A Narrative Review on Axonal Neuroprotection in Multiple Sclerosis

Dimethyl fumarate (DMF) showed a 21% reduction in the course of brain atrophy compared to placebo in the DEFINE study (the 240 mg twice daily regimen only) and produced only marginal but beneficial effects in BVL reduction in the CONFIRM study [92, 93]. A pilot study of 20 patients with RRMS showed a protective effect of DMF treatment in whole brain atrophy (PBVC − 0.37 ± 0.49% vs. − 1.04 ± 0.67%, p = 0.005) and putamen atrophy (− 0.06 ± 0.22 vs. − 0.32 ± 0.28 ml, p = 0.02), but no effect on other subcortical volumes or total GM atrophy [94].

For fingolimod, data concerning the evolution of cerebral atrophy (measured by SIENA) extracted from phase III FREEDOMS 1 and 2 and TRANSFORMS trials found an annual decrease in brain volume of between 0.3% and 0.5%, present from the first year of treatment and maintained for the following year [95]. The progression of this atrophy was more marked as the patients presented MRI characteristics showing disease activity at the time of inclusion. The evolution of thalamic atrophy was 0.82% the first year of treatment and 0.51% the second year [96]. Prospective studies comparing the evolution of atrophy with fingolimod and natalizumab found comparable data in patients without any inflammatory activity on brain MRI [97].

The study of neuroprotection under natalizumab shows that the evolution of WM atrophy and to a lesser extent GM atrophy is quite low and presents the risk of the effects of atrophied structures being masked by the effects of those which are not. On the other hand, the analysis of the literature shows that the evaluation of thalamic atrophy seems to be the best marker of the neuroprotective effect in patients with MS [98‐101]. These data can be extracted from the standard sequences. The increase of this atrophy in patients taking natalizumab was around 1.5% after 48 weeks of treatment and then seemed to be relatively stable and around 0.5% per year after this period. Note that this picture is close to the natural course of cerebral atrophy, the annual rate of which is 0.4% in healthy subjects.

For ocrelizumab, the data from the pivotal studies OPERA I and II and its extension phase made it possible to identify a change in cerebral atrophy of 0.6% over 18 months from week 24. Thalamic atrophy was 2% in the first year, then around 0.4% each year for 5 years of follow-up [102]. Lastly, ocrelizumab (vs. IFNβ1a) was associated with a reduced risk of composite clinical disease activity (hazard ratio [HR] 0.67) and confirmed PIRA (HR 0.78) and relapse-associated worsening (HR 0.47) events [44].

These data show that we should observe the same neuroprotective effect with fingolimod, ocrelizumab, and natalizumab in stable patients without recent inflammatory activity on MRI.

Ocrelizumab has been demonstrated as a highly efficacious drug in RRMS, with a significant reduction of both relapse rate and progression of the disease compared with IFNβ1a subcutaneously three times a week, in two parallel studies (OPERA I and II) [104]. However, the effect was essentially attributed to the anti-inflammatory mechanism of ocrelizumab rather than a mixed effect on both the inflammatory process, expressed by relapse, and progression. A third study, focusing on PPMS, showed a significant effect on EDSS progression against placebo [46]. This study was the first time we found a drug working on PPMS, even if the impact value (reduction of 24% of the disability progression) was not very impressive. The results were also positive for MRI markers such as new T2 lesions and brain atrophy that are usually attributed to neurodegeneration. In addition, ocrelizumab treatment increased the proportion of patients with PPMS maintaining NEP from baseline to week 120 by 47% compared to placebo. Very recently, an extrapolation study evaluated from the ORATORIO study the mean time to requiring a wheelchair, which may be considered as a good indirect marker of the neurodegenerative process [105]. The study found a mean time to requiring a wheelchair of 12 years on placebo against 19 years on ocrelizumab.

Siponimod is an S1P drug derived from the same class of drugs as fingolimod but with a small difference of S1P receptor affinity. The latter binds with nanomolar affinity as an agonist at four of the five S1P receptors, namely, S1P₁, S1P₃, S1P₄, and S1P₅. Siponimod may have a direct neurobiological effect in the CNS, independent from its effects on peripheral lymphocytes, through selective modulation of S1P₁ on astrocytes and S1P₅ on oligodendrocytes [106]. Fingolimod was not able to demonstrate any effect on PPMS in a phase III, placebo-controlled study even with stratification of some subgroups [107]. A specific study, EXPAND, was designed for SPMS with siponimod; the results were positive, with a reduction of 21% of the confirmed disability progression, but this effect was mainly driven by the subgroup of patients with active SPMS [103]. The FDA and EMA approved this new drug for active SPMS but not for non-active SPMS, which remains a clear unmet need in this field.

BTK was initially identified in human X-linked agammaglobulinemia, an inherited disorder characterized by a very low level of immunoglobulins. A defect in the BTK gene impairs normal B cell development and maturation [108]. Indeed, BTK is a cytoplasmic non-receptor tyrosine kinase modulating several intracellular signaling cascades, such as phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and nuclear factor-κB (NF-κB). These key pathways regulate activation, proliferation, survival, and differentiation, dysregulation of which participates in the development of B cell malignancies and autoimmune diseases, including MS [109, 110]. Thus, the X-linked immunodeficient (XID) mouse line lacking functional BTK exhibited milder clinical signs after induction of EAE [111]. Further analysis demonstrated that myeloid cells also undergo abnormalities during development in XID mice. Indeed, in addition to B cells, BTK is also expressed in other immune cells, including monocytes, macrophages, dendritic cells, granulocytes, mast cells, and platelets [112]. In physiological conditions, BTK-positive cells are barely detectable in the CNS. After lysophosphatidylcholine (LPC)-induced demyelination of murine organotypic cerebellar slices, BTK was detected in microglia, i.e., a resident myeloid immune cell of the CNS, and to a lesser extent in astrocytes [113]. High expression of BTK was also found in microglia associated with MS lesions in postmortem patient brain samples [114]. Both B cells and microglia are important actors of MS pathogenesis as mentioned in Sect. “Axonal Lesions and the Progressive Phase of Multiple Sclerosis”. Consequently, BTK appears as a valuable therapeutic target in the treatment of this disease. Twenty-four BTK inhibitors (BTKi) are currently under clinical evaluation [115]. Preclinical studies indicated that treatment of EAE mice with the BTKi tyrphostin AG126 improved the clinical course of the disease, with a reduced immune cell infiltration, milder myelin damage, and attenuation of microglia activation. Cell culture experiments showed that AG126 affects microglial functions directly [116]. Recent data have highlighted the implication of microglia during neurodegenerative processes [117]. Notably, genetic data from patients with MS have revealed an enrichment of susceptibility variants in microglial cells [118]. Microglia, the resident macrophages of the CNS, can adopt a diversity of polarization states after activation, but the precise protective or detrimental role of microglia in neurodegenerative diseases is far from being elucidated [119]. Specific microglial phenotypes have been identified in cortical lesions during the progressive phases of MS [120]. Studies have highlighted the essential role of microglia in remyelination by promoting oligodendrocyte differentiation [121], a process dependent on phagocytosis [122]. Microglia–axon interaction at the level of the nodes of Ranvier has been shown to contribute to remyelination [123]. Thus, modulation of microglial activation by BTKi appears to be a potential treatment for MS as the support given in remyelination would promote neuroprotection. Ibrutinib, the first approved BTKi, suppressed the proinflammatory polarization in microglia and decreased microglial phagocytic activity [124, 125]. Despite these promising results, the lack of specificity of ibrutinib precluded its evaluation in autoimmune diseases and led to the development of new BTKi, such as evobrutinib, a highly selective covalent inhibitor [126].

Evobrutinib has been successfully tested in several EAE models [112, 127] (Table 1). A phase II trial (NCT02975349) concluded on its safety and efficacy to reduce the total cumulative number of T1 gadolinium-enhancing lesions in patients with relapsing MS [128]. In comparison to other trials realized on MS, participants included in this study were older, with a longer disease duration, and experienced fewer relapses within the 2 years before baseline, which might have indicated the inclusion of patients with SPMS. Thus, longer and larger trials were required to investigate the effect and risks of this molecule in patients with MS. Evobrutinib has now entered two phase III trials on relapsing MS (EVOLUTION RMS 1 and 2) expected to be completed by September 2023.

The data obtained with evobrutinib represented the proof of concept that inhibiting BTK in patients with MS can improve clinical and MRI outcomes. To further enhance efficacy during MS, BTKi drugs have to better penetrate the CNS. Tolebrutinib (SAR442168) has been shown to cross the BBB efficiently and reach pharmacologically relevant concentrations in the CNS [129]. This phase IIb study reported good drug tolerability and a dose-dependent reduction in the number of new gadolinium-enhancing lesions after 12 weeks of treatment. The reduction of inflammation monitored by MRI in this study was comparable to that reported in other trials related to DMTs that were subsequently shown to be highly effective in clinical practice. The most efficacious and well-tolerated dose of tolebrutinib, i.e., 60 mg daily, was selected for further investigations. Phase III trials are currently recruiting patients with relapsing MS (GEMINI 1 and 2 trials), PPMS (PERSEUS trial), and SPMS (HERCULES trial), to compare efficacy and safety of tolebrutinib to placebo or teriflunomide for relapsing MS. BTK modulators were initially developed to target B cells. No investigation has so far analyzed the combination of anti-CD20 antibody treatments and BTK inhibitors. A phase II study (NCT04742400) aims to evaluate the effects of tolebrutinib on the paramagnetic rim of chronically inflamed white matter lesions following anti-CD20 antibody treatment in patients with MS. Orelabrutinib is another potent, orally active, irreversible, and highly selective BTKi developed to treat B cell malignancies and autoimmune diseases. This promising drug received its first approval in China for the treatment of patients with lymphomas at the end of 2020 [130]. Patients with RRMS are currently being recruited for a phase II trial (NCT04711148).

Ibrutinib, evobrutinib, tolebrutinib, and orelabrutinib have all been shown to bind covalently and irreversibly to BTK residues. Fenebrutinib represents a new type of inhibitor that nestles into a pocket of BTK, from where it is slowly released. This non-covalent and selective interaction triggers a conformational change in the BTK protein that prevents the chemical modifications required for enzyme activation. Preclinical studies indicated that prophylactic treatment of EAE mice with fenebrutinib significantly reduced the clinical scores in a dose-dependent manner, in association with an attenuated microglial activation [131]. Safety data from phase II trials conducted on patients with several inflammatory diseases (rheumatoid arthritis, systemic lupus erythematosus, and chronic spontaneous urticaria) concluded that fenebrutinib was generally well tolerated [132‐134]. Indeed, the high selectivity of the drug may limit off-target effects [135].

Phase III trials are currently recruiting patients to test fenebrutinib in MS. Two identical trials will investigate the effect of this molecule on the relapsing form (FENhance 1 and FENhance 2) and a third on the primary progressive form (FENtrepid). Most current DMTs work outside the CNS and aim to prevent peripheral immune cells from attacking the myelin. During the progressive forms of MS, resistance to the effective DMTs could be partly due to compartmentalized inflammation. Using drugs able to cross the BBB, such as the newly developed BTKi, should show higher efficacy as it would help to fight the endogenous immune attack against myelin directly in the CNS.

Ibudilast is a non-selective phosphodiesterase (PDE) inhibitor (PDE3, 4, 10, and 11), preventing the hydrolysis of cGMP or cAMP and in some cases both [136]. It is approved in Asia for the treatment of asthma and chronic obstructive pulmonary disease, and is used in ischemic stroke. Its peripheral anti-inflammatory effect results from the decrease in leukotriene release and tumor necrosis factor alpha (TNFα) or IFNγ production from peripheral white blood cells. At the central level, its anti-inflammatory effect is based on the attenuation of kainate-induced oligodendrocyte cell toxicity and astrocyte apoptosis in vitro, and on the dose-dependent decrease of microglial activation [137]. In vivo, ibudilast attenuates rat EAE, by reducing spinal cord inflammation and modulating the severity of clinical signs in a dose-dependent manner, after prophylactic administration [138]. In a genetic model of Krabbe’s disease, ibudilast (10 mg/kg/day, with a daily intraperitoneal injection) decreases the number of TNFα-labeled cells, reduces the number of oligodendrocytes undergoing apoptosis, and attenuates demyelination [139]. Among the neuroprotective mechanisms mentioned, PDE inhibition reduces inflammatory activities of several non-neuronal cell types: it reduces the production of ROS, suppresses TNFα release from the astrocytes and microglia cells [140], and other neurotoxic mediators (interleukin-6 [IL-6], nitric oxide [NO]) that can damage both neurons and oligodendrocytes [141]. Indeed, roflupram, a PDE4 inhibitor, suppresses inflammasome activation through autophagy in microglial cells [142], and it attenuates lipopolysaccharide (LPS)-induced neuroinflammatory responses in microglial cells in both the hippocampus and cortex [143]. Roflupram also prevents the increase of IL-1β and suppresses microglial reactivity in the hippocampus of mice subjected to the chronic unpredictable mild stress mouse model of depression [144]. The neuroprotective effects of PDE inhibitors have been demonstrated in PC12 cell lines, where concomitant treatment with epidermal growth factor (EGF) and cAMP is required to induce morphological differentiation and cell survival [145].

The passage of ibudilast across the BBB is difficult to demonstrate in clinical practice. The presence of ibudilast in the spinal cord and brain of rats 7 h after an intraperitoneal injection of ibudilast 5 mg/kg suggests its passage through the BBB, although concentrations in the CSF appeared to be low, probably equivalent to the unbound fraction in plasma [146].

The first proof-of-concept study suggesting a neuroprotective effect of ibudilast is based on a phase II study, comparing two doses of ibudilast (30 and 60 mg/day, for 12 months), in 297 patients with RRMS and significant disease activity. Although ibudilast treatment showed no beneficial effect on the primary endpoint (brain MRI lesion development, the volume of enhancing lesions) and relapses, post analysis highlighted the reduction in the brain atrophy rate. Furthermore, over 2 years, the percentage of patients with EDSS score progression was lower in those on active treatment (10.4%) versus placebo (21%, p = 0.026) [147] (Table 2). These data suggest that ibudilast has a neuroprotective effect rather than an anti-inflammatory action but, because for the second year of evaluation neither the patients nor the investigators were blinded, this needs to be confirmed in another large study, with a better primary endpoint. In the SPRINT-MS, phase II trial, ibudilast reduced the rate of brain atrophy by 48% compared to placebo, i.e., approximately 2.5 mL less brain tissue loss over 96 weeks, but at the expense of some adverse events, such as gastrointestinal side effects and depression [148]. A second analysis of the SPRINT-MS trial suggests that ibudilast has more effect on neurodegenerative processes in patients with PMS than on the inflammatory ones, by reducing the gray matter atrophy (p = 0.038) and slowing progression of whole brain atrophy by SIENA (p = 0.08), but without effect on new or enlarging T2 lesions, or new T1 lesions, and without a change in either serum or CSF NfL [149, 150].

However, in the SPRINT-MS trial, the absence of a positive effect of ibudilast on all the evaluation criteria still raises questions about either the mechanism of action or the extrapolation of brain atrophy or diffusivity results in post hoc longitudinal analyses performed with different hardware [151]. Despite very promising preclinical results, the repositioning of ibudilast in progressive MS remains in its infancy (only two phase II studies have been conducted, with a limited sample size, mixing different diagnoses of MS and with different ongoing treatments) and needs more evaluation of predictive clinical markers of neurodegeneration.

As part of a repositioning strategy in MS for old molecules administered orally and having proven their safety, minocycline, a second-generation tetracycline antibiotic used to treat acne and suppress inflammation, was tested as a drug candidate. In vitro, minocycline reduces matrix metalloproteinase 9 (MMP-9) production and attenuates T cell transmigration across a fibronectin barrier [152]. The anti-apoptotic effect of minocycline was studied in mixed spinal cord (glial and microglial cells) cultures treated with glutamate or kainate for 24 h. Minocycline inhibited excitotoxin-induced neuronal death and proliferation of microglial cells by inhibiting the release of nitric oxyde (NO) metabolites and IL-1β [153]. Moreover, minocycline inhibited microglial activation, in brain cell cultures treated with IFNγ and LPS, and promoted remyelination by enhancing the oligodendrocyte precursor cells and immature oligodendrocytes [154]. In vivo, minocycline pre-treatment delays the course of the disease in EAE mice, with minimal signs of inflammation and demyelination in the CNS, leading to improvements in motor coordination [152]. The neuroprotective effect of minocycline was confirmed in a rat model of EAE induced by myelin oligodendrocyte glycoprotein (MOG), where functional and histopathological data of retinal ganglion cells and optic nerves revealed neuronal and axonal protection, when administration was started on the day of immunization and also when it started on the day of disease onset [155]. Minocycline-induced neuroprotection is the consequence of the induction of anti-apoptotic intracellular signaling pathways (upregulation of Bcl2 and decrease of Bax) and of the decrease in glutamate excitotoxicity. Recently, in an EAE mouse model, the injection of minocycline into the dentate gyrus prevented microglial activation, dentate gyrus neurodegeneration, and memory impairment [156]. Minocycline, by inhibiting microglial activation and nicotinamide adénine dinucléotide phosphate (NADPH) oxidase, reversed synaptic and learning deficits in a chronic relapsing EAE mouse model, without preventing the development of disease [157]. Finally, Chen et al. demonstrated that minocycline could upregulate the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in both the cerebral cortex and the lumbar spinal cord of EAE mice [158]. Prior to clinical testing, minocycline was combined with other immunomodulatory therapies for MS. The experimental treatment, combining IFNβ-secreting mesenchymal stem cells with minocycline in a mouse model of EAE, reduced the clinical severity by maintaining the microvascular integrity of the BBB, and by inhibiting the production of MMP-2 and MMP-9 in the spinal cord, leading to a beneficial effect on neuroinflammation [159]. Another combination therapy involving minocycline and corticoids alleviated clinical scores and improved MRI outcome in the EAE mouse model. In the same model, the association with prednisolone reduced phenotype severity, inflammation, and demyelination, by preventing the reduction of BDNF and NGF mRNA expression in cerebral cortex [160]. Also, association with methylprednisolone improved severe clinical deficit and suppressed histopathological events in C57Bl/6 EAE mice by reducing the levels of IFNγ and increasing IL-4 expression/production in the splenocyte culture supernatants and brains of EAE mice [161]. Finally, the combination of minocycline and glatiramer acetate significantly reduced the severity of the disease by attenuating inflammation, axonal loss, and demyelination, through alleviation of neuronal T cell toxicity [162].

Regarding its pharmacokinetic properties, minocycline is a highly lipophilic molecule that crosses the BBB, with a good oral bioavailability of 95–100% and seems to be well tolerated in humans at 200 mg/day, which was the only dose tested in phase II trials [163].

Three open-label consecutive studies were conducted to evaluate the potential of minocycline in the same 10 patients with RRMS [164‐166] (Table 3). In the first study, minocycline treatment (100 mg twice daily for 6 months) reduced the number of gadolinium-enhancing lesions, compared to baseline [164]. In the second study, minocycline treatment at the same dosage for 24 months confirmed the reduction in the mean of gadolinium-enhancing lesions, and no relapses occurred after 18 months of treatment, despite a moderately high pretreatment annualized relapse rate [165]. These clinical and MRI outcomes are supported by systemic immunological changes: a decrease in MMP-9 activity and an increase in levels of the p40 subunit of IL-12, which might antagonize the proinflammatory IL-12 receptor. The third study, evaluating minocycline treatment over 36 months, highlighted a reduction of the ARR, and the proportion of active scans was lower during the first 6 months of treatment (5.6%, p < 0.001) and during the extension phase (8.7% p = 0.002) than during the 3-month run-in period (47.5%) [166]. Moreover, with minocycline administration for over 3 years, T2 lesion volume tended to remain stable and an attenuation of the brain volume impairment was observed, suggesting a possible neuroprotective role of minocycline in patients with RRMS. In a randomized, double-blind, placebo-controlled clinical trial, minocycline treatment, as an add-on therapy to glatiramer acetate, tended to reduce the total number of T1 gadolinium-enhanced lesions (mean 1.47 versus 2.95, p = 0.08), the total number of new and enlarging lesions (mean 1.84 versus 5.14, p = 0.06), and the total T2 disease burden (p = 0.10) [167]. The authors also reported a lower risk of relapse in the combination arm. The treatment was safe and well tolerated, but the lack of statistical significance makes it impossible to conclude on the efficacy of this association (minocycline 100 mg twice daily with glatiramer acetate) and could be attributed to an underpowered clinical trial. Another, larger, randomized, double-blind, multicentric, placebo-controlled clinical trial evaluated the effectiveness and safety of minocycline (100 mg twice daily, for 96 weeks), added to IFNβ1a therapy [168]. No differences were observed for primary (time to first relapse) and secondary (annualized relapse rate, number of new/enlarging T2 lesions, change in brain volume) outcomes between the two groups. Finally, in a Canadian multicentric trial, minocycline significantly reduced the risk of conversion from a clinically isolated syndrome to MS, compared to placebo, after 6 months of treatment, but not over 24 months [169]. This study is seeking to evaluate an early role for minocycline treatment; however, some potential biases limit this interpretation (small sample size, short study duration, lack of MRI scans for more than one-third of patients after 3 months, and higher risk of conversion in the placebo group patients since they had a greater number of baseline MS lesions). In addition, the preclinical results of minocycline suggest a protective effect in patients with progressive multiple sclerosis (SPMS and PPMS), a population not yet evaluated in well-conducted clinical trials with sufficient power.

Statins are powerful inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase, the rate-limiting enzyme of hepatic cholesterol synthesis [170]. This cholesterol-lowering treatment is indicated for the prevention of cardiovascular-related morbidity and mortality in individuals with coronary artery disease, with good tolerance [171] In the mid-1990s, the lower incidence of rejection episodes and mortality in heart transplant patients treated with pravastatin led to the investigation of a potential immunomodulatory effect of statins [172]. In fact, experimental and clinical studies have shown that statins can modulate immune responses through mevalonate pathway-dependent and -independent mechanisms. The pleiotropic effect of statins could be related to the inhibition of post-translational protein modifications, known as isoprenylation, by blocking the synthesis of isoprenoid compounds, such as farnesylpyrophosphate and geranylpyrophosphate, thus preventing the activation of small G proteins, such as Ras (farnesylated proteins), Rho and Rab (geranylated proteins). Proof of concept has been demonstrated in the EAE mouse model, where lovastatin treatment inhibited leucocyte migration into the CNS and significantly attenuated the development of both acute and relapsing clinical disease by inhibiting the Rho-mediated transendothelial T cell migration [173]. The immune effects of statins were analyzed in vitro on the pathogenic cascade of immune peripheral cells obtained from patients with MS. The inhibition of mononuclear cell transmigration/penetration across the BBB, the inhibition of dendritic cell maturation, the decreased T cell activation, inhibition of glutamate-mediated excitotoxicity, and alteration of the NO production by endothelial cells were observed [174]. In murine models, statins inhibit MHC class II-restricted antigen presentation, downregulate T cell activation and proliferation, and induce a shift from a proinflammatory Th1 to a Th2 phenotype. Statins also block adhesion molecule expression and inhibit leucocyte migration through the BBB, supporting their therapeutic use in early MS and their inconsistent effect depending on the inflammatory stage [175]. Among the described central neuroprotective effects induced by statins, atorvastatin, in primary cortical neurons, significantly protected from glutamate-induced excitotoxicity, independently of HMG-CoA reductase inhibition [176]. Moreover, after traumatic brain injury, simvastatin has been shown to induce the expression of BDNF. Some authors suggest that the neuroprotective effects may derive from statin-induced immunomodulatory effects, protecting oligodendrocytes from Th1 (TNFα) and Th17 (IL-17) phenotype cytokine toxicity in vitro, by inhibiting small Rho GTPases, via a peroxisome proliferator-activated receptor alpha (PPARα)-dependent mechanism, which in turn increases ROS detoxifying defense [177]. Statins induce central neuroprotection by inhibition of NO and glutamate synthesis in the CNS. Indeed, lovastatin inhibits the expression of inducible NO synthase and cytokines (IL-1β, TNFα, IL-6) in rat astrocytes, microglia, and macrophages [178]. In astrocytes, NO secretion appears to be dependent on the tropomyosin receptor kinase B (TrkB), a neurotrophin receptor whose expression is induced in white matter lesions of people with MS. Stimulation of astrocytes with BDNF, an agonist of TrkB, leads to neuronal death [179], as NO impairs the energy metabolism of these cells [180]. A similar mechanism connects microglia to the neuro-axonal degeneration that leads to irreversible MS progression [181].

Finally, a vascular effect could also contribute to the neuroprotective effects of statins by modulating endothelial cell eNOS activity, thereby improving cerebral vasomotor reactivity and protecting against long-term hypoxic damage [170]. Since vascular comorbidity is a risk factor for disability in MS, the benefit could also come from a reduction in total cholesterol levels.

The first clinical evidence of both immunomodulatory and neuroprotective effects of statins comes from three open-label studies in patients with RRMS, demonstrating positive effects on both the number and the size of MRI gadolinium-enhancing lesions, compared to baseline, after 12, 6, and 9 months of treatment, respectively, with a high-dose of statins [182‐184] (Table 4). Despite the beneficial effects, the analysis design is questionable and is exposed to the risk of probabilistic phenomena, such as regression to the mean, i.e., an expected decrease in disease activity in a population of patients with high initial activity [184]. In subsequent randomized, placebo-controlled trials, atorvastatin or simvastatin was consistently combined with IFNβ in patients with RRMS, at different doses. However, in view of the inconsistency of the results, some showing a worsening of the disease, an antagonistic effect of this association (statin and IFNβ) has been suggested [185]. The interaction between statin and interferon on blocking the signal transducer and activator of transcription 1 (STAT-1) phosphorylation and so the induction of IFNβ-stimulated genes was evaluated in the SIMCOMBIN trial, to remove any doubt about a possible antagonistic effect of the combination [186]. The evaluation of in vivo IFNβ bioactivity was assessed by measuring mRNA expression of the biomarkers IL-10, TNFSF10, MX1, and IRF7. All patients treated with IFNβ1a 30 µg/week and simvastatin had a full in vivo response, thereby providing reassurance on the concomitant use of the combination. The ACTIVE, SWABIMS, SIMCOMBIN, and ARIANNA [186‐188] studies investigated the long-term treatment effect of statins (atorvastatin or simvastatin), between 12 and 24 months, in combination with IFNβ in RRMS. They were all negative on their primary endpoint [186, 188, 189]. A post hoc analysis of the SENTINEL trial, and thus a retrospective study, found no significant changes on clinical (ARR, disability progression) or MRI (number of Gd-enhancing lesions [GELs], number or new enhancing T2-hypertensive lesions) endpoints between the 40 patients treated with statins, at doses used to treat hypercholesterolemia, and IFNβ1a, compared to the 542 patients treated with IFNβ1a alone, at 1 and 2 years [190]. Despite some biases (retrospective study, non-identical dose, two statins used, different statin treatment durations), the observation of musculoskeletal pain, as well as hepatic enzyme abnormalities, in the statin-treated group suggests that the dose received was sufficient to induce an effect on MS [190]. Even if the comparison of the results between these different studies is made difficult by the different endpoints and by the selection of patients being specific to each trial (years since onset of MS symptoms, EDSS score, number of relapses within previous year), there is no strong evidence of adapted doses of atorvastatin (low or high) or simvastatin, nor of effective treatment duration in add-on therapy with IFNβ, in patients with RRMS. The variable response to statins in MS trials may be explained by an inhomogeneous enrolled population with different inflammatory and progression stages.

In view of these contradictory results, a meta-analysis including eight clinical trials, five on RRMS and one on SPMS, showed no significant effect of statins when added to IFNβ therapy in RRMS. However, statins may be beneficial in SPMS in reducing brain atrophy and disability progression, but they have no effect on relapse rate [191]. This hypothesis was confirmed in the MS-STAT study, where a high dose of simvastatin (80 mg/day, for 2 years) attenuated brain atrophy and disability in patients with SPMS, supporting a real effect on disease progression. The lack of effect of simvastatin on five immune markers (IFNγ, IL-4, IL-10, IL-17, and CD4 Fox P3 levels) did not confirm a neuroprotective effect despite the clinical evidence [192]. Another post hoc analysis of the same study (MS-STAT) highlighted the positive effect of simvastatin on frontal lobe function and physical quality of life [193].

Blocking cation channels, such as sodium and calcium channels, has long been used as a neuroprotective approach in clinical situations such as stroke. This approach makes sense if we consider a potential mechanism of neurodegeneration involving these channels. Thus, it appears that NO, a mediator of inflammation, could strongly slow down or even stop the propagation of action potential in demyelinated fibers [194, 195]. Shrager et al. showed that this NO effect depends on the axonal environment, in their study a sciatic nerve [194]. Surprisingly, Chen and Schofield showed that NO could activate calcium channels in cervical ganglion neurons through a cGMP-dependent mechanism [196, 197]. Thus, NO would block nerve conduction, not by blocking depolarizing channels but by causing a depolarizing block of conduction [194]. The same is true for sodium channels which are activated by NO [198, 199]. Demyelinated neurons in MS are particularly sensitive to this effect since they overexpress sodium channels, most likely to compensate for the conductive deficits linked to demyelination [198]. Increased concentration of intracellular sodium in neurons in patients with MS was recently demonstrated using triple quantum filtered ²³Na magnetic resonance imaging at 7 T [199]. A second toxic effect placing NO at the center of the conduction disorders observed in MS concerns its mitochondrial toxicity. In mitochondria, NO is produced by NO synthase. It reacts with superoxide anion generated by the mitochondrial oxidation chain to form peroxynitrite. In brain mitochondria, NO and peroxynitrite decrease mitochondrial respiration and thus ATP production [200]. In this general schema, MS is seen as a systemic disease with mitochondria as a central player [201]. This reduction may ultimately lead to a decrease in the activity of all active transporters, including Na⁺/K⁺ ATPase and calcium pumps, and thus to a sodium/calcium overload in neurons. Therefore, massive sodium entry due to overexpression of sodium channels and reduced calcium and sodium ion output capacity produces a state of depolarization with conductive blockade and irreversible damage to demyelinated fibers [202].

Myelination is a fundamental physiological process that highlights the interactions between neurons and oligodendrocytes. Moreover, oligodendrocytes receive synaptic connections from neurons and express voltage-dependent channels such as sodium and potassium channels that seem to play a key role in the demyelination process [203]. Interestingly, glatiramer acetate, used as an immunomodulator by acting on B lymphocytes, affects the expression of K⁺ and Cl⁻ channels and the entry of Ca²⁺ into the cells [204]. The effect of drugs affecting ion channels must therefore be considered in a broad way by taking into account the effects on neurons and glial and immune cells. This multicellular impact greatly complicates interpretations and the transition from in vitro studies to in vivo results, especially in clinical trials.

In oligodendroglial cells, numerous Na⁺ (Nav), Ca²⁺, and K⁺ channels are expressed [203]. Voltage-dependent Na⁺ channels have been proposed as links between demyelinated axonal sections and oligodendrocyte progenitors [205]. In demyelinating diseases, Na⁺ channels appear to play opposing pathophysiological roles. Thus, Nav1.2 appears to support conduction by means of rapidly activating and inactivating currents. In contrast, Nav1.6 produces sustained depolarizations that can cause Ca²⁺ overload and all the associated intracellular deleterious effects [206, 207]. This means that targeting of Na⁺ channels in MS must be selective to reduce the risk of antagonistic actions. This led to the testing of PF-01247324, a selective blocker of Nav1.8, in mice with EAE. This compound was able to improve locomotor coordination disorders and the cerebellar syndrome [208]. Regarding voltage-dependent Ca²⁺ channels (Cav), all families are expressed in oligodendrocytes (L, T, N, and P/Q) [209]. Their roles are fundamental in the proliferation, migration, and maturation processes of oligodendrocyte progenitor cells (OPCs). Moreover, invalidation of Cav1.2 in these OPCs reduces their maturation and myelination in the mouse brain and disrupts remyelination in a non-inflammatory demyelination model induced by cuprizone [210, 211]. Thus, Ca²⁺ channel blockade appears to be deleterious. Nevertheless, here again, there would appear to be a balance between the different cell types since genetic invalidation of Cav1.2 in astrocytes decreases the activation and proliferation of astrocytes and microglia, as well as the inflammatory response measured by the production of TNFα, IL-1β, and TGFβ1. These effects are reproduced by nimodipine, a brain-tropic calcium antagonist [212]. Concerning this particular calcium channel antagonist, the origin of its neuroprotective effect appears elusive. Schampel et al. tested nimodipine in experimental autoimmune encephalomyelitis and demonstrated that this drug attenuates clinical signs and spinal cord degeneration and promotes remyelination [213]. This protection is due to the induction of microglia apoptosis. This effect is reproduced with high nimodipine concentrations (5 and 10 µM) in two microglia cell lines (N9 and BV-2) but not by nifedipine, another dihydropyridine calcium channel antagonist. The mechanism by which nimodipine induces such a neuroprotection is still unknown but seems drug-specific and not related to calcium channels. With respect to K⁺ channels, there is also a balance among various cell types. The main K⁺ channels opening at the resting membrane potential are the inward-rectifier Kir4.1 and the two-pore K⁺ channels [209]. Genetic knockout of Kir4.1 strongly disrupts oligodendrocyte maturation and myelination during development, an effect originating in mature oligodendrocytes since selective invalidation in these cells reproduces the phenotype [214, 215]. Interestingly, antibodies against Kir4.1 have been identified in patients with MS, suggesting a pathophysiological role associated with the blockade of this channel [216]. Thus, K⁺ channel blockade seems to be a poor therapeutic option to slow down demyelination or stimulate remyelination. However, at the neuronal level, demyelination leads to alterations in the expression and distribution of K⁺ channels [217]. This delocalization could disrupt neuronal function. Overall, the contribution of cationic channels to the pathophysiology of demyelination and remyelination is far from clear, nor is its contribution to the maintenance of neuronal functionality in MS. Nevertheless, several molecules have been evaluated in clinical trials.

Gray matter damage occurring early in the disease course contributes to clinical disabilities [226, 227]. Neuronal loss occurring in normal appearing gray matter emphasizes the need to explore neuronal endangering mechanisms independent of demyelination, such as synaptic dysfunctions [228, 229]. In inflammatory bursts of MS, experimental models have identified actors detrimental for synapses such as components of complement C1q and C3 [230], TNFα [231], IL-1β and oxidative stress [232]. Besides, proinflammatory cytokines released in the lesion disturb neurotransmission by increasing glutamate-mediated transmission and reducing GABA transmission. Glutamate accumulates in the synaptic cleft and induces excitotoxic damage [233]. Moreover, the maintenance of this excess of excitatory input observed during–remission phases of EAE could alter the establishment of neuronal connections [234]. Indeed, when long-term plasticity is impaired in EAE models, disability recovery is also strongly reduced [157].

Because of its potent reversibility, pharmacological treatment of synaptopathy remains attractive. Targeting of the glutamatergic pathway has therefore been tried with many drugs because of its role in the pathophysiology of MS. Increased glutamate concentration in the CSF correlates with relapsing phases of MS or disabling secondary progressive forms [235]. Therapeutic strategies have been tested through the targeting of glutamate ionotropic receptors (N-methyl-d-aspartate receptor [NMDAR], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor [AMPAR], kainate receptor) as well as metabotropic receptors. Already known for its toxic effect on neurons, glutamate also affects oligodendrocytes, astrocytes, endothelial cells, and immune cells. In inflammatory lesions, the immune cells involved in extracellular glutamate release are monocytes, macrophages, microglia, and dendritic cells via the cysteine/glutamate antiporter Xc^– [236‐238]. Altered glutamate transport likely contributes to this dysregulation, as seen for the glutamate transporter GLT-1, whose expression in oligodendrocytes decreases around active MS lesions [239].

AMPA receptors can lead to motor neuron damage through an excessive increase of calcium in cytosol and mitochondria resulting in oxidative stress [240]. Therefore, in the Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) chronic viral model of MS, the NBQX (2,3-dioxo-6-nitro-7-sulfamoylbenzo[f]quinoxaline) AMPA/kainate receptor antagonist reduced the clinical score and axonal damage visualized by lower dephosphorylation of the heavy chain of neurofilament H [241]. An alternative targeting of AMPA receptors was performed with a TAT-fusion peptide disrupting the interaction between the AMPA receptor subunit GluR2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [242]. Zhai et al. observed increased GluR2/GAPDH complexes in plaques of MS. Already shown to prevent neuronal damage, this peptide successfully reduced the clinical score and axonal damage [243].

Extrasynaptic NMDARs are mediators of neuronal damage whereas synaptic NMDARs are mediators of plasticity, thus complicating neuroprotective approaches [244, 245]. However, preclinical studies using NMDAR antagonists were promising. When inhibited by NMDAR antagonists memantine or amantadine, rats subjected to an EAE protocol developed lower clinical scores [244, 246]. Conversely, antagonists of group I metabotropic glutamate receptors, mGluRs (LY 367385 and MPEP) failed to demonstrate a beneficial effect [246].

To date, clinical trials have failed to demonstrate any significant effect of memantine on patients with MS whatever the parameter investigated: fatigue, cognitive performance, or spasticity [247‐251] (Table 6). Worse still, adverse events have been reported in several studies. Trials regarding treatment of fatigue by amantadine similarly have failed to reach a consensus [252, 253]. The first studies found a reduction of fatigue [254], but this effect was not found in more recent studies [255]. Ketamine, another NMDAR antagonist, emerged recently as a potential modulator of mood disorders with a rapid and sustained antidepressant effect when infused at low doses in patients suffering from treatment-resistant depression. A clinical trial conducted on participants with bipolar disorders showed also a strong and prolonged reduction in fatigue scores, indicating that ketamine may be a valuable approach in fatigue management in multiple diseases [256]. Thus, in patients with MS, ketamine infusions, which were shown to be safe and well tolerated, led to a decrease in fatigue severity over a long period [257]. Riluzole constitutes another approach via inhibition of glutamate release. It reduced inflammation, demyelination, axonal damage, and clinical score in a MOG-induced murine EAE model [258]. Despite encouraging data in a pilot study [259], phase II trials turned out to be disappointing since riluzole failed to reduce brain atrophy progression in early MS or in SPMS [260‐262]. However, drug efflux transporters present in the BBB like P-glycoprotein (P-gp) and breast cancer-resistant protein (BCRP) could limit riluzole’s effect. One possible solution has been successfully experimented in a mouse model where P-gp/BCRP inhibitor rescued riluzole’s effect [263].

Steroids synthesized by peripheral glands act on several tissues in the body, including the peripheral and central nervous systems, since free steroids are capable of crossing the BBB. Indeed, steroid hormones (progesterone, estrogens, androgens, and corticosteroids) that are lipophilic molecules may control several processes in the CNS by crossing the BBB (by simple diffusion or via influx transporters) or by targeting BBB cells, which in turn affect the brain parenchyma by modulating inflammatory and oxidative mechanisms [264, 265]. Sex steroids exert protective effects on the BBB or restore its integrity and permeability in experimental models of stroke or LPS-induced inflammation in rodents [266, 267]. Decreased levels of circulating estrogens during aging have been correlated with increased BBB permeability while testosterone depletion is associated with glial cell (microglia and astrocytes) activation and increased inflammation and BBB permeability [268]. These parameters are improved by testosterone treatments [269]. Moreover, progesterone administration exhibited positive effects on the BBB physiology after stroke and traumatic brain injury [270, 271]. Altogether, these data strongly support the rationale for the use of steroidal hormones to tackle the central disorders observed in MS. Furthermore and more importantly, it is also demonstrated that in MS, female to male prevalence is approximately 3:1, primarily in RRMS forms [272]. Various hormone-related physiological conditions in women significantly impact both the frequency and the course of disease. Numerous studies have pointed to the fact that steroid hormones exert a large array of biological effects, including the modulation of diverse biological processes such as neuroprotection and myelination [273‐279]. In terms of immune response, decreased concentrations of sex steroids are associated with higher serum levels of proinflammatory cytokines such as TNFα and INFγ [280, 281]. The influence of sex hormones on the immune system has also been observed in the EAE mouse model. Sex hormones (estrogens, progesterone, and androgens) likely play a role in the complex mechanism of the course of the disease. Indeed, the relapsing forms of MS are more frequent in young women; moreover, the relapse rate decreases during late pregnancy as hormonal estrogen secretions increase. These effects could be related to the anti-inflammatory properties of estrogens [282]. The influence of this hormone on the immune system has also been observed in the EAE mouse model. When administered to mice prior to inoculation of EAE, exogenous estriol reduces disease activity [283]. In EAE, both females and males had a decreased proinflammatory cytokine profile with estriol treatment [281].

Progesterone is believed to have neuroprotective, promyelinating, and anti-inflammatory effects in the nervous system [284]. In EAE mice, progesterone attenuates the clinical severity, decreases demyelination, neuronal dysfunction, and the inflammatory response [281, 285]. More importantly, it has been well demonstrated that the progesterone derivative 3α,5α-tetrahydroprogesterone, also called neurosteroid allopregnanolone, critically modulates neuroinflammatory processes and MS symptoms. Gas chromatography–mass spectrometry assessment evidenced reduced levels of allopregnanolone in the brain of patients with MS [255]. Interestingly, allopregnanolone treatment after EAE induction decreased the disease development in mice [286‐288]. Recently, we also combined various cellular and biochemical methods to show a direct effect of allopregnanolone on microglial morphology and phagocytic function, suggesting that allopregnanolone-based treatment may be of interest for the development of effective neuroprotective strategies against neurological disorders, including MS, that are evoked by microglia-related abnormalities [289].

We have previously reviewed extensively the key role played by testosterone in the modulation of immunodulatory, neuroprotective, and promyelinating mechanisms—a role that makes this sex steroid a potentially important candidate for the development of a hormone-based therapeutic strategy against MS [290]. Indeed, low testosterone has been reported in 40% of men with MS, correlating with physical and cognitive disability [291] and worse clinical outcomes [292]. Furthermore, in EAE models, there also seems to be a protective role of testosterone. EAE is less severe in female mice pretreated with dihydrotestosterone. In castrated mice the symptoms are more severe and can be ameliorated with testosterone replacement [283]. Administration of testosterone to men with MS could prevent the progression of the disease because of its potential neuroprotective and promyelinating effects [290].

On the basis of their neuroprotective, promyelinating, and/or anti-inflammatory effects, estrogen and testosterone have been considered as good candidates for MS treatment [290]. Therefore, clinical studies were conducted to investigate their therapeutic potential in MS (Table 7). The first clinical trial using estrogen was conducted as a pilot assay in 10 women with MS in order to mimic the protective effects observed during pregnancy. The resulting study showed a reduced ARR in the patients treated with estriol compared to the placebo group [293]. A phase III study entitled POPART’MUS (Prevention of postpartum relapsing with progestin and estradiol in MS) was also conducted in pregnant women with MS with the objective of preventing postpartum MS attacks. Unfortunately, no beneficial effect was found on either the relapse rates or on the MRI data [294]. Very recently, the group POPART’MUS designed a new clinical trial in postpartum women treated with nomestrol acetate (NOMAc) and 17-beta-estradiol. After 12 weeks, no treatment efficacy was observed compared to the placebo, likely as a result of the slow rate of inclusions [295]. However, another clinical trial in phase II on women with RRMS showed a beneficial effect of estrogens that decreased the brain lesions or reduced the relapse rates [296, 297].

In parallel to estrogens, some clinical trials using androgens, and in particular testosterone, for MS therapy have been reported. Indeed, Sicotte et al. conducted the first pilot study which tested the therapeutic action of testosterone in 10 men with RRMS [298]. The neuroprotective effects of the treatment resulted in an improvement of cognitive performance and a slowing of brain atrophy. This original study was extended to include other endpoints showing an immunomodulatory effect of testosterone [299]. A significant GM increase was also reported as the result of testosterone treatment [300]. Very recently, a phase II trial protocol was initiated with the aim of evaluating the beneficial effects of testosterone supplementation in testosterone-deficient men with RRMS [301].

Brain injuries induce the expression of growth factors within lesions, some of which can be measured in CSF and serum of patients. Long known for their trophic support of neurons, growth factors display their effect through pleiotropic target cells, including oligodendrocytes and immune cells.

In the development of new therapeutic strategies to treat MS, EPO is the most advanced growth factor. Essential for red blood cell production, EPO has multifunctional protective effects, due to its anti-inflammatory properties [327], blocking of ROS production and related apoptosis, neuroprotective effects, and stimulation of neurogenesis [328, 329]. EPO stimulates proliferation of OPCs and maturation of oligodendrocytes in vitro [330, 331]. In a rat model of EAE, recombinant human EPO (rhEPO) reduced inflammatory cytokines and infiltration of immune cells within lesions [332]. In a mouse model of EAE, rhEPO treatment led to a reduction of inflammatory infiltrates as well as a better functional recovery associated with an increase of OPC proliferation and BDNF-positive cells [333]. In a MOG-induced mouse model with optic neuritis, EPO combined with a high dose of methylprednisolone protected the optic nerve by reducing axonal damage and demyelination [334]. A double-blind, placebo-controlled phase II study evaluated the therapeutic potential of EPO on optical neuritis as add-on therapy (NCT00355095; VISION PROTECT). After 16 weeks of EPO treatment, retinal nerve fiber layer thinning was less apparent and visual evoked potentials were significantly shorter [335] (Table 8). A phase III study was then conducted and results have just been published [336]. However, the study did not confirm previous results and displayed neither functional nor structural neuroprotection in the visual pathways after optic neuritis. Further developments in EPO engineering deserve attention, such as EPO-derived small peptide with neuroprotective activity without hematologic effects, which in EAE models showed a protective effect, with lower clinical scores and decreased astrogliosis [337] or engineering with chimeric antibody targeting the transferrin receptor to improve EPO BBB penetrability [338].

Coenzyme Q10 is the main endogenous ubiquinone and acts as an essential cofactor of the electron transport chain. It is synthesized in the inner mitochondrial membrane and plays an essential role in the mitochondrial respiratory chain. Coenzyme Q10 is one of the most potent lipophilic antioxidants, acting as a catalytic antioxidant when chemically reduced from the ubiquinone to the ubiquinol form [339]. In progressive MS, oxidative stress and mitochondrial injury occur as a consequence of chronic inflammation and have a harmful impact on neurons and axons [340, 341].

Sanoobar et al. evaluated the effect of coenzyme Q10 supplementation on serum level of antioxidant factors in patients with RRMS (IRCT201102052602N5) (Table 9). Coenzyme Q10-treated patients had a significant increase in superoxide dismutase (SOD) activity and a decrease in malondialdehyde (MDA) levels compared with controls [342]. Coenzyme Q10 supplementation did not affect glutathione peroxidase activity. Moreover, the authors reported that coenzyme Q10 appears to decrease the inflammatory markers (TNFα, IL-6, and MMP-9) and improve fatigue and depression quantified by means of the fatigue severity scale and the Beck depression inventory [343, 344].

Moccia et al. reported that coenzyme Q10 in patients with RRMS improved various markers of scavenging activity (uric acid, bilirubin), oxidative damage (intracellular ROS, oxidative DNA damage), and induced a shift towards a more anti-inflammatory milieu in the peripheral blood [345].

Coenzyme Q10 exhibits limited oral bioavailability. For this reason, several synthetic analogues of coenzyme Q10 such as idebenone and mitoquinone have been developed and proposed in numerous diseases with mitochondrial injury. They are currently being investigated as therapeutic options in MS.

Idebenone, (10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone), is a synthetic analogue of coenzyme Q10 described as a potent antioxidant. However, discrepancies between achievable tissue levels in brain and the doses required to show the proposed effects call into question the current proposed mechanism of action (MoA) [346]. Recent findings reviewed by Gueven et al. provide new insight into the MoA of idebenone. The authors hypothesize that to explain its pleiotropic effects, idebenone may modulate distinct signaling pathways, including inhibition of p52Shc, increased Lin28A expression, activation of Akt, and the subsequent transcriptional changes [346]. In an EAE mouse model of MS, idebenone failed to affect disease incidence or onset when applied preventively, or to reduce disease severity when applied therapeutically [347]. In the clinical trial conducted by Kosa et al. (NCT00950248), idebenone was well tolerated but the change the area under the curve of the Combinatorial Weight-Adjusted Disability Score (CombiWISE) between idebenone and placebo did not reach statistical significance, attesting that idebenone does not inhibit disability progression in PPMS [348].

Mitoquinone (MitoQ) is a quinone moiety linked to a triphenylphosphonium moiety by a 10-carbon alkyl chain [349]. The positive results in an EAE mouse model indicated that MitoQ could be a candidate for investigation in human MS [350]. The authors reported that MitoQ can exert protective effects on neurons and reduce axonal inflammation and oxidative stress. MitoQ is currently under investigation in the “MitoQ for fatigue in MS” phase I/II placebo-controlled trial (NCT04267926). In this trial, the investigators hypothesize that mitochondria dysfunction and resultant neuronal energy depletion may be an important contributor to fatigue in MS. The study is still recruiting patients with MS with an EDSS score of 2–8, persistent fatigue (at least 2 months), and a Modified Fatigue Impact Scale (MFIS) score of 38 or greater. The primary outcome is change in fatigue impact as measured by the MFIS at 12 weeks.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors activated by small lipophilic molecules. Once activated by their ligand, these receptors bind to DNA sequences and regulate gene expression by transcriptional co-activation [351, 352]. More specifically, activated PPARs form a complex in the cytoplasm with the retinoid X receptor-α [353]. The complex finally targets the peroxisome proliferator response element in the promoter gene [354]. This is what causes the regulatory cascades. Pioglitazone is a member of the family of thiazolidinediones (TZDs). These drugs act as agonists of the PPARγ. Owing to their insulin sensitizing effect, two TZDs were originally marketed for the treatment of type 2 diabetes: pioglitazone and rosiglitazone. TZDs promote the differentiation of mesenchymal stem cells into adipocytes and lipogenesis in peripheral adipocytes, and they decrease hepatic and peripheral triglycerides and decrease visceral adipocyte activity [355]. These main effects of TZDs significantly improve insulin resistance and metabolic syndrome and decrease insulin requirements. However, TZD use has been limited because of concerns about safety issues and side effects. In particular, as a result of an increased risk of bladder cancer, pioglitazone was withdrawn by the French and German health authorities in 2011. However, a subsequent follow-up study in the USA involving over 193,000 patients aged 40 years and older found no correlation between bladder cancer and pioglitazone use. Bladder cancer may no longer be a significant issue [356]. Other side effects, such as edema, congestive heart failure, and bone fractures, require careful selection of patients who could benefit from these treatments [355].

Other effects of PPARγ agonists include reduction of TNFα, IL-1β, and inducible nitric oxide synthase (iNOS). PPARγ agonists also exert antiproliferative action, block cytokine production, and induce apoptosis in T cells [357‐366]. Because of the anti-inflammatory properties of PPARγ agonists, several authors have hypothesized that the repositioning of pioglitazone in EAE could reduce clinical and histological manifestations. In a mouse model of EAE, pioglitazone reduced the incidence and severity of chronic single-phase disease [367]. The suppression of clinical signs was accompanied by a reduction in lymphocyte infiltration, a decrease in demyelination, a reduction in chemokine and cytokine expression, and an increase in inhibitor of kappa B expression in the brain. Pioglitazone also reduced antigen-dependent IFNγ production from EAE-derived T cells.

As a result of the neuroprotective and anti-inflammatory effects of pioglitazone and the encouraging results obtained from preclinical experiments in EAE models, a clinical trial dedicated to pioglitazone evaluation in RRMS was initiated. In this randomized, placebo-controlled phase I trial (NCT00242177), Stefoski et al. reported that pioglitazone was well tolerated, with a similar incidence of non-serious adverse events in the placebo and treatment groups (10 and 11 patients, respectively). After 1 year, there were no significant differences in clinical symptoms as assessed by EDSS. The authors reported that MRI showed a significant reduction in gray matter atrophy and that pioglitazone could reduce conversion of normal appearing white matter to lesions [368, 369]. Using a different approach, the TRAP-MS study (NCT03109288), a phase I/II open-label trial, is currently recruiting patients with progressive MS to evaluate whether signs of inflammation in CSF help predict a person’s response to different drugs, one of which is pioglitazone. The primary outcome is the change in CombiWISE progression rate at the end of the monotherapy plus combination therapy period in comparison to projected baseline disability progression. Clinical data on the use of pioglitazone in MS remain particularly limited to date. Pioglitazone concentrations reaching the brain could be an issue. Some BBB transporters restrict pioglitazone delivery to the brain and higher amounts than the usual diabetic dose could be required [370]. In the current state of knowledge, the therapeutic positioning of pioglitazone in MS is particularly unpredictable. Further studies are required to assess the interest of this molecule in MS.