**3. Other neuroprotective strategies**

**FDAapproved DMDs**

Teriflunomide (Aubagio)

Dimethyl fumarate (Tecfidera)

Alemtuzumab (Lemtrada)

**Indication Primary**

First-line therapy for RR-MS

286 Trending Topics in Multiple Sclerosis

First line therapy for RR-MS

Second line therapy for RR-MS

**mechanisms of action**

Inhibits the activation and proliferation of stimulated lymphocytes

Reduce transendothelial migration of activated leukocytes

Lymphocyte B and T depletion; decrease of pro-inflammatory

cytokines

**Table 1.** The neuroprotective effects of FDA-approved DMD.

**Neuroprotective effects—results from basic research studies**

Antioxidative effects by activation of

Nrf2 [19, 20]

effects Induction of neurotrophin producing lymphocytes Preservation of axonal conductance Stabilizes blood–brain barrier [21]

Abbreviations: LTi―lymphoid tissue inducer; IGF-2—insulin growth factor; MPB—myelin-basic protein;

BDNF―brain-derived neurotrophic factor; GDNF—glial cell-derived nerve factor; Nrf2—nuclear factor erythroid 2 related factor; BBB—blood–brain barrier, RR-MS―relapse remitting MS; SP-MM—secondary progressive MS, CIS clinical-isolated syndrome; PML—progressive multifocal leukoencephalopathy; LTi—lymphoid tissue inducer.

In addition to the currently FDA-approved DMDs, some promising new agents are already in ongoing late-phase clinical trials, such as laquinomid, ozanimod, ponesimod, siponimod, ocrelizumab, ofatumumab, masitinib, and cladribine. Few data related to the mechanisms of action of these drugs are currently available. Of these compounds, laquinimod is the only one that appears to have neuroprotective properties, and laquinimod is currently being tested in patients with RR-MS in a third phase III trial, CONCERTO [23]. Basic research studies suggest that in addition to its neuromodulatory and anti-inflammatory effects, laquinimod also displays neuroprotective effects through several mechanisms, including reducing excitotox‐

Anti-inflammatory

**Neuroprotective effects—results from clinical research studies**

– – Hepatotoxicity,

It significantly decreases the T2 weighted lesion burden compared to IFNβ [22]

**Severe adverse effects**

peripheral neuropathy, hyperkalemia, transient acute renal failure, severe skin

reactions

Infusion-associated reactions, infections, auto-immunity

– Lymphopenia

In addition to DMDs, there are many additional potential neuroprotective agents, including ion channel modulators, glutamate antagonists, growth factors, sex hormones, statins, and immunophilin ligands. Most of these were tested only in experimental studies as a means to target molecular pathways involved in neurodegeneration or, in contrast, to stimulate endogenous defense mechanisms. There is increasing interest in pleiotropic molecules such as 5-HTR3 antagonists [27], polymerized nano-curcumin [28], and tyrphostin AG 126 [29]; in molecules that can modulate the kynurenine pathway [30]; in cannabinoid compounds [31– 33]; and in combination therapies of DMDs with pleiotropic molecules.

One of the factors that contributes to the persistence of inflammation in MS is sustained activation of the transcription of nuclear factor kappa B (NFκB), which is an important hub for several molecular mechanisms involved in apoptosis and in immune and inflammatory responses. Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-responsive protein that binds the p65 unit of NFκB and thus can reduce the immuno-inflammatory response. In cell cultures, a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ suppressed NFκB activation and prevented glutamate neurotoxicity [34]. Additionally, in an in vitro study, intraperitoneal administration of GILZ-P modulated the Th1/Th2 balance and ameliorated the symptomatology of experimental autoimmune encephalomyelitis (EAE) [35]. The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is another signaling molecule that triggers lymphocyte activation through NFκB signaling and also acts as a cysteine protease. To test the hypothesis that MALT1 inhibitors could be used to treat lymphocyte-mediated pathologies, the therapeutic potential of mepazine (a recently identified MALT1 inhibitor) was studied in mice with EAE. When mepazine was prophylactically administered, it significantly reduced clinical disease symptoms and histo‐ pathological parameters. Moreover, its therapeutic administration clearly promotes disease remission [36].

The nuclear receptor-related 1 protein (Nurr1) is a member of the class of steroid nuclear hormone receptors, and its activity is significantly downregulated in neurodegenerative disorders such as MS; its levels are also negatively correlated with EDSS progression. In mice with EAE, the administration of isoxazolo-pyridinone, an activator of the Nurr1 signaling pathway, delays EAE onset and reduces its severity. Therapeutic administration of isoxazolopyridinone also reduced neuro-inflammatory and histopathological alterations in the spinal cord but not the course of EAE [37].

KV1.3, the third member of the shaker-related subfamily of voltage-gated potassium channels, is known to modulate calcium signaling to induce T cell proliferation (effector memory T cells —TEM), immune system activation and cytokine production. Toxins derived from animal venoms can target ion channels, including KV1.3, and offer a means to diminish the activation and proliferation of TEM cells and to improve of the pathology underlying autoimmune diseases. For example, in a rat acute EAE model, ADWX-1, an analog of scorpion toxin, reduced the number of T cells and the secretion of inflammatory factors. These toxic peptides could be used to obtain better clinical results without neurological impairment [38]. There is increasing interest in bee venom therapy, which experimental studies have shown can ameliorate the symptomatology of EAE by decreasing inflammation and demyelination [39]. However, additional clinical evidence is needed.

The mitochondrial permeability transition pore (PT pore) is a drug target for neurodegenera‐ tive conditions and for ischemia-reperfusion injury. Cyclophilin D (CypD) is a positive regulator of the pore, and its downregulation improves outcomes in animal models of stroke. However, this isomerase is not selective and may have toxic effects. A new synthesized mitochondria-targeting CypD inhibitor, JW47, displayed selective cellular inhibition and reduced cellular toxicity. In an EAE model, JW47 significantly protected axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition could become a viable therapeutic strategy for MS [40].

Granzyme B (GrB) is a serine protease released from the granules of cytotoxic T cells, which can induce cell death by disrupting a variety of intra/extracellular protein substrates. GrBexpressing T cells were identified in close proximity to oligodendrocytes and demyelinating axons in acute MS lesions and were thus associated with neuronal loss. The GrB inhibitor serpina3n, which was isolated from mouse Sertoli cells, can inhibit the enzymatic activity of this protease. The administration of serpina3n attenuated disease severity in an animal model of MS by reducing T cell-mediated neuronal death and axonal injury. These observations suggest that serpina3n could be used to decrease inflammation-mediated neurodegeneration [41].

Experimental studies have shown that fasudil—an inhibitor of Rho kinase (ROCK)—can suppress experimental EAE when administered via multiple, short-term injections. Later, a novel ROCK inhibitor that can be delivered intranasally was developed. This inhibitor, FSD-C10, reaches the CNS faster and in a much lower dose. FSD-C10 reduced EAE severity and CNS inflammatory infiltration and promoted neuroprotection by inducing CNS production of IL-10, NGF, and BDNF and by inhibiting the production of multiple pro-inflammatory cytokines [42].

Eriocalyxin B (EriB) is a diterpenoid extracted from Isodon eriocalyx, a perennial herb from southwest China that is used as an anti-inflammatory remedy in traditional Chinese medicine. EriB has been reported to induce apoptosis in leukemia and lymphoma by elevating the intracellular levels of reactive oxygen species and by suppressing the NFκB pathway. In an EAE model, EriB alleviated symptoms, delayed disease onset, decreased T cell populations, inhibited the NFκB pathway and reduced CNS inflammation and demyelination, improving the course of the disease [43]. Adenanthin, which is also a diterpenoid isolated from the leaves of Isodon adenanthus, displays preventive and therapeutic effects in EAE, as demonstrated by improved clinical scores as well as by reduced infiltration of inflammatory cells and demyelination in the CNS [44, 45].

—TEM), immune system activation and cytokine production. Toxins derived from animal venoms can target ion channels, including KV1.3, and offer a means to diminish the activation and proliferation of TEM cells and to improve of the pathology underlying autoimmune diseases. For example, in a rat acute EAE model, ADWX-1, an analog of scorpion toxin, reduced the number of T cells and the secretion of inflammatory factors. These toxic peptides could be used to obtain better clinical results without neurological impairment [38]. There is increasing interest in bee venom therapy, which experimental studies have shown can ameliorate the symptomatology of EAE by decreasing inflammation and demyelination [39]. However,

The mitochondrial permeability transition pore (PT pore) is a drug target for neurodegenera‐ tive conditions and for ischemia-reperfusion injury. Cyclophilin D (CypD) is a positive regulator of the pore, and its downregulation improves outcomes in animal models of stroke. However, this isomerase is not selective and may have toxic effects. A new synthesized mitochondria-targeting CypD inhibitor, JW47, displayed selective cellular inhibition and reduced cellular toxicity. In an EAE model, JW47 significantly protected axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective

Granzyme B (GrB) is a serine protease released from the granules of cytotoxic T cells, which can induce cell death by disrupting a variety of intra/extracellular protein substrates. GrBexpressing T cells were identified in close proximity to oligodendrocytes and demyelinating axons in acute MS lesions and were thus associated with neuronal loss. The GrB inhibitor serpina3n, which was isolated from mouse Sertoli cells, can inhibit the enzymatic activity of this protease. The administration of serpina3n attenuated disease severity in an animal model of MS by reducing T cell-mediated neuronal death and axonal injury. These observations suggest that serpina3n could be used to decrease inflammation-mediated neurodegeneration

Experimental studies have shown that fasudil—an inhibitor of Rho kinase (ROCK)—can suppress experimental EAE when administered via multiple, short-term injections. Later, a novel ROCK inhibitor that can be delivered intranasally was developed. This inhibitor, FSD-C10, reaches the CNS faster and in a much lower dose. FSD-C10 reduced EAE severity and CNS inflammatory infiltration and promoted neuroprotection by inducing CNS production of IL-10, NGF, and BDNF and by inhibiting the production of multiple pro-inflammatory

Eriocalyxin B (EriB) is a diterpenoid extracted from Isodon eriocalyx, a perennial herb from southwest China that is used as an anti-inflammatory remedy in traditional Chinese medicine. EriB has been reported to induce apoptosis in leukemia and lymphoma by elevating the intracellular levels of reactive oxygen species and by suppressing the NFκB pathway. In an EAE model, EriB alleviated symptoms, delayed disease onset, decreased T cell populations, inhibited the NFκB pathway and reduced CNS inflammation and demyelination, improving the course of the disease [43]. Adenanthin, which is also a diterpenoid isolated from the leaves of Isodon adenanthus, displays preventive and therapeutic effects in EAE, as demonstrated

CypD inhibition could become a viable therapeutic strategy for MS [40].

additional clinical evidence is needed.

288 Trending Topics in Multiple Sclerosis

[41].

cytokines [42].

Regarding sex hormones, 2-methoxyestradiol (2ME2)—the endogenous metabolite of estra‐ diol and an antimitotic and antiangiogenic cancer drug—was found to suppress the develop‐ ment of mouse EAE, as it inhibited lymphocyte activation, cytokine production, and proliferation in a dose-dependent manner [46]. Other studies have shown that estrogen and estrogen receptor agonists reduce the severity of EAE in animals when they are administered after disease onset; these agents inhibit several inflammatory cytokines, induce apoptosis in T cells, and also regulate the expression of adhesion and accessory molecules by endothelial cells, altering leukocyte migration [47]. In addition, the β estrogen receptor has been demonstrated to modulate microglial activity. The β estrogen receptor agonist LY3201 can suppress activated microglia and NFκB activation in both microglia and T cells. All of these outcomes can be achieved without negative effects on the pituitary gland, mammary glands, or uterus [48].

Nevertheless, in animal models of demyelination, progesterone and synthetic progestins have been observed to attenuate myelin loss and to reduce clinical symptom severity. One study showed that progesterone and Nosterone (a synthetic 19-nor-progesterone derivative) promoted remyelination and attenuated inflammatory responses in female mice with severe chronic demyelinating lesions. The remyelinating effect of progesterone was receptordependent and began in the corpus callosum. Moreover, it enhanced the number of mature oligodendrocytes and their progenitors as well, indicating that these hormones could represent promising therapeutic agents for demyelinating diseases [49].

Statins are widely used to treat vascular diseases, but they also have immunomodulatory and neuroprotective properties that could make them possible treatment candidates for neurode‐ generative disorders. Lovastatin has been found to improve clinical symptoms associated with EAE as well as to reduce neuroinflammatory mediators such as iNOS, TNF-α and interferon gamma (IFNγ). Similarly, atorvastatin has also been shown to ameliorate EAE symptomatol‐ ogy by modulating T cell immunity [50]. One double-blind, controlled trial used simvastatin in patients with secondary progressive MS. High-dose simvastatin reduced the rate of wholebrain atrophy by 43% compared with placebo and was safe and well tolerated. Furthermore, differences between the simvastatin-treated and control groups were consistently observed over 12 and 25 months. A small but significant improvement in disability outcomes and a nonsignificant reduction in T2 lesion accumulation were also observed [51].

SWABIMS was a multi-center, randomized, parallel-group, rater-blinded study conducted in 8 Swiss hospitals that evaluated the efficacy, safety, and tolerability of daily administration of 40 mg atorvastatin and subcutaneous IFNB-1b compared to monotherapy with IFNB-1b. At the end of the study, both groups had an equivalent number of patients with new lesions on T2-weighted MRI images. Additionally, none of the secondary endpoints, including the number of new lesions and total lesion volume on T2-weighted images, the total number of new Gd-enhancing lesions on T1-weighted images, total brain volume, grey matter volume, white matter volume, EDSS, relapse rate and number of relapse-free patients, did showed any significant differences, suggesting that atorvastatin did not have a beneficial effect on relapsing-remitting MS [52].

Recent data from an established rat model of MS suggest that inhibiting excitatory glutama‐ tergic neurotransmission may have neuroprotective effects. One of these studies investigated whether drugs such as amantadine and memantine (antagonists of NMDA glutamate recep‐ tors), LY 367385 (a selective mGluR1 antagonist) or MPEP (an mGluR5 antagonist) could improve the condition of rats with EAE. On the one hand, amantadine and memantine reduced the development and duration of neurological deficits and modified all of the assessed parameters. On the other hand, LY 367385 and MPEP did not influence the condition of treated animals when they were administered alone or in conjunction with NMDA antagonists [53]. Another study evaluated if selective antagonism of the NR2B subtype of NMDA receptors (which are considered to play a more pivotal role in neurodegeneration) could be more effective than memantine in EAE mice. Therapeutic administration of RO25-6981 (a selective inhibitor of NR2B) caused a more significant decrease in neurological deficits, inflammation, myelin degradation, and degeneration of axons from the spinal cord, suggesting that this drug may be an effective treatment strategy to slow down the clinical deterioration that causes disability in MS [54].

The metabotropic glutamate receptor 4 (mGluR4) has immunomodulatory properties, such that a positive allosteric modulator of the receptor, ADX88178, protects mice with relapsingremitting EAE. ADX88178 is a newly developed drug with high selectivity and potency, optimal pharmacokinetics, good brain penetrance, and almost no toxicity. Its administration in EAE converted the disease into a form of mild chronic neuroinflammation that remained stable for two months after the drug treatment was discontinued [55].

Recent studies have demonstrated that atypical antipsychotic agents (antagonists of dopamine D2 and serotonin 5-HT2a receptors) have immunomodulatory properties, both peripherally and within the CNS. In an EAE animal model, chronic oral administration of risperidone improved disease severity, decreased both the size and the number of spinal cord lesions and substantially reduced antigen-specific interleukins such as IL-17a, IL-2, and IL-4 and the activation of microglia and macrophages in the CNS. In addition, another antipsychotic agent, clozapine, showed a similar ability to modify macrophages and to reduce disease severity. Together, these studies indicate that atypical agents could treat immune-mediated diseases such as MS [56].

Polyphenolic flavonoids and non-flavonoids have potent antioxidant abilities, but they can also target different molecules and affect multiple signaling pathways. Resveratrol, a phenol found in grapes and red wines, is considered to have neuroprotective effects. In EAE, it induces the apoptosis of activated T cells in the periphery and suppresses pro-inflammatory responses. Another plant-derived substance, oleanolic acid (a triterpenoid), is known to have potent antiinflammatory properties. Treatment with oleanolic acid has been reported to prevent EAE by suppressing peripheral inflammation and preventing CNS infiltration of inflammatory cells (due to blockade of the NF-κB pathway [45]. Other studies have shown that flavonoids are naturally immunomodulatory compounds that can limit demyelination, reduce neuroinflam‐ mation, and downregulate immune functions. For example, luteolin provides neuroprotection by reducing axonal damage and, together with quercetin and fisetin, is able to decrease the amount of myelin phagocytosed by macrophages; thus, luteolin may help prevent MS [57].

Polyphenolic curcuminoids are the mixtures of curcumin, desmethoxycurcumin, and bisde‐ methoxycurcumin, which are derived from turmeric (*Curcuma longa*). Both the mixtures and the individual components have been suggested to influence inflammatory and apoptotic genes and the regulation of signal transduction pathways that lead to the activation of transcription factors. In EAE, treatment with curcumin modulates pro- and anti-inflammatory responses, prevents the differentiation of neural antigen-specific T cells, decreases oxidative stress, improves remyelination and promotes neurogenesis [28]. However, despite the promising therapeutic potential of curcumin, its poor water solubility, fast degradation profile and poor bioavailability are significant hurdles for its clinical use.

Recent data from an established rat model of MS suggest that inhibiting excitatory glutama‐ tergic neurotransmission may have neuroprotective effects. One of these studies investigated whether drugs such as amantadine and memantine (antagonists of NMDA glutamate recep‐ tors), LY 367385 (a selective mGluR1 antagonist) or MPEP (an mGluR5 antagonist) could improve the condition of rats with EAE. On the one hand, amantadine and memantine reduced the development and duration of neurological deficits and modified all of the assessed parameters. On the other hand, LY 367385 and MPEP did not influence the condition of treated animals when they were administered alone or in conjunction with NMDA antagonists [53]. Another study evaluated if selective antagonism of the NR2B subtype of NMDA receptors (which are considered to play a more pivotal role in neurodegeneration) could be more effective than memantine in EAE mice. Therapeutic administration of RO25-6981 (a selective inhibitor of NR2B) caused a more significant decrease in neurological deficits, inflammation, myelin degradation, and degeneration of axons from the spinal cord, suggesting that this drug may be an effective treatment strategy to slow down the clinical deterioration that causes

The metabotropic glutamate receptor 4 (mGluR4) has immunomodulatory properties, such that a positive allosteric modulator of the receptor, ADX88178, protects mice with relapsingremitting EAE. ADX88178 is a newly developed drug with high selectivity and potency, optimal pharmacokinetics, good brain penetrance, and almost no toxicity. Its administration in EAE converted the disease into a form of mild chronic neuroinflammation that remained

Recent studies have demonstrated that atypical antipsychotic agents (antagonists of dopamine D2 and serotonin 5-HT2a receptors) have immunomodulatory properties, both peripherally and within the CNS. In an EAE animal model, chronic oral administration of risperidone improved disease severity, decreased both the size and the number of spinal cord lesions and substantially reduced antigen-specific interleukins such as IL-17a, IL-2, and IL-4 and the activation of microglia and macrophages in the CNS. In addition, another antipsychotic agent, clozapine, showed a similar ability to modify macrophages and to reduce disease severity. Together, these studies indicate that atypical agents could treat immune-mediated diseases

Polyphenolic flavonoids and non-flavonoids have potent antioxidant abilities, but they can also target different molecules and affect multiple signaling pathways. Resveratrol, a phenol found in grapes and red wines, is considered to have neuroprotective effects. In EAE, it induces the apoptosis of activated T cells in the periphery and suppresses pro-inflammatory responses. Another plant-derived substance, oleanolic acid (a triterpenoid), is known to have potent antiinflammatory properties. Treatment with oleanolic acid has been reported to prevent EAE by suppressing peripheral inflammation and preventing CNS infiltration of inflammatory cells (due to blockade of the NF-κB pathway [45]. Other studies have shown that flavonoids are naturally immunomodulatory compounds that can limit demyelination, reduce neuroinflam‐ mation, and downregulate immune functions. For example, luteolin provides neuroprotection by reducing axonal damage and, together with quercetin and fisetin, is able to decrease the amount of myelin phagocytosed by macrophages; thus, luteolin may help prevent MS [57].

stable for two months after the drug treatment was discontinued [55].

disability in MS [54].

290 Trending Topics in Multiple Sclerosis

such as MS [56].

The kynurenine pathway is known to have a regulatory function in the immune system. Alterations of this pathway have been described in preclinical and clinical investigations of MS. These data led to the identification of potential therapeutic targets, such as synthetic tryptophan analogs, endogenous tryptophan metabolites, structural analogs, indoleamine-2, 3-dioxygenase inhibitors, and kynurenine-3-monooxygenase inhibitors [30]. Additionally, high levels of a by-product of the kynurenine pathway, quinolinic acid, were found in EAE mice and MS patients. Sundaram et al. demonstrated two possible strategies to limit quinolinic acid gliotoxicity: by neutralizing quinolinic acid's effects with monoclonal antibodies or by inhibiting quinolinic acid production using specific KP enzyme inhibitors. These observations could represent a novel therapeutic approach in MS [58].

Cannabidiol (CBD) is a non-psychotropic cannabinoid constituent of *Cannabis sativa* that is known to possess anti-inflammatory and immunosuppressive properties. In a viral model of MS, CBD decreased the transmigration of blood leukocytes by downregulating the expression of VCAM-1, chemokines and the cytokine IL-1β and by attenuating the activation of microglia. Its administration had long-lasting effects and ameliorated motor deficits during the chronic phase of the disease, demonstrating the significant therapeutic potential of this compound [59]. Another study of CBD as a topical 1% cream also had surprisingly good results too. The daily treatment, initiated at the time of symptomatic disease onset, displayed neuroprotective effects against EAE, diminishing clinical disease scores (EDSS) by recovering hind limb paralysis and by ameliorating lymphocytic infiltration and demyelination in spinal cord tissues [60]. However, when the CUPID trial investigated if oral dronabinol (Δ<sup>9</sup> -tetrahydrocannabinol) might slow the course of progressive MS, it had no overall effect on disease progression, although there were no serious safety concerns [61].

Epigallocatechin-3-gallate (EGCG), one of the major polyphenolic extracts of green tea, has been shown to exhibit neuroprotective effects against toxic insults and neuronal injury. In an EAE animal model, the administration of EGCG attenuated clinical symptoms and leukocyte infiltration and demyelination in the CNS. Moreover, EGCG inhibited the NF-κB-mediated transactivation of inflammatory mediators, reducing the production of interferons, IL-17, IL-6, IL-1, and tumor necrosis factors [62]. These results were corroborated by other studies, which demonstrated that EGCG, due to its antioxidative properties, could reduce the clinical severity of EAE by limiting brain inflammation and reducing neuronal damage [63]. In addition, GA and EGCG combination therapy had synergistic protective effects in vitro and in vivo, with good results and no unexpected adverse events [64].

Ginseng has been used in traditional medicine for over 2000 years due to its antianxiety, antidepressant, and cognition-enhancing properties. Moreover, its effects on the brain are related to glutamatergic and monoaminergic transmission, estrogen signaling, nitric oxide production, neuronal survival, apoptosis, neural stem cells, and neuroregeneration. The efficacy of ginsenoside Rd has been studied in mice with EAE. The results were promising because the ginsenoside reduced the permeability of the blood–brain barrier, regulated the secretion of INF-gamma and IL-4 and decreased disease severity [65].

Based on the observational studies that showed that low levels of vitamin D represent a risk factor for the development of MS [66, 67], treatment with vitamin D has become increasingly attractive and has been tested in both experimental and clinical trials. Vitamin D appeared to modulate upon immune responses and inflammation, but clinical studies have not yet shown a clear benefit [68, 69].

In addition to pharmaceutical compounds, clinical and basic research studies have also highlighted that voluntary exercise can promote both neuroprotection and neuroregeneration [70, 71]. An experiment conducted in mice with EAE showed that the exercising mice (on a running wheel) presented a less severe neurological disease score, later disease onset and a significant reduction of inflammatory cell infiltration and demyelination in the ventral white matter tracts of the lumbar spinal cord [71]. Studies of patients with MS also support these observations, physical excesses determining not only improvement of muscle function and walking endurance, but also of cognitive abilities [72–75].
