These transposable elements include transposons and retrotransposons, such as endogenous retroviruses, which can essentially “cut and paste” into any portion of the human genome. In humans, the human endogenous retrovirus-K (HERV-K) is the most well studied and has been found to be increased in multiple degenerative disease states [266]. In ALS, expression of HERV-K and its envelope protein are found in higher concentrations in cortical pyramidal cells and spinal anterior horn cells and are specifically not seen in the other spinal cord tracts or nonmotor cortical areas [267].

Neurotrophic Factors and Stem Cells

Neurotrophic factors are proteins that promote a neuroprotective environment to slow neurodegeneration. They are endogenously used in response to injury or disease to enhance neuron survival, axonal growth, synapse formation, and plasticity [268]. The goal of investigational neurotrophic agents is to promote motor neuron growth, repair, and survival regardless of which cellular mechanisms are damaged. Examples of endogenous neurotrophic factors include BDNF, insulin-like growth factor-1, erythropoietin, granulocyte-colony stimulating factor, and hepatocyte growth factor, among others [269]. Many of these factors have been studied in ALS mouse models and found to reduce motor neuron degeneration and improve survival [268]. There are several investigational agents in clinical trials utilizing this approach (Table 5).

Table 5.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials involving neurotrophic factors

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
Nerve Growth Factor NCT06391645 Not yet recruiting	2/3	Small molecule	Nerve growth factor encapsulated with 2-MPC nanocapsules	Neurotrophic: enhances neuron regeneration, showing prolonged survival time in SOD1 mouse model.		All	12 weeks	Open label	60 (est)
VM202 (engensis) NCT04632225 Completed	2	Biologic	Small circular DNA delivering hepatocyte growth factor gene	Neurotrophic: hepatocyte growth factor delivered to muscle induces blood vessel and nerve growth, prevents muscle loss.		All	4 months	Randomized, blinded	18	Safe and well tolerated in phase 2a study. Awaiting further trial analysis to determine next steps in drug development.	[372]
MRG-001 (plerixafor and low-dose tacrolimus) NCT06315608 Not yet recruiting	2	Repurposed (lympoma, multiple myeloma; organ transplant rejection)	CXC chemokine receptor type 4 antagonist and calcineurin inhibitor	Neurotrophic: mobilizes bone marrow stem cells to sites of injury, stimulating tissue repair.	Neuroinflammation: mobilizes immunoregulatory cells to reduce inflammation at sites of injury.	All	3 months	Open label	10 (est)		[373]
Aleeto NCT06181526 Not yet recruiting	1	Small molecule	Protein polymers secreted by stem cells under stress	Neurotrophic: enhances neuron repair.		All	66 days	Randomized, blinded	24 (est)

Abbreviations: est, estimated; MPC, methacryloyloxyethyl phosphorylcholine

There is incredible interest surrounding the role of stem cell therapy for the treatment of ALS [270]. In early trials, stem cells were transplanted into patients with ALS with the goal of generating new motor neurons. However, they were unable to integrate due to a diseased microenvironment and long distance required for axon growth. Thus, while upstream stem cells do have the ability to differentiate into diverse cell types, in ALS, stem cells therapies are now intended to secrete neurotrophic factors, differentiate into astrocytes and microglia, or synapse with diseased motor neurons (Table 6) [271]. Stem cells can be isolated from embryonic stem cells, mesenchymal stem cells (derived from umbilical cord blood), and neural progenitor cells [272]. Challenges to use of stem cells include optimizing the correct source, dose, and delivery method [270].

Table 6.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials involving stem cells

Drug name NCT Study status	Phase	Drug category	Stem cell type	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
Human neural stem cells NCT06344260 Recruiting	2	Stem cell	Human neural stem cells	All	Single dose	Randomized, blinded	30 (est)
Mononuclear cells NCT04849065 Unknown	2	Stem cell	Autologous bone marrow mononuclear cells (including stem cells)	All	Single dose	Randomized, blinded	100 (est)		[374]
hUC-MSC-sEV-001 NCT06598202 Recruiting	1/2	Stem cell	Exosomes derived from human umbilical cord blood mesenchymal stem cells	All	14 days (phase 1), unknown (phase 2)	Randomized, blinded	38 (est)
Mesenchymal stem cells NCT04651855 Unknown	1/2	Stem cell	Mesenchymal stem cells isolated from Wharton’s jelly	All	60 days	Open label	20 (est)
CNS10-NPC-GDNF NCT05306457 Recruiting	1	Stem cell	Human neural progenitor cells differentiated into astrocytes with glial cell line-derived neurotrophic factor	All	3 weeks to 3 months	Open label	16 (est)		[375]

Abbreviations: est, estimated

Oxidative Stress

Oxidative stress plays a key role in neuronal injury and aging by altering the structure of cellular components (lipids, proteins, DNA, and RNA), leading to mitochondrial damage, and ultimately, cell death [8, 273]. It is mediated by reactive oxygen species (ROS) formed by the partial reduction of oxygen, including superoxide, hydrogen peroxide, and hydroxyl radical [273]. Mitochondria are a prominent site of superoxide production [274]. This occurs due to oxygen reduction necessary for ATP generation [275]. Oxidative stress is also produced outside of the mitochondria. In microglia, myeloperoxidase, a heme-containing enzyme involved in microbial response, utilizes hydrogen peroxide to create hypochlorous acid, an extremely potent reactive halogen species. Hypochlorous acid produced in excess causes cytotoxic cellular damage [276, 277].

Under normal conditions, ROS generation is balanced with antioxidant mechanisms, known as “free radical scavengers.” Antioxidant enzymes include both superoxide dismutase (as implicated in SOD1 ALS), which detoxicates superoxide into the less reactive hydrogen peroxide, and peroxisomal enzymes, which then reduce hydrogen peroxide to water [278].

In a healthy cell, ROS are necessary and have roles in cell signaling [277]. However, in ALS, oxidative stress overwhelms typical antioxidant responses. Increased levels of oxidative stress have been identified in patients with ALS [273]. For example, due to high energy demand and impaired glucose metabolism in ALS, neurons and astrocytes switch to fatty acid beta-oxidation as a key energy source. This incurs risk of creating ROS [279]. Increased ROS leads to several downstream effects including TDP-43 mislocalization and aggregation, mitochondrial dysfunction, and excitotoxicity from astrocyte-mediated glutamate release [8]. Additionally, oxidative stress leads to peroxidation of cellular membrane phospholipids (polyunsaturated fatty acids), leading to motor neuron damage via multiple mechanisms [280].

Current investigational drugs target pro-oxidant enzymes, lipid peroxidation, and antioxidant mechanisms (Table 7).

Table 7.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting oxidative stress

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
Verdiperstat NCT04436510 Completed	2/3	Small molecule	Irreversibly inhibits myeloperoxidase enzyme	Oxidative stress: inhibits pro-oxidant enzyme in microglia.	All	24 weeks	Randomized, blinded	167	Failed to meet primary endpoint (ALSFRS-R) or secondary endpoints.	[276, 376]
PTC857 (utreloxastat) NCT05349721 Active, not recruiting	2	Small molecule	Inhibits 15-lipooxygenase	Oxidative stress: inhibits an enzyme activated by reactive oxygen species that otherwise increases lipid peroxidation and cell death.	All	24 weeks	Randomized, blinded	307		[377]
RT001 NCT04762589 Unknown	2	Small molecule	11, 11 di-deuterated linoleic ethyl ester	Oxidative stress: makes membrane PUFAs resistant to lipid peroxidation.	All	24 weeks	Randomized, blinded	40 (est)	Nonsignificant signals of clinical benefit in ALSFRS-R (primary endpoint).	[280, 378]
Tetramethylpyrazine nitrone NCT04667013 Not yet recruiting	1	Small molecule	Free radical scavenger	Oxidative stress: improves mitochondrial antioxidant activity.	Healthy volunteers	7 days	Randomized, blinded	16 (est)	Previous phase 2 trial of 155 patients showed slower decline in grip strength but not in ALSFRS-R.	[379, 380]

Abbreviations: ALSFRS-R, ALS functional rating scale revised; est, estimated; PUFAs, polyunsaturated fatty acids

Neuromuscular Junction and Axonal Transport

The neuromuscular junction (NMJ) is the crucial connection between the motor neuron axon terminal and the muscle. Terminal Schwann cells (TSCs) are also a critical part of the NMJ, aiding in their development and maturation [281]. NMJ dysfunction and instability is a frequently encountered electrophysiologic finding in the evaluation of ALS [282]. In SOD1 mouse models, endplate denervation was seen prior to clinical weakness and motor neuron loss, suggesting that NMJ compromise occurs early in the pathophysiology of ALS and is often the inciting event prior to lower motor neuron injury (“dying back” hypothesis) [283].

Impaired presynaptic vesicle pool storage and ineffective or malformed presynaptic terminal formation have been demonstrated in multiple animal models of hereditary ALS [284, 285]. Axonal transport is essential to move cellular proteins to and from the NMJ, which requires an eloquent combination of neurofilaments, actin, microtubules, and motor proteins. High levels of vesicles are found in the NMJ of patients with ALS, suggesting dysfunctional axonal transport [286]. A key protein in axonal transport is Stathmin 2 (STMN2), which aids in axonal regrowth after injury and is found to be altered specifically by the toxic accumulations of TDP-43 and is found in lower quantities in spinal cords of mouse models of ALS [202, 287].

Postsynaptic NMJ changes, even preclinically, have also been identified in ALS [288, 289]. Dysregulation of TSCs in ALS is thought to lead to impaired and erroneous NMJ recovery and plasticity [290]. Early in disease, local signaling via miR-206-related pathways may allow for compensatory reinnervation until a certain threshold of functional muscle loss [291]. Ultimately, it seems as though injury to the NMJ is a culmination of mutant protein aggregation at the presynaptic nerve terminal, presynaptic cholinergic dysfunction, impaired axonal transport, reduced synaptic acetylcholinesterase, and diseased muscle itself, among others [292]. Each mechanism of dysfunction at the pre-synaptic, post-synaptic, and peri-synaptic level offers itself as a target for intervention to improve stabilization, junctional signaling, and synapse integrity [213], potentially even before symptomatic weakness in those with genetic susceptibilities. Current therapies under investigation (Table 8) are targeting muscle-specific kinase (MuSK), which promotes post-synaptic clustering, synaptogenesis via F-actin branching and dendritic proliferation [293], and reduction of aberrant sphingolipid regulation to promote NMJ stability [294].

Table 8.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting the neuromuscular junction and axonal transport

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	References
ARGX-119 NCT06441682 Recruiting	2	Biologic	Monoclonal antibody	NMJ: activates MuSK to promote maturation and stabilization of neuromuscular junctions.		All	100 weeks	Randomized, blinded	60 (est)	[381]
Darifenacin NCT06249867 Recruiting	2	Repurpose (overactive bladder)	M3 muscarinic receptor antagonist	NMJ: blocks muscarinic receptors to trigger perisynaptic schwann cells (glial cells) to repair the NMJ.		All	24 weeks	Randomized, blinded	30 (est)
Ambroxol NCT05959850 Recruiting	2	Repurpose (cough medication outside US)	Inhibits glucocerebrosidase 2 among others	NMJ: prevents breakdown of glucosylceramide (sphingolipid) to preserve NMJs in SOD1 mouse models.	Neuroinflammation: inhibits microglial-mediated inflammation. Proteostasis: clears misfolded proteins from ER during ER stress and enhances lysosomal function.	All	24 weeks	Randomized, blinded	50 (est)	[294]
SPG302 NCT05882695 Active, not recruiting	1	Small molecule	Pegylated benzothiazole derivative	NMJ: increases neural synapses that use glutamate, including motor neurons. Regulates actin cytoskeleton and increases dendritic spine density.		All	28 days for 3–12 cycles	Randomized, blinded	112 (est)	[293]
QRL-201 NCT05633459 Recruiting	1	Gene therapy	ASO vs stathmin-2 transcript	Axonal transport: modulates splicing of stathmin-2, a microtubule binding protein in motor neurons critical for axon stability. Stathmin-2 expression is controlled by nuclear TDP-43 and is therefore reduced in ALS.		Sporadic or C9orf72	Unknown	Randomized, blinded	64 (est)	[202]

Abbreviations: ASO, antisense oligonucleotide; ER, endoplasmic reticulum; est, estimated; MuSK, muscle-specific kinase; NMJ, neuromuscular junction; TDP-43, transactive response DNA binding protein

Mitochondrial Function and Energy Metabolism

Energy metabolism is dysregulated early in the course of ALS as a result of decreased appetite, increased energy expenditure, and hypothalamic dysfunction, among other complex factors [295–299]. Hypermetabolism and weight loss are associated with faster disease progression [300, 301]. One strategy to ameliorate this involves introducing a high-calorie diet, which was shown to extend survival in ALS mouse models and patients with fast-progressing ALS [302]. Other strategies focus on optimizing mitochondrial function, as they are the main source of energy production (adenosine triphosphate, ATP) within the cell.

In a healthy cell, mitochondria utilize the electron transport chain for oxidative phosphorylation, the main source of ATP production. Nicotinamide adenine dinucleotide plus hydrogen (NADH) produced during glycolysis (anaerobic glucose metabolism in the cytosol) and the citric acid cycle (in mitochondria) donates electrons to initiate the electron transport chain via NADH dehydrogenase. Increased cellular energy demand leads to calcium influx into the mitochondria, driving increase in the respiratory chain and ATP synthesis [303].

In fasted states, including ALS, skeletal muscles increase utilization of fatty acid oxidation, which in turn reduces glucose metabolism [304, 305]. Additionally, mitochondria are directly compromised in ALS due to multiple mechanisms, including direct effect of protein aggregates or sequestration of mitochondrial microRNAs and proteins. The result of mitochondrial dysfunction includes excess ROS generation, altered energy production, and impaired calcium buffering. Many investigational agents have targeted mitochondrial function, including TUDCA [8, 149].

Current drugs in development target mitochondrial metabolism, including NAD+/NADH ratio, calcium influx, and fatty acid oxidation. There is also an ASO in development for genetic ALS caused by a mitochondrial protein mutation (Table 9).

Table 9.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting mitochondrial function

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
CNM-Au8 NCT04414345 Completed	2/3	Small molecule	Gold nanocrystals catalyze oxidation of NADH to NAD+	Mitochondrial function: increases ATP production through glycolysis and oxidative phosphorylation via the mitochondrial electron transport chain to improve energy metabolism.	Proteostasis: reduced TDP-43 aggregation in rodent spinal neurons exposed to excitotoxic glutamate levels.	All	24 weeks	Randomized, blinded	161	After 76-week open label extension, neurofilament levels reduced by 28% and there was 60% decreased risk of death (secondary endpoint) versus controls.	[382, 383]
Pridopidine NCT04615923 Completed	2/3	Small molecule	Activates sigma-1 receptor	Mitochondrial function: promotes calcium influx from ER to mitochondria under stress conditions to increase ATP and energy production.	Excitotoxicity: modulates NMDA receptor function to reduce glutamate-mediated calcium influx. Proteostasis: induces autophagy to clear protein aggregates.	All	24 weeks	Randomized, blinded	163	Planning phase 3 trial. Failed to meet primary endpoint (ALSFRS-R) or secondary endpoints. Improved speech and articulation rate at 6 months; 41% slowing in ALSFRS-R decline in fast progressors with < 1.5 years of symptoms.	[384, 385]
Trimetazidine Dihydrochloride NCT04788745 Completed	2	Repurpose (angina pectoris outside US)	Inhibits long-chain fatty acid oxidation via long-chain mitochondrial ACAA2	Mitochondrial function: inhibits fatty acid uptake to improve glucose utilization and mitochondrial ATP production (energy metabolism).		All	12 weeks	Open label	21		[304]
nL-CHCHD-001 NCT06392126 Active, not recruiting	1/2	Gene therapy	ASO versus CHCHD10 transcript	Mitochondrial dysfunction: targets CHCHD10 mutation, which causes toxic-gain-of function in this mitochondrial protein with unknown function.		CHCHD10	Unknown	Open label	1		[204]

Abbreviations: ACAA2, acetyl-CoA acyltransferase 2; ALSFRS-R, ALS functional rating scale revised; ASO, antisense oligonucleotide; ATP, adenosine triphosphate;  ER, endoplasmic reticulum; NADH, nicotinamide adenine dinucleotide hydride; NMDA, N-methyl-D-aspartate

RNA Metabolism

RNA metabolism involves a complex interplay of RNA splicing, transport, export, and degradation [306]. RNA-binding proteins (RBPs) bind RNA transcripts to regulate their stability, localization, and translation [307]. Messenger RNA (mRNA) is transported from the nucleus to code for proteins and non-coding microRNAs, which can further regulate gene expression [306].

Multiple aspects of mRNA and microRNA metabolism are dysregulated in ALS. TDP-43 and FUS are RBPs and their alteration leads to downstream effects on mRNA stability [8]. Other ALS gene mutations can lead to changes in RNA splicing [308]. Stress granules, which can become insoluble due to ALS protein aggregates, can sequester mRNAs and stall protein translation [306]. Investigational agents involving RNA metabolism are currently targeting microRNA maturation and processing (Table 10).

Table 10.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting RNA metabolism

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Additional drug mechanism(s)	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
PrimeC (ciprofloxacin and celecoxib) NCT05357950 Active, not recruiting	2b	Repurpose (fluoroquinolone and nonsteroidal anti-inflammatory drug)	Inhibits bacterial DNA gyrase and inhibits cyclooxygenase-2	RNA metabolism: regulates microRNA processing, important in RNA interference and degradation, and chelates iron.	Neuroinflammation: reduces prostaglandin synthesis, which mediates the inflammatory pathway.	All	6 months	Randomized, blinded	68	Reduced disease progression by 36% and improved complication-free survival by 53% (secondary endpoints). Seeking approval in Canada. Celecoxib was previously not found to be beneficial as a single agent in ALS.	[172, 386]
Enoxacin NCT04840823 Completed	1/2	Repurpose (fluoroquinolone)	Inhibits bacterial DNA gyrase	RNA metabolism: enhances microRNA maturation, important in RNA interference and RNA degradation. Found to delay neurological symptoms in ALS mice.		All	30 days	Randomized, blinded	8	No serious adverse events. Global increase in plasma and CSF microRNA levels at all post-treatment timepoints	[387, 388]

Abbreviations: CSF, cerebrospinal fluid

Disrupted nucleocytoplasmic RNA transport found with C9orf72 and TDP-43 pathology is another important ALS mechanism. Mislocalization of TDP-43 to the cytoplasm sequesters nuclear pore complexes and transport factors [309]. C9orf72 mRNA sequesters a nucleocytoplasmic transport regulator in the cytoplasm, altering transport multiple RNAs and proteins through the nuclear pore [310]. No drugs in development currently target nuclear export specifically.

Excitotoxicity

Depolarization of upper motor neurons triggers glutamate release into the synapse, activating post-synaptic glutamate receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA, and N-methyl-D-aspartate, NMDA) [311]. Increased glutamate release leads to motor neuron hyperexcitability, aberrant firing, increased intracellular calcium, and cell death [312]. Primary mechanisms of glutamate excitotoxicity include increased glutamate release, upregulation of AMPA receptors, and reduced astrocyte glutamate reuptake, among others [311]. Other downstream effects include reduced glutathione antioxidant levels leading to oxidative stress, increased intracellular calcium, and ER stress leading to protein aggregation of TDP-43, SOD1, and FUS [8, 311]. Notably, while riluzole mediates glutamate excitotoxicity, not many drugs in development are targeting this pathway. Thus, no drugs with a primary mechanism acting on excitotoxicity are included in a table.

Gut Microbiota

The gut microbiome includes the trillions of bacteria found in the human stomach and small and large intestines. These microbes participate in host metabolism, prevent colonization by pathogens, and facilitate inflammatory and immune functions [313]. Changes to gut microbiome in ALS include increased gut permeability and low levels of butyrate-producing bacteria [314, 315]. Supplementation with butyrate improved survival time in an ALS mouse model [316]. It is also hypothesized that molecules produced by gut microbes could weaken the blood–brain barrier and promote systemic neuroinflammation [313]. Drugs in development target gut permeability (Table 11).

Table 11.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting gut microbiota

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
PLL001 NCT06513546 Not yet recruiting	1/2	Small molecule	Poly-L-lysine conjugated with small chain fatty acids (acetate, butyrate, lactate, propionate)	Microbiome: replaces gut microbiome and restores gut and blood–brain barrier permeability.	All	24 weeks	Randomized, blinded	141 (est, phase 2 arm)		[389]
MaaT033 NCT05889572 Active, not recruiting	1b	Biologic	Gut microbiota	Microbiome: replaces gut microbiome and restores gut permeability and gut–brain axis.	All	56 days	Open label	15 (est)	Safe and well tolerated.	[313, 390]

Abbreviations: est, estimated

Multiple Mechanisms

Some investigational drugs have broad cellular targets, affecting multiple pathways implicated in ALS pathogenesis (Table 12). It is possible that targeting many pathophysiological mechanisms at once could be necessary to make significant progress in slowing disease progression.

Table 12.

Investigational therapeutics in ALS phase 1, 2, and 3 clinical trials targeting multiple mechanisms

Drug name NCT Study status	Phase	Drug category	Drug target	Drug mechanism	Genetic or sporadic ALS	Treatment period	Trial design	Number of participants	Results	References
NX210c NCT06365216 Recruiting	2	Small molecule	Glycoprotein derived from subcommissural organ-spondin	Multiple: involved in neuron maturation during embryogenesis. Restores blood–brain barrier, reduces glutamate excitotoxicity, and enhances neurotransmission.	All	26 days	Randomized, blinded	80 (est)	Tolerability proven in phase 1b study.	[391, 392]
Clenbuterol NCT04245709 Completed	2	Repurpose (asthma outside US)	Beta-2 agonist	Multiple: affects multiple downstream signaling pathways in neurons, microglia, and muscle to induce neurotrophic factors, inhibit glutamate excitotoxicity, reduce microglial neuroinflammation, and preserve NMJ.	All	24 weeks	Open label	25	13 of 25 participants withdrew due to adverse events. Slowing in ALS FRS-R decline (secondary endpoint) but with limited sample size.	[393, 394]

Abbreviations: ALSFRS-R, ALS functional rating scale revised; est, estimated; NMJ, neuromuscular junction

Future Directions, Gaps, and Needs

Currently, much of ALS care focuses on symptomatic management using medications described above. Very few drugs are approved to slow disease progression in ALS. Ongoing research is needed to clarify the complex and interacting pathophysiological mechanisms at play, as well as to identify upstream triggers for disease (such as environmental exposures). Of note, many investigational agents have previously been found to be ineffective to slow ALS progression, highlighting the potential need to target multiple pathophysiologic mechanisms simultaneously [205].

Additionally, it is important to optimize strategies to bring new disease-modifying drugs to market. Improvements in drug development, clinical trials, and outcome measures can help bring drugs to market faster. Important strategies to accelerate general drug development include discovering new ALS biomarkers that can be used as clinical trial endpoints; enhancing data sharing between trials; and performing natural history studies in diverse ALS populations, which may be able to serve as controls for future trials [317]. Separately, research to make ALS a more livable disease is also an important future need [318]. Specifically pertaining to clinical trials, the use of platform trials, which use a single control group for multiple interventions, can help streamline evaluation of multiple investigational agents [53]. Finally, artificial intelligence (AI) is starting to be used to optimize multiple stages of drug development.

Artificial Intelligence in Drug Development

AI is being used more frequently in the drug development pipeline in ALS and beyond. Use of AI can be particularly beneficial with rare diseases or targeted therapies, where profitability of a clinical trial is less certain [319]. AI can be implemented in nearly all stages of drug development, including drug screening, biomarker discovery, clinical trials, and data analysis [320].

Preclinically, AI and machine learning can be used to discover new biomarkers and molecular underpinnings in ALS subtypes [321, 322]. In drug development, it can be used in high throughput drug screening. This technique uses computational biology to screen up to millions of potential compounds for predicted effect on target binding, drug metabolism, and central nervous system penetrance. This reduces the need for resource-intensive animal modeling [197].

Within clinical trials, AI can be used to create a synthetic control arm, eliminating the need for a placebo group [323]. AI has been used to stratify patients with ALS by clinical subgroup and to predict disease progression [324, 325]. Regarding recruitment, AI can comb through electronic medical record data to find patients meeting inclusion criteria, summarize eligibility criteria [323], and disseminate information regarding trials to the broader public, improving fair access. In data analysis, AI can determine a heterogeneity effect on outcome measures (e.g., quickly progressing subgroup) or impute missing data [319]. AI can continue to analyze post-market surveillance data to ensure safety and efficacy of approved drugs.

Limitations of AI in ALS drug development currently include lack of high-quality training data for machine learning models and lack of a global regulatory framework [319, 320].

Conclusions

The pharmacologic management of ALS continues to evolve on the basis of approved therapies, symptomatic management, and ongoing clinical trials. Given the incurable nature of ALS, there is incredible hope that new, effective, and clinically meaningful therapeutics are on the horizon. Unfortunately, data showing a disease-modifying effect for several recently approved drugs have not been replicated in larger trials. Nonetheless, ALS therapeutic development is highly active with a large number of ongoing clinical trials. Advancements in ALS outcomes, ALS clinical trials, and drug discovery have the potential to speed drug development for this fatal disease.

Declarations

Funding

This work was funded by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS120926 and R01NS127188 to S.A.G.), the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (R01TS000344 and R01TS000327 to S.A.G.), and James and Margaret Hiller and Linda and Eric Novak (S.A.G.).

Conflicts of Interest

S.A.G. is listed as inventor on a patent, issue number US10660895, held by University of Michigan titled “Methods for Treating Amyotrophic Lateral Sclerosis” that targets immune pathways for use in ALS therapeutics. Scientific consulting for Evidera. S.E.Q. and K.H.Q. have no relevant financial or non-financial interests to disclose.

Ethics Approval

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Data availability

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Code availability

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Author contributions

S.A.G. conceived the idea for the article. S.E.Q. and K.H.Q. performed the literature search, and all authors drafted, reviewed, and critically revised the final manuscript.