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4 Failed Clinical Trials in Progressive Multiple Sclerosis

4.1 Neuroprotective Approaches

4.1.1 Acid‑Sensing Ion Channels—Amiloride

4.1.1.1 Background

A certain group of ion channels linked to Na+- and Ca2+-dependent neuroaxonal injury is the family of acid-sensing ion channels (ASICs). These are proton-gated cationic channels activated by an acidic pH leading to an influx of Na+ and Ca2+ ions [175]. Amiloride, well-known as a diuretic, turned out to be an unspecific inhibitor of ASICs and already proved efficacy in animal models of PD [176], stroke [177], and Huntington’s disease [178]. Consistently, amiloride-induced neuroprotection alleviated axonal degeneration as well as clinical symptoms of acute EAE [179, 180]. More importantly, amiloride treatment also ameliorated clinical signs of EAE performed in Biozzi ABH mice, a model resembling characteristics of progressive MS [179, 180]. Although ASICs are also expressed on several immune cells (e.g., B cells, T cells, and macrophages), compelling evidence suggests that beneficial efects of amiloride in EAE are mediated independently from immunomodulation [180].

Beyond insights gathered from animal experiments, there are also clinical data supporting the hypothesis of the involvement of ASICs in MS pathophysiology. First, SNPs located in a gene coding for ASICs are associated with enhanced susceptibility to MS [181]. Moreover, axonal ASIC expression is upregulated at the border of acute MS lesions [179]. Importantly, amiloride treatment was already tested in an uncontrolled pilot study in PPMS [182]. There, it slowed brain atrophy and alleviated worsening of tissue damage related to markers of DTI.

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4.1.1.2 Studies

Amiloride in AON In the ACTION trial, amiloride was evaluated in patients with the first episode of unilateral AON [183]. In this double-blind Phase II study, 48 patients were randomly assigned to either daily treatment with 10 mg amiloride or placebo. Treatment was initiated within 28 days after symptom onset and continued for five months. Steroid treatment after AON onset was allowed but not mandatory. The pSE evaluated the change of the peri-papillary RNFL thickness after six months. However, this endpoint was not reached. In addition, analysis of secondary endpoints, including structural, visual, and electrophysiological criteria, did not show an advantage of amiloride treatment. Patients of the active treatment group even dis- played a signifcantly prolonged peak time of VEPs.

Amiloride in SPMS Following the positive outcome of the mentioned pilot study in PPMS [182], the efficacy of amiloride treatment in SPMS was tested in a multi-arm, double-blind Phase IIb trial, in parallel to fluoxetine and riluzole (MS-SMART) [184]. For a period of 96 weeks, 223 patients were included to receive either 10 mg/day amiloride or placebo. However, amiloride treatment failed to induce any difference concerning the pSE, i.e., the PBVC after 96 weeks. Moreover, secondary MRI (PBVC after 24 weeks, new or enlarging T2 lesions after

96 weeks) and clinical endpoints (e.g., changes in EDSS) were not met.

4.1.1.3 Comment

Besides the negative outcomes of the ACTION and MS-SMART trials, large registry-based cohort studies could not detect an association between amiloride treatment and a decreased risk for MS or MS-related hospitalization, further questioning the efficacy of amiloride in MS [185].

Moreover, as the pathophysiology of AON is mainly mediated by inflammatory processes [186], selectively targeting the neuroaxonal loss without affecting the immunological response might be insufficient. Notably, 20% of patients in the placebo but only 5% in the treatment group received steroids after AON onset in the ACTION study [183]. Considering the improvement of visual functions by corticosteroid treatment in AON [187], the higher frequency of steroid administration might have influenced the outcome in favor of the placebo group. This connection is especially important given the extended time frame of treatment within 28 days after onset, as crucial damage might have already occurred before the initiation of therapy [183]. Future studies should, therefore, investigate amiloride treatment in obligate combination with high-dose IVMPS rapidly after AON onset. Concomitant immunosuppressive strategies might, for instance, enhance the efficacy of amiloride by ameliorating the inflammatory milieu and immune-mediated neuroaxonal injury.

Notably, there are also uncertainties related to the mechanism of action of amiloride in MS. ASIC opening is mediated by an acidic environment. Strikingly, extra-cellular acidosis was solely demonstrated in infanta- tory lesions in EAE [180]. In MS, acidosis was merely assumed, as inflammatory lesions exhibited a higher lactate concentration [188]. However, this increase of lactate seemed to correlate with the degree of infammation and was already reduced in inactive plaques, further doubting the impact of amiloride particularly in progressive MS. In line with this, ASIC expression in MS lesions was pre-dominantly observed at the lesion border, which is associated with a more inflammatory milieu [179]. A recent study evaluates imaging-based measurements of pH in the brain tissue of MS patients and might thus shed light on this issue [189].

4.1.2 Fluoxetine

4.1.2.1 Background

Selective serotonin reuptake inhibitors (SSRIs) are well-known therapeutics for the treatment of psychiatric disorders [190]. One of these SSRIs is fluoxetine. Despite its traditional use, a report in 1991 mentioned

20 MS patients who experienced clinical improvement under fluoxetine treatment [191]. Therefore, SSRIs were repurposed in the field of neuroinflammation and neurodegeneration. Repurposing of SSRIs was further supported by insights derived from EAE experiments. There, both the prophylactic and therapeutic application of fluoxetine alleviated clinical EAE progression and enhanced disease remission [192, 193]. Several mechanisms underlying EAE amelioration are discussed. Foremost, a neuroprotective mode of action is considered. Fluoxetine was shown to stimulate astrocytic glycogenolysis and lactate release, thereby providing energy supply to neurons [194, 195]. In addition, fluoxetine enhances the production of neurotrophic factors [196] and inhibits voltage-gated calcium [197] as well as sodium channels [198]. In this way, fluoxetine prevents Ca2+- and Na+-induced neurotoxicity [197, 198]. Beyond neuroprotection, several models indicate a stabilizing impact of fluoxetine on the BBB through restoring tight junction molecules [199, 200]. Finally, fluoxetine has several implications concerning the inflammatory response. Among these are an inhibited microglial activation [200], impaired glial antigen presentation [201], and subsequently, a reduced release of pro-inflammatory cytokines by T cells [202].

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4.1.2.2 Studies

Starting in 2012, a randomized, double-blind Phase II trial evaluated the impact of fluoxetine on disease progression in 77 PPMS and 55 SPMS patients (FLUOX- PMS) [203]. Participants received either 40 mg/day fluoxetine or placebo over 108 weeks. The pSE assessed the time to a 12-week confirmed 20% increase in the T25FW or 9HPT. However, no significant improvement was observed in the analysis of primary and secondary objectives (e.g., cognitive tests and MRI criteria).

Fluoxetine was further investigated in the already described multi-arm Phase IIb MS-SMART trial at a dose of 40 mg/day [184]. One hundred and eleven SPMS patients were allocated to the fluoxetine group, and 112 received placebo. However, fluoxetine failed to afect the pSE, namely the PBVC, after 96 weeks. On the contrary, PBVC was even enhanced compared to placebo after 24 weeks. In terms of secondary objectives, fluoxetine treatment reduced the number of new or enlarging T2 lesions. Other secondary endpoints (e.g., changes in EDSS) were not met.

4.1.2.3 Comment

In conclusion, fluoxetine treatment did not lead to positive outcomes in two Phase II trials. While the MS-SMART study was sufficiently powered [184], an unexpectedly low rate of disease progression was observed in the FLUOX-PMS trial, strongly reducing its power [203]. Additionally, a double-blind, placebo-controlled trial in 42 patients with PPMS or SPMS did not demonstrate an advantage of fluoxetine treatment [204]. Taking together the failure of fluoxetine in all three trials, a convincing beneficial efect in progressive MS is unlikely.

When looking at the mode of action, the initially assumed predominant neuroprotective role of fluoxetine might be outweighed by its anti-inflammatory impact. Fluoxetine treatment results in a variety of immunomodulatory processes, which were observed in both animal and human studies [193, 205]. Along the same lines, fluoxetine proved efficacy in animal models characterized by strong inflammatory pathophysiology [192, 193]. The use of animal models hallmarked by pronounced inflammatory pathophysiology may also explain why promising data of fluoxetine treatment in pre-clinical experiments were not translatable into clinical trials including patients with progressive MS. Possibly, an animal model of progressive MS, such as EAE in non-obese diabetic mice, would have been more appropriate to study the efects of fluoxetine on the pathophysiology of progressive disease courses.

Further support for a predominant anti-inflammatory role of fluoxetine derives from two clinical studies. First, escitalopram, another SSRI, reduced the cumulative risk for relapses compared to controls in an open-label trial [206]. Also, in a double-blind, placebo-controlled study in RRMS and SPMS patients, fluoxetine itself decreased the formation of GELs [207]. In line with a putative use of fluoxetine in relapsing MS is the reduction of T2 lesions in the MS- SMART trial, although this observation needs to be confirmed considering that post-gadolinium scans at baseline were missing [184]. Taken together, fluoxetine potentially provides benefits to patients with relapsing rather than progressive MS.

4.1.3 Riluzole

4.1.3.1 Background

Glutamate excitotoxicity is a widely discussed mechanism contributing to the pathophysiology of neurodegeneration [208]. Underlying processes of glutamate excitotoxicity involve axonal damage and neuronal cell death [209, 210]. Furthermore, glutamate excitotoxicity leads to oligodendrocyte damage and subsequent demyelination [208]. Therefore, glutamate antagonists gained inter- est as neuroprotective therapies.

One well-known glutamate antagonist is riluzole, which is commonly used in the therapy of amyotrophic lateral sclerosis [211]. Besides suppressing the release of glutamate from nerve terminals, riluzole stabilizes sodium channels in an inactivated state [211]. In line, EAE experiments indicated neuroprotective efects induced by riluzole application [210]. Moreover, a pilot study performed in 16 patients with progressive MS showed a reduction in the rate of cervical cord atrophy due to treatment with riluzole [212].

4.1.3.2 Studies

Following the mentioned pilot study in progressive MS, riluzole was further investigated in the multi-arm Phase IIb MS-SMART trial [184]. Two hundred and twenty-three SPMS patients were included to receive either 100 mg/day riluzole or placebo for a period of 96 weeks. However, the active treatment group failed to meet the pSE (PBVC after 96 weeks) and all secondary outcome parameters (e.g., changes in EDSS or MSFC).

4.1.3.3 Comment

Besides the MS-SMART trial and the pilot study, riluzole was further evaluated in combination with weekly administered interferon-β1a [213]. This randomized placebo-controlled Phase II trial included 43 patients with RRMS or clinically isolated syndrome. Of note, treatment with riluzole did not reduce brain atrophy in this study. In line with the results of riluzole, treatment with the NMDA antagonist memantine failed to improve cognitive impairment in relapsing and progressive MS patients [214]. Given the lack of an efect of riluzole and memantine treatment on both CNS atrophy and clinical parameters, the question arises to what extent glutamate excitotoxicity is relevant to neurodegenerative processes in MS. On the other hand, the high number of glutamate-dependent cell targets and mechanisms of action lead to pleiotropic effects of these glutamate antagonists [208]. Eventually, further identification of relevant signaling pathways involved, together with the design of highly specifc compounds, could advance our understanding of the role of glutamate excitotoxicity in neurodegeneration during MS.

4.1.4 Ubiquinone—Idebenone

4.1.4.1 Background

Ubiquinone (CoQ) is an anti-oxidant and lipophilic electron carrier in the mitochondrial electron transport chain. However, low water solubility makes CoQ impracticable for clinical application [215]. Idebenone, a water-soluble short-chain analog of CoQ [216], is therapeutically used in the treatment of Friedreich’s ataxia and Leber’s hereditary optic neuropathy, both caused by mitochondrial dysfunction [217]. Just like CoQ, idebenone administration was shown to detoxify free radicals and inhibit lipid peroxidation [218]. Moreover, treatment with idebenone regenerates mitochondrial function potentially through restoring the electron flow using a bypass mechanism [219, 220]. As oxidative stress and mitochondrial dysfunction also contribute to neuroaxonal damage in neuroinflammation and neurodegeneration [8], idebenone is considered a putative agent in MS [215].

Beyond its neuroprotective features, idebenone also has anti-infammatory capabilities. In microglia, it suppressed the production of pro-inflammatory factors (e.g., interleukin (IL)-1β, tumor necrosis factor-α) and induced a shift towards an M2 phenotype that is related to anti-infammation and regeneration [221]. Given the neuroprotective and immunomodulatory properties, idebenone could be a promising medication in MS.

4.1.4.2 Studies

In a double-blind, placebo-controlled Phase I/II trial, idebenone was evaluated in 77 PPMS patients (IPPoMS) [222]. A 1-year pre-treatment period was followed by an active phase of 2 years with a daily application of 2250 mg idebenone. The pSE investigated the change in the Combinatorial Weight-Adjusted Disability Score (CombiWISE) consisting of EDSS, T25FW, 9HPT, and Scripps Neurological Disability Scale [223]. However, treatment had no impact on the CombiWISE. Additionally, evaluation of secondary objectives, including changes in the enlargement of ventricular volume as well as differences in the single categories of the CombiWISE, did not reveal notable improvements induced by idebenone.

Afterward, all patients who had completed the IPPoMS trial were invited to an open-label extension study [224]. There, all patients should receive idebenone for 1 additional year. Unfortunately, the publication of the extension study is missing.

4.1.4.3 Comment

The shortage of available clinical data hampers the analysis of the role of idebenone in MS. Moreover, pre-clinical insights are sparse. Idebenone was once tested in EAE, failing to afect disease incidence, onset, and severity [225]. Further, idebenone had no impact on neuroinflammation or axonal damage.

It can be speculated that the failure of idebenone was related to the dependency of its function on cytoplasmic NAD(P)H: quinone oxidoreductase 1 (NQO1). NQO1 catalyzes the reduction of idebenone into idebenone [220]. While idebenone itself exhibits oxidative properties [226] and impairs the electron transport chain [227], the favorable actions are attributed to idebenone [215]. Unfortunately, NQO1 expression is mainly restricted to astrocytes and a subset of oligodendrocytes, while it is rare in neurons [228]. Therefore, the potential for direct neuroprotection is limited [229]. However, as NQO1 expression is inducible, combination with agents selectively upregulating NQO1 in neurons might enforce idebenone treatment in neurodegeneration [229].

Conclusively, the paucity of data impedes an assumption of the impact of idebenone on neuroinflammation. Nonetheless, the underwhelming performance in the IPPoMS trial and insights derived from animal experiments contradict a beneficial role of idebenone in PPMS, at least when used as monotherapy.

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4.2 Neuroregenerative Approaches

4.2.1 Erythropoietin

4.2.1.1 Background

Erythropoietin (EPO) is an internal body hormone stimulating erythropoiesis. Interestingly, a tissue expression study identified EPO and its receptor (EPOR) in neurons, glial, and also endothelial cells of the CNS [230–232]. Emerging evidence describes the EPO-EPO-R- axis as an endogenous neuroprotective system getting activated in response to neuronal damage in terms of hypoxia, metabolic stress, or infammation, just as observed in EAE [233, 234]. Accordingly, exogenously administered EPO ameliorated clinical signs of neuroinflammation in mice [235–237]. Clinical improvement depends at least partly on the induction of remyelination. In vitro, for instance, EPO promotes OPC differentiation and oligodendrocyte maturation [230, 238]. Further, it enhances the expression of myelin markers after cuprizone-induced demyelination and proliferation of OPCs during EAE [239, 240].

Besides remyelination, EPO prevents different hallmarks of autoimmune neuroinflammation, such as BBB breakdown or axonal injury [235–237]. The underlying mechanisms of neuroprotection involve neurotrophic [241], anti-oxidative [242], and anti-apoptotic processes [243]. Even more, EPO modulates the inflammatory response, leading to increased expansion of regulatory T cells and suppressed differentiation of TH 17 cells [236].

In addition to the aforementioned pre-clinical observations, a small exploratory open-label Phase I/IIa trial on high-dose EPO treatment demonstrated a long-lasting significant increase in walking distance and improvements of cognitive impairment in PPMS and SPMS [244]. Prolonged EPO treatment was further assessed in a small study in an SPMS cohort, indicating beneficial efects of EPO in terms of neurophysiological criteria (e.g., increased intracortical facilitation) and a trend of ameliorated fatigue [245].

4.2.1.2 Studies

Following the frst promising clinical results, a randomized, double-blind Phase II trial was performed in 18 PPMS and 34 SPMS patients (without relapses within the last 2 years) [246]. The mean disease duration varied between 16.7 years in the EPO and 14.9 years in the placebo group. Participants received either 48,000 IU EPO or placebo for 24 weeks (in the frst half weekly, afterward biweekly) followed by a 24-week observation period. IVMPS 1 g was applied prior to the frst and second infusion of EPO or placebo. The pSE investigated the change of a composite score consisting of the maximum gait dis-

tance, hand dexterity, and cognition after 24 weeks. Patients treated with EPO demonstrated a slight increase in the composite score, indicating clinical improvement. However, this trend was not significant and could not be observed after 48 weeks. Analysis of clinical parameters (e.g., EDSS and MSFC), MRI assessment (e.g., T2 lesion volume and PBVC) as well as patient-reported outcomes (36-Item Short-Form Health Survey [247]) did not reveal any advantage of EPO treatment. Concerning the safety profle, most patients in the active treatment group needed blood-lettings due to increased hematocrit and experienced hypertensive episodes more frequently.

4.2.1.3 Comment

While an frst exploratory pilot study indicated improvements under EPO treatment, especially in terms of motor functions [244], these results could not be reproduced in the reviewed Phase II trial, even though a positive trend was observed [246]. An underlying reason for failure might be the mean disease duration of over 16 years. Indeed, remyelination declines with MS duration and is less present in chronic lesions [6, 87]. Thus, agents promoting remyelination might be more effective in ear-lier disease courses, since an efect on long-lasting, inac- tive lesions is doubtful. In line with this assumption, a small double-blind study supported efficacy of EPO-treatment in RRMS patients with an acute relapse [248]. The frst promising results of EPO in AON in a placebo-controlled Phase II trial further support this hypothesis [249]. Conclusively, the selection of patients in advanced stages of disease progression might have masked potentially existing beneficial efects of EPO treatment.

Nonetheless, the potential use of EPO in MS is hampered by side efects. Administration of EPO not only induces erythropoiesis but has several other implications such as vasoconstriction or thrombocythemia, thereby enhancing the risk for thromboembolic events and hypertension [233]. Thus, patients with cardiovascular risk factors, a history of thromboembolic events, or immobility were excluded from clinical trials [246, 249, 250]. Moreover, EPO can cause rare but potentially fatal pure red cell aplasia [251]. Due to the expression of EPO and EPO-R on several malignant tumours [252] and reports of tumour expansion under EPO treatment [253, 254], patients with a history of malignancy should not receive this drug either. Taken together, a remarkable proportion of patients, especially that suffering from multimorbidity, would not be eligible for treatment even if EPO were to be efective in MS.

On the other hand, tissue-protective processes may not only be mediated via the classical EPO-R. Foremost, these efects seem to be induced by the interaction of a distinct EPO region with a heteromeric receptor consisting of one EPO-R subunit and the β-common chain shared by members of the IL-3 receptor family [233, 255]. Thus, derivates of EPO were shown to exhibit neuroprotection without afect- ing erythropoiesis [235]. Among these derivates is JM-4 [256]. This peptide protected mice from demyelination and alleviated the pro-infammatory response, thereby amelio- rating clinical signs of EAE [256]. Of note, an early Phase I study was recently initiated to evaluate the safety and efficacy of JM-4 in the context of MS [257].

4.2.2 Carboxylase Enzymes—Biotin

4.2.2.1 Background

Biotin is a ubiquitous water-soluble vitamin, acting as an essential co-enzyme for several carboxylases [258]. Based on a case report on the efficacy of biotin in SPMS, the frst studies were introduced to evaluate biotin as a potential target in progressive MS [259]. Reduced levels of biotin observed in the cerebrospinal fluid (CSF) and serum of MS patients gave further rise to repurpose biotin in the context of neuroinflammation [260]. Additionally, deficiency of biotinidase, an enzyme required for biotin recycling [261], leads to demyelination and neurological symptoms that are comparable to MS [262].

The rationale behind the use of biotin is based on the hypothesis of a ‘virtual hypoxia’ in MS [258]. It is assumed that mitochondrial dysfunction and an increased need for energy due to demyelination result in a mismatch of energy supply and demand. Consequently, this mismatch would lead to neuroaxonal injury. Biotin potentially addresses both reduced energy production and increased demand. First, being a critical co-enzyme of three carboxylases involved in the tricarboxylic acid cycle, increased concentrations of biotin might foster neuronal ATP supply [263]. Second, bio- tin is required in oligodendrocytes for the activity of two carboxylases that produce a substrate of fatty acid synthesis [263]. In this way, biotin might enhance remyelination due to increased levels of these fatty acids, which are needed for myelin production [264]. By restoring saltatory conduction and thus, restricting membrane excitation to the nodes of Ranvier, biotin might reduce the energy demand [265].

Further evidence supporting a beneficial role of bio- tin in MS derives from a small, uncontrolled study in 23 PPMS and SPMS patients, reporting improved visual and motor functions in almost all patients [266]. Following this pilot study, a frst Phase III trial in 154 PPMS and SPMS patients (SPI) showed alleviation of disability through biotin treatment [267].

4.2.2.2 Studies

A second double-blind Phase III trial was initiated in 2016 (SPI2) [268], in which 227 PPMS and 415 SPMS patients without relapses in the previous 2 years were randomly assigned to either 300 mg/day biotin or placebo. All patients remained in the double-blind, placebo-controlled phase of the trial until the last-entered participant reached Month 15. At that time, all patients switched to biotin treatment at the next scheduled visit. Therefore, the placebo-controlled part of the study lasted between 15 and 27 months (mean duration: 20.1 months). Continuation of treatment with DMTs was permitted throughout the whole trial. The pSE evaluated differences in the proportion of patients with a confirmed improvement in EDSS score or T25FW after 12 months. Unfortunately, biotin failed to pro-vide significant benefits in terms of the pSE. Furthermore, the study did not meet any secondary endpoint (e.g., time to EDSS progression, change in T25FW).

In addition to the SPI2 trial, two studies were conducted in real-world settings. The frst was performed in seven PPMS and 36 SPMS patients, including also relapsing patients [269]. Using an open-label design, all patients received 300 mg/day biotin for 1 year as an add-on medication in the case of a pre-existing DMT. One-third of the patients reported subjective worsening of MS, leading to an increased EDSS score in two cases. Only two participants experienced clinical improvements, which were, however, not sufficient to decrease EDSS. Strikingly, only 24 of 43 patients completed the whole study duration. The main reasons for withdrawal included a lack of efficacy and worsen- ing of symptoms.

The second open-label study within a real-world setting included 84 PPMS and 94 SPMS patients [270]. Concomitant intake of DMTs was permitted. The pSE assessed the improvement of disability after 12 months of high-dose bio- tin treatment, measured by a decrease in EDSS. A reduction of the EDSS score was observed in only six patients. Secondary outcome parameters, analyzing disability, processing speed, and radiological activity, revealed no benefits other than a significant improvement in the pain and discomfort dimension in a patient questionnaire. Although the ARR was not enhanced compared to the time prior to biotin treatment, MRI assessment demonstrated radiological activity in about 30% of patients with MRI scans.

4.2.2.3 Comment

Not only failure in clinical trials but also inconclusiveness of the propagated role of biotin in auto-immune neuroinflammation question further application. Clinical improvement through biotin substitution in the case of biotinidase deficiency appears consistent in the light of severe shortness of biotin. However, it seems unlikely that the mere increase of fatty acid supply enhances the highly complex processes of remyelination in MS. Moreover, recent pre-clinical data provide evidence contradicting a beneficial impact of biotin on autoimmune neuroinflammation. Buonvicino et al reported that biotin failed to increase ATP levels in murine cortical neurons [271]. Further, biotin was unable to protect neurons from glutamate-induced excitotoxicity and oligodendrocytes from cuprizone-mediated injury. Even more, biotin treatment did not afect EAE progression in non-obese diabetic mice, resembling progressive MS [271]. To the best of our knowledge, biotin was also never shown to promote remyelination in vitro or in vivo. Given the lack of beneficial efects of biotin treatment on brain atrophy and serum neurofilament levels in the SPI2 trial [268], a neuroprotective efect is also highly unlikely.

Besides the doubts about its mechanism of action, some studies reported increased rates of relapses and MRI lesions under high-dose biotin treatment even in patients without previous relapses [267, 269, 270, 272–276]. A possible biotin-mediated influence on BBB integrity has not been assessed. Furthermore, the immunomodulatory effects of high-dose biotin are not sufficiently clarified. Investigation of PBMCs obtained from biotin-treated patients with clinical or MRI worsening indicated changes in immune cell frequencies [277]: a decrease in the overall number of lymphocytes, a reduction of CD4+ as well as CD8+ T cells, and an increase of class-switched memory IgD-CD27+ B cells were reported. Unfortunately, subpopulations of CD4+ T cells were not further characterized, limiting conclusions about biotin-mediated effects on the inflammatory response.

However, the SPI2 trial did not confirm the fear of a higher relapse rate [268]. Additionally, a large retrospective study (IPBio-SeP) investigates the incidence of relapses in biotin-treated patients [278]. Intermediate analysis of 1279 biotin-receiving patients and 483 controls did not show a difference in the frequency of relapses after 16 months.

One reason for the different outcomes of biotin treatment in the SPI compared to the SPI2 trial may have been the markedly higher placebo response rate in the SPI2 trial strongly reducing the power of this study [268]. However, given the much higher number of patients in the SPI2 trial, it is more likely that the beneficial efects observed in the SPI trial resulted from a type 1 error [268]. In addition, the rate of disability progression was reduced in both groups of the SPI2 trial. This, in turn, may have caused the observation period to be too short to depict the neuroregenerative efects. A striking point in the study design of the SPI2 trial is the pSE as it evaluated the improvement of disability. However, a delay of disease progression seems to be much more realistic than a significant improvement of clinical symptoms.

Altogether, given the negative results of the SPI2 trial and two studies in real-world settings, a beneficial efect of biotin in progressive MS is unlikely.

4.3 Other Approaches

4.3.1 Glycogen Synthase Kinase‑3—Lithium

4.3.1.1 Background

Being one of the frst FDA-approved drugs, lithium is a mood-stabilizer essential for the treatment of bipolar disorders [279]. Pre-clinical data obtained from several EAE models give rise to the use of lithium in neuroinflammatory conditions [280, 281]. Although lithium affects diverse targets, amelioration of neuroinflammation seems to be mainly mediated by suppression of glycogen synthase kinase-3 (GSK-3) [280]. Inhibition of GSK-3, for instance, interferes with the generation of TH 1 [282] and TH 17 cells [283]. Suppression of GSK-3 might also preserve BBB integrity through upregulation of the WNT/β-catenin pathway [284]. There is further evidence for the involvement of lithium and GSK-3 in MS as serum lithium levels are reduced in RRMS patients [285], while expression of GSK-3β was upregulated in patients suffering from PPMS [286].

4.3.1.2 Studies

Following promising pre-clinical results, an open-label, rater-blinded Phase I/II trial was performed in 3 PPMS and 20 SPMS patients [287]. Disease duration varied from 3 to 43 years. The study was conducted in a cross-over design, with random assignment to lithium treatment in either the frst or second year. Lithium doses varied between 150 and 300 mg/day. Simultaneous treatment with DMTs (natalizumab, interferon-β, GA) was permitted. The application of lithium resulted in positive but non-significant trends for the pSE, i.e., the PBVC. Moreover, lithium treatment failed to afect secondary clinical objectives (e.g., change in EDSS, MSFC) but signifcantly improved patient-reported outcomes in terms of the mental domains of the MS Quality of Life-54 questionnaire [288].

4.3.1.3 Comment

Besides the open-label design, heterogenous baseline characteristics in terms of divergent dis- ease durations and simultaneous application of DMTs in some patients hinder interpretation of the results. Unfortunately, there are no data on correlations between outcome results and DMT intake or patients’ lithium serum levels, which might have been interesting given the low doses administered.

Further, due to the variety of affected targets, lithium-induced aggravation of neuroinflammation cannot be excluded. Among the possibly involved mechanisms is the WNT/β-catenin-mediated dysfunction of remyelination [289]. Another example is the activation of the pro- tein kinase Akt-1 by lithium, as recent data demonstrate a detrimental role of Akt-1 in EAE [290]. In line with this, a retrospective analysis of lithium treatment including 101 US veterans with all types of MS revealed increased relapse rates. The worsening of EDSS, however, was signifcantly slowed [291].

Given the small number of participants, methodological limitations, and the lack of other prospective trials, the role of lithium in MS remains elusive.

5 Discussion

In this review, we give an update on clinical trials evaluating remyelination-promoting strategies, neuroprotective treatments, and other approaches that either failed or were interrupted for other reasons. Thereby we continue our series of previous reviews on failed trials in the context of MS [292–295]. Of note, a current update on failed or interrupted studies of immunomodulatory agents has been recently given in a separate article [9].

The most important lesson learned from the failed trials is the demand for further research. Many of the pathways involved are known. However, we do not ultimately understand their interplay resulting in remyelination and, more important, the mechanisms underlying its insufficiency [6]. There is a lack of comprehension of why remyelination fails in some patients, while others display a high proportion of remyelinated lesions [87]. Further insights into the crucial processes of remyelination are required to address them sufficiently. Accordingly, given the heterogeneity of lesions, the variety of disease courses, and the complexity of MS, it seems unlikely that targeting single pathways will be efficient [296]. Therefore, a combination of approaches that promote not only OPC proliferation and differentiation but also establish a favorable microenvironment seems to be more promising [297]. Moreover, the combination with immunomodulatory drugs might create a less inflammatory but more favorable milieu enhancing remyelination and providing a further benefit by tackling MS pathophysiology more widely. Of the reviewed drugs that were tested in combination with immunomodulatory agents, most agents were examined as add-on therapeutics to interferon-β [22, 60, 71, 98, 120, 169]. Due to the plethora of interferon-mediated mechanisms, however, there is a high risk for (unforeseen) interactions [38]. Considering these unpredictable interactions, the use of compounds in combination with interferon-β might lead to antagonistic efects. In the case of atorvastatin, for instance, combined use with interferon-β has potentially antagonized interferon-mediated suppression of MMPs and phosphorylation of STAT 1 [36, 37]. This may have attenuated beneficial clinical efects in the active treatment group. On the other hand, the combination of interferon-β with compounds that induce comparable processes might cover differences between the active treatment and the control group, as described for inosine (see 3.1.4) and minocycline (see 3.3.2). In contrast, monoclonal antibodies such as natalizumab could be a more suitable alternative, as they provide several advantages over interferon-β. First, the more selective mechanisms of action facilitate combinations with promising compounds since interactions at the molecular level are easier to predict [298]. Second, monoclonal antibody therapies provide a higher degree of disease control as well as a stronger suppression of the inflammatory activity making them a favorable partner in add-on studies with neuroregenerative agents [299].

We also observed a trend to repurpose well-known therapeutics that are licensed for other diseases in the context of autoimmune neuroinflammation. Thereby, investigators not only minimize the risk of unforeseen adverse reactions but also reduce costs and accelerate the progression through the diferent phases of clinical trials to enter the market. How- ever, none of these drugs has been resoundingly successful [22, 142, 169, 183, 184, 203, 222, 246, 287]. Along with a lack of efficacy, in some cases, the initial indication for approval turns out to cause side efects limiting the use in MS, as seen for EPO [233]. Nevertheless, the latter example shows that designing new and more specifc derivates might be a way to reduce side efects but still benefit from the initial rationale.

Given the frequent failure of approaches that proved efficacy in preclinical experiments, it becomes obvious that animal models help study specifc facets of CNS autoimmunity but cannot resemble the whole intricacy of MS. The cuprizone model, for instance, gives valuable insights into processes of remyelination but neglects the inflammatory impact [110]. Another useful tool for studying neuroregenerative abilities is a model using injections of the white matter gliotoxin lysolecithin [300]. This acute injury in a localized area allows remyelination to be assessed in the absence of ongoing tissue damage [301]. However, this injury occurs without the chronic involvement of lymphocytes [296]. EAE, on the other hand, shares many immunological and histological characteristics with MS. Still, the intensity of the ongoing inflammatory response might impede the investigation of remyelination [296].

Moreover, it is highly relevant to select animal models according to the presumed mechanism of action of the tested compounds. For example, agents treating neurodegeneration should be studied in models that share pathophysiological features of progressive MS, such as EAE in non-obese diabetic MS mice [271, 302]. Nevertheless, promising agents need to be evaluated in not just one animal model but different in vivo and in vitro approaches, covering a variety of aspects involved in MS.

Another important aspect involves frequently observed problems regarding the reproducibility of pre-clinical data between diferent laboratories. These problems are partly based on the strong dependence of experiments on factors such as the strain or genetic background of the animals, the environment, or the diet [303]. A hopeful approach to improve the reproducibility of insights gained from animal experiments may be pre-clinical randomized controlled trials (pRCTs) that are conducted in a number of diferent laboratories under standardized conditions [304]. These RCTs could provide a higher degree of comparability and validity of pre-clinical data. Therefore, these pRCTs could be an important step to bridge the gap between pre-clinical studies and Phase I clinical trials.

Furthermore, it needs to be discussed to what extent compounds in early clinical trials should necessarily be evaluated solely on the basis of the achievement of statistical significance of study endpoints. In some cases, such as GSK239512 [120], the efect size may have been overestimated. Therefore, possibly also in light of the already high costs, many studies were likely underpowered. Nevertheless, these studies can provide important insights into the mechanisms of action of the evaluated compounds. More importantly, subgroup analyses can be used to select patients who may benefit from treatment in subsequent clinical trials. Therefore, promising candidates should not be discarded just because they failed to meet the pSE.

6 Conclusions

The key conclusion is the critical requirement of appropriate study designs. Most importantly, there is still a need for reliable and sensitive outcome parameters in trials investigating neuroregenerative therapeutics. To date, there is no marker available that can distinguish between pre-existing and remyelinated myelin [6]. In addition, it is not clear how to properly quantify remyelination. A variety of imaging techniques, including MTR, DTI, or positron emission tomography, have emerged and have already been proven to depict the extent of remyelination [305–307]. However, these methods are not widely used or sufficiently validated [308]. In the trials reviewed, only the study on GSK239512 made use of one of these techniques as per [120]. Another approach to detecting remyelinating efects is the measurement of multifocal VEPs, a technique that uses simultaneous stimulations of the visual field [96]. In this way, multifocal VEPs provide a higher degree of sensitivity and specificity in detecting regeneration of the anterior visual pathway compared to conventional VEPs [309].

In terms of studies investigating neuroprotective approaches, future trials might also include neurofilament light chain (NFL) measurement. Neurofilaments are part of the neuronal cytoskeleton and are particularly enriched in axons. Thus, neurofilaments are released into the CSF and serum upon neuroaxonal injury [310] making NFL measurement a promising tool elucidating neuroprotective efects. Although not being MS-specifc, NFL was shown to be a biomarker of disease activity and predictor of the long-term outcome in MS tracking both neuroinflammatory and neurodegenerative damage [310–312].

As well as these parameters, the demand for adequate clinical outcome criteria is even more critical. Compounds will only be successful therapeutic options for patient care if they not only improve paraclinical parameters but provide a relevant benefit to patients. Therefore, sensitive clinical endpoints that depict the effects of neuroregeneration (e.g., improvements of cognitive symptoms) are urgently required. In particular, integrated clinical scores refecting diferent clinical dimensions, as used in the SYNERGY [98] and IPPoMS trials [222], seem to be favorable to assess the clinical efects of neuroprotective and neuroregenerative agents.

Further, the beneficial effects of neuroprotection are mainly attributed to regeneration from axonal injury and reduced accumulation of neuronal damage over time [6]. Thus, clinical effects are unlikely to be observed after a single relapse. They might rather be seen after years in terms of delayed disability and disease progression, thereby claiming for long-term trials [308]. Yet, most of the current trials do not meet this need for an extensive follow-up period.

Another important aspect with regard to the study design, especially of remyelinating therapies, is the age of the patients included. Taking the subgroup analysis of the RENEW study as an example, age-dependent differences in the efficacy of remyelinating approaches become clear [106]. Remyelination capacities, for instance, decline with age [107]. Future studies need to show, whether this decline leads to a decreased success of remyelinating therapies or possibly, on the contrary, to an improved outcome of remyelinating therapies due to a wider margin of improvement. Therefore, the age of the participants needs to be considered in the study design of these therapies in form of stricter age restrictions and age matching approaches. In addition to the influence of age, the disease duration also appears to have a major impact on the efficacy of neuroregenerative as well as neuroprotective therapies. Underlying reasons seem to involve advanced damage of neuroaxonal structures. First, the presence of functionally intact axons is a mandatory prerequisite for remyelination [6]. Second, the integrity of neuronal networks seems to be signifcantly reduced in advanced stages of the disease, reducing the potential success of neuroprotective approaches [111]. Hence, a more accurate consideration of disease duration is essential for the study design of future trials.

Contrasting results of the same agent in diferent disease entities, as observed in trials on atorvastatin (see 3.1.1) or EPO (see 4.2.1), underline the importance of a strict separation of these entities in clinical studies. Together with the relevance of the diferent pathophysiological concepts underlying the disease courses [3], this observation high- lights the importance of a careful selection of the appropriate patient population for the success of clinical trials.

Finally, his review further emphasizes the need for the publication of failed clinical studies. Although these trials did not measure up to the expectations, they nevertheless provide essential information on MS pathophysiology. In addition, they help to understand issues concerning the translation of insights gained from preclinical studies and are indispensable for advancements in study design. Therefore, investigators and journals should be further encouraged to publish negative outcomes.

Cistanche has a very good neuroprotective effect

Declarations

Funding Open Access funding enabled and organized by Projekt DEAL.

Conflicts of interest Niklas Huntemann: declares no conflicts of interest. Leoni Rolfes declares no conflicts of interest. Marc Pawlitz- ki: declares no conflicts of interest. Tobias Ruck: received personal fees from Alexion, Biogen, Merck Serono, Sanofi-Genzyme, Roche, and Teva, grants from Alexion and Sanofi-Genzyme, and nonfinancial support from Merck Serono. Stefen Pfeufer: received travel re- reimbursements from Sanofi Genzyme and Merck Serono, honoraria for lecturing from Sanofi Genzyme, Biogen, and Mylan Healthcare, and research support from Merck Serono, Diamed, and the German Multiple Sclerosis Society North Rhine-Westphalia. Heinz Wiendl: received grants from the German Ministry for Education and Research

(BMBF), Deutsche Forschungsgesellschaft (DFG), Else Kröner Fresenius Foundation, Fresenius Foundation, the European Union, Hertie Foundation, NRW Ministry of Education and Research, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, Biogen, Glaxo- SmithKline GmbH, Roche, and Sanofi-Genzyme; consulting fees from AbbVie, Actelion, Argenx, Biogen, EMD Serono, Idorsia, IGES, Immunic, Merck, Novartis, Roche, Sanofi-Aventis, the Swiss Multiple Sclerosis Society, and UCB; support for travel to meetings for other purposes from Alexion, Biogen, Cognome, F. Hofmann-La Roche Ltd., Hertie Foundation, Merck Serono, Novartis, Roche, Genzyme,Teva, and WebMD Global; fees for participation in review activities such as data monitoring boards from Polpharma Biologics; payment for lectures from Alexion, Biogen, Cognome, F. Hofmann-La Roche Ltd., Hertie Foundation, Merck Serono, Novartis, Roche, Genzyme,Teva, and WebMD Global; honorarium for expert testimony from the Drug Commission of the German Medical Association. Sven G. Meuth: received honoraria for lecturing and travel reimbursement for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS, and Teva. His research is funded by the German Ministry for Education and Re- search (BMBF), Bundesinstitut für Risikobewertung (BfR), Deutsche Forschungsgemeinschaft (DFG), Else Kröner Fresenius Foundation,Gemeinsamer Bundesausschuss (G-BA), German Academic Exchange Service, Hertie Foundation,Interdisciplinary Center for Clinical Stud- ies (IZKF) Muenster, German Foundation Neurology and Alexion,Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, HERZ Burgdorf, Merck Serono, Novartis,ONO Pharma, Roche, and Teva.

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Author contributions NH: study concept and design, acquisition of data, analysis and interpretation of data, writing of the manuscript. LR: study concept and design, acquisition of data, writing of the manuscript. MP: critical revision of the manuscript for intellectual content. TR: critical revision of the manuscript for intellectual content. SP: critical revision of the manuscript for intellectual content. HW: critical revision of the manuscript for intellectual content. SGM: study concept and design, critical revision of the manuscript for intellectual content.

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