World-wide around 2.8 million people are affected by multiple sclerosis (MS), a common and disabling immune-mediated and neurodegenerative disease of the central nervous system (CNS) which results in focal and diffuse demyelination and neuroaxonal damage (1). Onset of disease happens during (young) adulthood in the majority of cases (2, 3) and the subsequent disease course may be relapsing-remitting (RRMS) or progressive (PMS). There is no strict cut-off point between relapsing and progressive MS, leading to a recently suggested redefining of the disease course in a more biological manner (4–6). Neurodegeneration is believed to be triggered by sustained demyelination and an exhaustion of compensatory mechanisms, leading to a cascade of events including oxidative stress, mitochondrial injury, energy failure and ionic dysregulation (7). Despite the numerous immunomodulating therapies for MS that have proven benefits in reducing inflammatory disease processes, a considerable number of individuals experience disability progression. This is also referred to as silent progression, smoldering MS, or more recently, progression independent of relapse activity (PIRA). More than relapse-associated worsening, PIRA is considered a major contributor to long-term disability accumulation in MS. PIRA has been associated with brain and spinal cord atrophy and chronic active lesions on MRI (8–12). Paramagnetic rim lesions (PRLs) and slowly expanding lesions (SELs) are markers of chronic active lesions, associated with accumulation of tissue damage. PRLs are visualized using susceptibility weighted images (SWI) based on the presence of iron-containing macrophages and/or microglia at the lesion edge. SELs are detected on longitudinal T1 and T2 weighted images and correspond to non-enhancing T2 lesions with greater microstructural damage and ongoing inflammation. Co-localization of these lesions corresponds with the most destructive type of chronic lesions (13, 14). PMS places a significant hardship on patients, not only by diminishing their quality of life and limiting their involvement in the workforce and community activities, but also by imposing a burden on the healthcare system (cost) and caregivers (strain) (10, 15, 16).

Currently approved disease-modifying therapies (DMTs) mainly focus on controlling the malfunctioning adaptive immune responses that are driving the early disease process but are unable to halt disease progression in PMS. High-efficacy DMTs such as anti-CD20 treatments do not completely resolve PRLs, which are regarded as surrogate markers for chronic active lesions in multiple sclerosis (17). In fact, multiple highly relevant PMS disease related mechanisms, such as chronic microglial inflammation, oxidative stress and failure of remyelination remain unaddressed by currently approved DMTs (11, 18). Two approved DMTs for active PMS, ocrelizumab and siponimod, do exhibit enhanced effectiveness in patients with ongoing disease activity. However, their impact on clinical disease progression remains limited, yielding reductions of only 24% and 21% respectively when compared to a placebo (19). Promising results have been observed in a clinical study with alpha lipoic acid and high dose simvastatin, while other clinical trials investigating treatment with Bruton’s tyrosine kinase inhibitors (BTKis) in PMS are ongoing (20). However, recently it was announced that two phase III clinical trials of the BTKi evobrutinib versus teriflunomide in RMS did not meet their primary endpoints, dampening the high hopes for the clinical use of evobrutinib and BTKis in general to treat MS. Results of clinical trials investigating other BTKis are therefore eagerly awaited. Several other therapeutic approaches explored in the past, such as amiloride, fluoxetine, rilozole, and high-dose biotin, were proven unsuccessful (21–24). The unmet and urgent medical need for a neuroprotective and restorative treatment in PMS patients is clear and well recognized by the international research community as specified in a perspective by the Progressive MS Alliance (25).

To tackle this major unmet need, remyelination, which leads to restoration of axonal conduction and holds the key for clinical recovery in MS, is an interesting focus. Remyelination is an endogenous process that occurs naturally, but that declines with ageing and is compromised in the context of MS. In a healthy CNS, oligodendrocyte precursor cells (OPCs) generate myelin-producing oligodendrocytes (ODCs), which are responsible for forming the myelin sheaths around axons. However, within MS lesions, OPCs are present but fail to differentiate and form new myelin sheaths, resulting in the unveiling of the underlying axonal membrane, subsequently causing a disruption in axonal conduction. In demyelinated axons, sodium channels are redistributed resulting in an influx of extracellular sodium and forcing the energy-demanding Na/K pump to work harder to maintain proper ionic balance and cell polarization. To generate this energy, axons highly depend on mitochondria. However, inflammatory processes involving immune cells producing reactive oxygen species (ROS) and nitric oxide (NO), severely affect the integrity of mitochondria. In the end, the imbalance between heightened energy demands and reduced mitochondrial metabolic function can initiate certain processes that ultimately lead to neurodegeneration (7, 26).

Hence, stimulation of endogenous remyelination may have a significant therapeutic impact, since it will safeguard the axons from being damaged (27–29). In this context, metformin, traditionally used in the treatment of patients with type 2 diabetes mellitus, is a promising agent. It has been put forward as a potential anti-ageing drug, due to its pleiotropic properties, among which antioxidant, antiproliferative, antifibrotic and immunomodulatory effects have been demonstrated (30–32).

In the last two decades, metformin has been investigated in rodent models of MS and demyelination, including the MOG-induced Experimental Autoimmune Encephalomyelitis (EAE)- and cuprizone- model (33–35). In the EAE model, metformin has proven its ability to reduce inflammation, improve functional outcomes of disease symptoms and decrease demyelination (36). Additionally, functioning as an agonist of AMP-activated protein kinase (AMPK), the drug has exhibited protective effects on OPCs and ODC lineage dynamics (37, 38). One of the most compelling preclinical studies to support the use of metformin in MS investigated the effects of ageing on OPCs. When aged OPCs were transplanted into neonatal CNS of rats, they exhibited the same proliferation capabilities as neonatal OPCs, while conversely, neonatal OPCs ceased to proliferate when transplanted into aged rat brains. This suggests that, rather than intrinsic OPCs-factors, it is the local environment that impacts OPCs function. Therefore, rejuvenating the environment of OPCs may lead to improved remyelination. One well known modulator of ageing is fasting and when aged animals are subjected to a fasting regime, remyelination capacities comparable with those seen in young animals can be observed. Although intermittent fasting was deemed to be difficult in clinical practice, researchers found that the effect in the described experiment could be perfectly mimicked through the use of metformin. Indeed, metformin rejuvenated the OPCs and made them responsive to differentiation factors once again, allowing them to proliferate (10, 39–41). The importance of AMPK signaling, leading to improved mitochondrial homeostasis and protection of ODCs, was confirmed in the cuprizone model. A recent study discovered that metformin led to a dose dependent increase in AMPK activation in healthy ODCs, which resulted in both promotion of changes in ODC bioenergetics and acceleration of ODC differentiation. A knockdown approach in this study confirmed the findings of Neumann et al. (39) that metformin does need an active AMPK complex in order to promote OPC differentiation. Additional in vivo work in this study confirmed the enhanced ODC differentiation and remyelination promotion in a cuprizone model, particularly in white matter regions (38). Additional mechanisms of metformin include antioxidant mechanisms related to the mammalian target of rapamycin (mTOR) pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as well as reduced microgliosis by down-regulation of Mac-3 mRNA, a marker of pro-inflammatory microglia/macrophages (42–44).

Based on preclinical evidence, metformin has shown neuroprotective effects in other neurodegenerative disorders (Parkinson’s disease, Alzheimer’s disease, Huntington disease). Metformin is able to cross the blood brain barrier and exert anti-apoptotic, antioxidant and regulatory effects on neuroinflammation, microglial activity, brain derived neurotrophic factor mediation, neurotransmission and neurogenesis (45, 46). A clinical study within a cohort of individuals diagnosed with type 2 diabetes mellitus explored the impact of metformin treatment on the likelihood of developing dementia. In this population, treatment with metformin could be linked to a dose-dependent decrease in the risk of dementia (47). Another population-based cohort study confirmed these findings: individuals with type 2 diabetes initiating metformin exhibited a markedly reduced risk of dementia compared to individuals who were not prescribed any anti-diabetes medication, even though the latter group had superior glycemic profiles and cardiovascular health at the study’s onset (48).

In addition, promising results of metformin use in patients with MS have been revealed through clinical research. In a pilot study in adult obese MS patients with metabolic syndrome (N = 50), treatment with metformin (dose range of 850-1500 mg/day) led to a reduction in new T2 hyperintense brain lesions on MRI in 20 patients. This positive impact was accompanied by immunological changes, supporting an anti-inflammatory effect of metformin in MS. Notably, the treatment was well tolerated (49). In a cross-sectional study investigating comorbidities in MS (N = 453), it was noted that diabetes type 2/insulin resistance was associated with a reduced risk of disability (Expanded Disability Status Score (EDSS) ≥ 6.0) in 87 PMS patients. Interestingly, 72% of these individuals were using metformin as therapy (50). It is important to highlight that vascular comorbidities, such as diabetes, are associated with clinical and imaging markers of both neurological dysfunction and neurodegeneration in MS patients (51). However, despite the unfavorable prognoses associated with these comorbidities, metformin demonstrated positive effects in this specific subset of MS patients. Another exploratory prospective single-center, phase II, randomized, open-label clinical trial investigated the safety and efficacy of metformin as adjuvant to interferon-beta-1a treatment in relapsing remitting MS patients during one year. The study revealed a statistically significant change in serum malondialdehyde, an indicator of oxidative stress. However, no significant effects were found for additional immunological, imaging (T2 lesions) and clinical (EDSS) outcome measures (52). It is well known that EDSS is less sensitive to change than the Timed 25 Foot Walk test (T25FW) in order to detect disability progression in PMS (53). The sensitivity of T25FW as a clinical outcome measure for capturing intervention effects on progression in MS in pharmacological and rehabilitation trials has been confirmed in various clinical trials (54–57).

Several other clinical trials are currently ongoing and are summarized in Table 1 . One single-center, phase I, randomized clinical trial in Canada is recruiting pediatric and young adult MS patients to investigate safety, feasibility and visual outcome measures of metformin add-on treatment. Another single center, phase IIa, randomized placebo-controlled clinical trial will evaluate the effect of combination therapy of clemastine 1,34 mg prolonged release tablets with metformin 500 mg prolonged release tablets on safety, visual evoked potentials and MRI outcome measures, in patients with relapsing MS who are on a disease-modifying therapy. In the USA a single-center, phase I, placebo-controlled, randomized clinical trial in PMS patients has started recruiting patients. Here they investigate the safety and efficacy of metformin add-on treatment. A large phase II/III multi-center, multi-stage, multi-arm, randomized, placebo-controlled clinical trial (OCTOPUS) will investigate efficacy and safety of multiple drugs (including metformin and alpha-lipoic acid) in both primary and secondary progressive patients. The main outcomes are whole brain atrophy on MRI and disability progression. In Australia, the PLATYPUS study will follow the same protocol as the OCTOPUS clinical trial.

In conclusion, the time is right to start a clinical trial which can confirm preliminary but promising findings from human studies by evaluating the efficacy of metformin in PMS.

A summary of baseline data with comparison of demographics (age, sex, ethnicity), clinical tests at baseline, concomitant therapies and disease duration between the metformin and placebo arm will be presented. For the health economic analysis, marital status, household composition/living situation and educational level will be recorded. In addition, a CONSORT flow diagram (74) will be produced to summarize the number of patients at each stage (eligibility, randomization, allocation, discontinuation and follow-up). Adverse events (AE), Serious adverse events (SAE) and Suspected Unexpected Serious Adverse Reaction (SUSAR) will be summarized per arm using descriptive statistics.

Analysis will be conducted based on the intention-to-treat population. If the data exhibit a normal distribution, the primary analysis will entail in first instance (to align with sample size calculation) an independent samples t-test to compare the alteration in T25FW from baseline to the end of the 96-week treatment period between the metformin and placebo arm. A secondary sensitivity analysis will include a linear regression model with 96-week T25FW as outcome and baseline T25FW as predictor, plus a linear mixed model with T25FW at all-time points as outcome (both models can take other confounders into account) to get a more precise estimate of the treatment effect. Similar linear regression and linear mixed models will be used to analyze the secondary and clinical tertiary outcomes at 96 weeks and over time. The cost effectiveness analysis will use the societal perspectives, including both direct and indirect, medical and non-medical costs, collected through questionnaires. Main health outcome will be Quality Adjusted Life-Years (QALYs), but other health outcomes including MSIS-29 will also be considered. The return of investment will be assessed using the incremental cost-effectiveness ratio and incremental net monetary benefit.

A per protocol-analysis will be done to evaluate the sensitivity of the main results. Use of at least ≥80% of study drug will be regarded as being compliant to study drug. Study drug adherence will be reported as mean with SD or median with interquartile range.

An electronic case report form (eCRF) in Redcap will be used by all participating sites to collect the individual patient data in accordance with the trial protocol. This data will be stored on a dedicated storage server located at the Sponsor’s premises and retained for 25 years. Clinical trial monitoring will be performed by the study sponsor according to a prespecified monitoring plan.

Presented here is the study protocol of a phase II clinical trial aimed at evaluating the effectiveness and safety of metformin as add-on treatment in patients with non-active PMS. This patient group holds a substantial unmet need for treatments that promote restoration and neuroprotection. Recently, metformin was shortlisted by the UK MS society expert consortium as a highly promising repurposed drug and recommended for immediate use in clinical trials focusing on PMS, due to its rejuvenating properties (75). Previous studies have provided preliminary data on mechanisms of action, safety and feasibility of the use of metformin in MS. These studies encompass preclinical animal models, RRMS patients and RRMS patients who presented either metabolic syndrome and/or type 2 diabetes mellitus (49, 50, 52).

Currently, several trials aiming to further investigate the safety and efficacy of metformin in multiple sclerosis are in the recruitment phase ( Table 1 ). Our study aims to provide evidence for the efficacy of metformin specifically in PMS patients, by focusing on both clinical and cost-related outcome measures. By also collecting MRI data and blood samples, we aim to improve our understanding of the potential modes of action of metformin in MS. Contrary to other ongoing trials, we aim to explore remyelination, neuroprotection and smoldering inflammation in PMS patients using advanced imaging techniques, including DTI and SWI.

This study holds a notable advantage in its utilization of metformin as add-on treatment, allowing patients who are already on stable disease-modifying therapies to continue their treatment. This reflects the common clinical practice where patients with PMS can continue using a DMT, despite clinical progression without visible inflammatory disease activity on MRI. The use of T25FW as the primary outcome measure is an additional strength, as it is more sensitive to detect changes over time than the EDSS. Another advantage is the investigation of the cost-effectiveness of the intervention. While great effort is being made in investigating new molecules to slow down disease progression, development of pharmaceutics remains a time-consuming and costly undertaking. By repurposing metformin, an off-patent drug with evidence of safety in humans, blood-brain barrier penetrance and demonstrated efficacy in preclinical studies, clinical trials can be conducted quickly and affordably. Additionally, considering the low cost of metformin, even a small effect-size will prove the cost-effectiveness of this treatment.

In conclusion, MACSiMiSE-BRAIN has the potential to demonstrate the clinical benefit of metformin as an add-on treatment in PMS patients. Considering the lack of treatment options to halt disability progression, the aim of this study is to investigate a potential safe and effective treatment option for PMS, while also unravelling the underlying disease-mechanisms of disability progression.

All subjects have to provide written informed consent prior to inclusion in the study. The clinical trial was approved by regulatory authorities and ethical committee after submission to the European Clinical Trials Informations System (EUCT number: 2023-503190-38-00, Date of approval: April24, 2023).

A-VD: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing, Validation. EV: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing. VP: Conceptualization, Investigation, Supervision, Writing – original draft, Writing – review & editing. LW: Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review & editing. MC: Conceptualization, Investigation, Supervision, Writing – original draft, Writing – review & editing. GL: Conceptualization, Investigation, Supervision, Writing – original draft, Writing – review & editing. MD’H: Conceptualization, Investigation, Supervision, Writing – original draft, Writing – review & editing. MB: Formal analysis, Supervision, Writing – original draft, Writing – review & editing. ER: Conceptualization, Formal analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing. ML: Formal analysis, Supervision, Writing – original draft, Writing – review & editing. MBD’H: Investigation, Supervision, Writing – original draft, Writing – review & editing. JD: Conceptualization, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. TR: Conceptualization, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. BW: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.