A perspective on therapies for amyotrophic lateral sclerosis: can disease progression be curbed?

The mammalian target of rapamycin (mTOR) pathway is the best-characterized regulator of autophagy initiation. Rapamycin, a widely used autophagy enhancer by inhibiting the mTOR pathway, has presented controversial effects in different genetic animal models of ALS, which limits its practical use in ALS treatment [74‐76].

Trehalose is a natural disaccharide that induces mTOR-independent autophagy. Administration of trehalose has been shown to prolong the lifespan and delay the disease onset in SOD1^G86R mice, accompanied by reduced SOD1 accumulation and enhanced MN survival in the spinal cord [77]. Similar therapeutic effects have been demonstrated in a SOD1^G93A mouse model of ALS [78], but the efficacy diminishes during later stages of the disease [79].

Although effective in delaying the disease onset and extending the lifespan in different ALS mouse models by regulating the mTOR-independent pathway [80‐82], lithium failed to show therapeutic benefits in ALS patients in previous clinical trials [83‐87]. However, a recent meta-analysis on the different responses between genotypes has found that the treatment effect of lithium is not homogenous across patients. Patients carrying UNC13A (protein unc-13 homolog A) mutation could benefit more from lithium treatment than C9orf72 mutation carriers, with 12-month survival probability improving from 40.1% to 69.7%. This result provides a new insight for future clinical trials and suggests that we should start focusing on genotype-targeted therapies and standardize genotyping due to the heterogeneity of ALS [88].

N-butylidenephthalide is the main component of a traditional Chinese medicine Danggui. It can regulate autophagy by mediating ER stress [89]. Oral administration of 250 mg/kg (bid) n-butylidenephthalide before the onset of the disease has shown a better effect on survival than riluzole in an ALS mouse model [90]. Furthermore, administration of n-butylidenephthalide could decrease MN loss and restore the gastrocnemius function of SOD1^G93A mice. These neuroprotective effects may be associated with the inhibition of autophagy through the Akt/mTOR signaling pathway, and anti-apoptosis, anti-inflammation and antioxidative effects [91].

Carbamazepine is a well-known anti-epileptic drug that has been reported to stimulate autophagy by decreasing the intracellular level of inositol [92]. Carbamazepine could activate autophagy via the mTOR-independent pathway and rescue the motor dysfunction of frontotemporal lobar dementia (FTLD-U) mice with TDP-43 proteinopathies [75]. In SOD1^G93A mice, oral administration of carbamazepine (200 mg/kg per day) could delay the disease onset and significantly extend survival. In addition, carbamazepine increases the clearance of mutant SOD1 aggregates via the AMPK-ULK1 pathway, which plays a protective role in the preservation of MNs [93].

W. somnifera is a perennial herb containing the component Withaferin A which has been reported to have a beneficial effect in both TDP-43 and SOD1^G93A mouse models [94, 95] and increase autophagosomes [96]. Recent studies have proven that W. somnifera has the same effect as Withaferin A and can serve as an autophagy-inducer [97, 98].

Previous studies have demonstrated that the elevated level of cytosolic Ca²⁺ in ALS MNs is associated with autophagy regulation in a mTOR-independent manner. Verapamil is a L-type Ca²⁺ channel blocker used clinically for cardiovascular diseases. It can activate autophagy and improve autophagy influx by reducing intracellular Ca²⁺. In the SOD1^G93A mouse model, intraperitoneal injection with verapamil (25 mg/kg per day) could delay the disease onset and extend survival by improving autophagy influx and reducing SOD1 aggregation [99].

Stress granules play an important role in regulating the formation of insoluble aggregates in pathological conditions [100]. Recently, Torkinib, a selective mTOR inhibitor, has been demonstrated to reverse the pathological changes of stress granules in induced pluripotent stem cell (iPSC)-derived neurons with P525L FUS mutation, which is one of the most severe mutations of ALS [101].

Bosutinib and ropinirole are two candidate anti-ALS drugs recently identified in iPSC-based drug screens and are now under clinical investigation [102]. Bosutinib, an inhibitor of Src/c-Abl, has been found to increase the survival of ALS iPSC-derived MNs by inducing autophagy and reducing misfolded SOD1 and TDP-43 proteins. Furthermore, bosutinib has been shown to delay disease onset and prolong survival of SOD1-mutant mice [103]. A 12-week phase I dose-escalation trial has been initiated in ALS subjects (UMIN000036295) to evaluate the safety and tolerability of bosutinib at 4 levels (100, 200, 300 or 400 mg/day) [20]. Ropinirole, an agonist of dopamine D2 and D3 receptor (D2R/D3R), has shown a mitochondrion-targeted antioxidant effect in iPSC-derived ALS MNs. Recent reports have demonstrated that agonists of D2R/D3R might promote autophagy through a Beclin-1-dependent pathway [102]. It would be interesting to determine whether the ropinirole-induced D2R/D3R activation leads to degradation of abnormal RNA–protein complexes via autophagy activation in ALS MNs.

Pathologic TDP-43 accumulation is one of the typical pathophysiological manifestations in ALS. Tamoxifen has been validated to enhance autophagy through the mTOR-dependent pathway by inhibiting AKT/PKB and increase the clearance of TDP-43 aggregates. A small phase II clinical trial initiated to explore the efficacy of tamoxifen in ALS patients has been completed in Taipei (NCT02166944). Among 18 patients, 10 of them were randomly assigned to receive tamoxifen treatment (40 mg daily for 1 year). According to the result from the first 6-month follow-up, a slower decline rate of ALSFRS-R score was observed in the tamoxifen group, though there was no difference in the ALSFRS-R score between the two groups at the 12-month follow-up. This outcome demonstrated a modest effect of tamoxifen in slowing progression during 6-month follow-up. Given the small-scale of this trial, larger-scale trials are required for a robust conclusion on tamoxifen efficacy [21].

HDAC6 plays an essential role in the regulation of mutant SOD1 aggregation [104, 105] and controls the fusion of autophagosomes to lysosomes [106]. While some studies have demonstrated detrimental effects associated with HDAC6 inhibition, a recent study showed that overexpression of HDAC6 in ALS mice could prolong the lifespan by inducing the formation of autolysosomes and the degradation of mutant SOD1 protein aggregates [107]. In another study, genetic ablation of HDAC6 increases the number of remaining neurons in the ventral horn of the spinal cord, along with a significant improvement in the survival of SOD^G93A mice [108].

In a recent study, clinical-grade human NSCs (hNSCs) were bilaterally transplanted into the anterior horns of the lumbar spinal cord of SOD1 mutant rats (4 sites, L3–4 segment), and 40 days after the transplantation, the hNSCs integrated extensively within the cord, presented with neural phenotypes, and migrated from the injection site for 3.77 ± 0.63 cm. More importantly, the transplantation delayed the decrease of body weight and the deterioration of motor performance, reduced the level of astroglial and microglial activation, and increased the density of MNs [112]. In another study, transplantation of hNSCs engineered to secrete glial cell line-derived GDNF into the SOD1^G93A ALS rat cortex induced a delay of disease pathology and prolongation of survival without adverse effects. This demonstrates that the motor cortex could also be a transplant site in addition to the spinal cord [113]. Previous clinical studies have confirmed the safety and feasibility of intrathecal NSC transplantation [22‐24]. The combined lumbar (L2–L4) and cervical cord (C3–C5) injections at 20 sites (400,000 cells/injection) have proven to be safe and well-tolerated in 15 participants without acceleration of the disease progression, which indicates that intrathecal transplantation of hNSCs can be safely accomplished at high doses (NCT01730716) [24]. Consistently, a recent phase I clinical trial (NCT01640067) using a highly standardized cell drug product has obtained the same results and underscored good reproducibility and homogeneity of stable hNSCs lines. The potential therapeutic effects of hNSCs have provided sufficient promise for future phase II trials, which are currently in preparation by the same group [25]. Once the therapeutic effect and safety of this standardized cell drug product are firmly verified, it is promising to address thorny issues related to ethics and source restrictions in the future.

MSCs can be obtained from bone marrow, peripheral blood, umbilical cord blood and adipose tissue. The relative ease of in vivo harvesting and expansion of patient-derived MSCs has allowed their wide application by cell transplantation in studies of neurologic diseases with less risk of rejection and fewer ethical issues [112]. Multiple intracerebroventricular transplantation of MSCs (250,000 hUC-MSCs resuspended in 8 μl of sterile PBS) isolated from the human umbilical cord protected MNs but had no beneficial effects on the survival of ALS mice [114]. The safety and feasibility of MSC transplantation in the central nervous system have been demonstrated by a phase I clinical trial. In that trial, expanded autologous MSCs suspended in the autologous CSF were injected into the thoracic spinal cord. After transplantation, no structural or pathologic change was observed in the central nervous system (CNS) as revealed by MRI scanning [115]. Further phase I and II clinical studies have been conducted to investigate the safety of repeated intrathecal injection of MSCs from human bone marrow. Results have shown increased TGF-β and IL-10 as well as reduced MCP-1 expression, which are related to MN injury, suggesting that intrathecal injection of MSCs may modulate immunoinflammation in ALS patients [26, 27]. In the phase II study (NCT01363401), two repeated treatments with intrathecal autologous bone marrow-derived MSCs (1 × 10⁶ cells/kg with a 26-day interval) showed significant therapeutic benefits with safety in ALS patients [27]. MSCs can be induced to offer enhanced secretion of neurotrophic factors (GDNF, BDNF, VEGF), and the treated MSCs were developed as a cell product called MSC-NTF or NurOwn by BrainStorm Cell Therapeutics in 2007 [116]. In a phase I/II and a IIa clinical trials (NCT01051882 and NCT01777646), intramuscular or intrathecal administration of NurOwn is safe and well-tolerated. Furthermore, possible clinical benefits were observed in patients with intrathecal administration, or combined intrathecal and intramuscular administration [28]. Recently, a phase II RCT (NCT02017912) tested the single-dose transplantation of MSC-NTF cells, and found a higher proportion of treated participants (combined intrathecal and intramuscular administration) with ≥1.5 points/month ALSFRS-R slope improvement, increased neurotrophic factors and decreased inflammatory biomarkers after transplantation, in addition to a good safety, suggesting promising efficacy of NurOwn transplantation [29]. Another phase I/II clinical trial (NCT02286011) selected the tibialis anterior muscle as a transplant site, and found that single injection of BM-MSCs (ranging 206 × 10⁶–1086 × 10⁶ cells) was safe and caused a higher D50 index, a parameter used to quantify the compound muscle action potential scan curve which decreases with disease severity [30]. As muscle is predominantly affected in ALS, retarding the progressive loss of motor units and denervation atrophy may improve the functional outcome or survival. Therefore, direct intramuscular implantation combined with intrathecal injection may be more effective and need to be assessed in future trials.

Respiratory dysfunction is the most common cause of death in ALS patients [3, 6]. Numerous microhemorrhages caused by microvasculature impairment have been observed in SOD1^G93A mice at the late stage, which may cause respiratory complications in ALS [117]. Microvasculature impairment is relevant to the damage of microvessel endothelial cells. In SOD1^G93A mice with hemorrhagic damage in the lung, intravenous transplantation of BM-MSCs attenuates endothelium damage through the re-establishment of vascular integrity by BM-MSCs. The transplanted cells could also release VEGF, angiogenin, and vesicles to promote angiogenesis in the lung and mediate intercellular communication [117].

Neuroinflammation characterized by activation of neuroglia cells and infiltration of peripheral monocytes and lymphocytes is increasingly being recognized as a key pathogenic feature of ALS [118]. Regulatory T lymphocytes (Tregs) are a subpopulation of immunosuppressive T lymphocytes, and their dysfunction has been demonstrated in ALS patients [119, 120]. HSCs are multipotent cells that have the potential to differentiate into all mature cell types in blood, and the transplantation of HSCs has been proven to suppress inflammation and modulate immune response [116, 121]. HSC transplantation has been used for clinical treatment for a long time, and the ease of collection from peripheral blood or bone marrow, together with the non-invasive nature of administration, has made it a reliable treatment for ALS [116, 122]. Transplantation of bone marrow which could reconstitute Tregs has exerted positive effects on survival in an ALS mouse model by modulating the trophic/cytotoxic balance of glia [123]. Recently, a phase I, first-in-human study (NCT03241784) found that autologous infusion of expanded Tregs was safe and well-tolerated in all three patients. After a total of 8 infusions of expanded autologous Tregs (1 × 10⁶ cells/kg) with concomitant subcutaneous IL-2 injections (once a week, 2 × 10⁵ IU/m²) that help enhance the proliferation and function of infused Tregs, slowing of progression rate was observed at both early and later stages of the disease. Meanwhile, the increased suppressive function of Tregs showed a positive correlation with the slowing of clinical progression, supporting the value of Tregs suppressive function as an indicator of clinical status [31].

Despite the numerous preclinical and clinical studies demonstrating safety and therapeutic benefits in ALS patients and animal models, large, prospective RCTs with long-term follow-up are still needed to identify the safety, efficacy and optimal dose of these therapies [124, 125].

ASOs are short oligonucleotide sequences that selectively target and bind mRNA to interfere with its processing or transduction in many different ways, thereby preventing or modifying the expression of toxic protein. ASOs can induce cleavage of RNA by activating endogenous, intranuclear RNase H or preventing the interaction with specific RNA-binding proteins, thereby modulating its splicing [127]. In 2016, nusinersen (Spinraza) for spinal muscular atrophy became the first FDA-approved ASO-based therapy for neurodegenerative diseases [128]. In a phase I trial (NCT01041222), an ASO designed to inhibit SOD1 expression (ISIS 333611) was delivered by a single intrathecal infusion (L3–4) for 11 h and 22 min using an infusion pump at increasing doses (0.15 mg, 0.5 mg, 1.5 mg, 3 mg), and results showed that it was safe and well-tolerated [32]. ASOs can also effectively decrease the expression of SOD1 mRNA and protein in the CSF and brain of SOD1^G93A rat model [129]. Recently, a single dose of next-generation SOD1 ASOs has shown a positive effect on the survival of SOD1^G93A rats and mice, which was prolonged by 50 days and 40 days, respectively. Furthermore, it turned out that the initial loss of compound muscle action potential could also be reversed in SOD1^G93A mice [130]. Hexanucleotide expansions in C9ORF72 are a common genetic cause for ALS and frontotemporal dementia (FTD). Previous studies have shown that ASOs could suppress RNA foci formation and gene expression and abrogate the C9ORF72 RNA expansion-dependent pathology, the aggregation of RNA-binding proteins and the glutamate-mediated excitotoxicity in fibroblasts and human-derived iPSC neurons [127, 131, 132]. In C9ORF72 transgenic mice, cerebroventricular injection of single-dose ASOs could reduce RNA foci and dipeptide-repeat proteins, and ameliorate behavioral deficits [133]. Ataxin 2 gene is also associated with the risk of ALS. Administration of ASOs targeting ataxin 2 in the CNS of TDP-43 mice could greatly extend the survival and improve the motor performance of the mice [134].

The ASO-based therapy has shown great potential in preclinical studies. More recently, a phase I-II ascending-dose trial (NCT02623699) was conducted to evaluate the safety, pharmacokinetics and pharmacodynamics of tofersen in ALS cases carrying SOD1 mutation [33]. Tofersen is an ASO that reduces SOD1 protein synthesis by mediating the degradation of SOD1 mRNA. In that trial, 48 participants were randomly assigned to receive different doses of tofersen (20 mg, 40 mg, 60 mg, or 100 mg) or placebo by lumbar intrathecal bolus injection on days 1, 15, 29, 57, and 85. The total SOD1 protein concentration in the CSF was reduced in the four dose cohorts. The change of CSF SOD1 concentration at day 85 from that at baseline in the tofersen groups differed from the placebo group by 2%, − 25%, − 19%, and − 33%, respectively. Moreover, in the fast-progression subgroup, the ALSFRS-R score of the 100 mg tofersen group seemed to decrease slower than the placebo group [33]. For further investigation of the safety and efficacy of tofersen, a phase III, randomized, double-blind, placebo-controlled trial (NCT02623699) and its long-term extension study (NCT03070119) are currently underway.

RNAi is another approach against RNA-mediated gain of toxicity, in which long double-stranded RNA duplexes are first processed within the cell and loaded into an RNA-induced silencing complex (RISC). The binding of RISC and the targeted cellular mRNA will then silence the mRNA by RNase-mediated degradation or translational repression [135]. RNAi can be induced by small RNA duplexes, and the three most common duplexes are artificial microRNA (miRNA), short interfering RNAs, and short hairpin RNAs (shRNAs) [135]. Due to the blood-brain barrier (BBB), viral vectors derived from lentivirus and adeno-associated virus (AAV) are used for therapeutic gene delivery [136]. In an animal study, neonatal SOD1^G93A mice receiving injection of a single-stranded AAV9 vector encoding an artificial mRNA against human SOD1 in the cerebral lateral ventricles showed improvements in multiple parameters including survival, number of MNs, the extent of neuroinflammation, diameter of ventral root axons and pulmonary function [137]. Similar results have been demonstrated in another study [138]. A recent study has demonstrated that the AAV5-delivered artificial miRNAs targeting C9orf72 could reduce the accumulation of repeat-containing C9orf72 transcripts in both iPSC-derived neurons and ALS mouse model [139, 140]. Although an allele-specific silencing effect has been observed through intrathecal or intraventricular delivery of the vector, there are still some limitations in the delivery method. To improve the penetrability of the viral delivery vectors, high titers are required, which limits the feasibility, effectiveness, and safety of this gene silencing therapy. An excellent work using a spinal subpial method to deliver AAV9-mediated shRNA targeting SOD1 has provided a potent approach for transduction of neurons and surrounding glia and showed higher efficiency in reducing SOD1^G37R-encoding mRNA than the intrathecal injection method [141]. Additionally, the spinal subpial delivery method shows an impressive effect in delaying disease onset and stopping disease progression in SOD1^G37R mice [141]. Therefore, seeking appropriate delivery methods should also be viewed as an important part of future studies.

The safety and efficacy of RNAi-based therapies has been tested predominantly in preclinical models of ALS. Recently, two ALS patients with SOD1 mutation were treated with a single intrathecal infusion of AAV encoding a miRNA targeting SOD1 [142]. Unfortunately, patient 1 died from respiratory failure 15.6 months after the initiation of treatment. Autopsy results of patient 1 revealed lower SOD1 levels in the spinal cord than in the untreated patients. In addition, transient and slight reduction of SOD1 levels in the CSF was observed in patient 1. Although having no change in the CSF SOD1 level, the patient 2 had stable functional status and vital capacity for 12 months [142]. In general, this study demonstrated that intrathecal miRNA might be a promising treatment for ALS. However, due to the heterogeneity of ALS and the insufficient sample size, confirmative conclusions on treatment effects could not be drawn.

The CRISPR/Cas9 genome editing system is emerging as a promising tool to disrupt the expression of mutant genes at the genomic level. CRISPR/Cas9 is a type II CRISPR/Cas system with guide RNA to target specific DNA sequences and with Cas9 as a nuclease [143]. CRISPR/Cas9 has already been successfully used to establish gene-corrected ALS iPSCs [144]. Moreover, when delivered by the AAV vector in SOD1^G93A mice, CRISPR/Cas9 could disrupt expression of mutant SOD1 and reduce the protein level of mutant SOD1 in the spinal cord, accompanied by prolonged survival and improved motor function [126, 143]. RNA-targeting Cas9 has been reported to be able to reduce RNA foci and polyglutamine protein products in the C9orf72-linked ALS patient cells [145]. In the C9orf72-linked ALS cell line, transcriptional inhibition mediated by deactivated Cas9 could rescue the splicing defects and block the repeat-associated non-ATG translation to reduce toxic dipeptide polymers [146]. In addition, increased expression of the GluA1 AMPA receptor (AMPAR) subunit observed in iPSC-derived MNs with C9ORF72 mutations, but not in iPSC-derived cortical neurons, is rescued by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs, together with the abolishment of increased Ca²⁺-permeable AMPAR expression and MN vulnerability to excitotoxicity caused by the increased GluA1 subunit [130]. Andrade et al. have reported that dipeptide repeat proteins (DPRs) encoded by C9ORF72 through a non-canonical translation mechanism are neurotoxic and can increase the frequency of DNA double-strand breaks (DSBs) by inhibiting the key DNA DSB repair pathways. It is noteworthy that the accumulation of DNA DSBs is reduced and the dysregulation of single-strand annealing is improved after deletion of C9ORF72 expansion by CRISPR/Cas9 in C9ALS iPSC lines [147]. These results indicate that genome editing is the best approach to correcting disease-causing mutations. However, the safety, target specificity, immunogenicity as well as ethical concerns need to be addressed before translation to clinical use.

ALS is a lethal neurodegenerative disorder without effective treatment. Heritability research has demonstrated that about 60% of the risk of ALS is genetically determined [9, 38] and mutations in more than 25 genes have been found in patients with or without a family history [148, 149]. In recent years, the development of gene therapy techniques and clinical application of the nusinersen-based therapy targeting spinal muscular atrophy have demonstrated great potentials of gene therapies in treatment of neurological disorders, suggesting that the gene editing approach may hold promise for ALS treatment. To date, applications of gene therapies in ALS have mainly focused on C9ORF72 correction. C9ORF72 repeat expansion is identified as the most common genetic cause for ALS and FTD. Abnormal translation of the expanded repeat will result in loss of function, RNA toxicity, and DPR protein toxicity that are implicated in pathogenesis, thus gene-editing techniques targeting causal mutations to cut repeat expansions, inhibit transcription, or selectively reduce the repeat-containing RNAs might be promising in retarding disease progression. The effectiveness of the ASO and RNAi therapies in reducing RNA foci has been observed in vitro and in vivo [127, 131, 139, 140]. Based on these results, a phase I clinical trial of ASOs targeting the sense strand of C9ORF72 in C9 ALS patients has been conducted (NCT03626012). In addition, genetic correction of C9ORF72 repeat expansions has been seen in patient iPSCs [130, 147]. These encouraging results suggest that gene editing is a promising tool for treating ALS patients carrying a single gene mutation. However, previous reports of oligogenic inheritance in ALS indicate that the early-onset patients may carry more than one ALS gene mutation [150]. In this situation, the effect of gene therapies targeting an individual mutation might become suboptimal. More importantly, the genetic heterogeneity of ALS and the limited numbers of identified genes in sALS patients make the personalized gene therapy more challenging and it is impossible to design clinical trials targeting each specific gene. However, the genetic mutations can be categorized into several subgroups that share a common mechanism of pathology, providing an opportunity for therapies. On the other hand, in the loss-of-function ALS subgroups, gene delivery strategies aiming to normalize the function of the mutated protein may be within reach. In addition, safety issues such as off-target effects and inactivation of the normal gene copy remain to be solved before clinical use. Currently, there is no effective treatment for ALS, and the inspiring results from ASOs and RNAi-based therapies in both preclinical and clinical studies are raising hope for ALS treatment.

Mito Q is an antioxidant that has been shown to mitigate oxidative damage in neurodegenerative disease models. Mito Q can accumulate within mitochondria and improve mitochondrial function in different neuronal cells exposed to oxidative stress [151]. Mitochondrial dysfunction is involved in the pathogenesis of ALS, and impaired mitochondrial function has been observed in the SOD1 mouse model. Previous studies have shown that pretreatment of SOD1^G93A astrocytes with Mito Q prevents the toxicity of mutant SOD1 and improves ATP generation in MNs [39, 152]. Moreover, Mito Q orally administered (500 μM) in SOD1^G93A mice since the presentation of early symptoms significantly preserves the neuromuscular junctions and increases the strength of hindlimbs by improving mitochondrial function and decreasing nitroxidative damage in the lumbar spinal cord and the quadriceps muscle. The extension of lifespan suggests that administration of Mito Q may slow the disease process [153].

Methylcobalamin, physiologically equivalent to vitamin B12, shows a protective effect against glutamate-induced cytotoxicity in cultured cortical neurons [154]. Multivitamin therapy with vitamin B12 and folic acid significantly delays disease onset and prolongs the average lifespan of SOD1^G93A transgenic mice [155]. In addition, a retard in the progression of motor symptoms and neuropathological changes has been observed in wobbler mice after intraperitoneal treatment with high-dose methylcobalamin [156]. In a recently reported phase II/III clinical trial (NCT00444613) on the safety and efficacy of intramuscular ultra-high-dose methylcobalamin in ALS patients, primary endpoints of survival and ALSFRS-R change both failed to show a difference between methylcobalamin (25 and 50 mg) and placebo groups. However, post-hoc analyses demonstrated that 50 mg methylcobalamin prolonged survival and retarded symptomatic progression when administered at an early stage (≤12 months after symptom onset) [34].

Rasagiline, a monoamine oxidase B inhibitor with a symptomatic efficacy in Parkinson’s disease, prolongs survival in preclinical animal studies of ALS, both alone and in combination with riluzole [157]. In a small US study with 80 ALS patients and 177 historical placebo controls, rasagiline (2 mg/day) alone neither altered disease progression nor showed evidence for biomarker engagement [35]. Albert Ludolph and colleagues conducted a phase II RCT (NCT01879241) of rasagiline (1 mg/day) as an add-on treatment to riluzole in 252 ALS patients. No difference in the primary outcome of survival was observed between the rasagiline and placebo groups. However, post-hoc stratifications revealed a possible survival benefit and slower functional decline in a subset of patients with an initial slope of ALSFRS-R greater than 0.5 points per month [36].

Masitinib, a targeted anticancer drug, has shown therapeutic potential in SOD1^G93A mice via its immunomodulatory properties targeting microglia as well as macrophage activity in both CNS and peripheral nervous system. The efficacy of masitinib as an add-on therapy to riluzole has been assessed in a phase II/III clinical trial over a 48-week treatment period (NCT02588677), in which 394 ALS patients were randomly assigned to receive riluzole (100 mg/day) plus placebo or masitinib (3.5 or 4.5 mg/kg per day). Compared with the placebo group, riluzole plus masitinib (4.5 mg/kg per day) showed a significant 27% slowing in rate of functional decline [37]. Significant results were also found for secondary endpoints such as ALSAQ-40, FVC and time-to-event analysis [37].

AMX0035, a combination of sodium phenylbutyrate and taurursodiol, was designed to attenuate neuronal death by mitigating ER stress and bioenergetic dysfunction. In a multicenter, phase 2, placebo-controlled trial (CENTAUR) that evaluated the efficacy of oral AMX0035 in ALS patients, there was a significant difference of 0.42 points per month between the active-drug group and the placebo group in the mean rate of change in the ALSFRS-R total score over 6 months, representing an approximately 25% slowing of disease progression [158]. In the open-label extension of CENTAUR, random assignment to AMX0035 at baseline resulted in a 6.5-month survival advantage compared with the placebo assignment [159].