Evaluating emerging drugs in phase II & III for the treatment of amyotrophic lateral sclerosis

ABSTRACT

Introduction

Amyotrophic Lateral Sclerosis is a rapidly progressive motor neuron disorder causing severe disability and premature death. Owing to the advances in uncovering ALS pathophysiology, efficient clinical trial design and research advocacy program, several disease-modifying drugs have been approved for treating ALS. Despite this progress, ALS remains a rapidly disabling and life shortening condition. There is a critical need for more effective therapies.

Areas covered

Here, we reviewed the emerging ALS therapeutics undergoing phase II & III clinical trials. To identify the investigational drugs, we searched ALS and phase II/III trials that are active and recruiting or not yet recruiting on clinicaltrials.gov and Pharmaprojects database.

Expert opinion

The current pipeline is larger and more diverse than ever, with drugs targeting potential genetic and retroviral causes of ALS and drugs targeting a wide array of downstream pathways, including RNA metabolism, protein aggregation, integrated stress response and neuroinflammation.

We remain most excited about those that target direct causes of ALS, e.g. antisense oligonucleotides targeting causative genes. Drugs that eliminate abnormal protein aggregates are also up-and-coming. Eventually, because of the heterogeneity of ALS pathophysiology, biomarkers that determine which biological events are most important for an individual ALS patient are needed.

KEYWORDS:

• Amyotrophic lateral sclerosis (ALS)
• clinical trials
• Antisense oligonucleotide (ASO)
• protein aggregation
• neuroinflammation
• antiretroviral therapy

1. Background

Amyotrophic Lateral Sclerosis (ALS), or Lou Gehrig’s disease, is a fatal neurodegenerative disease affecting upper motor neurons and lower motor neurons [1]. Patients develop progressive limb weakness, impaired speech and swallowing functions, and ultimately, respiratory failure and death. We now recognize that ALS is also a multisystem disease [2]: multiple population-based studies in different genetic backgrounds showed about 15% of patients have concomitant frontotemporal dementia (FTD), while approximately 50% of patients have some degree of cognitive impairment manifested as executive dysfunction, language impairment and behavioral changes [3–5], which challenges decision-making and end-of-life planning. Compared to other neurodegenerative diseases, ALS is known for its relentless progression, and most ALS patients survive for less than 5 years despite the recent advances in disease-modifying treatments and more accessible multidisciplinary supportive care [6]. Understandably, ALS poses a tremendous burden and emotional and financial strain to patients and their caregivers [7,8].

ALS is considered a rare disease with a global incidence rate of 1–2 per 100,000 person-years and low prevalence rate of 4–8 per 100,000 due to rapid disease progression and death, but the data varies significantly between geographic locations and ancestral backgrounds [9–11]. It is anticipated the prevalence will increase significantly in the next few decades [12], probably due to the aging population, improved ALS therapeutics and the implementation of quality care measures.

One major challenge for developing ALS therapeutics is related to its heterogeneous pathophysiology. Approximately 10% of the ALS population have familial ALS, and thus far, over 30 genes have been identified to cause ALS directly [13]. A total of 5–10% of sporadic ALS cases (patients who do not have a family history) are also caused by gene mutations [14]. The two most common mutated genes are hexanucleotide repeat GGGGCC expansion in chromosome 9 open reading frame 72 (C9ORF72) and Cu-Zn superoxide dismutase 1 (SOD1) point mutations, leading to toxic SOD1 protein and dipeptide repeat protein aggregations [15,16]. Ataxin-2 intermediate-length polyglutamine (polyQ) expansion (27–33 glutamines) is also found to be a genetic risk factor for developing ALS [17,18]. Though TDP43 gene mutations account for less than 5% of familial ALS, TDP43 protein aggregation in the brain and spinal cord is a universal pathologic feature in ALS [19,20], excluding SOD1 and FUS-ALS. In the past two decades, there have been significant advances in understanding the ALS pathophysiology, which are largely derived from decoding these genes and their disease-causing mechanisms [21]. To date, a myriad of mechanisms has been proposed to drive progression in ALS, including glutamate excitotoxicity, oxidative stress, the gain of toxic function from protein aggregates, impaired protein degradation, disturbance of RNA synthesis, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and neuroinflammation [22–25].

Another substantial hurdle for therapeutic development is the existence of tremendous phenotypic variations [26]: the site of onset, progression rate and survival time. The onset site, sex and genetics can all influence disease progression. Typically, bulbar and respiratory onset ALS progresses faster than limb onset ALS. Progression is commonly measured using the Revised ALS Functional Rating Scale (ALSFRS-R). Studies have shown remarkable heterogeneity in ALS progression rate [27–29]. Considering this, ALS diagnostic criteria have been developed and repeatedly modified to ensure recruiting of a relatively homogenous population during clinical trials and to reduce noise related to phenotypic variation [30–32].

2. Medical need and existing treatment

Riluzole modulates neurotransmitter glutamate synaptic transmission and reduces excitotoxicity [33]. In randomized, double-blind, placebo-controlled trials (RCT), Riluzole was shown to have a modest effect in prolonging survival time but did not significantly slow down ALS functional decline [34,35]. It received FDA approval in 1995 as the first disease-modifying treatment for ALS. Riluzole is taken 50 mg twice daily, is generally well-tolerated, and the most common side effects are asthenia, gastrointestinal symptoms and mild transaminitis.

It was not until two decades later that a second medication, intravenous Edaravone, was approved in the US for treating ALS. Edaravone is a free radical scavenger that improves poststroke functional outcomes and has been approved to treat stroke patients in Japan. The first phase 3, 24-week RCT trial in ALS enrolled 102 patients in the Edaravone group and 104 patients in the placebo group whose disease duration was less than 3 years and forced vital capacity (FVC) was >70% of predicted and ALSFRS-R decline of −1 to −4 per month during the 3 months of observation before randomization. The result did not show a significant slowing of ALS functional decline in the Edaravone-treated group compared to placebo [36]. A subsequent phase 3 trial was conducted in a well-defined subset of ALS patients who had preserved motor functions, defined by having at least 2 points on all 12 items of ALSFRS-R, a disease duration of less than 2 years, and FVC > 80% of predicted. Sixty-eight patients were enrolled in the Edaravone group, and 66 were in the placebo group, and the result revealed a 33% slowing of ALSFRS-R decline during the 24-week trial period [37]. Intravenous (IV) Edaravone is administered 60 mg daily for 14 days during the first cycle, followed by 14 days of drug-free period and subsequent repeated cycles − 2 weeks on (10 days of drug administration) and 2 weeks off. The side effect profile includes bruising, gait difficulty, headache and rare hypersensitivity reactions. It is not widely used due to the lack of demonstrated efficacy for the broader ALS population in the real world [38], the burden on patients and families and accessibility issues from frequent IV infusions.

To overcome the burden of IV infusion, an open-label study was conducted to compare 105 mg Edaravone oral suspension with 60 mg IV formulation in healthy adults, and the results showed these two formulations are bioequivalent [39]. Since then, Edaravone oral suspension has been approved for ALS treatment in North America and Asia but not in Europe (except in Switzerland). Recently, a 48-week phase III ADORE clinical trial (NCT05178810) of a different edaravone formulation, FAB122, was completed in Europe but failed to achieve the primary and secondary endpoints (https://www.ferrer.com/en/results-study-ADORE-ALS). FAB122 is a granule formulation of edaravone developed by Ferrer International and was administered as a 100 mg daily dose in the clinical trial. Given the differences in the formulation, dosing, and the clinical trial design, comparing FAB122 directly with edaravone IV and ORS by Mitsubishi Tanabe Pharma can be difficult.

A fixed-dose coformulation of Sodium Phenylbutyrate 3 g-Taurursodiol g (PB/TURSO) was designed to target two distinct proposed disease mechanisms- ER stress and mitochondrial dysfunction. In a phase-2 RCT trial, 89 patients were enrolled in PB/TURSO treatment group and 48 patients in the placebo and at the end of the 24-week randomized phase, PB/TURSO treatment significantly slowed down ALS functional decline; there was a 2.32 absolute difference of ALSFRS-R between PB/TURSO and placebo groups [40]. The participants then entered a 6-month open-label extension phase where everyone received PB/TURSO, but the initial placebo group started it 6 months later. The result revealed a mean survival time of 25 months in patients who were originally assigned to the treatment arm vs. a mean survival time of 18.5 months in patients who were initially assigned to placebo, suggesting PB/TURSO treatment at early disease phase provides better survival benefit [41]. These led to conditional approval in Canada and full approval for treatment of ALS in the US. In the meantime, a Phase 3, 48-week trial has completed its enrollment of over 600 ALS patients across the US and Europe in early 2023. It will offer more insight into its safety and efficacy. PB/TURSO comes in as a packet containing 3 g PB and 1 g TURSO mixed powder and needs to be dissolved in 8 oz water and taken within 1 hour and twice daily. Patients complain of an unpleasant taste. The large volume poses a challenge to patients who already have dysphagia, though this issue resolves after feeding tube is placed. The most common side effects are gastrointestinal symptoms including nausea, diarrhea and abdominal pain, which can be severe and lead to cessation of treatment in some patients in our practice.

Tofersen is the first antisense oligonucleotide (ASO) being developed to target ALS caused by SOD1 gene mutations. It functions by reducing SOD1 mRNA production and protein synthesis. Current data shows approximately 10% of familial and 1% sporadic patients have SOD1 ALS. So far, approximately 200 different point mutations have been identified [42].

In the recently completed 28-week randomized phase 3 trial [43], 108 SOD1 ALS participants were randomized with a 2:1 ratio to treatment and placebo. Intrathecal bolus infusion of 100 mg Tofersen or placebo was administered as 3 loading doses every 14 days followed by monthly for 5 doses. Only 60 participants with fast progression were included in the primary endpoint analysis. At 28 weeks, 39 participants who received Tofersen had an ALSFRS-R decline of 6.98 points compared to 8.14 in 21 participants who received placebo, and the primary endpoint did not reach statistical significance. However, SOD1 protein levels in the cerebrospinal fluids were reduced by 29% in the Tofersen group and increased by 16% in placebo. The mean concentration of neurofilaments light chain (NfL) was reduced by 60% in Tofersen-treated group and increased by 20% in placebo. Further, the reduction of SOD1 protein and NfL levels was sustained through open-label extension phase, and at 52 weeks, there was 3.5-point difference in ALSFRS-R between the two groups. The slow progression SOD1 ALS participants were not included in the primary endpoint analysis; nonetheless, a similar pattern of SOD1 protein and NFL reduction after Tofersen treatment was observed. Tofersen was approved by FDA for SOD1 ALS in April 2023 and is still under review in Europe. Tofersen may cause severe side effects such as myelitis, radiculitis, elevated intracranial pressure, and aseptic meningitis in some patients. The most common adverse effects are procedural pain, fatigue, arthralgia, myalgia, CSF pleocytosis and elevated protein. Patients require close monitoring of these severe adverse events, and prompt management or discontinuation of the drug should be considered. Tofersen is also limited by only benefiting approximately 1% of the ALS population. A trial of Tofersen in asymptomatic SOD1 mutation carriers is underway.

A study of the efficacy of ultrahigh dose methylcobalamin in early-stage ALS was conducted in Japan. They enrolled 130 ambulatory patients with disease duration of less than 12 months, moderate progression rate, and FVC > 60% of predicted value. Participants received either 50 mg methylcobalamin or placebo intramuscularly twice weekly for 16 weeks. The results showed methylcobalamin, when given at the early phase, slowed ALSFRS-R decline by 1.97 at 16 weeks [44]. No severe side effects were directly related to methylcobalamin treatment. However, ultrahigh dose methylcobalamin did not show efficacy in the previous phase 2/3 trial in patients with disease duration of 3 years [45], limiting its use only in early-phase ALS. It is unclear to date whether methylcobalamin will be submitted to FDA for approval and the outcome is not guaranteed.

3. Current research goals

It is encouraging to see several ALS medications become available to ALS patients in just the past 2 years, the result of advances in understanding ALS pathophysiology but also improvements in trial designs. For example, trial inclusion criteria have been developed that maximize sensitivity for detecting small changes in ALSFRS-R scores over 6-month studies [37,44]. NfL appears promising as a biomarker that predicts future clinical benefit [43]. An adaptive platform trial paradigm has been established to accelerate therapeutic development [46,47]. This model involves building a long-term clinical trial infrastructure that is used to test multiple investigational products in the pipeline to increase the efficiency of drug development. Further, placebo data from each regimen can be shared with other regimens, thereby decreasing the number of participants that need to be randomized to the placebo group. People living with ALS are partners in drug development now, facilitated by a training program called ALS clinical research learning institute. Since the program was launched in 2011, it has trained over 600 ALS research ambassadors, who have been advocating to raise funds and change laws that benefit ALS research, help sponsors create trials that are more patient-centric, and testifying at FDA advisory committees [48].

In spite of this progress, ALS remains a rapidly disabling and life shortening condition. There is a critical need for more effective therapies.

4. Scientific rationale for current therapeutic targets

Glutamatergic excitotoxicity and oxidative stress were among the earliest hypotheses for ALS pathogenesis [25,49,50], which led to the discoveries of the first two ALS disease modifying treatment, Riluzole and Edaravone. The rapid advances in identifying ALS causal genes and pathophysiology lead to many potential targets for therapeutic development, such as protein homeostasis, neuroinflammation and stem cells.

A hallmark of ALS pathology is abnormal protein accumulation in the brain and spinal cord. Cytoplasmic TDP43 aggregation is found in most familial (including C9ORF72-ALS) and sporadic ALS cases except SOD1-ALS and FUS-ALS, where SOD1 or FUS aggregates were found [19,20,51–53]. C9ORF72 hexanucleotide repeat expansion mutation further leads to the abnormal translation of dipeptide repeat protein [16,54]. These protein products lead to toxic gain-of-function effects by disrupting the normal protein translation and causing endoplasmic reticulum stress. Further, the loss-of-function of transcription factor TDP43 affects at least several hundreds of other downstream gene transcription, which are likely caused by aberrant RNA splicing, the inclusion of cryptic exons leading to frameshift mutations, or the introduction of alternative polyadenylation sites [55–57].

Increasing evidence has emerged to support the significant role of neuroinflammation in the pathophysiology of ALS [58]. Microglia, the innate immune cells in the central nervous system, are activated in ALS patients and play an important role in disease progression [59]. Replacing the SOD1+ microglia with normal microglia slowed progression in SOD1 ALS mice [60]. Immune profiles of ALS patients’ peripheral blood samples showed increased proinflammatory cytokines (e.g. IL-6, IL-17, IFN- γ, TNF-α) and decreased anti-inflammatory cytokines (e,g., IL-4, IL-10, TGFβ) [61]. Recent research has shown haploinsufficiency of C9ORF72 contributes to proinflammatory state in ALS [62].

However, ALS clinical trials using broad immunosuppressant were futile [63–65], which underscores the complexity of the neuroinflammatory network in ALS. Recently, T regulatory cells (Treg), a subgroup of CD4+T cells that expresses transcription factor Foxp3+, have gained interest. Treg cells can suppress the pro-inflammatory T effector lymphocytes. Emerging data in ALS animal models and human studies show a reduced number of Treg cells and increased proinflammatory T cells in ALS patient’s peripheral blood, and the level of peripheral Treg inversely correlates with disease progression [58].

Finally, the chronic stress imposed by the loss of cellular homeostasis and accumulation of toxic proteins in motor neurons can activate integrated stress response (ISR). Though ISR is an essential mechanism to restore homeostasis, chronic activation may lead to cell death [66]. ISR activation has been found in ALS rodent models and is associated with accelerated motor neuron death [67]. ISR markers have been detected in the spinal cord of ALS patients [68].

5. Competitive environment

To identify the investigational drugs, we searched ALS and phase II/III trials that are active and recruiting or not yet recruiting on clinicaltrials.gov and Pharmaprojects database.

Competitive environment table.

Compound	Company	Structure	Stage of development	Mechanism of Action
RAPA-501	RAPA therapeutics	Regulatory T clls	Phase 2/3	Suppress pro-inflammatory T effector cells.
Aldesleukin	ILTOO Pharma	Low dose IL-2	Phase 2	Activate regulatory T cells
BIIB105	Biogen	Antisense oligonucleotide	Phase1/2	Block Ataxin-2 gene expression
AIT-101	AI therapeutics	Apilimod dimesylate	Phase 2a	PIKfyve inhibitor, clear toxic protein aggregates
ABBV-CLS-7262	Calico and Abbvie	eIF2B activator	Phase 2/3	Activate eIF2B, reduce ISR
DNL343	Denali	eIF2B activator	Phase 2/3	Activate eIF2B, reduce ISR
Masitinib	AB Sciences	Tyrosine kinase inhibitor	Phase 3	Inhibit microglia in CNS and macrophages and mast cells in PNS.
ION363	Ionis Pharmaceuticals	Antisense oligonucleotide	Phase 1–3	Block FUS gene expression
ZYIL1	Zydus	NLRP3 inflammasome inhibitor	Phase 2	Inhibit inflammasome pathway in innate immune system
Utreloxastat	PTC Therapeutics	Ferroptosis inhibitor	Phase 2	Reduce oxidative stress
Prime C	NeuroSense Therapeutics	Fixed dose coformulation of Ciprofloxacin and celecoxib	Phase 2b	Target neuroinflammation, iron accumulation and microRNA dysregulation.
Rapamycin	PharmAust	mTOR pathway inhibitor	Phase 2	Protein homeostasis, autophagy, anti-neuroinflammation
Monepantel	Pharmaust	mTOR pathway inhibitor	Phase 2	Protein homeostasis, autophagy, anti-neuroinflammation
Ibudilast	MediciNova	Phosphodiesterase inhibitor	Phase 2b/3	Suppress microglia activation and proinflammatory cytokines
TUDCA	European Commission	hydrophilic bile acid	Phase 3	inhibit cell apoptosis, neuroprotection
Abacavir/lamivudine/dolutegravir	Macquarie University, Australia	Antiretroviral therapy	Phase 3	Inhibit HERVK expression

The table includes a list of drugs in Phase II & III development for ALS, the companies or entities that sponsor the clinical trials, drug structure, their developmental status and mechanisms of action.

Abbreviations: PIKfyve, phosphatidylinositol 3-phosphate 5 (PI3P5) kinase; eIF2B, eukaryotic translation initiation factor 2B; FUS, Fused in Sarcoma; NLRP3, nucleotide-binding domain and leucine-rich repeat protein 3; mTOR, mammalian Target of Rapamycin; HERVK, human endogenous retrovirus K.

5.1. RAPA-501

RAPA-501 is an autologous T cell therapy developed by RAPA therapeutics. The cells are generated by reprogramming patients’ peripheral blood cells in culture to enrich anti-inflammatory Treg and Th2 cells that express FOXP3 and GATA3 transcription factors (markers). An open-label, non-randomized, multi-center phase 2/3 autologous hybrid Treg/Th2 cells (NCT04220190) study is open at 10 sites across the US and is actively recruiting patients. The estimated completion date is 1 July 2025. During the initial phase 1/2 study on different dosing regimens, no adverse events directly related to RAPA-501 were reported. The primary outcome of the phase 2/3 open-label study is the safety of the highest dose RAPA-501 (80×10^{^}6 cells per infusion), and secondary outcomes include whether the intervention increases circulating Treg/Th2 cells and reduces proinflammatory Th1 cells and cytokines: IL-1β, IL-6 and TNFα, as well as the effects on serum NfL concentration and ALSFRS-R decline.

5.2. Aldesleukin

Low-dose IL-2 enhances Treg number and its function [69]. Aldesleukin is developed by MIROCALS consortium. A phase-2 RCT trial of 5-day cycles of 1–2 million international units of aldesleukin administration showed a statistically significant increase of % Treg cells in the CD4+ T lymphocyte population [70,71]. Aldesleukin causes more injection site reactions and flu-like symptoms compared to placebo. ILTOO Pharma announced signing a license agreement with MIROCALS for development of low-dose IL-2 for the treatment of ALS in March 2023 (http://www.iltoopharma.com/wp-content/uploads/2023/05/MIROCALS-ILTOO-Joint-Press-release-9-May-20239-May-2023-Licence-agreement-1.pdf) and is preparing to launch a phase-3 clinical trial. Both RAPA-501 and aldesleukin are promising therapeutic developments in ALS.

5.3. BIIB105

Ataxin-2 gene intermediate-length polyQ (27–33 Qs) expansion is a genetic risk factor for ALS. In preclinical studies, increasing ataxin-2 gene expression enhanced TDP43 protein toxicity. Conversely, deleting ataxin-2 gene reduced TDP43 aggregation and significantly slowed motor neuron death and prolonged survival in TDP43 ALS rodent models [72]. Ataxin-2 ASO (BIIB105), developed by Biogen, is designed to reduce the level of Ataxin-2 protein in motor neurons. A phase1/2 multiple ascending-dose RCT study is to investigate the safety, tolerability and efficacy of BIIB105 in PALS with or without polyQ expansion in Ataxin2 gene (NCT04494256). The primary outcome is the number of patients with adverse effects, and the second outcome is pharmacokinetics and changes in serum NfL and ALSFRS-R. The first 6 months is a placebo-controlled study followed by a 3-year long-term open-label extension phase. The study started in September 2020 and will be completed by July 2026. BIIB105 is the first drug in the pipeline that could potentially target toxic TDP43 aggregation, a near universal pathological hallmark of ALS. Therefore, it may have a broad impact in therapeutic development for both familial and sporadic ALS.

5.4. ION363

Fused in Sarcoma (FUS) is an RNA processing protein and mutations of the gene lead to abnormal FUS protein accumulation in the cytoplasm of motor neurons and have been identified as rare genetic causes of ALS and FTD [52,53]. FUS ASO (ION363), developed by Ionis Pharmaceuticals, was previously studied in one ALS patient with FUS^P525L mutation [73]. The patient received 12 intrathecal infusions, which were initiated at 20 mg and titrated to 120 mg maximum dose. There were no severe side effects, but the patient died of ALS progression 1 year after initiation of ION363. The following postmortem analysis of the patient’s brain and spinal cord revealed a broad distribution of ION363 in the central nervous system and a remarkable reduction of FUS protein aggregates in neurons [73].

Based on this promising result, a phase 1–3 clinical trial sponsored by Ionis was started in 2021 and is currently actively recruiting FUS-ALS patients (NCT04768972). The Part 1 study is a randomized control study for 60 weeks, followed by Part 2, in which participants receive ION363 for 80 weeks. Participants who complete Part 2 can enter Part 3 and continue the treatment for 156 weeks. The primary outcome of Part 1 study is the change in functional impairment, measured by a combined assessment of ALSFRS-R, time of discontinuation from Part 1 and entering Part 2 due to deterioration, and ventilation assistance-free survival.

5.5. Apilimod dimesylate (AIT-101)

Apilimod dimesylate is an oral phosphatidylinositol 3-phosphate 5 (PI3P5) kinase inhibitor (PIKfyve inhibitor) developed by AI therapeutics. It helps convert PI3P to PI (3,5) P2 and regulate cellular vesicle fusion. In the cultured motor neurons derived from sporadic ALS patients, PIKfyve inhibition by Apilimod stimulates exocytosis of various misfolded cytoplasmic proteins, including TDP43 and dipeptide repeat proteins, which in turn improves motor neuron survival [74]. Further, in TDP43 mouse model, PIKfyve inhibition reduces TDP43 aggregation and prevents motor neuron death in the ventral spinal cord [74].

A phase 2a, multicenter and biomarker-driven clinical trial was started to evaluate the safety, tolerability, and biological effect of Apilimod dimesylate in approximately 12 adults with C9ORF72-associated ALS (NCT05163886). This is a 12-week randomized, placebo-controlled study followed by either a 12-week or a 36-week open-label extension study. The study was sponsored by AI Therapeutics and launched in December 2021 and will be completed in May 2024. Apilimod has the potential to restore protein homeostasis in diseased motor neurons and represents a promising therapy for the broad ALS population.

5.6. ABBV-CLS-7262 and DNL343

ABBV-CLS-7262 is an oral eukaryotic translation initiation factor 2B (eIF2B) activator under development by Calico and Abbvie, and DNL343 is a similar drug under development by Denali Therapeutics. In ALS, stress response triggered by misfolded proteins can lead to phosphorylation of eIF2α and thereby reduce global translation and protein synthesis, a process that is central to ISR. ABBV-CLS-7262 and DNL343 are designed to restore eIF2B function, protein translation and overrule ISR. However, it is worth pointing out that the effects of ISR in ALS pathogenesis are complex, and contradictory data has also emerged. For example, preclinical studies using SOD1 ALS models showed enhanced ISR delays in ALS onset and prolonged survival [75]. Conversely, pharmacological activation of eIF2B and inhibition of ISR accelerated disease progression [76].

A phase 2/3 RCT trial of ABBV-CLS-7262 was recently initiated in February 2023 as regimen F of the Healey PLATFORM trial (NCT05740813). It is administered orally once daily, and two different doses will be tested for their effects on ALSFRS-R changes. The most common side effects reported during the phase I trial were nausea, itchiness, constipation, dizziness and headache (https://www.massgeneral.org/assets/mgh/pdf/neurology/als/regimenf_calicosabbv-cls-7262drugsciencewebinar_2023.pdf).

A phase 2/3 RCT trial of DNL343 was initiated in April 2023 as regimen G of the Healey PLATFORM trial (NCT05842941). DNL343 is also administered orally once daily and was well tolerated during the phase 1 trial (https://www.massgeneral.org/news/press-release/healey-center-dnl-343-first-patient-dosed). Both ABBV-CLS-7262 and DNL343 demonstrated efficient CSF penetration.

5.7. Masitinib

Masitinib is an oral tyrosine kinase inhibitor under development by AB Sciences. It targets innate immune cells in the central and peripheral nervous systems, including microglia, macrophages and mast cells. In the SOD1-ALS model, increased mast cell number and degranulation correlate with the degeneration of neuromuscular junctions and paralysis progression. Masitinib has shown a neuroprotective effect in the peripheral nervous system by significantly downregulating mast cell and macrophage infiltration [77]. Such findings represent a novel pathogenic mechanism in ALS that is shown to be therapeutically targeted by masitinib, in addition to its primary mechanism of action on aberrant microglia of the central nervous system.

The phase 2/3 international multicenter 48-week RCT trial (NCT02588677) enrolled 394 ALS patients and compared the efficacy and safety of 3 mg/kg/d, 4.5 mg/kg/d masitinib and placebo. The difference in ALSFRS-R score between 4.5 mg/kg/d and placebo groups at the end of 48 weeks was 3.39 points (a 27% slowing in decline in the masitinib group), and it was statistically significant [78]. Adverse effects associated with masitinib were maculopapular rash, peripheral edema and transaminitis. One autoimmune hepatitis was reported in the high-dose masitinib treatment arm, which resolved after the discontinuation of masitinib and treatment with prednisone and azathioprine. A phase 3 RCT trial was started in 2017 by AB Science (NCT03127267) and continues to recruit patients. Masitinib is a promising therapeutic product as it regulates the innate immune cells in both central and peripheral nervous systems. It may provide broader protection for motor neurons and axon degeneration, which is particularly relevant given various studies have found NMJ denervation and axonal transportation deficit predating motor neuron degeneration, supporting the axon dying back mechanism for ALS [79,80].

5.8. ZYIL1

ZYIL-1 is an oral small-molecule inhibitor of nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain-containing 3 (NLRP3), and the latter is a key component of the inflammasome pathway in the innate immune system. It is under development by Zydus. In the first-in-human phase I study [81], 3 ascending doses (12.5, 50, 100 mg daily for 14 days) of ZYIL1 was tested in 18 healthy controls. Common side effects were constipation, headache, pyrexia, glycosuria, nasopharyngitis, low neutrophil count, and an increase in transaminases and triglycerides. Among them, two events of low neutrophil counts and high triglyceride were determined to be severe. A phase 2, proof-of-concept, multi-center RCT study was recently started in July 2023 to evaluate the efficacy, safety, tolerability, pharmacokinetics and pharmacodynamics of ZYIL1.

5.9. Utreloxastat

Utreloxastat (PTC-857), developed by PTC Therapeutics, is a small molecule that inhibits 15-lipoxygenase, a key enzyme that mediates oxidative stress and ferroptosis, i.e. iron-dependent cell death. It also appears to have anti-inflammatory effects, as in Parkinson’s disease model, Utreloxastat reduced pro-inflammatory IL-1ß cytokine production by microglial cells. Utreloxastat is under development by PTC Therapeutics. There were no safety concerns when it was given orally to healthy controls at 500 mg per day for 14 days or one time 1000 mg dose [82]. PTC Therapeutics initiated a 24-week, phase 2 RCT study of its efficacy, safety and biomarker effects in April 2022 (NCT05349721).

5.10. Ciprofloxacin-celecoxib

Ciprofloxacin-celecoxib (Prime C) is a fixed-dose coformulation of ciprofloxacin and celecoxib under development by NeuroSense Therapeutics. Ciprofloxacin is a widely used fluoroquinolone antibiotic that can also regulate microRNA and gene expression [83], and microRNA dysregulation signatures were found in C9ORF72 associated ALS and frontotemporal dementia (FTD) as well as in sporadic ALS [84,85]. Celecoxib is a selective COX-2 inhibitor and a nonsteroidal anti-inflammatory drug, which itself does not benefit ALS [86]. However, the preclinical study showed that the combined ciprofloxacin and celecoxib have a synergistic effect in promoting motor function [87]. In an open-label phase IIa study, Ciprofloxacin-celecoxib was administered three times daily to 15 ALS patients for 12 months. Four participants experienced mild to moderate, drug-related adverse events, including gastrointestinal, dizziness and insomnia. A phase IIb RCT study was started in April 2022 to evaluate its safety, tolerability, and efficacy (NCT05357950).

5.11. mTOR pathway inhibitors

The mammalian Target of Rapamycin (mTOR) pathway plays a critical role in stress granule formation in the diseased motor neurons, which then serves as a nidus for protein aggregation [88]. Rapamycin, an mTOR pathway inhibitor, is shown to promote autophagy and have immunomodulatory effects. In TDP43-ALS models, rapamycin reduced TDP43 accumulation in the cytoplasm, restored its nuclear localization, and ameliorated neurodegeneration [89,90]. However, conflicting data was reported in the SOD1-ALS model, suggesting rapamycin exacerbates motor neuron degeneration, possibly due to its off-target effects [91].

In a phase 2 RCT trial, two different doses of rapamycin 1 mg/m²/day, 2 mg/m²/day or placebo were tested in 63 ALS participants for 54 weeks; the primary outcome was Treg cell count. Though there was a trend of increasing Treg cells in the 1 mg/m²/day rapamycin treatment arm, this did not achieve statistical significance [92]. Frequent adverse events were reported in rapamycin treatment arms, which include skin and subcutaneous tissue disorders, gastrointestinal and respiratory disorders and headache. Further trial of rapamycin in ALS has not been announced. An alternative mTOR pathway inhibitor, monepantel, is currently under phase I trial in ALS (NCT04894240). Monepantel is a widely used veterinary drug and is under development by Pharmaust for cancer and ALS. Studies found that monepantel shows off-target activity, inhibiting the mTOR pathway and enhancing autophagy [93].

5.12. Ibudilast (MN-166)

Ibudilast is a phosphodiesterase pathway inhibitor that downregulates several proinflammatory factors, such as macrophage migration inhibitory factor and tumor necrosis factor α, and suppresses CNS microglia activation in preclinical studies [94,95]. It is an orally administered small molecular drug under development by MediciNova. During the 6-month, phase 2 RCT study (NCT02238626) and an open-label study in ALS [96], no severe adverse effects were directedly associated with Ibudilast 60 mg-100 mg per day. The common adverse events were GI disorders and fatigue. The phase 2b/3 12-month RCT to evaluate the efficacy and safety of Ibudilast was started in August 2019 and is actively enrolling patients (NCT 04057898).

5.13. Tauroursodeoxycholic acid

Tauroursodeoxycholic acid (TUDCA) is a hydrophilic bile acid that has been shown to inhibit cell apoptosis and provide neuroprotection [97] and is widely available as a dietary supplement. In the phase 2 RCT study, 34 ALS patients taking Riluzole were randomized to TUDCA (1 g twice daily) and placebo. In total, 87% of the TUDCA group were classified as responders compared to 43% of the placebo group (p = 0.021) [98]. The responders were defined as an improvement of at least 15% in the ALSFRS-R slope during the treatment period compared to the lead-in period. Mild diarrhea was reported in the TUDCA group. A phase 3 RCT trial (NCT03800524), sponsored by European Commission, was completed in October 2023. The primary outcome of this 18-month trial is to identify the treatment responders, which is defined as a minimum 20% improvement in the ALSFRS-R slope during the treatment period. The clinical trial results have yet to be released.

5.14. Antiretroviral therapy

Studies suggest that human endogenous retrovirus K (HERV-K) reactivation may contribute to ALS. A study has shown that 50% of ALS patients enrolled in the study had elevated retroviral enzyme reverse transcriptase activity in the serum, while only 7% of healthy control did [99]. Increased HERV-K expression has also been found in postmortem ALS patients’ brain tissue [100], but a separate study in a different ALS cohort did not confirm this finding [101]. In the completed phase 2a, 24-week open-label study of antiretroviral combination therapy (Abacavir 600 mg, lamivudine 300 mg, dolutegravir 50 mg), there were no treatment-related severe adverse events, and one patient withdrew due to elevated liver enzymes. The most common adverse events were upper respiratory tract infection, urinary tract infection, nausea, diarrhea and headache [102]. A phase 3 RCT study (NCT05193994), sponsored by Macquarie University, Australia was launched in 2022 and will be completed in 2026.

6. Potential development issues

Aside from its complex and ever-evolving disease mechanisms, ALS has a markedly variable clinical presentation, which poses a substantial hurdle for therapeutic development. Notably, both Edaravone and ultrahigh dose methylcobalamin failed the initial phase 3 RCT trials. They only succeeded in the second phase 3 trials in a more homogeneous early-stage disease population defined by stringent inclusion criteria. While careful selection of trial inclusion criteria has been helpful in finding approvable signals, it can have negative consequences from a standpoint of insurance approval and thus patient access. The prognostic and therapeutic biomarker research might facilitate the identification of the most suitable participants for specific products. Among them, the most studied is neurodegenerative biomarker NfL. Studies showed baseline NfL was more strongly correlated with ALSFRS-R changes than the pre-randomization slope [103]. SOD1 protein and NfL biomarkers have been successfully used in the development and final FDA approval of Tofersen [43]. However, NfL and ALSFRS-R changes do not always align with each other. In the CENTAUR trial, PB/TURSO slowed down ALSFRS-R decline but did not affect NfL level [40]. An urgent need exists to develop targeted biomarkers, aimed at individual ALS pathogenetic mechanisms, in order to improve therapeutic outcome measures.

7. Conclusion

ALS is a relentlessly progressive motor neuron disorder, defined by its convoluted pathophysiology and the lack of effective treatment. The last few years have seen a rapid uptaking of therapeutic development owing to the significant advances in uncovering the disease pathogenesis. In this context, many investigational products entered phase 2/3 RCT trials recently. These products will tackle a myriad of disease mechanisms: the deranged protein homeostasis and RNA metabolism; the chronic cellular stress response to neurodegeneration and its negative impact on protein translation; the dysregulation of CNS innate immune system and peripheral immune system leading to neuroinflammation. In order to match the burgeoning investigational products in the pipeline, ALS research community has adopted a new platform trial to build a long-term infrastructure and increase the efficiency of drug development. We remain optimistic that these collective efforts will bring more effective treatments of PALS.

8. Expert opinion

ALS has been a particularly challenging disease to develop treatments for because of its rarity, its different origins and pathophysiologies, its phenotypic heterogeneity, and a lack of sensitive and objective measures of disease progression. But gains are being made as it becomes more common, as we understand it better, and as we learn to do better trials. The current pipeline is larger and more diverse than ever before, with drugs targeting genetic and retroviral causes of ALS and drugs targeting a wide array of downstream pathways. Currently, we see no drug that universally stops or reverses ALS progression. Thus, we strive for ‘rational polypharmacy:’ to prescribe a collection of drugs with different mechanisms of action whose small individual effects we hope will add up to something larger. But insurance barriers currently limit our ability to do this for most patients.

We remain most excited about pipeline drugs that target potential direct causes of ALS. Indeed, Tofersen, the first of these, is the only drug we are aware of for which long-term treatment appears to result in stabilization or gains in strength for some patients [43]. Ataxin-2 ASO (BIIB105) is another antisense drug in development, and it potentially has a broader therapeutic impact in the ALS treatment landscape because of the synergistic effects of Ataxin-2 and TDP43 in ALS pathology and the preclinical data suggesting loss of function of Ataxin-2 leading to less TDP 43 protein aggregation. The challenges of this type of gene therapy are frequent intrathecal drug administration, which is technically difficult to set up and is associated with significant side effects such as elevated intracranial pressure which requires close monitoring. Compared to ASO, RNAi requires one or a few doses to achieve a sustained reduction of target gene expression. A phase I study of SOD1 RNAi (ARO-SOD1) (NCT05949294) is underway. Retrovirus has long been suggested to be a cause or trigger of ALS. The phase III trial of the widely used antiviral combination drug – abacavir, lamivudine and dolutegravir – in ALS may provide a definitive answer.

While it is difficult to know the order of the downstream events, we believe protein aggregation may be closer to the top. Notably, several promising products in the pipeline target TDP43, DRP or all abnormal protein aggregates in ALS, including BIIB105, AIT101 and mTOR pathway inhibitors.

Eventually, we will be able to rely on biomarkers that show us which downstream events are most important in an individual patient. For instance, an anti-neuroinflammation treatment will benefit patients who have pronounced microglia activation in CNS. In this sense, new PET tracers such as18F Hydroxyl Dendrimer are being developed to help select patients for clinical trials and eventually for clinical use.

Article highlights

• Amyotrophic lateral sclerosis is a rapidly disabling, life-shortening neurodegenerative disease.

• We now understand some of the causes and some of the downstream pathways driving progression.

• We have a small number of diseae modifying drugs approved for use.

• A large number of drugs with diverse mechanisms are now in the research pipeline.

• Sorting throught hese will be facilitated by biomarkers such as neurofilmaent lightchain, by more efficient trial designs and by working with patient-partners.

• In the future we anticipate being able to attack the causes of ALS in more patients with new therapies.

• For those in whom we cannot find the cause, we hope to be able to combine drugs with different mechansisms but insurance barriers may limit this strategy.

Declaration of interest

R Bedlack has research support from ALS Association, Healey Center and Medicinova. He has other financial support from AB Science, American Institute of Biological Sciences, Amylyx, Annexon, Betty’s Brigade, Biogen, Carespace, Clene Nanomedicine, Eikonoclastes, Eisenhower Medical Center, General Dynamics, GenieUS, Guidepoint Global, Kaplan, MJH Life Sciences, Neuroscense, Novartis, PTC Therapeutics, Prime Education, Roon, Springer Publishing, State of Maryland, CM Group, Uniqure and Webb MD. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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Funding

This paper was not funded.