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3.2 Neuroregenerative Approaches

In MS lesions, remyelination is a naturally occurring repair mechanism, restoring neuroaxonal functions [86]. Through the re-establishment of saltatory conduction, remyelination induces recovery from clinical symptoms [6]. Of note, insufficient myelin repair has been described in all courses of MS, making remyelination-promoting therapies a hopeful approach for both relapsing and progressive forms of MS [87].

3.2.1 LINGO‑1—Opicinumab

3.2.1.1 Background

About pre-clinical data, one of the most promising remyelination-influencing targets is leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein-1 (LINGO- 1). LINGO-1 is a highly conserved transmembrane protein expressed selectively on neurons, oligodendrocytes, and OPCs [88].

Inhibition of LINGO-1 is associated with axonal regeneration, terminal OPC differentiation, and increased survival of neurons as well as oligodendrocytes [89]. Consequently, blockade of LINGO-1-mediated pathways led to clinical improvement and remyelination in several animal models that cover distinct pathophysiological aspects of MS [90–92]. Among these are AON, EAE as well as non-inflammatory models such as cuprizone-induced demyelination. Apart from myelin repairing processes, results from adoptive transfer EAE experiments indicate that anti-LINGO-1-in- reduced beneficial effects (e.g., preservation of neuroaxonal integrity) are independent of immunomodulation [92].

To reinforce remyelination, the human monoclonal IgG antibody ipilimumab (BIIB003) was designed to suppress LINGO-1-mediated pathways via direct binding [93]. Two-Phase, I study assessing ipilimumab treatment in healthy volunteers and relapsing MS patients indicated a tolerable safety profile [94].

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3.2.1.2 Studies

Opicinumab in AON Started in 2012, a double-blind Phase II study (RENEW) was conducted in 82 patients with first unilateral AON [95]. Following standard high-dose treatment with intravenous methylprednisolone (IVMPS), participants were randomized to receive either

100 mg/kg ipilimumab or placebo within 28 days after symptom onset. Treatment was administered every four weeks up to Week 20, followed by a 12-week observation period. The pSE assessed the recovery of the affected optic nerve conduction in terms of visually evoked potentials (VEPs) after 24 weeks. Although ipilimumab treatment ameliorated P100 latency, this difference was not signified- cant. Significance was only reached in the per-protocol population at Week 32. Moreover, ipilimumab treatment did not improve secondary endpoints such as VA or preservation of RNFL thickness assessed by optical coherence tomography.

Thirty-nine patients were further enrolled in a substudy in which multifocal VEP measurements were used to examine optic nerve repair [96]. Non-significant trends towards reduced latency prolongation and increased recovery of VEP amplitude were observed in the active treatment group.

Recently, the first data of a 2-year follow-up study of the RENEW trial were announced (RENEWED) [97]. Of all participants of the RENEW trial who received at least one dose of ipilimumab or placebo, and thus could join this trial, 52 attended the study. Investigation of the pSE (VEP latency) indicated that the observed positive trend in the ipilimumab group was maintained over 2 years. However, this trend was not significant.

Opicinumab in RRMS and SPMS The double-blind Phase II SYNERGY trial included 330 RRMS and 89 SPMS patients with relapses who were randomly assigned in a 1:2:2:2:2 ratio to either 3, 10, 30, or 100 mg/kg ipilimumab or placebo [98]. Patients received active treatment or placebo every four weeks for 72 weeks as an add-on medication to interferon- β1a. The pSE comprised the proportion of participants with confirmed improvement in EDSS score, Timed 25-Foot Walk (T25FW) [99], Nine-Hole Peg Test (9HPT) [100], or 3s Paced Auditory Serial Addition Test (PASAT-3) [101]. No significant benefit was demonstrated regarding treatment with 3, 10, and 100 mg/kg ipilimumab [98]. A signifcantly higher proportion of participants with an improvement of disability was detected only at a dose of 30 mg/kg topic- numb compared to placebo. However, a significant dose-linear improvement could not be observed. Evaluation of the secondary (percentage of patients with confirmed disease worsening measured by the same criteria) and tertiary end-point (overall response score comprised of the mentioned criteria) showed several mild effects, favoring ipilimumab treatment with 10 or 30 mg/kg.

In 2017, 263 RRMS and SPMS patients were enrolled in a placebo-controlled, randomized, double-blind Phase II trial (AFFINITY) [102, 103]. This study evaluated the administration of 750 mg ipilimumab, corresponding to a dose of 10 mg/kg [104], as an add-on to DMTs. The pSE included the integrated response score already used in the SYNERGY study. In October 2020, Biogen announced that the AFFINITY trial failed to meet the pSE [105].

3.2.1.3 Comment

Inhibition of LINGO-1 showed impressive results concerning OPC differentiation and remyelination in animal models [90–92] but so far failed to effectively improve clinical parameters in patients. The example of ipilimumab prototypically depicts the challenges regarding the translation of remyelination-promoting approaches into a human application. First, the appropriate timing for initiating these therapies is still elusive. It seems that remyelination starts immediately after the onset of demyelination and finishes within several weeks or months [6]. Therefore, it can be speculated that remyelination-promoting strategies are most effective when administered instantly after demyelination, and thus at symptom onset. This hypothesis is further supported by a post hoc analysis of the RENEW trial, revealing that patients who started ipilimumab treatment earlier after AON onset gained more benefit [106]. In the same trial, the mean duration between the beginning of visual impairment and the first application of ipilimumab comprised 24 days [95]. At this time, however, most retinal thinning has already occurred, limiting potential improvements induced by anti-LINGO-1 treatment [95].

Moreover, it is still not clear how remyelination can be reliably measured both clinically and para-clinically.

Therefore, a new pSE was used in the SYNERGY study. Strikingly, this endpoint assessed the improvement of disability rather than a delay or arrest of disease progression [98]. Thus, an integrated score as used as the tertiary objective in the SYNERGY study and as pSE in the AFFINITY trial seems to be more suitable [102]. In addition, the SYNERGY study made use of innovative imaging techniques such as magnetization transfer ratio (MTR) or diffusion tensor imaging (DTI) for specific assessment of remyelination as exploratory objectives [98]. However, the validity of these methods has not been adequately verified. As a consequence of the aforementioned lack of experience with (para-)clinical parameters for remyelination and uncertainties regarding the effect size, it is not surprising that the discussed studies (SYNERGY and RENEW) were also underpowered [95, 98].

Last, there is a lack of experience concerning the appropriate selection of patients who might profit from remyelination-promoting approaches. In the RENEW study, post hoc analysis revealed that ipilimumab provided benefit to older AON patients (aged ≥ 33 years) and those with more severe visual impairment at baseline [106]. Most likely, this is due to a diminished intrinsic recovery potential without treatment in these subpopulations. Remyelination capacities, for instance, decline with age [107]. This decline seems to be caused by impaired differentiation of OPCs into sheath-forming oligodendrocytes rather than a lack in the number of OPCs [108]. Therefore, ipilimumab potentially restores differentiation of still available OPCs that might become insufficient more frequently in older patients. However, the observation that ipilimumab treatment provided the most benefit to patients with severe visual impairment at baseline, may also result from regression to the mean artifact.

Concerning relapsing MS, subpopulation analyses of the SYNERGY trial identified predictors for ipilimumab efficacy in RRMS and SPMS patients [98, 109]. These analyses suggest that especially patients with shorter disease duration (≤ 20 years since symptom onset) and MRI criteria, indicating preserved brain integrity at baseline (lower DTI radial diffusivity), benefit from ipilimumab. The mentioned predictors are in line with reports defining functional intact axons as a prerequisite for remyelination [6]. Furthermore, restoration of neuronal functionality not only depends on the degree of remyelination but also region-specific factors as well as the extent of irreversible neuronal network damage [110]. Most likely, patients with a shorter disease duration have higher integrity of neuronal networks compared to those in advanced disease stages [111] and might therefore benefit from remyelination-promoting approaches. However, in the case of ipilimumab, even a long observation period and the inclusion of patients with a short duration of disease did not lead to a positive outcome in the AFFINITY trial. The AFFINITY study included those patients who were shown to have the greatest benefit during the SYNERGY trial. Therefore, the failure of this study raises the question of whether the positive results demonstrated in the subgroup analyses may be rather related to statistical errors in terms of multiple testing.

Given the failure of ipilimumab in the mentioned clinical trials, a beneficial effect of ipilimumab in relapsing MS is very unlikely. Inline, Biogen announced the discontinuation of the development of ipilimumab in MS in 2020 [105].

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3.2.2 Histamine H3 Receptor—GSK239512

3.2.2.1 Background

The histamine H3 receptor (H3R) is an inhibitory G protein-coupled receptor predominantly expressed in the CNS [112]. Activation of H3R inhibits the release of histamine as well as other neurotransmitters such as acetylcholine, norepinephrine, dopamine, and serotonin [113]. Based on this function, H3R became an attractive target for the treatment of neuropsychiatric disorders [114]. Due to the high level of spontaneous signaling of H3R, (pre-)clinical research focused on inverse agonists of this receptor [115]. Inverse agonists are agents that not only inhibit the binding of agonists but, even more, induce opposite effects by inhibiting the constitutive, i.e., spontaneous receptor activity [116]. Thus, the selective H3R inverse agonist GSK239512 entered clinical Phase II trials for the therapy of cognitive alterations in schizophrenia [117] and AD [118].

In the field of neuroinflammatory research, H3R gained attention, as Chen et al demonstrated that inverse agonists of this receptor promote OPC differentiation [115]. Moreover, they observed an increased expression of H3R on oligodendrocytes in demyelinated human MS lesions. Interestingly, the administration of an H3R inverse agonist resulted in enhanced remyelination accompanied by preserved axonal integrity in a murine model of demyelination. Further support for the involvement of H3R in MS pathogenesis derives from the association between an exonic single nucleotide polymorphism (SNP) in the human gene coding for H3R and an increased susceptibility to MS [115].

3.2.2.2 Studies

After showing an acceptable safety profile in a Phase I study [119], a double-blind Phase II trial on GSK239512 was performed in RRMS [120]. The study included 131 patients with disease activity within the previous year (≥ 1 GELs on MRI or reported relapses). GSK239512 was tested as an add-on treatment to interferon- β1a or glatiramer acetate (GA). Once daily, patients received either placebo or GSK239512, up-titrated within 4–5 weeks to a maximum tolerable dose of up to 80 µg/day. The final dose corresponded to an H3R occupancy in the brain of more than 90% [121]. The co-pSEs assessed the mean changes in MTR in newly developed GELs or Delta-MTR-defined lesions. After 43–44 weeks of treatment, however, GSK239512 missed the target effect size of 0.5. Moreover, there were no relevant improvements of secondary clinical endpoints (e.g., EDSS score, performance at CogState battery [122], ARR). Analysis of MRI scans even favored placebo regarding the development of new or enlarging T2 lesions. Finally, while only three patients receiving placebo discontinued the study (none were due to AEs), 14 of the treatment group dropped out (seven due to AEs, two at the investigator’s discretion).

3.2.2.3 Comment

Besides a short observation time, one explanation for the small effect sizes might be the small number of participants completing the trial. One underlying reason was the high drop-out frequency in the active treatment group [120]. Also, considering that only patients with a high baseline disease activity were included, an unexpectedly high number of patients did not develop new MRI lesions. Only 55 out of 114 participants contributed lesions to the analysis. Therefore, disease activity in the study cohort was insufficient to evaluate the potential effects of GSK239512 treatment adequately. Taking together the high drop-out rate and the low rate of new MRI lesions, the study was most likely underpowered. Furthermore, the target effect size of 0.5 needs to be questioned in general. Given the complexity of the underlying mechanisms, the reduced sample size, and the heterogeneity of the cohort, the target effect size may have been overestimated. Therefore, GSK239512 should not be discarded solely based on failing to meet this effect size.

Moreover, the pathophysiological role of H3R in neuroinflammation is still controversially discussed. Contradict- ing a beneficial role of H3R inverse agonists in MS, mice deficient for H3R displayed an earlier and more intense disease progression in active EAE [123]. This aggravation of symptoms was accompanied by increased permeability of the BBB, leading to more severe inflammatory infiltrate- on. Correspondingly, data acquired in models of cerebral malaria [124] and spinal cord injury [125] indicate a protective influence of H3R on BBB integrity. These observations might also explain the increased rate of T2 lesions in patients treated with GSK239512 [120]. In line with these results, the treatment of mice with an agent activating H3R ameliorated EAE symptoms and reduced CNS infiltration [126]. Thus, even agonists of H3R are considered therapeutic options in MS [127].

Another striking point is the impact of H3R modulation on glial cells. In microglia, for instance, H3R suppresses the production of pro-inflammatory cytokines, chemotaxis as well as phagocytosis [128]. Regarding the influence on astrocytes, H3R contributes to the astrocyte-induced reduction of pro-inflammatory cytokines and secretion of neurotrophic factors [129]. In addition, the application of an H3R agonist reduced the expression of co-stimulatory molecules of dendritic cells (DCs) and inhibited DC-induced differentiation of pathogenic T helper 1 (TH 1) and 17 (TH 17) cells [126]. Therefore, H3R blockade in glial cells and DCs might enhance rather than alleviate neuroinflammation.

Given the shortcomings in study design and conflicting pre-clinical results, the role of GSK239512 in MS remains elusive.

3.3 Other Approaches

3.3.1 ATP‑sensitive K+ Channels—Diazoxide

3.3.1.1 Background

Potassium channels are promising targets ameliorating CNS autoimmunity [130, 131]. In the last years, adenosine triphosphate (ATP)-sensitive potassium (KATP) channels gained increasing interest due to their neuroprotective capacities [132]. KATP channels are located both in the plasma (sarcKATP) and mitochondrial membrane (mitoKATP) [133]. One well-known activator of KATP channels is diazoxide, a selective agonist of mitoKATP channels when administered in low doses [133]. Diazoxide appears to be an attractive agent for treating neurological disorders as it induces anti-inflammatory and neuroprotective processes in animal models of stroke [134], PD [135], and AD [136]. Neuroprotection seems to be mediated through maintenance of mitochondrial homeostasis, reduction of oxidative stress, protection against excitotoxicity as well as the implementation of a favorable energy profile [137–139]. Moreover, diazoxide was also evaluated in EAE [140, 141]. There, both the prophylactic and therapeutic application resulted in alleviated signs of neuroinflammation. This amelioration was mediated by inhibition of microglial pro-inflammatory activities, restriction of antigen presentation by DCs, and diminished astrocytic activity.

3.3.1.2 Studies

A multicentre, double-blind Phase IIa study evaluated the efficacy and safety of diazoxide in 102 patients with RRMS [142]. Patients were randomized in a 1:1:1 ratio to receive either placebo or diazoxide in a dose of 0.3 or 4 mg/day. Following 24 weeks of treatment, patients could join an additional 24-week observation period. The cumulative number of new T1 GELs was investigated as pSE. Secondary endpoints included further MRI (e.g., new or enlarging T2 lesions and PBVC) and clinical criteria (e.g., EDSS and relapse-free status). Notably, patients treated with diazoxide showed a significantly higher rate of new GELs and a trend towards more frequent new or enlarging T2 lesions. Also, a slight trend of stronger disease progression related to MRI criteria was observed in the 4 mg/day group compared to patients treated with 0.3 mg/day diazoxide. However, treatment signifcantly reduced the loss of brain volume and displayed a trend of diminished evolution of T2 lesions into black holes. No differences regarding clinical outcome parameters were observed.

3.3.1.3 Comment

Due to methodological limitations, conclusions drawn from this trial need to be interpreted with caution. Negative results may at least partially result from inhomogeneous baseline characteristics. The treated groups displayed an increased disease activity, indicated by more and enlarged T2 lesions at baseline [142]. On the other side, the placebo group showed a higher volume of black holes. Thus, patients in the placebo group might have been in more advanced stages of the disease, associated with dominant neurodegenerative rather than neuroinflammatory characteristics. However, this might not fully explain the higher rate of new lesions and reduced brain atrophy observed in the active treatment group. Concerning brain atrophy, other explanations might include fluid shifts and a direct vasodilating effect of diazoxide [142]. In addition, diazoxide possibly has a more prominent role in neurodegeneration than neuroinflammation, considering its mentioned impact in models of AD and PD along with extensive defects on neuronal preservation [135, 136].

The authors of this trial also hypothesized a modulation of BBB integrity by diazoxide as a mechanism of action. Strikingly, the beneficial actions of diazoxide related to the BBB were not shown pre-clinically. Moreover, diazoxide- treatment did not affect lymphocytic CNS infiltration in EAE [140]. Also, while an inhibitory influence of diazoxide on succinate dehydrogenase was demonstrated [143], earlier studies suggest a protective effect of the latter on BBB function [144]. Conclusively, especially considering the higher frequency of GELs in the treatment group, a beneficial impact of diazoxide on BBB integrity is highly doubtable.

Furthermore, data from animal studies imply that diazo- ide-mediated effects highly depend on the exact dosage. In several experiments, lower doses of diazoxide resulted in a better outcome, potentially due to more selective binding to mitoKATP channels [137, 141]. The slight trend of stronger MRI disease activity observed in patients treated with 4 mg/ day compared to those receiving 0.3 mg/day [142] gives further rise to reconsider the chosen dosage.

In addition, diazoxide was shown to promote OPC proliferation and oligodendrocyte differentiation, leading to enhanced myelination in vivo [145, 146]. Unfortunately, remyelination was not assessed in the reviewed trial.

As mentioned before, other types of potassium channels such as voltage-gated potassium (VK) channels raised interest as potential mediators of neuroinflammation and neurodegeneration [147]. One well-known blocker of VK channels, 4-aminopyridine (4-AP), is already approved to treat ambulatory disability in MS [148]. Beyond symptomatic treatment, recent data indicate that 4-AP might also prevent neuroaxonal loss. For instance, 4-AP administration seemed to alleviate clinical signs of chronic EAE and retinal neurodegeneration during experimental AON [149]. In a retrospective trial, including 103 MS patients, the same authors reported an amelioration of macular RNFL loss following 2 years of 4-AP intake without a significant benefit in terms of peripapillary RNFL thickness or total retinal preservation [149]. Contradicting a beneficial impact of 4-AP on neuroaxonal injury, however, Göbel et al and Moriguchi et al did not observe any effect of 4-AP treatment incomparable models of chronic EAE [150, 151]. Moreover, Ruck et al and others could not detect a significant improvement in the MS Functional Composite (MSFC) [152] in 4-AP-treated MS patients, further questioning an amelioration of disability progression induced by 4-AP [153–155]. These conflicting results and the ambiguous performance of diazoxide high- light the critical need for ongoing research to take advantage of the therapeutic potential of potassium channel modulators in MS.

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3.3.2.1 Background

Minocycline is a second-generation antibiotic of the tetracycline class. It first raised interest as a neuroprotective agent in 1998 when it was shown to induce beneficial effects in ischaemic brain injury [156]. Since then, minocycline was tested in various neurodegenerative diseases [157]. In EAE, minocycline treatment resulted in clinical and histological improvements when administered both as a single agent [158] or in combination with approved DMTs such as interferon-β [159] or GA [160]. These improvements are mediated by a plethora of actions. First, minocycline is known to be a potent inhibitor of microglial [161] and astrocytic activation [162]. Second, it reduces the activity of MMPs [163]. Despite other detrimental effects, MMPs degrade extracellular matrix proteins around blood vessels [158, 163]. Thereby, activation of MMPs facilitates BBB breakdown and inflammatory CNS infiltration [158]. Third, minocycline was shown to induce a shift in the immune response from TH 1 towards TH2 cells [162]. Apart from the mentioned effects, minocycline is also capable of reducing apoptosis [162], glutamate excitotoxicity [164], and oxidative stress [165].

Further support for a neuroprotective and immunomodulatory role of minocycline derives from clinical studies. These trials show a reduced conversion of clinically isolated syndrome into MS and decreased numbers of GELs in RRMS patients [166–168].

3.3.2.2 Studies

The role of minocycline in RRMS was further tested in a double-blind, randomized Phase II trial (RECYCLING) [169]. Following a run-in period of three months, 149 and 155 patients received minocycline or placebo, respectively, both in combination with interferon-β1a for 96 weeks. The pSE evaluated the time to first qualifying relapse, while secondary end-points included the ARR, number of new or enlarging T2 lesions as well as changes in brain volume. How- ever, the trial met none of these endpoints. Only a trend towards a reduced ARR in the minocycline group was observed, while results of disability progression and brain volume change tended to favor placebo. Notably, the study was terminated prematurely due to a halt of minocycline production. However, merely two patients were affected by study termination.

3.3.2.3 Comment

Most striking in the methodology of the RECYCLING study is the unexpectedly low rate of relapses. Only 23% of all patients experienced a relapse within 96 weeks of treatment [169]. Therefore, evaluation of the pSE, namely the time to first qualifying relapse, is hampered.

Despite this limitation, one could also doubt long-term improvements induced by minocycline. Suppression of astrocytes and microglia, as well as reduced MMP activity, are beneficial during the acute inflammatory response. However, these cells and proteins have crucial functions in the resolution of inflammation and tissue repair [6]. Removal of myelin, for instance, is a prerequisite for remyelination [170]. Moreover, microglia and astrocytes supply essential factors and create a favorable microenvironment critically required for remyelination [171, 172]. The same is true for MMPs. MMP9, for instance, facilitates remyelination through removing the proteoglycan neural/glial antigen 2 [173]. Thus, unselective inhibition of microglia, astrocytes, and MMPs might counteract neuroregeneration. Impaired tissue repair and remyelination might also partially explain the trend towards increased disability progression following minocycline treatment in the RECY- CLINE study [169].

Last, interferon-β might not be the optimal co-medication for minocycline treatment. Just like minocycline, beneficial effects of interferon-β depend at least partially on reduced MMP activity [37] and a shift of T cell differentiation towards a TH2 response [174]. A combination with therapeutics inducing other processes than minocycline might be more suitable to address a variety of targets.