Formulation of glibenclamide and low-dose metformin for the prevention or treatment of neurodegenerative diseases

Abstract

The present invention provides a formulation of metformin and glibenclamide for the prevention and / or treatment of neurodegenerative diseases, preferably chosen among Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia, Huntington's disease and Alzheimer's disease.

Description

DescriptionTitle of Invention: |Formulation of glibenclamide and low- dose metformin for the prevention or treatment of neurodegenerative diseases. |

[0001] The present invention provides a formulation of metformin and glibenclamide for the prevention and / or treatment of neurodegenerative diseases, preferably chosen among Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia, Huntington’s disease and Alzheimer’s disease.Technical Field

[0002] The technical domain of the invention is the treatment of neurodegenerative diseases.Background Art

[0003] Neurodegenerative diseases are a group of disorders characterized by the progressive degeneration and loss of function of neurons in the nervous system. These diseases primarily affect the brain and spinal cord, leading to a decline in cognitive, motor, and sensory functions. The most well-known neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). While the specific causes and symptoms vary among these conditions, they share common pathological mechanisms and present significant challenges for medical research and treatment.

[0004] One of the hallmarks of neurodegenerative diseases is the accumulation of abnormal proteins in the brain. In Alzheimer's disease, for instance, the buildup of amyloid-beta plaques and tau tangles disrupts neuronal communication and leads to cell death.

[0005] Neuroinflammation is another common feature across neurodegenerative diseases. The immune response in the brain, primarily mediated by microglia and astrocytes, can become dysregulated, leading to chronic inflammation that exacerbates neuronal damage. Oxidative stress, caused by an imbalance between free radicals and antioxidants, also plays a significant role in neuronaldegeneration by damaging cellular components such as DNA, proteins, and lipids.

[0006] Genetic factors contribute to the development of many neurodegenerative diseases, although the extent varies. For example, mutations in specific genes like APP, PSEN1 , and PSEN2 are linked to familial forms of Alzheimer's disease, while mutations in the HTT gene cause Huntington's disease. In contrast, the majority of Parkinson's disease cases are sporadic, with only a small percentage attributed to genetic mutations, such as those in the LRRK2 and SNCA genes. Environmental factors, including exposure to toxins, head trauma, and lifestyle choices, also influence the risk of developing neurodegenerative diseases.

[0007] Neurodegenerative diseases have a profound impact on individuals, families, and society. As these diseases progress, they lead to a gradual loss of independence, requiring increasing levels of care and support. The cognitive decline seen in Alzheimer's disease, for example, severely impairs memory, reasoning, and language abilities, while Parkinson's disease leads to motor symptoms like tremors, rigidity, and bradykinesia (slowness of movement). These conditions often result in a reduced quality of life and can lead to complications such as infections and injuries that further shorten life expectancy.

[0008] The economic burden of neurodegenerative diseases is substantial, encompassing healthcare costs, lost productivity, and the emotional toll on caregivers. As the global population ages, the prevalence of these diseases is expected to rise, making the need for effective treatments and interventions even more urgent.

[0009] Research into neurodegenerative diseases is focused on understanding the underlying mechanisms, identifying biomarkers for early diagnosis, and developing treatments to slow or halt disease progression. Currently, treatments are primarily symptomatic, aiming to manage symptoms rather than address the root cause of neuronal degeneration. For example, cholinesterase inhibitors and NMDA receptor antagonists are used to alleviate cognitive symptoms in Alzheimer's disease, while dopamine replacement therapies help manage motor symptoms in Parkinson's disease.

[0010] However, significant challenges remain, including the complexity of the diseases, the difficulty of delivering therapies across the blood-brain barrier, and the need for personalized approaches to treatment.

[0011] Metformin has a triple effect in neuroprotection. First, by lowering glucose levels, it decreases the availability of serum glucose, which is transformed into cyclic 1 ,3-bisphosphoglycerate during glycolysis. Second, it forces glycerol excretion, thus detoxicating the intermediary metabolites responsible for the accumulation of cyclic 1 ,3-bisphosphoglycerate. Finally, by promoting neurogenesis (Mor et al. 2020b; Markowicz-Piasecka et al. 2017; Wang et al. 2012).

[0012] Metformin could therefore be of interest for the treatment of neurodegenerative diseases. The major drawback concerning this molecule lies in the tolerance issues. Metformin is mainly cleared through kidney ultrafiltration. In case of insufficient kidney function, it accumulates and causes a lifethreatening increase in serum lactate, resulting in lactic acidosis. Thus, the molecule is contraindicated for use in patients above 65 years of age when renal function ascertained by blood clearance drops below 30mL / min.

[0013] However, patients concerned by neurodegenerative diseases are mostly aged, and have a tendency towards cachexia, which makes them at risk for metformin use (Barichella, Cereda, et Pezzoli 2009).

[0014] Based on the hypothesis that neuroprotection comes from preventing cyclic 1 ,3-bisphosphoglycerate accumulation inside dopaminergic neurons from the substantia nigra, the ideal treatment would have the properties of metformin without the side effects endangering the target population.

[0015] Taking into consideration the persistent need for medication to slow down the progression of neurodegenerative diseases, the applicant identified a synergistic and original combination of molecules which surprisingly combine efficacy and low side-effects, thanks to a specific dosage of metformin and glibenclamide, allowing for the safe chronic treatment of a vast majority of patients, and which efficiently cross the blood-brain barrier.Summary

[0016] The present invention aim is to provide a formulation allowing for a therapeutic dosage of metformin and glibenclamide relevant to the treatment of neurodegenerative diseases.

[0017] The present invention relates to a pharmaceutical composition comprising metformin and glibenclamide for use in the treatment of neurodegenerative diseases.

[0018] Said pharmaceutical composition comprises a therapeutically effective amount of metformin and glibenclamide.

[0019] In an embodiment, said formulation allows for differential release of metformin and glibenclamide enabling combined administration (once a day dosing or more) with parallel pharmacokinetic profile.

[0020] once-a-day dosing and parallel pharmacokinetic profiles.

[0021] In an embodiment, said metformin is present in an amount providing an effective dose of between 0.025 mg / day and 450 mg / day.

[0022] In an embodiment, said metformin is present in an amount providing an effective dose of between 0.025 mg / day and 250 mg / day.

[0023] Preferably, said metformin is present in an amount providing an effective dose of 0.025 mg / day, or 0.050 mg / day, or 0.075 mg / day, or 0.1 mg / day or 0.125 mg / day, or 0.150 mg / day, or 0.175 mg / day, or 0.200 mg / day, or 0.225 mg / day, or 0.250 mg / day, or 0.275 mg / day, or 0.300 mg / day, or 0.325 mg / day, or 0.350 mg / day, or 0.375 mg / day, or 0.400 mg / day, or 0.425 mg / day, or 0.450 mg / day, or 0.475 mg / day, or 0.500 mg / day, or 0.525 mg / day, or 0.550 mg / day, or 0.575 mg / day, or 0.600 mg / day, or 0.625 mg / day, or 0.650 mg / day, or 0.675 mg / day, or 0.700 mg / day, or 0.725 mg / day, or 0.750 mg / day, or 0.775 mg / day, or 0.800 mg / day, or 0.825 mg / day, or 0.850 mg / day, or 0.875 mg / day, or 0.900 mg / day, or 0.925 mg / day, or 0.950 mg / day, or 0.975 mg / day, or 1 mg / day, or 1 ,25 mg / day, or 1.50 mg / day, or 1.75 mg / day, or 2 mg / day, or 2,25 mg / day, or 2.50 mg / day, or 2.75 mg / day, or 3 mg / day, or 3,25 mg / day, or 3.50 mg / day, or 3.75 mg / day, or 4 mg / day, or 4,25 mg / day, or 4.50 mg / day, or 4.75 mg / day, or 5 mg / day, or 6 mg / day, 7 mg / day, 8 mg / day, 9 mg / day, or 10 mg / day, or 12,5 mg / day, or 15 mg / day, or 17,5 mg / day, or 20 mg / day, 22,5 mg / day, or 25 mg / day, 27,5 mg / day or 30 mg / day, or 32,5 mg / day, or 35 mg / day, or 37,5 mg / day, or 40 mg / day, or42,5 mg / day, or 45 mg / day, or 47,5 mg / day, or 50 mg / day, or 60 mg / day, or 70 mg / day, or 80 mg / day, or 90 mg / day or 100 mg / day, or 125 mg / day, or 150 mg / day, or 175 mg / day, or 200 mg / day, or 225 mg / day, or 250 mg / day, or 275 mg / day, or 300 mg / day, or 325 mg / day, or 350 mg / day, or 375 mg / day, or 400 mg / day, or 425 mg / day, or 450 mg / day.

[0024] More preferably, said metformin is present in an amount providing an effective dose of about 50 mg / day.

[0025] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 5 mg / day.

[0026] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 2 mg / day.

[0027] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 1 mg / day.

[0028] Preferably, said glibenclamide is present in an amount providing an effective dose of 0.001 mg / day, or O. 005 mg / day, or 0.010 mg / day, or 0.015 mg / day, or 0.020 mg / day, or 0.025 mg / day, or 0.030 mg / day, or 0.035 mg / day, or 0.030 mg / day, or 0.035 mg / day, or 0.040 mg / day, or 0.045 mg / day, or 0.050 mg / day, or 0.055 mg / day, or 0.060 mg / day, or 0.065 mg / day, or 0.070 mg / day, or 0.075 mg / day, or 0.080 mg / day, or 0.085 mg / day, or 0.090 mg / day, or 0.095 mg / day, or 0,1 mg / day, or 0.15 mg / day, or 0.2 mg / day, or 0.25 mg / day, or 0.3 mg / day, or 0.35 mg / day, or 0.4 mg / day, or 0.45 mg / day, 0,5 mg / day, or 0.55 mg / day, or 0.6 mg / day, or 0.65 mg / day, or 0.7 mg / day, or 0.75 mg / day, or 0.8 mg / day, or 0.85 mg / day, or 0.9 mg / day, or 0.95 mg / day, or 1 mg / day, or 1 .05 mg / day or 1 , 1 mg / day, or 1 .15 mg / day, or 1 .2 mg / day, or 1 .25 mg / day, or 1 .3 mg / day, or 1 .35 mg / day, or 1 .4 mg / day, or 1 .45 mg / day, 1 ,5 mg / day, or 1.55 mg / day, or 1 .6 mg / day, or 1 .65 mg / day, or 1 .7 mg / day, or 1 .75 mg / day, or 1 .8 mg / day, or 1 .85 mg / day, or 1.9 mg / day, or 1.95 mg / day, or 2 mg / day, or 2.05 mg / day, or 2.10 mg / day, or 2.15 mg / day, or 2.20 mg / day, or 2.25 mg / day, or 2.30 mg / day, or 2.35 mg / day, or 2.40 mg / day, or 2.45 mg / day, or 2.50 mg / day, or 2.55 mg / day, or 2.60 mg / day, or 2.65 mg / day, or 2.70 mg / day, or 2.75 mg / day, or 2.80 mg / day, or 2.85 mg / day, or 2.90 mg / day, or 2.95 mg / day, or 3.00 mg / day, or 3.05 mg / day, or 3.10 mg / day, or 3.15 mg / day, or 3.20 mg / day, or 3.25 mg / day, or 3.30 mg / day, or 3.35mg / day, or 3.40 mg / day, or 3.45 mg / day, or 3.50 mg / day, or 3.55 mg / day, or 3.60 mg / day, or 3.65 mg / day, or 3.70 mg / day, or 3.75 mg / day, or 3.80 mg / day, or 3.85 mg / day, or 3.90 mg / day, or 3.95 mg / day, or 4.00 mg / day, or 4.05 mg / day, or 4.10 mg / day, or 4.15 mg / day, or 4.20 mg / day, or 4.25 mg / day, or 4.30 mg / day, or 4.35 mg / day, or 4.40 mg / day, or 4.45 mg / day, or 4.50 mg / day, or 4.55 mg / day, or 4.60 mg / day, or 4.65 mg / day, or 4.70 mg / day, or 4.75 mg / day, or 4.80 mg / day, or 4.85 mg / day, or 4.90 mg / day, or 4.95 mg / day, or 5.00 mg / day.

[0029] In an embodiment, said glibenclamide is present in an amount providing an effective dose of 0.5 mg / day.

[0030] In an embodiment, said neurodegenerative disease is chosen among: Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, Frontotemporal dementia, Lewy body dementia, Huntington’s disease or Alzheimer’s disease.

[0031] In an embodiment, the pharmaceutical composition according to the invention further comprises at least one other acceptable active pharmaceutical ingredient, and / or at least one acceptable pharmaceutical excipient or carrier.

[0032] In a preferred embodiment, said active pharmaceutical ingredient is another agent for preventing or treating a neurodegenerative disease, preferably an agent for preventing or treating Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, Frontotemporal dementia, Lewy body dementia, Huntington’s disease and / or Alzheimer’s disease.Definition

[0033] “Pharmaceutical composition” or “pharmaceutical formulation” refers to the formation of a pharmaceutical product in which different chemical substances, including the active drugs, are combined to produce a final medicinal product.

[0034] “Combination” in the present invention refers to the association of two compounds, e.g., metformin and glibenclamide, to be administered to the same subject. According to the present invention, said two compounds can be administered concomitantly or sequentially. Thus, according to the present invention, the administration of the first compound does not necessarily overlap with the administration of the second compound.

[0035] “Concomitantly” refers to an administration of metformin and glibenclamide at the same time, the administration of the first compound overlapping with the administration of the second compound.

[0036] “Sequentially” refers to an administration of metformin and glibenclamide which is not done at the same time. Glibenclamide or metformin is administered before the other molecule, and the administration of the first compound does not overlap with the administration of the second compound.

[0037] “Subject”, “individual” or “patient” refers to a mammal, preferably a human. In one embodiment, the subject is diagnosed with a neurodegenerative disease. In one embodiment, the subject is a patient, preferably a human patient, who / which is awaiting the receipt of, or is receiving, medical care orwas / is / will be the subject of a medical procedure or is monitored for the development or progression of a neurodegenerative disease. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0038] “Therapeutically effective amount” or “therapeutically effective dose” refer to the amount or dose of metformin, to the amount or dose of glibenclamide, or to the amount or dose of both, in a combination according to the invention, that is aimed at, without causing significant negative or adverse side effects to the subject, slowing down or stopping the progression, aggravation, or deterioration of neurodegenerative disease affecting the subject.

[0039] “Efficient crossing of the blood brain barrier” corresponds to the dosage allowing for an optimal concentration inside the brain compartment.

[0040] “Prevention” or “preventing” or “prophylactic treatment” refers treatment that reduce the risk of developing a specific disease or condition. This type of treatment is administered before the onset of symptoms or exposure to the disease-causing agent. Its primary purpose is to enhance the body's natural defenses, block or neutralize the disease agent, or alter conditions that might lead to the disease, thereby preventing the disease from occurring.

[0041] “Treating”, “treat” or “treatment” refers to therapeutic treatment, to prophylactic or preventative measures, or to both, wherein the object is to prevent or slow down the neurodegenerative disease. A subject is successfully “treated” if, afterreceiving a therapeutic amount of metformin and of glibenclamide in combination according to the present invention, the subject shows observable and / or measurable reduction in the consequences of a neurodegenerative disease, acknowledged by symptom reduction, restoration of previously lost or hindered symptomatic treatment efficacy.

[0042] “About” preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically disclosed.

[0043] “Idiopathic Parkinson’s Disease” is a multisystemic synucleinopathy of the human nervous system with functional consequences and a diagnostic potential that extend beyond the nigrostriatal system. Intracerebrally, only a few predisposed types of nerve cells develop the inclusion body pathology that appears in the form of Lewy neurites, Lewy bodies, and Lewy plaques. Melanoneurons and other projection cells generating long axons that are unmyelinated or sparsely myelinated are particularly susceptible. This selective vulnerability on the part of specific neuronal populations as well as anatomically and functionally interconnected brain regions results in a distinctive topographic distribution pattern of brain lesions that is nearly consistent across autopsy cases and, as such, makes it possible to predict the intracerebral progress of IPD and stage it neuropathologically. In stage 1 , the lesions are confined to predisposed induction sites: the brain stem dorsal visceromotor nucleus of the vagal nerve, intermediate reticular zone, and / or telencephalic bulbus olfactorius. In stage 2 cases, inclusion bodies begin to appear in portions of the caudal raphe nuclei (above all, the nucleus raphes magnus and obscurus), the gigantocellular reticular nucleus, and the coeruleus / subcoeruleus complex. The severity of the lesions in stage 1 typically increases in stage 2 cases, and the pathology at brain stem sites not only worsens throughout the following stages but is directed essentially upwards in the direction of the cerebral cortex. Neuronal damage begins in the mesencephalic substantia nigra, pars compacta in stage 3 and is accompanied by alterations in the tegmental pedunculopontine nucleus and in the prosencephalon (central subnucleus and basolateral complex of the amygdala, magnocellular nuclei of the basal forebrain, hypothalamic tuberomammillary nucleus). In stage 4, the disease process reaches the cerebralcortex (anteromedial temporal mesocortex) for the first time. During stage 4 and subsequent stages, IPD progresses into additional telencephalic regions, including chiefly the transentorhinal region, hippocampal formation, anterior cingulate mesocortex (all limbic loop structures), and insular and subgenual areas of the mesocortex (cortical components of the autonomic loop). The pathology that accrues in these and other nonsomatomotor system structures almost certainly leads to detectable olfactory impairment, deficits in responses to emotional stimuli, dysfunctions of visceromotor and endocrinal systems, and most probably diminished cognitive faculties, including, in some individuals, dementia. The reduced influence of input from limbic system high-order centers upon both the cerebral cortex and the brain stem reticular formation probably contributes to the affect-related deficits of the voluntary and emotional motor systems that typically become manifest in the course of IPD. In the final stages 5 and 6, the pathology advances until it occupies extensive stretches of the neocortex, beginning with the high-order sensory association and prefrontal areas, then the first order sensory and premotor fields, and eventually the primary sensory and primary motor fields of the mature neocortex. (Kelly del Tredici and Heiko Braak, Madame Curie Bioscience Database)

[0044] “Early onset Parkinson’s disease” or “young onset Parkinson’s disease (YOPD)” refers to a type of Parkinson’s disease where the diagnosis is made for someone who is 21-50 years old. While common symptoms of Parkinson’s may be similar no matter what age you are, the progression is often different: Young people often have more involuntary movement problems due to the most commonly prescribed Parkinson’s disease medication, levodopa. Other problems associated with Parkinson’s such as memory loss, confusion, and balance difficulties tend to be less frequent in young people with the disease.

[0045] “Parkinson’s disease associated with aberrant splicing” refers to Parkinson’s disease including aberrant splicing. At least PARK2, SNCAIP, LRRK2, SNCA, SRRM2, and MAPT are involved in aberrant AS events in PD patients (FU, Ru- Huei, et al. 2013).

[0046] PARK2 mutations are accompanied by an imbalance in programmed cell death systems in which non-apoptotic molecular mechanisms play the leading role (Konovalova et al. 2015). The PARK2 gene is responsible for almost half ofcases of autosomal recessive Parkinson's disease with early onset (Olga Corti 2014).

[0047] Mutations within the MAPT gene encoding the microtubule-associated protein tau result in the clinical phenotype of frontotemporal dementia with parkinsonism. Genome-wide association studies have implicated MAPT H1 as a significant risk factor for Parkinson’s disease (PD); however preliminary sub-haplotype analyses suggest that different genetic variants on the MAPT H1 haplotype associate with each of these parkinsonian disorders. (Ross Owen 2012).

[0048] Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common genetic causes of Parkinson’s disease (PD), and also one of the strongest genetic risk factors in sporadic PD. The LRRK2 protein contains a GTPase domain and a kinase domain, and several protein-protein interaction domains (Yulan Xiong et al. 2017).

[0049] Synphilin-1 (SNCAIP) is a presynaptic protein that associates with synaptic vesicles (Ribeiro et al. 2002). It is associated with Parkinson's Disease (PD) because it is an intrinsic component of Lewy bodies (Wakabayashi et al. 2000) and a mutation of the SNCAIP gene has been identified in some PD patients (Marx et al. 2003), suggesting that accumulation of SNCAIP and its interaction with SNCA may be relevant for Lewy body formation in PD. SNCAIP is ubiquitinated by several different E3 ubiquitin-ligases, including Parkin (PARK2)

[0050] The alpha-synuclein gene (SNCA) was indisputably considered as the first pathogenic gene responsible for autosomal dominant PD, supported by the fact that its protein aggregation is thought to be the primary pathological hallmark of the patients, though only a few mutations were identified (Polymeropoulos et al., 1997; Fields et al., 2019).

[0051] SRRM2 is a RNA splicing factor which has been reported as being consistently dysregulated in different PD neuronal sources (Shehadeh et al. 2010).

[0052] “Trauma-induced Parkinson’s disease” refers to Parkinson’s disease linked to traumatic brain injury. Traumatic brain injury (TBI) has been implicated as a risk factor for PD. This includes mild TBI (mTBI), which is known to be responsible fora 56% higher risk of developing PD in U.S. Veterans, the risk increasing with severity of injury (Delic et al. 2020).

[0053] “Vascular Parkinson’s disease” refers to Parkinson’s disease related to vascular or pseudovascular injuries. This denomination includes progressive ambulatory impairment and abnormal white matter (WM) signal visible on neuroimaging.

[0054] “Drug-induced Parkinson’s disease” refers to Parkinson’s disease which followed the take of a drug. This subtype is often reversible after withdrawal of the causative drug. All known causative drugs were prescribed in non- neurological departments and over one half were prescribed in non-psychiatric departments; most were prescribed to treat depression or abdominal discomfort (Shiraiwa et al. 2018).

[0055] “Parkinson’s disease in the elderly adult” refers to Parkinson’s disease on patients exceeding the age of 65.

[0056] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, Charcot disease, or motor neuron disease, is a progressive neurodegenerative disorder that affects nerve cells in the brain and spinal cord. ALS leads to the degeneration and eventual death of motor neurons, which are responsible for controlling voluntary muscle movement. As these motor neurons degenerate, the muscles they control weaken and atrophy, leading to symptoms such as muscle stiffness, weakness, twitching, and eventually paralysis.

[0057] There are many different motor phenotypes of ALS and they are mainly classified according to the relative involvement of UMNs (upper motor neurons) versus LMNs (lower motor neurons) and the regional distribution of involvement.

[0058] It is important to recognize the different motor phenotypes, as life expectancy varies considerably between ALS subtypes. In addition, varying degrees of cognitive and behavioral impairments may be present.

[0059] UMN signs groups hyper-reflexia, spasticity and slowness of movements.

[0060] LMN signs groups weakness, muscle muscular and fasciculation.

[0061] There is typically no single agreed-upon classification system for ALS types, but different classifications may refer to various aspects of the disease. Of all types, here are the main ones:

[0062] Sporadic ALS: This is the most common form of ALS, accounting for approximately 90-95% of all cases. Sporadic ALS occurs randomly and does not have a clear genetic link. It can affect anyone, regardless of family history.

[0063] Familial ALS: In about 5-10% of cases, ALS runs in families. Familial ALS is inherited, meaning it is caused by mutations in specific genes that are passed down from one generation to the next. Several genes have been associated with familial ALS, including SOD1 , C9orf72, FUS, and others.

[0064] Bulbar onset ALS: This type of ALS begins in the bulbar region of the brain, which controls functions such as speech, swallowing, and chewing. Symptoms often start with difficulty in speaking or swallowing, and patients may experience slurred speech, trouble swallowing, and muscle weakness in the face and throat.

[0065] Limb onset ALS: Limb onset ALS is characterized by initial symptoms affecting the limbs, such as weakness, muscle cramps, twitching, or difficulty with fine motor tasks. Over time, the weakness and atrophy may spread to other muscles, leading to more widespread impairment.

[0066] Progressive muscular atrophy (PMA): PMA is a rare form of ALS that primarily affects the lower motor neurons in the spinal cord. It leads to progressive muscle weakness and atrophy without significant involvement of upper motor neurons. PMA tends to progress more slowly than classical ALS and may have a better prognosis.

[0067] Primary lateral sclerosis (PLS): PLS is a rare variant of ALS that primarily affects the upper motor neurons in the brain and spinal cord. It is characterized by progressive stiffness, spasticity, and weakness, primarily in the legs. PLS typically progresses more slowly than classical ALS and does not usually lead to significant muscle atrophy or respiratory involvement.

[0068] Although ALS is considered a motor neuron disorder, neuroinflammation also plays an important role. Recent evidence in ALS disease models shows activation of the inflammasome and subsequent initiation of pyroptosis, an inflammatory type of cell death. In this study, the authors determined theexpression and distribution of inflammasome and pyroptosis effector proteins and the status of neuronal loss in the post-mortem brain and spinal cord of ALS patients and controls, as well as in TDP-43 mutant mice. NLRP3 expression containing the NOD, LRR and pyrin domains of gasdermin D (GSDMD) cleaved by the pyroptosis effector protein and IL-18 was detected in human ALS motor cortex and spinal cord microglia, indicating a canonical state of inflammasome- triggered pyroptosis activation. Similar results were obtained in TDP-43A315T mice, where microglial pyroptosis activation was significantly increased in the motor cortex from the onset of symptoms and correlated with neuronal loss (Van Schoor, E., Ospitalieri, S., Moonen, S., Tome, S. O., Ronisz, A., Ok, O., ... & Thai, D. R. (2022). Increased pyroptosis activation in white matter microglia is associated with neuronal loss in ALS motor cortex. Acta neuropathologica, 144(3), 393-411.).

[0069] In a healthy person, the cerebral distribution of glyburide 11C corresponds to the cerebral blood volume, suggesting negligible penetration of the blood-brain barrier (BBB). This clinical observation corroborates preclinical findings suggesting that local changes in BBB structure and function are necessary for targeted administration and favorable effects of glyburide on injured brain tissue while minimizing potential side effects on the healthy brain (Marie, S., Comtat, C., Caille, F., Becquemont, L., Bottlaender, M., & Tournier, N. (2019). 11C-glyburide PET imaging unveils the negligible brain penetration of glyburide in humans. Neurology, 92(17), 813-814.). Alterations of the blood-brain barrier (BBB) or the blood-brain barrier (BSCB) have been reported in ALS. These alterations may be involved in the complex etiology and pathogenesis of ALS (Alarcan, H., Al Ojaimi, Y., Lanznaster, D., Escoffre, J. M., Corcia, P., Vourc’h, P., ... & Blasco, H. (2022). Taking Advantages of Blood-Brain or Spinal Cord Barrier Alterations or Restoring Them to Optimize Therapy in ALS?. Journal of Personalized Medicine, 12(7), 1071 .). An extensive literature review of over 2000 articles has recently shown that BCNSB integrity is disrupted throughout disease progression in rodent models, starting before the onset of symptoms and detectable neurodegeneration. Increased permeability, drug resistance to up-regulated efflux transporters and morphological changes in BCNSB supporting cells, including pericytes, astrocytes and endothelial cells, were observed in animal models.Abnormalities of the BCNSB have also been demonstrated in post-mortem studies of ALS patients (Mirian, A., Moszczynski, A., Soleimani, S., Aubert, I., Zinman, L., & Abrahao, A. (2022). Breached barriers: a scoping review of bloodcentral nervous system barrier pathology in amyotrophic lateral sclerosis. Frontiers in Cellular Neuroscience, 16, 851563). Such alterations in BBB and BCNSB have the potential to impact CNS drug exposure in ALS, modulating the efficacy of drugs intended to reach the brain and the toxicity of drugs not intended to reach the brain (Pan, Y., & Nicolazzo, J. A. (2022). Altered blood-brain barrier and blood-spinal cord barrier dynamics in amyotrophic lateral sclerosis: Impact on medication efficacy and safety. British Journal of Pharmacology, 779(11), 2577-2588.).

[0070] Moreover, having a low dose of metformin allows to prevent lactic acidosis and B12 deprivation, two major side effects of this molecule.

[0071] This combination, in the case of ALS patient, is an advantage, limiting the metformin-related toxicity but retaining its intrinsic neuroprotective, antioxidant effects.

[0072] Multiple System Atrophy (MSA) is a rare, progressive neurodegenerative disorder characterized by dysfunction in the autonomic nervous system, motor control, and balance. MSA is classified into two main types: MSA-P (Parkinsonian type), featuring symptoms like slowness of movement, rigidity, and tremor; and MSA-C (Cerebellar type), marked by ataxia, impaired coordination, and balance problems.

[0073] Frontotemporal dementia is a progressive neurodegenerative disorder that primarily affects the frontal and temporal lobes of the brain, which are responsible for behavior, personality, language, and decision-making. FTD is caused by abnormal accumulations of proteins, such as tau or TDP-43, in the brain, leading to neuronal damage and loss. The disease is often inherited, with up to 40% of cases having a family history. The progression of FTD varies, but it generally leads to significant cognitive and physical decline, ultimately requiring full-time care.lt typically begins between the ages of 45 and 65, though it can occur earlier or later.

[0074] There are several subtypes of FTD, each presenting distinct symptoms:

[0075] Behavioral variant FTD (bvFTD): The most common form, bvFTD, is marked by drastic personality changes, inappropriate social behavior, impulsivity, and apathy. Individuals may lose empathy, display repetitive behaviors, or develop unusual eating habits. They may also struggle with planning, decision-making, and maintaining attention.

[0076] Primary progressive aphasia (PPA): This subtype primarily affects language skills. PPA can be further divided into:- Nonfluent / agrammatic variant: Characterized by difficulty in forming sentences and speaking fluently, often accompanied by grammatical errors.- Semantic variant: Involves a loss of understanding of words and objects, leading to difficulties in naming things and understanding speech.- Logopenic variant: Marked by slow speech and difficulty finding the right words, with relatively preserved grammar and comprehension.

[0077] FTD with motor neuron disease: Some individuals with FTD also develop symptoms similar to amyotrophic lateral sclerosis (ALS), including muscle weakness and atrophy.

[0078] Lewy body dementia (LBD) is a progressive neurodegenerative disorder characterized by the abnormal accumulation of protein deposits called Lewy bodies in the brain. These deposits primarily consist of alpha-synuclein, a protein that disrupts normal brain function, particularly in regions responsible for cognition, movement, and behavior. LBD is the second most common form of dementia after Alzheimer's disease, accounting for up to 20% of dementia cases.

[0079] LBD manifests in two closely related forms: dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). The distinction between the two lies in the timing of cognitive and motor symptoms. If dementia symptoms appear within a year of motor symptoms, the diagnosis is DLB. If motor symptoms precede dementia by more than a year, it is classified as PDD.

[0080] Huntington’s disease (HD) is a progressive, inherited neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the HTT gene. The disease typically manifests between ages 30 and 50, though juvenile-onset formsexist. HD is characterized by a triad of symptoms: involuntary choreiform movements (chorea), cognitive decline, and psychiatric disturbances such as depression, irritability, and apathy.

[0081] Pathologically, HD is marked by the selective degeneration of medium spiny neurons in the striatum, leading to motor dysfunction and behavioral changes. The mutant huntingtin protein forms toxic aggregates, disrupts cellular processes, and induces mitochondrial dysfunction, oxidative stress, and neuroinflammation. As the disease progresses, patients experience worsening motor impairment, dementia, and increasing dependence, with a typical disease course lasting 15- 20 years. There is currently no cure; management is symptomatic and multidisciplinary

[0082] Alzheimer’s disease is a progressive neurodegenerative disorder and the most common cause of dementia, accounting for 60-80% of dementia cases worldwide. It primarily affects older adults, with symptoms typically beginning after the age of 65, though early-onset forms can occur in younger individuals.

[0083] Alzheimer's disease is characterized by the gradual decline in cognitive function, memory, and behavior. The hallmark features of the disease are the accumulation of two abnormal protein structures in the brain: amyloid plaques and neurofibrillary tangles. Amyloid plaques are sticky clumps of beta-amyloid protein that build up between nerve cells, disrupting cell communication. Neurofibrillary tangles, composed of a protein called tau, form inside neurons and interfere with vital cellular functions, ultimately leading to cell death.

[0084] Symptoms of Alzheimer's disease typically begin with mild memory loss, particularly affecting recent events and new information. As the disease progresses, cognitive impairments worsen, affecting language, problem-solving, and decision-making abilities. Individuals may struggle with daily tasks, lose track of time, become disoriented in familiar places, and experience significant personality and behavior changes, such as increased irritability, depression, and social withdrawal.

[0085] “Acceptable pharmaceutical excipient or carrier” refers to an excipient or carrier that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, preferably a human. It includes any and allsolvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the regulatory offices such as the FDA or EMA.

[0086] “neurodegenerative disease agent” refers to a molecule known to prevent, treat or slow down neurodegenerative disease and / or its consequences.

[0087] “Anti-amyotrophic lateral sclerosis agent” refers to a molecule known to prevent, treat or slow down amyotrophic lateral sclerosis and / or its consequences.

[0088] “Administration” refers to the take of a drug, by whatever routes, in order to get through the target site in the patient’s body.

[0089] “Oral administration” refers to a route of administration whereby pharmaceutical compositions are taken through the mouth, swallowed, and then processed via the digestive system.

[0090] “Rectal administration” refers to a route of administration using the rectum. Pharmaceutical compositions are absorbed by the rectum’s blood vessels and flow into body’s circulatory system, distributing the drug to the target site.

[0091] “Intramuscular administration” refers to the injection of a substance into a muscle. Muscles have larger and more numerous blood vessels than subcutaneous tissue, leading to faster absorption than subcutaneous or intradermal injections.

[0092] “Intravenous administration” refers to an administration directly into a person’s veins.

[0093] “Subcutaneous administration” refers to the insertion of medications beneath the skin either by injection or infusion.

[0094] “Nasal administration” refers to the delivery of drugs or substances through the nose, where they are absorbed by the nasal mucosa.

[0095] “Transethmoidal administration” refers to the delivery of drugs or substances directly through the ethmoid bone, a porous bone separating the nasal cavity from the brain. This route allows drugs to bypass the blood-brain barrier, enabling direct access to the central nervous system (CNS).

[0096] “Coated” or “coating” refers to a layer applied to the surface of a tablet, capsule, or granule to protect the drug, to control the release of the active compound.

[0097] “Immediate release” of a compound refers to a formulation, for example in the form of a tablet, which disintegrate rapidly and get dissolved to release the medicaments.

[0098] “Extended-release” refers to a formulation that allows a reduction in dosage frequency as compared to an immediate-release (conventional) dosage form. Examples of extended-release dosage forms include controlled-release, sustained-release, and long-acting drug products.

[0099] “Delayed-release” refers to a formulation that comprises a combination of a portion of immediate release and a portion of extended release. An immediate portion of the formulation may be released promptly after administration, and the extended release portion is released later. Enteric-coated dosage forms are an example of delayed-release products.

[0100] “Weekly administration” refers to a dosing schedule in which the pharmaceutical composition, or each active ingredient thereof, is administered one or more times within each consecutive seven-day interval, optionally on the same or on different days in successive weeks. Weekly administration may consist of a single administration per week or of multiple administrations within the week, and may be implemented concomitantly or sequentially for the two active ingredients as defined herein. The cumulative amount administered over said seven-day interval corresponds to a therapeutically effective dose as defined herein.

[0101] “Biweekly administration” refers to a dosing schedule in which the pharmaceutical composition, or each active ingredient thereof, is administered one or more times within each consecutive fourteen-day interval (i.e., once every two weeks), without limitation as to the specific day within the interval. Biweeklyadministration may comprise a single administration perfourteen-day interval or multiple administrations within that interval and may be implemented concomitantly or sequentially as defined herein. The cumulative amount administered over said fourteen-day interval corresponds to a therapeutically effective dose as defined herein.

[0102] “Intermittent administration” refers to non-continuous dosing regimens that include planned drug-free periods between administrations that are greater than 24 hours, including, without limitation, weekly, biweekly, or cyclic regimens. Intermittent administration may, but need not, follow a fixed periodicity, and may be implemented concomitantly or sequentially for the two active ingredients as defined herein, provided that the aggregate exposure over the selected dosing interval corresponds to a therapeutically effective dose as defined herein.

[0103] “Dosing interval” refers to the predefined period over which the cumulative administered amount is determined for a given regimen, including, without limitation, a seven-day interval for weekly administration or a fourteen-day interval for biweekly administration.

[0104] “Cumulative administered amount” refers to the sum of the quantities of an active ingredient (or of the pharmaceutical composition) actually administered within a dosing interval, regardless of the number or timing of administrations within that interval.

[0105] “Drug-holiday period” refers to a planned drug-free period within a dosing interval during which no administration of one or both active ingredients occurs, without departing from therapeutic efficacy when considered over the relevant dosing interval.

[0106] ‘“mg / day” or the indication “ / day” refers to a conventional unit of expression of dose used for convenience and comparison across regimens. As used herein, “‘mg / day’” does not require daily administration; rather, it denotes a daily-equivalent exposure that may be achieved by intermittent (e.g., weekly or biweekly) administration, provided that the cumulative administered amount over the dosing interval corresponds to the therapeutically effective dose.Description of Embodiments

[0107] The present invention thus relates to a pharmaceutical composition comprising metformin and glibenclamide for use in the treatment of neurodegenerative diseases.

[0108] Metformin is the International Nonproprietary Name (INN) to designate the substance 1 ,1 -dimethylbiguanide (CAS 657-24-9). Metformin is a largely use medication for the treatment of type 2 diabetes, especially used in the form of its hydrochloride C4HnN5HCI. Classically, its role is to decrease the insulin resistance of the carbohydrate intolerant organism and to decrease the hepatic neoglucogenesis. Metformin can be prepared by known methods, or may be obtained from commercial sources (For example GLUCOPHAGE®, STAGID®, GLUMETZA® AND FORTAMET®). Metformin has the structure depicted below:

[0109] As used herein, the term “metformin” encompasses any prodrugs, pharmaceutically acceptable salts, hydrates and solvates thereof. In particular, the term “metformin” encompasses chlorhydrate and embonate salts, as well as the hydrochloride salts and mono-hydrochloride salt thereof, e.g., metformin hydrochloride and metformin mono-hydrochloride. The term “metformin” also encompasses the crystalline forms of said compound.

[0110] Glibenclamide, also known as glyburide, is an antidiabetic drug in a class of medications known as sulfonylureas, closely related to sulfa drugs. This molecule has a hypoglycemic effect, exhibit a good tolerance profile, is compatible for combined formulation, and exhibit synergy to allow for low dosing because of frequent polymedication in the target population. Glibenclamide can be prepared by known methods, or may be obtained from commercial sources (For example DAONIL®.) Glibenclamide has the structure depicted below:

[0111] The Applicant surprisingly showed that the combined administration of metformin and glibenclamide significantly and synergistically result in neuroprotection in the context of neurodegenerative diseases, and in situ by promoting the survival of neurons.

[0112] Moreover, having a low dose of metformin allows to prevent lactic acidosis and B12 deprivation, two major side effects of this molecule.

[0113] This combination, in the case of neurodegenerative diseases patients, and especially in elderly patients, is an advantage, limiting the metformin-related toxicity but retaining its intrinsic neuroprotective, antioxidant effects.

[0114] This combination of molecules works through a novel mechanism of action characterized by the Applicant.

[0115] A growing number of studies showed a strong link between metformin and the regulation of AMPK, mainly mediated by an inhibition, at the mitochondrial level, of the respiratory chain Complex I, as well as of Glycerol-3- Phosphate Dehydrogenase (Francesco Agostini et al., Int. J. Mol. Sci., 2022 and Vial et al., Front. Endocrinol., 07 May 2019). One of its main transporters, OCT1 , is expressed on the surface of a large number of cell types, including neurons and endothelial cells. These mechanisms applied to the treatment of neurodegenerative diseases, should therefore be studied with the greatest attention.

[0116] Then, metformin is likely to exert a neuroprotective role mainly by regulating mitochondrial function:- By preventing the accumulation of toxic protein aggregates via the inhibition of Glycerol-3-Phosphate Dehydrogenase activity, a decrease in gluconeogenesis, and ultimately abnormal protein glycation events.- By preventing protein missfolding, oxidative stress and protein synthesis, as well as promoting the process of autophagy (regulation of mitochondrial AMPK levels).

[0117] The NLRP3 inflammasome complex has been associated with Blood-brain barrier (BBB) permeability and neuroinflammation (Lawrence et al. 2022). NLRP3-inflammasome is an intracellular protein complex and a key mediator of inflammation in many pathologies. Its activation by various effectors leads to the release of the pro-inflammatory cytokines IL-1 B and IL-18, as well as the process of pyroptosis, a type of programmed cell death characterized by permeabilization of the plasma membrane by family members gasdermin proteins (Rebecca C. Coll et al..Trends in Pharmacological Sciences. 2022). A recent study showed that the use of glibenclamide is capable of improving the integrity of the BBB by reducing the activity of NLRP3 (mechanism explained by a reduction in cellular potassium efflux) and pyroptosis at the level endothelial cells (Fulin Xu et al., Brain Behav. 2019).

[0118] Then, Glibenclamide mainly mediates its neuroprotective effects by inhibiting the activity of the NLRP3 inflammasome and ultimately programmed cell death by pyroptosis. Two different places of action could constitute the targets of this mechanism:- Within the BBB, where it would help maintain the integrity of the barrier.- In-situ in the neuron, preventing the decrease in the cell population.

[0119] The concomitant use of the 2 molecules (metformin and glibenclamide) and the targeting of the previously mentioned mechanisms would result in neuroprotection in the context of neurodegenerative diseases by regulating the integrity of the BBB, and in situ by promoting the survival of neurons.

[0120] Metformin is known to cross the blood brain barrier through an active transport, while Glibenclamide, the other drug, was thought to be excluded from the brain compartment.

[0121] Metformin inhibits two major components responsible for the efflux of glibenclamide outside of the brain

[0122] The treatment of neurodegenerative diseases associating metformin and glibenclamide relies on an orthogonal mechanism of action of the two compounds. Metformin modifies the cell metabolism, especially related to glycolysis and oxidative stress, whilst glibenclamide decreases neuro inflammation and inhibits pyroptosis.

[0123] Metformin has a shorter half-life as compared to that of glibenclamide. The present invention describes a formulation allowing for parallel pharmacokinetic profiles of metformin and glibenclamide, ensuring maintenance of a therapeutically relevant concentration inside the brain compartment.

[0124] Metformin and glibenclamide exert a synergistic pharmacodynamic effect on neurodegenerative diseases.

[0125] Metformin could potentiate the cerebral uptake of glibenclamide as it inhibits efflux pumps such as the Pgp and BCRP.

[0126] The disposal of metformin and glibenclamide relies on different mechanisms and have different characteristics, which are described in Scheen, A. J. (1996). Clinical pharmacokinetics of metformin. Clinical pharmacokinetics, 30, 359-371 for metformin, and in Pearson, J. G. (1985). Pharmacokinetics of glyburide. The American Journal of Medicine, 79(3), 67-71 for glibenclamide.

[0127] Apparent terminal half-life is between 6.5 hours for metformin, versus 11 hours for glibenclamide. Thus, a daily coformulation must allow for a release of half the speed for metformin. A 95% elimination of an active principle takes five times the terminal half-life. Thus, metformin reaches 50% of its Cmax in 6.5h, 25% in 13h, 12.5% in 21 ,5h and 6.25% in 26h, whilst these values are reached in 11 , 22, 33 and 44h respectively for glibenclamide.

[0128] Glibenclamide alone has been described as unable to penetrate the blood brain barrier in healthy subjects. As a hydrophobic drug, virtually insoluble in water, it might cross the blood brain barrier through passive diffusion, but is a substrate to multiple efflux pumps such as multi drug resistance proteins, the breast cancer resistance protein, and the Pgp notably.

[0129] In our experiments, metformin and glibenclamide exert a synergistic, neuroprotective effect on 6-OHDA intoxicated primary mesencephalic neuron cultures containing 5% dopaminergic neurons. The single agents have little or noeffect on neuron viability or cell fitness which is evaluated through measurement of mean neurite length. This direct action requires that both drugs reach the dopaminergic neurons.

[0130] The BBB is thought to be subject to leakage in Parkinson’s disease (Al- Bachari, S., Naish, J. H., Parker, G. J., Emsley, H. C., & Parkes, L. M. (2020). Blood-brain barrier leakage is increased in Parkinson’s disease. Frontiers in physiology, 11 , 593026.) as well as in a number of other neurodegenerative diseases such as amyotrophic lateral sclerosis (Steinruecke, M., Lonergan, R. M., Selvaraj, B. T., Chandran, S., Diaz-Castro, B., & Stavrou, M. (2023). Blood- CNS barrier dysfunction in amyotrophic lateral sclerosis: proposed mechanisms and clinical implications. Journal of Cerebral Blood Flow & Metabolism, 43(5), 642-654.), Alzheimer’s disease (Chen, Y., He, Y., Han, J., Wei, W., & Chen, F. (2023). Blood-brain barrier dysfunction and Alzheimer’s disease: associations, pathogenic mechanisms, and therapeutic potential. Frontiers in Aging Neuroscience, 15, 1258640.), frontotemporal and Lewy body dementia (Janelidze, S., Hertze, J., Nagga, K., Nilsson, K., Nilsson, C., Wennstrdm, M., ...& Swedish BioFINDER Study Group. (2017). Increased blood-brain barrier permeability is associated with dementia and diabetes but not amyloid pathology or APOE genotype. Neurobiology of aging, 51, 104-112.).

[0131] It is known that metformin is actively transported (Koepsell, H. (2021). Update on drug-drug interaction at organic cation transporters: Mechanisms, clinical impact, and proposal for advanced in vitro testing. Expert Opinion on Drug Metabolism & Toxicology, 17(6), 635-653.), like dopamine (Boxberger, K. H., Hagenbuch, B., & Lampe, J. N. (2014). Common drugs inhibit human organic cation transporter 1 (OCTI)-mediated neurotransmitter uptake. Drug Metabolism and Disposition, 42(6), 990-995 ), through organic cation transporter channel type 1 across the BBB.

[0132] Glibenclamide inhibits the NLRP3 inflammasome complex, with several involvements, including on the BBB itself, tightening the intercellular junctions and decreasing the permeability (Xu, F., Shen, G., Su, Z., He, Z., & Yuan, L. (2019). Glibenclamide ameliorates the disrupted blood-brain barrier in experimental intracerebral hemorrhage by inhibiting the activation of NLRP3 inflammasome. Brain and behavior, 9(4), e01254).

[0133] Being substrate to efflux pumps and decreasing the leakages which were a means of crossing the blood brain barrier, increasing the striatal concentration of glibenclamide requires either improving BBB penetration or decreasing the active efflux of the drug.

[0134] Increasing doses of glibenclamide might increase bioavailability in the ventral striatum but will also result in hypoglycemia, which needs to be avoided in Parkinson’s disease (Guatteo, E., Marinelli, S., Geracitano, R., Tozzi, A., Federici, M., Bernardi, G., & Mercuri, N. B. (2005). Dopamine-containing neurons are silenced by energy deprivation: a defensive response or beginning of cell death?. Neurotoxicology, 26(5), 857-868.), in Alzeihmer’s disease (He, C., Li, Q., Cui, Y., Gao, P., Shu, W., Zhou, Q., ... & Zhu, Z. (2022). Recurrent moderate hypoglycemia accelerates the progression of Alzheimer’s disease through impairment of the TRPC6 / GLUT3 pathway. Jci Insight, 7(5).), in SLA (Lerskiatiphanich, T., Marallag, J., & Lee, J. D. (2022). Glucose metabolism in amyotrophic lateral sclerosis: It is bitter-sweet. Neural Regeneration Research, 17(9), 1975-1977.), and in FTD (Garrett, L. R., & Niccoli, T. (2022). Frontotemporal dementia and glucose metabolism. Frontiers in Neuroscience, 16, 812222.).

[0135] To overcome these technical biases, the combination according to the invention allow pharmacokinetic interaction of glibenclamide with metformin to induce Pgp and BCRP inhibition and prevent glibenclamide efflux, through parallel pharmacokinetic and a maintenance of efflux pump inhibition throughout the day.

[0136] Diffusion of glibenclamide is facilitated by the action of metformin on BCPR and PgP channels. Data in the literature demonstrate the ability of metformin to inhibit BCRP activity in drug-resistant epilepsy and PgP expression via AMPK activation in vivo in rats. Such processes, applied in the context of Parkinson's disease, provide a rational explanation for the synergy observed when using a metformin / glibenclamide combination in our studies.

[0137] A low dosage is required to prevent the neurotoxic action of the two molecules characterized at anti-diabetic dosages: (i) As described in the literature, metformin treatment at anti-diabetic doses can induce a neurotoxic effect bycausing vitamin B12 deficiency and an increase in homocysteine and methylmalonic acid. The use of the drug in Parkinson's disease in non-diabetic patients at usual doses is not relevant in the context of a neuroprotective effect, (ii) As also reported in the literature and based on the drug's public data, glibenclamide exerts neurotoxic effects at anti-diabetic doses via a peripheral hypoglycemic effect. In the central nervous system, glibenclamide is not able to cross the BBB in a non-pathological context. The use of low doses in the context of Parkinson's disease seems appropriate to limit the hypoglycemic effects also in situ in the neuron, a cell highly dependent on carbohydrate metabolism.

[0138] This treatment is intended for patients at all stages of neurodegenerative disorders, including early diagnosis, and patients presenting few to no symptoms. These patients are likely to be inobservant and require a daily dosing to ensure treatment compliance.

[0139] The present invention thus relates to a combination comprising metformin as described hereinabove and glibenclamide as described hereinabove for use in the prevention and / or treatment of neurodegenerative diseases.

[0140] A second object of the invention concerns a method of preventing and / or treating neurodegenerative diseases comprising the administration of metformin and glibenclamide.

[0141] According to a third object, the invention concerns the combined administration of metformin and glibenclamide, for use in the prevention and / or treatment of neurodegenerative diseases.

[0142] Another object of the present invention is a kit-of-parts comprising a first part comprising metformin and a second part comprising glibenclamide for use in the prevention and / or treatment of neurodegenerative diseases.

[0143] Another object of the present invention is the use of a combination comprising or consisting of metformin and glibenclamide as described hereinabove for the manufacture of a medicament for preventing and / or treating neurodegenerative diseases.

[0144] Another object of the present invention is a pharmaceutical combination comprising or consisting of metformin and glibenclamide as describedhereinabove, and at least one pharmaceutically acceptable excipient, for use in the prevention and / or treatment of neurodegenerative diseases.

[0145] Another object of the invention is a medicament comprising or consisting of a combination of metformin and glibenclamide as described hereinabove, or a pharmaceutical combination as described hereinabove, or a kit-of parts as described hereinabove, for use in the prevention and / or treatment of neurodegenerative diseases.

[0146] In an embodiment, neurodegenerative disease is chosen among: Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, Frontotemporal dementia, Lewy body dementia, Huntington’s disease or Alzheimer’s disease.

[0147] In an embodiment, the Parkinson’s disease is early onset Parkinson’s disease, idiopathic Parkinson’s Disease, Parkinson’s disease associated with aberrant splicing, trauma-induced Parkinson’s disease, vascular Parkinson’s disease, drug-induced or Parkinson’s disease in the elderly adult.

[0148] It will be understood that the total daily usage of metformin and the total daily usage of glibenclamide in combination according to the invention will be decided by the attending physician within the scope of sound medical judgment.

[0149] Said pharmaceutical composition comprises a therapeutically effective amount of metformin and glibenclamide.

[0150] Said therapeutically effective amount of metformin is low compared to the dosage usually administered for treating diabetes.

[0151] According to another embodiment of the invention, the pharmaceutical composition according to the invention is formulated for immediate release of metformin and glibenclamide.

[0152] According to another embodiment of the invention, the pharmaceutical composition according to the invention is formulated for extended-release of metformin and / or glibenclamide, preferably controlled-release, sustained-release, or long-acting drug products.

[0153] According to another embodiment of the invention, the pharmaceutical composition according to the invention is formulated for delayed-release of metformin and / or glibenclamide, preferably with an enteric-coating.

[0154] According to another embodiment of the invention, the pharmaceutical composition according to the invention is formulated for a delayed-release administration, preferably a combined administration of an immediate release form of metformin, an extended-release form of metformin, and glibenclamide.

[0155] According to another embodiment of the invention, the pharmaceutical composition according to the invention comprise a core containing coated particles of metformin and uncoated particles of glibenclamide.

[0156] In an embodiment, said core is surrounded by an outer layer containing uncoated particles of metformin and uncoated particles of glibenclamide.

[0157] In another embodiment, the pharmaceutical composition according to the invention comprise a core containing coated particles of metformin. Preferably, said core is surrounded by an outer layer containing uncoated particles of metformin and uncoated particles of glibenclamide.

[0158] In an embodiment, said particles can be particles, microparticles or nanoparticles.

[0159] In an embodiment, said coating is composed of a material or mixture of materials to slow, modify or control the release of metformin.

[0160] Preferably, said material is selected from the group consisting of acetate succinate, a polyvinyl derivative, polyethylene oxide, polyacrylic acid, modified starch, cross-linked high amylose starch, hydroxypropyl starch, hydroxypropyl methylcellulose phthalate, cellulose, microcrystalline cellulose, carboxymethylethyl cellulose, cellulose acetate, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, cellulose phthalate, cellulose acetate, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate succinate, cellulose acetate butyrate, cellulose acetate trimellitate, poloxamer, povidone, alginic acid, sodium alginate, polyethylene glycol, polyethylene glycol alginate, gums, polymethacrylate, a copolymer of methacrylic acid and ethyl acrylate, a copolymer of polymethyl vinyl ether and malonic acid anhydride, a copolymer of polymethyl vinyl ether and malonic acid or the ethyl-, isopropyl-, n-butylesters thereof, zein, and mixtures of any of the foregoing.

[0161] In an embodiment, said core contains fillers, binders, surfactants, antioxidants, glidants, lubricants or a mixture thereof.

[0162] In an embodiment, said outer layer contains fillers, binders, surfactants, antioxidants, glidants, lubricants or a mixture thereof.

[0163] In an embodiment, in the pharmaceutical composition according to the invention, glibenclamide is released over a period of at least 6 hours.

[0164] In an embodiment, in the pharmaceutical composition according to the invention, glibenclamide is released over a period of at least 12 hours.

[0165] In an embodiment, in the pharmaceutical composition according to the invention, glibenclamide is released over a period of at least 24 hours.

[0166] In an embodiment, in the pharmaceutical composition according to the invention, metformin is released over a period of at least 6 hours.

[0167] In an embodiment, in the pharmaceutical composition according to the invention, metformin is released over a period of at least 12 hours.

[0168] In an embodiment, in the pharmaceutical composition according to the invention, metformin is released over a period of at least 24 hours.

[0169] In an embodiment, said formulation allows for differential release of metformin and glibenclamide enabling once-a-day dosing and parallel pharmacokinetic profiles

[0170] In an embodiment, said metformin is present in an amount providing an effective dose of between 0.025 mg / day and 450 mg / day.

[0171] In an embodiment, said metformin is present in an amount providing an effective dose of between 0.025 mg / day and 250 mg / day.

[0172] Preferably, said metformin is present in an amount providing an effective dose of 0.025 mg / day, or 0.050 mg / day, or 0.075 mg / day, or 0.1 mg / day or 0.125 mg / day, or 0.150 mg / day, or 0.175 mg / day, or 0.200 mg / day, or 0.225 mg / day, or 0.250 mg / day, or 0.275 mg / day, or 0.300 mg / day, or 0.325 mg / day, or 0.350 mg / day, or 0.375 mg / day, or 0.400 mg / day, or 0.425 mg / day, or 0.450 mg / day, or 0.475 mg / day, or 0.500 mg / day, or 0.525 mg / day, or 0.550 mg / day, or 0.575 mg / day, or 0.600 mg / day, or 0.625 mg / day, or 0.650 mg / day, or 0.675 mg / day, or0.700 mg / day, or 0.725 mg / day, or 0.750 mg / day, or 0.775 mg / day, or 0.800 mg / day, or 0.825 mg / day, or 0.850 mg / day, or 0.875 mg / day, or 0.900 mg / day, or 0.925 mg / day, or 0.950 mg / day, or 0.975 mg / day, or 1 mg / day, or 1 ,25 mg / day, or 1.50 mg / day, or 1.75 mg / day, or 2 mg / day, or 2,25 mg / day, or 2.50 mg / day, or 2.75 mg / day, or 3 mg / day, or 3,25 mg / day, or 3.50 mg / day, or 3.75 mg / day, or 4 mg / day, or 4,25 mg / day, or 4.50 mg / day, or 4.75 mg / day, or 5 mg / day, or 6 mg / day, 7 mg / day, 8 mg / day, 9 mg / day, or 10 mg / day, or 12,5 mg / day, or 15 mg / day, or 17,5 mg / day, or 20 mg / day, 22,5 mg / day, or 25 mg / day, 27,5 mg / day or 30 mg / day, or 32,5 mg / day, or 35 mg / day, or 37,5 mg / day, or 40 mg / day, or 42,5 mg / day, or 45 mg / day, or 47,5 mg / day, or 50 mg / day, or 60 mg / day, or 70 mg / day, or 80 mg / day, or 90 mg / day or 100 mg / day, or 125 mg / day, or 150 mg / day, or 175 mg / day, or 200 mg / day, or 225 mg / day, or 250 mg / day, or 275 mg / day, or 300 mg / day, or 325 mg / day, or 350 mg / day, or 375 mg / day, or 400 mg / day, or 425 mg / day, or 450 mg / day.

[0173] More preferably, said metformin is present in an amount providing an effective dose of about 50 mg / day.

[0174] In one embodiment, the subject is a mammal, preferably a human, and said therapeutically effective dose of metformin is a daily dose to be administered in one take or in one injection.

[0175] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 5 mg / day.

[0176] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 2 mg / day.

[0177] In an embodiment, said glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 1 mg / day.

[0178] Preferably, said glibenclamide is present in an amount providing an effective dose of 0.001 mg / day, or O. 005 mg / day, or 0.010 mg / day, or 0.015 mg / day, or 0.020 mg / day, or 0.025 mg / day, or 0.030 mg / day, or 0.035 mg / day, or 0.030 mg / day, or 0.035 mg / day, or 0.040 mg / day, or 0.045 mg / day, or 0.050 mg / day, or 0.055 mg / day, or 0.060 mg / day, or 0.065 mg / day, or 0.070 mg / day, or 0.075 mg / day, or 0.080 mg / day, or 0.085 mg / day, or 0.090 mg / day, or 0.095 mg / day, or 0,1 mg / day, or 0.15 mg / day, or 0.2 mg / day, or 0.25 mg / day, or 0.3 mg / day, or0.35 mg / day, or 0.4 mg / day, or 0.45 mg / day, 0,5 mg / day, or 0.55 mg / day, or 0.6 mg / day, or 0.65 mg / day, or 0.7 mg / day, or 0.75 mg / day, or 0.8 mg / day, or 0.85 mg / day, or 0.9 mg / day, or 0.95 mg / day, or 1 mg / day, or 1 .05 mg / day or 1 , 1 mg / day, or 1 .15 mg / day, or 1 .2 mg / day, or 1 .25 mg / day, or 1 .3 mg / day, or 1 .35 mg / day, or 1 .4 mg / day, or 1 .45 mg / day, or 1 ,5 mg / day, or 1.55 mg / day, or 1 .6 mg / day, or 1 .65 mg / day, or 1 .7 mg / day, or 1 .75 mg / day, or 1 .8 mg / day, or 1 .85 mg / day, or 1.9 mg / day, or 1.95 mg / day, or 2 mg / day, or 2.05 mg / day, or 2.10 mg / day, or 2.15 mg / day, or 2.20 mg / day, or 2.25 mg / day, or 2.30 mg / day, or 2.35 mg / day, or 2.40 mg / day, or 2.45 mg / day, or 2.50 mg / day, or 2.55 mg / day, or 2.60 mg / day, or 2.65 mg / day, or 2.70 mg / day, or 2.75 mg / day, or 2.80 mg / day, or 2.85 mg / day, or 2.90 mg / day, or 2.95 mg / day, or 3.00 mg / day, or 3.05 mg / day, or 3.10 mg / day, or 3.15 mg / day, or 3.20 mg / day, or 3.25 mg / day, or 3.30 mg / day, or 3.35 mg / day, or 3.40 mg / day, or 3.45 mg / day, or 3.50 mg / day, or 3.55 mg / day, or 3.60 mg / day, or 3.65 mg / day, or 3.70 mg / day, or 3.75 mg / day, or 3.80 mg / day, or 3.85 mg / day, or 3.90 mg / day, or 3.95 mg / day, or 4.00 mg / day, or 4.05 mg / day, or 4.10 mg / day, or 4.15 mg / day, or 4.20 mg / day, or 4.25 mg / day, or 4.30 mg / day, or 4.35 mg / day, or 4.40 mg / day, or 4.45 mg / day, or 4.50 mg / day, or 4.55 mg / day, or 4.60 mg / day, or 4.65 mg / day, or 4.70 mg / day, or 4.75 mg / day, or 4.80 mg / day, or 4.85 mg / day, or 4.90 mg / day, or 4.95 mg / day, or 5.00 mg / day.

[0179] In an embodiment, said glibenclamide is present in an amount providing an effective dose of 0.5 mg / day.

[0180] In one embodiment, the subject is a mammal, preferably a human, and said therapeutically effective dose of glibenclamide is a daily dose to be administered in one take or in one injection.

[0181] According to the present invention, metformin and glibenclamide in a combination of the invention are to be administered either simultaneously, separately or sequentially with respect to each other.

[0182] According to one embodiment, said method for preventing and / or treating neurodegenerative diseases in a subject in need thereof comprises administering to the subject a combination comprising or consisting of metformin and glibenclamide as described hereinabove, wherein a therapeutically effective dose of metformin is administered once to the subject; then a therapeutically effectivedose of glibenclamide is administered to the subject, preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the administration of a therapeutically effective dose of metformin.

[0183] According to one embodiment, said method for preventing and / or treating neurodegenerative diseases in a subject in need thereof comprises administering to the subject a combination comprising or consisting of metformin and glibenclamide as described hereinabove, wherein a therapeutically effective dose of glibenclamide is administered once to the subject; then a therapeutically effective dose of metformin is administered to the subject, preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the administration of a therapeutically effective dose of metformin.

[0184] In an embodiment, the pharmaceutical composition may optionally be formulated to allow for differential release of metformin and glibenclamide. Such a configuration enables once-daily administration while maintaining parallel pharmacokinetic profiles of the active ingredients.

[0185] In an embodiment, the combination of metformin and glibenclamide is administered intermittently.

[0186] In an embodiment, the combination of metformin and glibenclamide is administered weekly, or biweekly.

[0187] In an embodiment, the therapeutically effective amounts of metformin and glibenclamide disclosed herein may be delivered over one or more administrations within a dosing interval (e.g., within a week), such that the aggregate administered quantity matches the targeted mg / day-based exposure on a cumulative basis. In certain embodiments, the regimen includes drug-holiday periods within each interval without departing from therapeutic efficacy.

[0188] Preferably, intermittent administration is compatible with any of the routes of administration described herein (e.g., oral, intravenous, subcutaneous, intramuscular, topical, or rectal), and may be implemented concomitantly or sequentially for the two actives, consistent with the scheduling options disclosed in this description.

[0189] In a preferred embodiment, when metformin and glibenclamide are administered sequentially, the interval between successive administrations may be selected within a window of days (for example between 1 and 10 days), and may coincide with a weekly or biweekly rhythm, thereby providing an intermittent pattern while maintaining therapeutically effective exposure.

[0190] This administration is made according to a weekly schedule or another dosing interval deemed appropriate by the attending physician, taking into account the patient’s condition, tolerance, and therapeutic objectives.

[0191] In an embodiment, the pharmaceutical composition according to the invention further comprises at least one other acceptable active pharmaceutical ingredient, and / or at least one acceptable pharmaceutical excipient or carrier.

[0192] In a preferred embodiment, said active pharmaceutical ingredient is another agent for preventing or treating a neurodegenerative disease.

[0193] In a preferred embodiment, said active pharmaceutical ingredient is another anti-Parkinson’s disease agent.

[0194] In a more preferred embodiment, said anti-Parkinson’s disease agent is selected from the group consisting of L-DOPA, dopamine agonists, MAO inhibitors, COMT inhibitors, amantadine, carbidopa-levodopa, dopamine agonists, istradefylline, inosine, isradipine, apomorphine, donepezil, varenicline, foslevodopa-foscarbidopa and anti-cholinergics.

[0195] In another embodiment, said active pharmaceutical ingredient is an antidiabetic agent, preferably chosen among: Repaglinide, Gliclazide, Glimepiride, Glipizide.

[0196] In a preferred embodiment, said active pharmaceutical ingredient is another amyotrophic lateral sclerosis agent.

[0197] Preferably, agents used in treating Amyotrophic Lateral Sclerosis (ALS) include Riluzole, which slows disease progression by reducing glutamate, and Edaravone, a free radical scavenger that may slow functional decline. Nuedexta is used for pseudobulbar affect, managing uncontrollable emotional expressions. Muscle spasticity can be treated with Baclofen or Tizanidine.

[0198] In a more preferred embodiment, said anti-Amyotrophic lateral sclerosis agent is selected from the group consisting of riluzole, edaravone, and / or masitinib.

[0199] Riluzole is believed to work by decreasing the release of glutamate, a neurotransmitter that is thought to be involved in the progression of ALS. Riluzole is thought to slow down the progression of ALS, although it does not cure the disease or reverse its effects. It may help prolong survival and delay the need for mechanical ventilation in some patients with ALS. Riluzole is usually taken orally in the form of tablets. Side effects of riluzole include dizziness, nausea, fatigue, and liver function abnormalities. Riluzole 50 mg twice daily has antiglutamatergic effects and prolongs average patient survival by 3-6 months.

[0200] Edaravone is believed to work as a free radical scavenger. Free radicals can cause damage to cells and contribute to the progression of ALS. By reducing oxidative stress caused by free radicals, edaravone may help slow down the progression of the disease and preserve motor function in individuals with ALS. The exact mechanism of action of edaravone in ALS is not fully understood, but clinical trials have shown that it can slow down the rate of functional decline in some patients with ALS. Edaravone is administered intravenously and is typically given in cycles of daily infusions for a set period, followed by a period of rest. Common side effects of edaravone may include bruising or injection site reactions, headache, and difficulty walking. Edaravone has been studied in ALS via a randomized, double-blind Phase III study of intravenous edaravone 60 mg / day for 2 weeks a month in selected ALS patients showed a significantly lower decline in ALSFRS-R scores after 6 months of treatment. To date, edaravone has been approved for the treatment of ALS in the USA, Canada, Japan, South Korea and Switzerland.

[0201] Masitinib works by inhibiting the activity of specific enzymes called tyrosine kinases. These enzymes play a crucial role in various cellular processes, including cell growth, proliferation, and survival. By blocking the activity of these enzymes, masitinib can interfere with the growth and spread of cancer cells and help to reduce inflammation in certain diseases. A randomized controlled trial using 4.5 mg / kg / day masitinib as an adjunct to riluzole suggested a positive effect on ALSFRS-R decline, at least in patients with typical disease progression, aneffect whose extent should be better defined in a confirmatory study (Masrori et al., Eur J Neurol, Oct. 2020).

[0202] In a preferred embodiment, said active pharmaceutical ingredient is another Frontotemporal dementia agent.

[0203] Currently, there are no specific medications approved to treat Frontotemporal Dementia (FTD). However, symptoms can be managed with several agents. Selective serotonin reuptake inhibitors (SSRIs) like sertraline or fluoxetine are often used to address behavioral symptoms, such as impulsivity, aggression, or compulsive behaviors. Antipsychotic medications like quetiapine may be prescribed cautiously for severe agitation or psychosis, though they carry risks. Cholinesterase inhibitors, commonly used in Alzheimer's, are generally not effective in FTD.

[0204] In a preferred embodiment, said active pharmaceutical ingredient is another Lewy body dementia agent.

[0205] Agents for treating Lewy Body Dementia (LBD) focuses on managing symptoms. Cholinesterase inhibitors like rivastigmine are commonly used to improve cognitive function and reduce hallucinations. Levodopa can help alleviate Parkinsonian motor symptoms, though it may exacerbate hallucinations. Antipsychotics, particularly atypical ones like quetiapine, are used cautiously for severe hallucinations or delusions, as LBD patients are highly sensitive to these medications. Melatonin or clonazepam may be prescribed for REM sleep behavior disorder. Antidepressants like SSRIs can help manage mood symptoms. Careful medication management is essential due to the high sensitivity and risk of side effects in LBD patients.

[0206] In a preferred embodiment, said active pharmaceutical ingredient is another multiple system atrophy agent.

[0207] Agents for treating multiple system atrophy focus on managing symptoms. For example, midodrine and fludrocortisone are used to treat orthostatic hypotension, a common autonomic symptom, by increasing blood pressure.

[0208] In a preferred embodiment, said active pharmaceutical ingredient is another Alzheimer’s disease.

[0209] Agents for treating Alzheimer’s disease includes cholinesterase inhibitors like donepezil, rivastigmine, and galantamine, which help improve memory and cognitive function in early to moderate stages. Memantine, an NMDA receptor antagonist, is used in moderate to severe cases to slow cognitive decline by regulating glutamate activity. Antidepressants like SSRIs may be prescribed to manage depression, anxiety, or agitation. Antipsychotics such as risperidone may be used cautiously for severe behavioral symptoms, though they carry significant risks. Additionally, vitamin E is sometimes used as an antioxidant, though its benefits remain unclear. Non-drug therapies and supportive care are also crucial.

[0210] According to one embodiment, metformin and glibenclamide, the combination or pharmaceutical combination thereof, medicament or kit-of-parts according to the invention will be formulated for administration to the subject.

[0211] In an embodiment, the pharmaceutical composition is formulated to be suitable for oral, nasal, transethmoidal, rectal, topical, intramuscular or intravenous or subcutaneous administration of the compounds.

[0212] In a preferred embodiment, the pharmaceutical composition is formulated to be suitable for nasal administration.

[0213] In an embodiment, the nasal administration is made by nasal sprays, nasal drops, nasal powders, nasal gels, metered-dose aerosols, nasal foams, nasal creams, nasal ointments, nasal implants, nasal films, nasal nebulizers.

[0214] In a preferred embodiment, the pharmaceutical composition is formulated to be suitable for transethmoidal administration.

[0215] In an embodiment, the transethmoidal administration is made by microneedles, catheters, micro-pumps, intranasal implants, direct infusions, nanoparticles, liposomes, hydrogels, drug-eluting stents.

[0216] In a preferred embodiment, the pharmaceutical composition is formulated to be suitable for oral administration.

[0217] In an embodiment, the oral administration is made by gelatin capsules, capsules, caplet, pill, compressed tablet, tablets, powders, granules, oral solutions, microgranules, multilayer tablets, gastroresistant tablets, extended release insoluble matrix tablets, or suspensions.

[0218] In one embodiment, metformin as described hereinabove is in an adapted form for an oral administration. In one embodiment, metformin as described hereinabove is in an adapted form for nasal administration. In one embodiment, metformin as described hereinabove is in an adapted form for transethmoidal administration. In another embodiment, metformin as described hereinabove is in an adapted form for an injection. Thus, in one, metformin as described hereinabove is to be injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion. In one embodiment, glibenclamide as described hereinabove is in an adapted form for an oral administration.

[0219] Thus, in one embodiment, glibenclamide as described hereinabove is to be administered orally to the subject, for example as a capsule or as a tablet. In one embodiment, glibenclamide as described hereinabove is in an adapted form for nasal administration. In one embodiment, glibenclamide as described hereinabove is in an adapted form for transethmoidal administration. In another embodiment, glibenclamide as described hereinabove is in an adapted form for an injection. Thus, in another embodiment, glibenclamide as described hereinabove is to be injected to the subject, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion, preferably by intravenous injection.

[0220] In one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for oral administration. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for oral administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered orally to the subject.

[0221] Examples of forms adapted for oral administration include, without being limited to, liquid, paste or solid compositions, and more particularly tablets, tablets formulated for extended or sustained release, capsules, caplet, compressed tablet, pills, dragees, liquids, gels, syrups, slurries, suspensions, and the like. In one embodiment, the combination, pharmaceutical combination,medicament or kit- of-parts according to the invention is in a form adapted for parenteral administration. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for parenteral administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered parenterally.

[0222] In one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for injection, such as, for example, for intravenous, subcutaneous, intramuscular, intradermal, transdermal injection or infusion. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for injection, such as, for example, for intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered by injection to the subject, such as, for example, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion.

[0223] Sterile injectable forms of metformin and glibenclamide, the combination or pharmaceutical combination thereof, or medicament according to the invention may be a solution or an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. In one embodiment, the combination, pharmaceutical combination, medicament or kit- of-parts according to the invention comprises metformin that is in a form adapted for oral administration and glibenclamide that is in a form adapted for injection, such as, for example, for intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin that is to be administered orally and glibenclamide that is to be administered by injection to the subject, such as, for example, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion. In one embodiment, the combination, pharmaceutical combination, medicament or kit- of-parts according to the invention comprises metformin that is in a form adapted for injection, such as, for example, for intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion and glibenclamide that is in a form adapted for oral administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin that is to be administered by injection to the subject, such as, for example, by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion and glibenclamide that is to be administered orally.

[0224] In another embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for topical administration. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for topicaladministration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered topically to the subject.

[0225] Examples of forms adapted for topical administration include, without being limited to, liquid, paste or solid compositions, and more particularly aqueous solutions, drops, dispersions, sprays, microcapsules, micro- or nanoparticles, polymeric patch, or controlled-release patch, and the like.

[0226] In another embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for rectal administration. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for rectal administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered rectally.

[0227] Examples of forms adapted for rectal administration include, without being limited to, suppository, micro enemas, enemas, gel, rectal foam, cream, ointment, and the like.

[0228] In another embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for nasal administration. In other words, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for nasal administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be administered by the nasal route.

[0229] Examples of forms adapted for nasal administration include sprays, drops, powders, gels, aerosols, foams, creams, ointments, implants, nebulizers.

[0230] In another embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention is in a form adapted for transethmoidal administration. In other words, the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are both in a form adapted for transethmoidal administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit-of-parts according to the invention comprises metformin and glibenclamide which are to be transethmoidal administered.

[0231] In one embodiment, metformin is to be administered after glibenclamide. In one embodiment, metformin is to be administered at least 1 , 2, 3, 4, 5, 6, 7, 8, or 10 days after glibenclamide.

[0232] It is understood that, although the doses are expressed in mg / day for convenience, the administration of the active ingredients according to the present invention is not necessarily continuous. The combination of metformin and glibenclamide may therefore be administered in a discontinuous manner, for example according to a weekly schedule or any other interval deemed appropriate by the attending physician, depending on the patient’s condition, tolerance, and therapeutic objectives.

[0233] In particular, the indication “ / day” should not be construed as imposing a strict daily administration. The invention also encompasses administration regimens that are fractionated or spaced, including weekly, biweekly, or otherwise intermittent dosing schedules, provided that the cumulative amount administered corresponds to a therapeutically effective dose.

[0234] In the description and in the following examples, unless otherwise indicated, ranges of values denominated as "between ... and ..." include the lower and upper limits specified.Brief Description of Drawings

[0235] Fig. 1 : Histogram presenting the impact of metformin (CXS001) and glibenclamide (CXS002) on dopaminergic neurons survival after 6-OHDA injury. It is shown that metformin (CXS001) and glibenclamide (CXS002) alone did not improve dopaminergic neuron survival in 6OHDA intoxication as compared to Brain Derived Neurotrophic Factor. CXS002 at high concentration seems to decrease neuron viability.

[0236] Fig. 2: Histogram presenting the impact of metformin (CXS001) and glibenclamide (CXS002) on neurites length after 6-OHDA injury. It is shown thatmetformin (CXS001) and glibenclamide (CXS002) alone did not improve dopaminergic neuron fitness (neurite length) in 6OHDA intoxication, as compared to Brain Derived Neurotrophic Factor.

[0237] Fig. 3: Histogram presenting the impact of metformin (CXS001) and glibenclamide (CXS002) on dopaminergic neurons survival after 6-OHDA injury. It is shown that metformin (CXS001) and glibenclamide (CXS002) at low dose improve dopaminergic neuron viability in 6OHDA intoxication. A tendency might be seen for metformin 10pM + glibenclamide 10pM, despite not being statistically significant at this dose.

[0238] Fig. 4: Histogram presenting the impact of metformin (CXS001) and glibenclamide (CXS002) on neurites length after 6-OHDA injury. It is shown that metformin (CXS001) and glibenclamide (CXS002) at low dose improves dopaminergic neuron fitness, estimated by mean neurite length, in 6OHDA intoxication.

[0239] Fig. 5: Representative pictures of the primary neuron cell cultures in standard conditions (A) or upon 6OHDA intoxication (B, C, D). Arrows point to viable dopaminergic neurons, differentially stained (dependent on tyrosine hydroxylase expression). Four conditions are represented: standard conditions (A); intoxication with 6OHDA (B); 6OHDA intoxication after 48-hour-pretreatment with metformin and glibenclamide (C); and 6OHDA intoxication after 48-hour- brain derived neurotrophic factor pretreatment (D).

[0240] Fig. 6: Effect of metformin (CXS001) and glibenclamide (CXS002) after 6- OHDA injury on survival of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, **** p<0.0001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0241] Fig. 7: Effect of metformin (CXS001) and glibenclamide (CXS002) in combination after 6-OHDA injury on survival of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, **** p<0.0001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0242] Fig. 8: Effect of metformin (CXS001) and glibenclamide (CXS002) in combination after 6-OHDA injury on survival of rat primary dopaminergic neuronsexpressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, *** p<0.001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0243] Fig. 9: Effect of metformin (CXS001) and glibenclamide (CXS002) after 6- OHDA injury on neurites length of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, *** p<0.001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0244] Fig. 10: Effect of metformin (CXS001 ) and glibenclamide (CXS002) in combination after 6-OHDA injury on neurites length of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, **** p<0.0001 , stats vs 6-OHDA 20uM; one-way ANOVA followed by Dunnett’s test).

[0245] Fig. 11 : Effect of metformin (CXS001 ) and glibenclamide (CXS002) in combination after 6-OHDA injury on neurites length of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; * p<0.05, *** p<0.001 , stats vs 6-OHDA 20uM; one-way ANOVA followed by Dunnett’s test)

[0246] Fig. 12: Effect of metformin (CXS001 ) and glibenclamide (CXS002) after 6- OHDA injury on neurites length of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; # N / A (reference), * p<0.05, *** p<0.001 , **** p<0.0001 ; stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0247] Fig. 13: Effect of Am broxol, lixisenatide, metformin (CXS001) and glibenclamide (CXS002) after 6-OHDA injury on neuron survival of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; # N / A (reference), * p<0.05, *** p<0.001 , **** p<0.0001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0248] Fig. 14: Serum titration of a single administration of 10mg immediate release metformin, 40mg extended release metformin and 1 mg glibenclamide in human in (ng / mL) vs hours.

[0249] Fig. 15: Evaluation of (A) number of cortical neurons and (B) total length of cortical neurons after 10pm A O intoxication. DMSO ; A O 10pM (cellsintoxication) ; Metformin 10pm (+ A O 10pM) ; Glibenclamide 1 pm (+ A O 10pM) ; Metformin 10pm + Glibenclamide 1 pm (+ A O 10pM) ; BDNF 50ng / ml (+ Apo 10pM) as a positive control. All value are expressed as mean ± s.e. mean.Statistical analyses have been done on the different conditions (ANOVA followed by Dunnett’s test).

[0250] Fig. 16: Evaluation of (A) number of motor neurons, (B) total length of motor neurons, (C and D) nuclear and cytoplasmic pTDP-43 inclusions after overexpressing TDP-43. pTDP-43 ; Metformin 10pm (+ pTDP-43) ; Glibenclamide 1 pm (+ pTDP-43) ; Metformin 10pm + Glibenclamide 1 pm (+ pTDP-43) ; All value are expressed as mean ± s.e. mean. Statistical analyses have been done on the different conditions (ANOVA followed by Dunnett’s test).

[0251] Fig. 17: Evaluation of (A) survival of mesencephalic neurons, (B) survival of GABAergic neurons. DMSO, Glutamate 40pM (cells intoxication) ; Metformin 10pm (+ Glutamate 40pM) ; Glibenclamide 1 pm (+ Glutamate 40pM) ; Metformin 10pm + Glibenclamide 1 pm (+ Glutamate 40pM) ; MK801 20pM (+Glutamate 40 pM) as a positif control (glutamate receptor inhibitor). All value are expressed as mean ± s.e. mean. Statistical analyses have been done on the different conditions (ANOVA followed by Dunnett’s test).

[0252] Fig. 18: Differences in initiation time test (IT) values (A) were assessed between Sham / vehicle and 6-OHDA / veh using a Mann-Whitney test. Number of rats (n) was 12-14 per group. A value of p < 0.05 was considered statistically significant. Differences in IT (B) were assessed between 6-OHDA / veh and all 6- OHDA treated groups using Kruskal-Wallis followed by Steel post hoc test. Number of rats (n) was 12-14 per group. 6-OHDA, 6-hydroxydopamine; %, percentage; RF, right forepaw.

[0253] Fig. 19: Differences in Stepping test (ST) values (A) were assessed between Sham / vehicle and 6-OHDA / veh using a Mann-Whitney test. Number of rats (n) was 12-14 per group. A value of p < 0.05 was considered statistically significant. Differences in ST (B) were assessed between 6-OHDA / veh and all 6-OHDA treated groups using Kruskal-Wallis followed by Steel post hoc test. Number of rats (n) was 12-14 per group. 6-OHDA, 6-hydroxydopamine; %, percentage; RF, right forepaw.Examples

[0254] Example 1 : Study of potential neuroprotector effect of 2 test compounds and a mix of these 2 compounds after 6OH-DA injury on rat primary dopaminergic neurons survival: analysis of the number of dopaminergic neurons:

[0255] 6-hydroxydopamine (6OH-DA) is a selective catecholaminergic neurotoxin that is not only used as a pharmacological agent able to trigger PD-like stigmata (Sauer and Ortel, 1994; Cass et al., 2002) but also likely corresponds to a natural dopaminergic catabolite that accumulates in Parkinson’s disease-affected brains and that appears to strongly contribute to this pathology (Jellinger et al., 1995). For this reason, 6OH-DA-induced dopaminergic neurotoxicity in mice is widely used as a model for Parkinson’s disease research. Moreover, 6OH-DA inducing neurodegeneration of dopaminergic neurons in vitro, provides a useful model of Parkinson’s disease.

[0256] This study will investigate the effect of 2 compounds and a mix of these 2 compounds at 3 concentrations on rat primary mesencephalic culture injured by 6OH-DA, a Parkinson’ disease in vitro model. For the Parkinson’s disease, total number of dopaminergic neuron survival. BDNF will be used as positive control in this study.

[0257] A first culture of dopaminergic neurons will be carried out to determine the toxic concentrations of the compounds alone (at 4 concentrations) and the mix of the compounds (at 12 concentrations) in order to determine the maximum nontoxic concentrations to be used for the rest of the study.

[0258] A second and third culture of dopaminergic neurons will be prepared to study the protection of neurons in the presence of compounds after intoxication with 6 OH DA.

[0259] Experimental Protocol

[0260] 1. Dopaminergic neuron cell culture

[0261] Rat dopaminergic neurons will be cultured as described by Schinelli et al., 1988. Briefly pregnant female rats of 15 days gestation will be killed by cervical dislocation (Rats Wistar; Janvier) and the foetuses will be removed from the uterus. The embryonic midbrains will be removed and placed in ice-cold medium.Only the ventral portions of the mesencephalic flexure will be used for the cell preparations as this is the region of the developing brain rich in dopaminergic neurons. The midbrains will be dissociated by trypsinisation. Dissociated cells will be re-suspended in a defined culture medium and will be seeded at a in 96 wellplates and will be cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere.

[0262] Half of the medium will be changed every 2 days with fresh medium. In these conditions, after 5 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation. Three to six percent, of the neuronal cell population, are dopaminergic neurons.

[0263] After 6 days of culture, the culture medium will be changed by culture medium without growth factor.

[0264] 2. Cytotoxicity of compound

[0265] A first culture of dopaminergic neurons will be prepared for cytotoxicity test. Briefly, on day 7 of culture, cells will be incubated with compounds at 4 concentrations and with the mix of these 2 compounds at 16 concentrations treated for 48h.

[0266] The following conditions will be done:Control medium (vehicle to be defined, 48h).Control medium CX001 at 1mM, 100 pM, 10 pM and 1 pM.Control medium CX002 at 100 pM, 10 pM, 1 pM and 0.1 pM.Control medium CX001 at 1mM + CX002 at 100 pM, 10 pM, 1 pM and0.1 pM.Control medium CX001 at 100pM + CX002 at 100 pM, 10 pM, 1 pM and 0.1 pM.Control medium CX001 at 10pM + CX002 at 100 pM, 10 pM, 1 pM and0.1 pM.Control medium CX001 at 1 pM + CX002 at 100 pM, 10 pM, 1 pM and0.1 pM.

[0267] One culture will be performed with six wells per condition and three 96-well plate.

[0268] 3. End point evaluation: measurement of total number of TH positive neurons.

[0269] After 2 days of incubation, cells will be fixed by a solution of 4% paraformaldehyde for 20 min at room temperature, the control conditions will be fixed as well following the same procedure. The cells will be then permeabilized and non-specific sites will be blocked with a solution of phosphate buffered saline containing (PBS) with saponin and FCS for 15 min at room temperature. Cells will be incubated with Mouse Monoclonal Anti-Tyrosine Hydroxylase antibody (anti- TH, Sigma) in PBS with saponin and FCS overnight at 4°C. This antibody will be revealed with an Alexa Fluor 488 goat anti-mouse IgG in PBS with 1 % FCS, 0.1 % saponin, for 1 h at room temperature. Nuclei of cells will be labelled by a fluorescent marker (Hoechst solution) in the same solution.

[0270] For each well of culture, 20 pictures per well will be taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. All the images will be taken in the same conditions. Analysis of cell bodies of TH positive neurons will be performed using Developer software (GE healthcare). A total of 6 data per experimental condition will be provided.

[0271] All values will be expressed as mean ± s.e.mean. Statistical analyses will be done on the different conditions (ANOVA followed by Dunnett ‘s test).

[0272] The highest non-toxic concentrations of the mixes will be chosen by the sponsor for the following studies.

[0273] 4. 6OH-DA exposure and drug treatment: protective protocol

[0274] A second and third first culture of dopaminergic neurons will be prepared for the 6OHDA challenge. On day 7 of culture, cells will be pre-treated for 1 h with test compound or reference compound then intoxicated with 6-OHDA (20pM) for 48h.The following conditions will be done:

[0275] The following conditions will be done:- Control medium (vehicle to be defined, 48h).- Control medium + 6OHDA (20 pM, 48h).- Control medium + CX001 at C1 , C2 and C3 + 6OHDA (20 pM, 48h).- Control medium + CX002 at C1 , C2 and C3 + 6OHDA (20 pM, 48h).- Control medium + CX001 at C1 + CX002 at C1 , C2 and C3 + 6OHDA(20 pM, 48h).- Control medium + CX001 at C2 + CX002 at C1 , C2 and C3 + 6OHDA (20 pM, 48h).- Control medium + CX001 at C3 + CX002 at C1 , C2 and C3 + 6OHDA (20 pM, 48h).- BDNF (50 ng / ml; as reference compound + 6OHDA (20 pM, 48h).

[0276] Two cultures will be performed with six wells per condition and two 96-well plates per culture.

[0277] 5. End point evaluation: measurement of total number of TH positive neurons after 6OH-DA intoxication.

[0278] After 2 days of incubation, cells will be fixed by a solution of 4% paraformaldehyde for 20 min at room temperature, the control conditions will be fixed as well following the same procedure. The cells will be then permeabilized and non-specific sites will be blocked with a solution of phosphate buffered saline containing (PBS) with saponin and FCS for 15 min at room temperature. Cells will be incubated with Mouse Monoclonal Anti-Tyrosine Hydroxylase antibody (anti- TH, Sigma) in PBS with saponin and FCS overnight at 4°C. This antibody will be revealed with an Alexa Fluor 488 goat anti-mouse IgG in PBS with 1 % FCS, 0.1 % saponin, for 1 h at room temperature. Nuclei of cells will be labelled by a fluorescent marker (Hoechst solution) in the same solution.

[0279] For each well of culture, 20 pictures per well will be taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. All the images will be taken in the same conditions. Analysis of cell bodies of TH positive neurons will be performed using Developer software (GE healthcare). A total of 6 data per experimental condition will be provided.

[0280] All values will be expressed as mean ± s.e.mean. Statistical analyses will be done on the different conditions (ANOVA followed by Dunnett ‘s test).[02811 Results

[0282] In vitro, metformin (CXS001 ) and glibenclamide (CXS002) were incubated both alone and in combination on primary cultures of rat neurons, containing approximately 5% of dopaminergic neurons (identified by tyrosine hydroxylase expression). The cultures, assessed for vitality (number of living dopaminergic neurons) and fitness (length of neurites) exhibited the results presented in figures 1-5:- metformin (CXS001) and glibenclamide (CXS002) alone did not improve dopaminergic neuron survival in 6OHDA intoxication as compared to Brain Derived Neurotrophic Factor. CXS002 at high concentration seems to decrease neuron viability.- metformin (CXS001) and glibenclamide (CXS002) alone did not improve dopaminergic neuron fitness (neurite length) in 6OHDA intoxication, as compared to Brain Derived Neurotrophic Factor.- metformin (CXS001) and glibenclamide (CXS002) at low dose improve dopaminergic neuron viability in 6OHDA intoxication. A tendency might be seen for CXSOOl 10pM + CXS002 10pM, despite not being statistically significant at this dose.- metformin (CXS001) and glibenclamide (CXS002) at low dose improves dopaminergic neuron fitness, estimated by mean neurite length, in 6OHDA intoxication.

[0283] Discussion

[0284] Metformin treatment has been associated with increased risk of developing Parkinson’s disease in large studies, and glibenclamide is known to be potentially neurotoxic through excessive hypoglycemic effect. This example shows that a treatment involving low doses of metformin and glibenclamide can protect dopaminergic neurons against 6OHDA intoxication. Metformin reaches the brain, transported through the BBB by a transporter of the OCT family whilst glibenclamide remains outside, in healthy subjects. Yet, the BBB is subject toleakage in PD, this mechanism contributing to disease progression. By inhibiting the NLRP3 / inflammasome complex on glial cells, glibenclamide inhibits their activation in the brain compartment in case of BBB hyper permeability. By inhibiting the NLRP3 / inflammasome on endothelial cells of the BBB themselves, glibenclamide strengthens it, thus preventing further leakage. This justifies discontinuous administration of glibenclamide in a combinatory treatment also involving metformin.

[0285] Example 2: Study of potential neuroprotective effect of the combination according to the invention alone or in combination after 6-OHDA injury on rat primary dopaminergic neurons survival. A model of Parkinson’s disease

[0286] The aim of this study was to investigate the neuroprotective effect of metformin (CXS001) and glibenclamide (CXS002) alone or in combination, at 3 different concentrations, on survival of rat primary dopaminergic culture injured by 6-Hydroxydopamine (6-OHDA), a Parkinson’s disease in vitro model. The total number and the neurites length of dopaminergic neurons was assessed. Brain Derived Neurotrophic factor (BDNF) was used has reference compound.

[0287] Experimental Protocol

[0288] Dopaminergic neurons cell culture

[0289] Rat mesencephalic neurons were cultured as described by Schinelli et al., 1988. Briefly pregnant female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar; Janvier Lab) and the fetuses removed from the uterus. The embryonic midbrains were removed and placed in ice- cold medium of Leibovitz 15 (L15; PanBiotech, Ref P04-27055, Batch: 1260323) containing 2% of Penicillin-Streptomycin (PS; PanBiotech, ref: P06-07100, Batch: 2585615) and 1 % of bovine serum albumin (BSA; Sigma, Ref: 810533, Batch: 73). Only the ventral portions of the mesencephalic flexure were used for cell preparation as this region of the developing brain is enriched in dopaminergic neurons. The midbrains were dissociated by 50rypsinization for 20 minutes (min) at 37°C (Trypsin EDTA 1X; PanBiotech, Ref: P10-023100, Batch: 8290223). The reaction was stopped by the addition of Dulbecco’s modified Eagles medium (DMEM; PanBiotech, Ref: P04-03600, Batch: 6041222) containing Dnase | grade II (0.1 mg / ml; PanBiotech, Ref: P60-37780100, Batch: H210916) and 10% of fetal calfserum (FCS; Invitrogen, Ref: 10270106, Batch: 2534381 ). Cells were then mechanically dissociated by 3 passages through a 10 mL pipette. Cells were then centrifuged at 180 x g for 10 min at +4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cell pellets were resuspended in a defined culture medium consisting of Neurobasal Plus (Gibco, Ref: A3582901 , Batch: 2537248) supplemented with 2% of B27 Plus (Gibco, ref: A3582801 , Batch: 2722550), L- glutamine (2 mM; PanBiotech, Ref: P04-80100, Batch: 7061121 ), 2% of PS, 10ng / mL of BDNF PeproTech, Ref: 450-02, Batch: 092361 ) and 1 ng / mL of Glial Derived Neurotrophic factor (GDNF; PeproTech, Ref: 450-10, Batch: 012364). Viable cells were then counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 40 000 cells / well in 96 well-plates coated with poly-L-Ornithine (Sigma, Ref: P4957, Batch: RNBL6953) and laminin (Sigma, Ref: L2020, Batch: 0000216258), and were cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium was changed every 2 days with fresh medium. In these conditions, after 5 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation. In this condition, 2 to 5 % of neurons are dopaminergic neurons.

[0290] 6-OHDA preparation, exposure and drug treatment Dopaminergic neurons cell culture

[0291] Briefly, 6-OHDA (Sigma, Ref: H4381 , Batch: MKCQ5002) was reconstituted in define culture medium at 40uM (stock solution). The control medium was prepared in the same conditions. After 7 days of culture, primary mesencephalic neurons were pre-treated for 1 hour with test compounds or reference compound (BDNF, 50ng / mL) and then intoxicated with 6-OHDA at a final concentration of 20uM for 2 days incubation in order to induce a neuronal cell death of about 40%. The following conditions were done:

[0292] Platel :- Control (0.05% DMSO)- Control + 6-OHDA (20pM, 2 days)- CXS001 at 10 pM, 3uM and 1 pM + 6-OHDA (20pM, 2 days)- CXS002 at 1 OpM, 3|JM and 1 pM + 6-OHDA (20pM, 2 days)- BDNF at 50ng / ml + 6-OHDA (20pM, 2 days)

[0293] Plate 2:- Control (0.05% DMSO)- Control + 6-OHDA (20|JM, 2 days)- CXS001 at 10|JM + CXS002 at 10pM, 3|JM and 1 pM + 6-OHDA (20pM, 2 days)- CXS001 at 3uM + CXS002 at 10pM, 3pM and 1 pM + 6-OHDA (20pM, 2 days)- BDNF at 50ng / ml + 6-OHDA (20pM, 2 days)

[0294] Plate 3:- Control (0.05% DMSO)- Control + 6-OHDA (20pM, 2 days)- CXS001 at 1 pM + CXS002 at 10pM, 3pM and 1 pM + 6-OHDA (20pM, 2 days)- BDNF at 50ng / ml + 6-OHDA (20pM, 2 days)

[0295] Two culture was performed with 6 wells per condition.

[0296] End point evaluation: measurement of total number of TH positive neurons and length of TH positive neurites

[0297] After 2 days of intoxication, cells were fixed by a solution of 4% paraformaldehyde (Alpha Aesar, ref J19943, Batch: 211457) for 20 min at room temperature, the control conditions were fixed as well following the same procedure. The cells were then permeabilized and non-specific sites were blocked with a solution of phosphate buffered saline (PBS; VWR; ref: L0615-500, Batch: MS01 MB) containing 0.1 % of saponin (Sigma; ref: S7900, Batch: BCBL8667V) and 1% FCS for 15 min at room temperature. Cells were incubated with a rabbit polyclonal anti-Tyrosine Hydroxylase antibody (TH, 1 / 1000, Sigma, ref: AB152, Batch: 3870479) in a solution of PBS overnight at 4°C.

[0298] Staining was revealed with the addition of an Alexa Fluor 568 goat anti-rabbit IgG (1 / 400, Molecular probe, ref: A11011 , Batch: 2500544) in PBS with 1 % FCS and 0.1 % saponin for 1 hour at room temperature. Nuclei of cells were labelled by a fluorescent marker (Hoechst, Sigma; ref: B1155, Batch: 046M4048V) in the same solution.

[0299] For each condition, 20 pictures per well were taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. Images of each culture well were taken in same condition. The number of dopaminergic neurons (TH) and the neurite length of dopaminergic neurons are automatically evaluated with Developer system analysis (GE Healthcare). A total of 6 data per experimental condition were provided.

[0300] Statistics

[0301] The data were expressed as mean + s.e.mean (of 6 data per condition, 1 culture). A global analysis of the data was performed using a one-way analysis of variance (ANOVA) following by Dunnett’s test. The level of significance is set at p<0.05.

[0302] Results

[0303] Effect of CXS001 and CXS002 on dopaminergic neurons survival after 6- OHDA injury (plate 1 )

[0304] As observed on Figure 6, a treatment with 6-OHDA at 20pM during 48 hours induces a significant decrease of dopaminergic neurons survival (54% of cellular death, p<0.0001). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hours is able to significantly rescue neurons from cell death (81 % of the control, p<0.05). These results allow validating the culture conditions.

[0305] A one-hour pre-treatment with CXS001 at 10pM, 3pM and 1 pM fails to significantly rescue dopaminergic neurons from cell death induced by 6-OHDA exposure during 48 hours (respectively 51 %, 44% and 43% of cellular death).

[0306] Moreover, a one-hour pre-treatment with CXS002 at 10pM, 3pM and 1 pM fails to significantly rescue dopaminergic neurons from cell death induced by 6- OHDA exposure during 48 hours (respectively 47%, 57% and 58% % of cellular death).

[0307] Effect of CXS001 and CXS002 on dopaminergic neurons survival after 6- OHDA injury (plate 2)

[0308] As observed on Figure 7, a treatment with 6-OHDA at 20|JM during 48 hours induces a significant decrease of dopaminergic neurons survival (55% of cellular death, p<0.0001). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hours is able to significantly rescue neurons from cell death (87% of the control, p<0.0001). These results allow validating the culture conditions.

[0309] A one-hour pre-treatment with CXS001 at 10pM in combination with CXS002 at 10pM and 3pM fails to significantly rescue dopaminergic neurons from cell death induced by 6-OHDA exposure during 48 hours (respectively 45% and 37% of cellular death). However, a co-treatment with CXS001 at 10pM and CXS002 at 1 pM is able to significantly rescue dopaminergic neurons from cell death (72% of the control, p<0.05).

[0310] Moreover, A one-hour pre-treatment with CXS001 at 3pM in combination with CXS002 at 10pM, 3pM and 1 pM fails to significantly rescue dopaminergic neurons from cell death induced by 6-OHDA exposure during 48 hours (respectively 45%, 45% and 47% of cellular death).

[0311] Effect of CXS001and CXS002 on dopaminergic neurons survival after 6- OHDA injury (plate 3)

[0312] As observed on Figure 8, a treatment with 6-OHDA at 20uM during 48 hours induces a significant decrease of dopaminergic neurons survival (46% of cellular death, p<0.001 ). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hours is able to significantly rescue neurons from cell death (83% of the control, p<0.05). These results allow validating the culture conditions.

[0313] A one-hour pre-treatment with CXS001 at 1 uM in combination with CXS002 at 10uM, 3uM and 1 uM fails to significantly rescue dopaminergic neurons from cell death induced by 6-OHDA exposure during 48 hours (respectively 41 %, 39% and 47% of cellular death).

[0314] Effect of CXS001 and CXS002 on neurites length of dopaminergic neurons after 6-OHDA injury (plate 1 )

[0315] As observed on Figure 9, a treatment with 6-OHDA at 20|JM during 48 hours induces a significant decrease of neurites length (54% of neurites loss, p<0.001). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hours is able to significantly rescue neurites length (79% of the control, p<0.05). These results allow validating the culture conditions.

[0316] A one-hour pre-treatment with CXS001 at 10pM, 3pM and 1 pM fails to significantly rescue neurites length of dopaminergic neurons after 6-OHDA exposure during 48 hours (respectively 55%, 43% and 38% of neurites loss).

[0317] Moreover, a one-hour pre-treatment with CXS002 at 10pM, 3pM and 1 pM fails to significantly rescue neurites length of dopaminergic neurons after 6-OHDA exposure during 48 hours (respectively 47%, 57% and 58% of neurites loss).

[0318] Effect of CXS001 and CXS002 on neurites length of dopaminergic neurons after 6-OHDA injury (plate 2)

[0319] As observed on Figure 10, a treatment with 6-OHDA at 20pM during 48 hours induces a significant decrease of neurites length (56% of neurites loss, p<0.0001). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hours is able to significantly rescue neurites length (76% of the control, p<0.05). These results allow validating the culture conditions.

[0320] A one-hour pre-treatment with CXS001 at 10pM in combination with CXS002 at 10pM, 3pM and 1 pM fails to significantly rescue neurites length of dopaminergic neurons after 6-OHDA exposure during 48 hours (respectively 47%, 42% and 42% of neurites loss).

[0321] Moreover, A one-hour pre-treatment with CXS001 at 3pM in combination with CXS002 at 10pM, 3pM and 1 pM fails to significantly rescue neurites length of dopaminergic neurons after 6-OHDA exposure during 48 hours (respectively 52%, 52% and 41 % of neurites loss).

[0322] Effect of CXS001 and CXS002 on neurites length of dopaminergic neurons after 6-OHDA injury (plate 3)

[0323] As observed on Figure 11 , a treatment with 6-OHDA at 20pM during 48 hours induces a significant decrease of neurites length (45% of neurites loss, p<0.001). As expected, the reference molecule, BDNF at 50ng / mL, applied during 48 hoursis able to significantly rescue neurites length (85% of the control, p<0.05). These results allow validating the culture conditions.

[0324] A one-hour pre-treatment with CXS001 at 1 pM in combination with CXS002 at 1 OpM, 3pM and 1 pM fails to significantly rescue neurites length of dopaminergic neurons after 6-OHDA exposure during 48 hours (respectively 40%, 46% and 43% of cellular death).

[0325] Conclusion

[0326] Model validation

[0327] As expected 6-OHDA applied at 20uM during 2 days induces a significant loss of dopaminergic neurons and their neurites. Moreover, the reference molecule, BDNF at 50ng / mL is able tosignificantly rescue these neurons from cell death.

[0328] Metformin (CXS001) and glibenclamide (CXS002)

[0329] A one-hour pre-treatment with CXS001 or CXS002 alone does not protect dopaminergic neurons from cell death and neurites from damages induced by 6- OHDA exposure during 48 hours.

[0330] A one-hour pre-treatment with CXS001 in combination with CXS002 does not significantly protect dopaminergic neurons from cell death or neurites from degradation induced by 6-OHDA exposure during 48 hours. However, the association of CXS001 at 10pM with CXS002 at 1 pM partially and significantly protects dopaminergic neurons following 6-OHDA exposure during 48 hours.

[0331] Example 3: Evaluation of the neuro-pharmacokinetic profiles of the compounds according to the invention, monitored by microdialysis in the ventral striatum, and plasma samples of awake rats

[0332] The objective of the study is to quantitate the levels of CXS001 and CXS002 in brain striatal microdialysates (interstitial fluid, ISF) and plasma samples following a single peroral administration of test compounds alone, and in combinations in awake rats. Concentrations of the test compounds in the microdialysis samples and plasma is measured by ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS).

[0333] Summary of the study design- 1 . Male, Sprague Dawley rats (300-320 g on the day of the experiment) will be used.- 2. Separate groups of rats fasted overnight will be administered perorally (p.o.) with the test compounds at two doses each, resulting in four combinations and two additional groups will be administered with each test compound alone at a higher dose.- 3. The study will include six groups, each consisting of 5 rats, 30 rats in total.- 4. Rats will be implanted with a microdialysis guide cannula in in the striatum and allowed to recover for 7 days. A day before the experiment, each rat will be implanted with one catheter into the jugular vein.- 5. The microdialysis experiments will be carried-out on awake rats treated acutely with the test compounds.- 6. The probes will be perfused with artificial cerebrospinal fluid (aCSF) at a flow rate of 0.1 pl / min and mixed at the probe outlet with a make-up buffer pumped at a flow rate of 1 pl / min giving a constant dilution ratio 1 :10. This setup allows near 100% in vivo recovery of the test substances.- 7. The microdialysis samples will be collected in 20-min intervals.- 8. Three microdialysis samples will be collected for estimation of blank (zero) levels, thereafter the test compounds will be administered and the microdialysis sampling will continue for additional 4 h. The total number of the post-treatment samples will be 12 / rat for the microdialysates.- 9. Blood samples will be collected simultaneously during the microdialysis sampling. Seven blood samples (~ 75-100 pl) will be collected from the jugular vein at time 0, 10, 20, 40, 80, 160, 240 min post-dosing.- 10. Concentrations of the test compounds in the microdialysis samples and plasma will be measured by UHPLC-MS / MS.

[0334] Materials and methods

[0335] Animals

[0336] The study is conducted at Pronexus Analytical AB, Sweden, a facility accredited by the Swedish Board of Agriculture, which is fully equivalent to AAALAC accreditation. All animal experiments and protocols are approved by the regional ethical committee at Stockholm County Court (Stockholms Norra djurfdrsdksetiska namnd) following the directives of the Swedish Animal Welfare Act 1988:534, complying with the Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, and The Guide for the Care and Use of Laboratory Animals, National Research Council, 2011 , 8th edition, The National Academies Press. All efforts are made to minimize animal suffering and the number of animals used for the study. The validation of the data is ensured through an internal quality control process. The results are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (McGrath et al., 2010).- Species: Rat- Strain: Sprague Dawley- Sex: male- Age range: 7-8 weeks- Body weight range: 300 - 350 g- Acclimatization period after arrival to the animal facility: 7 days- Supplier: Janvier Labs, France

[0337] Rats are maintained in a controlled environment (22 ± 1 °C; 45-50% relative humidity) on a 12-hour light (20 lux) / 12-hour dark cycle with the lights on at 7 AM. The naive rats are housed in groups of four in Eurostandard type IV cages (595 x 380 x 200 mm, LWH, floor area 1820 cm2) with wire lids (Tecniplast, Buguggiate, Varese, Italy), aspen bedding, and environmental enrichment including aspen gnawing bricks and aspen house / arcade (Tapvei, Estland). Once implanted with guide cannulae, the rats are housed individually in Eurostandard type III H cages (425 x 266 x 185 mm, LWH, floor area 800 cm2) with corresponding wire lids (Tecniplast). The rats have free access to standard lab chow (V1534-000, ssniffSpezialdiaten GmbH, Soest, Germany) and tap water during the housing periods and the microdialysis experiment. Only healthy animals are placed in the study. Each rat is randomly assigned across the treatment groups.

[0338] Groups and doses

[0339] The study includes six groups, each consisting of 5 rats, totaling 30 rats. The rats are fasted overnight and during the microdialysis experiment but have free access to water. The test compounds are administered p.o. at low (dose 1) and high (dose 2) doses in a volume of 10 ml / kg.

[0340] Microdialysis samples are collected in 20-minute intervals, starting 60 minutes before the treatment and continuing up to 240 minutes post-dose- Group 1 : CXS001 (dose 2)- Group 2: CXS002 (dose 2)- Group 3: CXS001 (dose 1) and CXS002 (dose 1 )- Group 4: CXS001 (dose 1 ) and CXS002 (dose 2)- Group 5: CXS001 (dose 2) and CXS002 (dose 1)- Group 6: CXS001 (dose 2) and CXS002 (dose 2)

[0341] Experimental procedures

[0342] Microdialysis : Microdialysis experiments are carried out on awake, freely moving rats following the protocol described elsewhere (Kehr, 1999; Kehr and Yoshitake, 2006; Pronexus SOP QW 03:1 ).

[0343] Surgery: The microdialysis experiments are carried out on awake rats. The initial stereotaxic surgery is performed under aseptic conditions following current recommendations for rodent surgery and analgesia (Flecknell, 2018; Hoogstraten-Miller and Brown, 2008; Lilley and Berdoy, 2017). The rats are anesthetized with isoflurane using a Univentor 400 anesthesia unit (AgnThos, Lidingd, Sweden) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). About 30-60 minutes before the start of the surgery, each animal receives carprofen (Rimadyl, Pfizer) 5 mg / kg subcutaneously (s.c.) and buprenorphine (Tegmesic, 0.05 mg / kg s.c.). The body temperature of the animal is controlled by a thermometer and a heating pad maintained at 37 °C during theoperation using a CMA / 105 temperature controller (CMA / Microdialysis, Stockholm, Sweden). A guide cannula (CMA / Microdialysis) is implanted into the striatum;. The coordinates are specified from bregma and the dural surface according to the atlas of Paxinos and Watson (2009). The guide cannula is firmly fixed to the skull surface using dental cement (Dentalon Plus, Heraeus Instruments, Hanau, Germany). One day after the surgery, the rats receive a second dose of 5 mg / kg carprofen s.c. and are allowed to recover over the following 6 days. During that period, no further treatment is given, and the general health status of the animals is monitored daily. A day before the microdialysis experiment, the rats are anesthetized with isoflurane and placed on a heating pad maintained at +37 °C as described above. The hair is shaved at the position of the jugular vein, and following an incision and exposure of the vein, a 10 cm piece of polyethylene tubing (PE50, Becton Dickinson, USA) is inserted (about 1 cm deep) into the vein. The tubing is secured in place, flushed, and filled with saline containing heparin and closed at its distal end; thereafter, the incision is closed with sutures, leaving about 5 cm of tubing exposed from the skin.

[0344] Microdialysis: On the day of the microdialysis experiment, the rat, fasted overnight in its home cage, is placed into the frame of a Rotating Animal Cage System, RACS (Microbiotech / se, Arsta, Sweden). The RACS allows a swivel-free connection of tubing to the syringe pump and to the fraction collector while also enabling brief recording of locomotor activation. A microdialysis probe is inserted into the guide cannula. The probe is connected with fluorinated ethylene propylene (FEP) tubing (0.1 mm I.D.) to the balancing arm of the RACS assembly. The probe is perfused at a constant flow rate of 0.1 pL / min with artificial cerebrospinal fluid (aCSF) solution (148 mM NaCI, 4 mM KCI, 0.8 mM MgCI2, 1 .4 CaCI2, 1 .2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.2) using a CMA / 100 Microinjection Pump (CMA / Microdialysis). A capillary T-union (Microbiotech / se) is mounted at the probe outlet and connected to a make-up buffer (the mobile phase A for the UHPLC-MS / MS method) pumped at a flow rate of 1 pL / min. Each rat is allowed to habituate to the new room environment for 120 to 150 minutes. Following this stabilization period, the microdialysis samples are collected at 20-m inute intervals using the refrigerated microdialysis fractioncollector (CMA / Microdialysis) operating at +4 °C. The first three samples are collected to determine blank levels. Thereafter, the test compound is administered, and samples are collected for the next 240 minutes. Blood samples are collected from the jugular vein catheter simultaneously during the microdialysis sampling. Typically, about 75-100 pL of blood is withdrawn, with eight blood samples collected at the following time intervals: 0, 10, 20, 40, 80, 160, and 240 minutes post-dosing. After the experiment is finalized, the animals are sacrificed by an overdose of isoflurane and dislocation of the neck.

[0345] Collecting blood and brains: Blood is collected into microcentrifuge tubes containing K2EDTA and kept on ice. Within 15 minutes, the tubes are centrifuged at 10,000 rpm for 10 minutes in a refrigerated centrifuge (Biofuge Fresco; Heraeus Instruments, Germany). Plasma is carefully aspirated and stored at - 80°C in the freezer. The brains are rapidly removed, frozen on dry ice, and stored at -80°C for additional analysis or histological verification of the microdialysis probe placement.

[0346] Quantification of the test compounds by UHPLC-MS / MS: The concentrations of the test compounds in the microdialysis samples are measured by ultra-high- performance liquid chromatography with tandem mass spectrometry (UHPLC- MS / MS). Briefly, the UHPLC-MS / MS system includes an Elute HTG binary gradient UHPLC pump, an Elute column oven, and an EVOQ Elite triple quadrupole mass spectrometer (all from Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) source operating in positive mode. The samples are injected using a PAL autosampler (CTC Analytics, Switzerland) equipped with a 10-pL sample loop, and the samples are kept in a PAL stack cooler for 6 microtiter plates, operating at +6 °C. The system is controlled by Compass 2.0 / HyStar 4.0 software (Bruker), and the compound screening and quantitation are performed using TASQ 2.2 data acquisition and processing software (Bruker). The mass spectrometer is supplied with nitrogen and air generated by a Genius 3045 nitrogen / air generator (Peak Scientific Instruments, Inchinnan, Scotland, UK). The mass spectra are scanned in MRM mode to find the optimal collision energies for the test compound and its respective precursor and product ions. In addition, a suitable substance to serveas an internal standard is identified, and the respective calibration curves for the test compound in aCSF and plasma matrices are constructed.

[0347] Data presentation and analysis

[0348] Raw data are entered into the data files using a standard spreadsheet program (Microsoft Excel), and statistical analysis is performed using Prism 6 (GraphPad Software, USA) software. The values are presented as mean ± standard error of mean (SEM), and differences are considered statistically significant at the P<0.05 level. The raw data, statistics, graphs, and report are scrutinized through the internal QC / QA process.

[0349] Results

[0350] The results show that metformin partially reaches the inside of the brain cavity as single agent as ascertained by the measured concentration inside the striatum. On the contrary, glibenclamide as a single agent is not retrieved inside the striatal area. Combination of glibenclamide and metformin allows for the retention of glibenclamide inside the brain compartment, as ascertained by the concentration increase upon combination with metformin.

[0351] Example 4: Evaluation of the effect of metformin (CXS001 ) and glibenclamide (CXS002) after 6-OHDA injury on neurites length and neuron survival of rat primary dopaminergic neurons, in order to determine the target concentrations of metformin and glibenclamide inside the brain.

[0352] The aim is to determine the target concentrations for metformin and glibenclamide inside the brain, in a primary rat neuron culture Parkinson model using 6OHDA as a toxic:

[0353] Material and Methods

[0354] Dopaminergic neurons cell culture

[0355] Rat mesencephalic neurons were cultured as described by Schinelli et al., 1988. Briefly pregnant female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar; Janvier Lab) and the fetuses removed from the uterus. The embryonic midbrains were removed and placed in ice- cold medium of Leibovitz 15 (L15; PanBiotech, Ref P04-27055, Batch: 1260323) containing 2% of Penicillin-Streptomycin (PS; PanBiotech, ref: P06-07100, Batch: 2585615) and1 % of bovine serum albumin (BSA; Sigma, Ref: 810533, Batch: 73). Only the ventral portions of the mesencephalic flexure were used for the cell preparation as this region of the developing brain is enriched in dopaminergic neurons. The midbrains were dissociated by 63rypsinization for 20 minutes (min) at 37°C (Trypsin EDTA 1X; PanBiotech, Ref: P10-023100, Batch: 8290223). The reaction was stopped by the addition of Dulbecco’s modified Eagles medium (DMEM; PanBiotech, Ref: P04-03600, Batch: 6041222) containing Dnase | grade II (0.1 mg / ml; PanBiotech, Ref: P60-37780100, Batch: H210916) and 10% of fetal calf serum (FCS; Invitrogen, Ref: 10270106, Batch: 2534381 ). Cells were then mechanically dissociated by 3 passages through a 10 mL pipette. Cells were then centrifuged at 180 x g for 10 min at +4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cell pellets were resuspended in a defined culture medium consisting of Neurobasal Plus (Gibco, Ref: A3582901 , Batch: 2537248) supplemented with 2% of B27 Plus (Gibco, ref: A3582801 , Batch: 2722550), L- glutamine (2 mM; PanBiotech, Ref: P04-80100, Batch: 7061121 ), 2% of PS, 10ng / mL of BDNF PeproTech, Ref: 450-02, Batch: 092361 ) and 1 ng / mL of Glial Derived Neurotrophic factor (GDNF; PeproTech, Ref: 450-10, Batch: 012364). Viable cells were then counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 40 000 cells / well in 96 well-plates coated with poly-L-Ornithine (Sigma, Ref: P4957, Batch: RNBL6953) and laminin (Sigma, Ref: L2020, Batch: 0000216258), and were cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium was changed every 2 days with fresh medium. In these conditions, after 5 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation. In this condition, 2 to 5 % of neurons are dopaminergic neurons.

[0356] 6-OHDA preparation, exposure and drug treatment Dopaminergic neurons cell culture

[0357] Briefly, 6-OHDA (Sigma, Ref: H4381 , Batch: MKCQ5002) was reconstituted in define culture medium at 40uM (stock solution). The control medium was prepared in the same conditions. After 7 days of culture, primary mesencephalic neurons were pre-treated for 1 hour with test compounds or reference compound (BDNF, 50ng / mL) and then intoxicated with 6-OHDA at a final concentration of20uM for 2 days incubation in order to induce a neuronal cell death of about 40%. The following conditions were done:

[0358] Platel :- Control (0.1 % DMSO)- Control + 6-OH DA (20uM, 2 days)- CXS001 at 10|JM and CXS002 at 10pM, 3|JM or 1 pM + 6-OHDA (20pM, 2 days)- BDNF at 50ng / ml + 6-OHDA (20pM, 2 days)

[0359] Two culture was performed with 6 wells per condition.

[0360] End point evaluation: measurement of total number of TH positive neurons and length of TH positive neurites

[0361] After 2 days of intoxication, cells were fixed by a solution of 4% paraformaldehyde (Alpha Aesar, ref J19943, Batch: 211457) for 20 min at room temperature, the control conditions were fixed as well following the same procedure. The cells were then permeabilized and non-specific sites were blocked with a solution of phosphate buffered saline (PBS; VWR; ref: L0615-500, Batch: MS01 MB) containing 0.1 % of saponin (Sigma; ref: S7900, Batch: BCBL8667V) and 1% FCS for 15 min at room temperature. Cells were incubated with a rabbit polyclonal anti-Tyrosine Hydroxylase antibody (TH, 1 / 1000, Sigma, ref: AB152, Batch: 3870479) in a solution of PBS overnight at 4°C.

[0362] Staining was revealed with the addition of an Alexa Fluor 568 goat anti-rabbit IgG (1 / 400, Molecular probe, ref: A11011 , Batch: 2500544) in PBS with 1 % FCS and 0.1 % saponin for 1 hour at room temperature. Nuclei of cells were labelled by a fluorescent marker (Hoechst, Sigma; ref: B1155, Batch: 046M4048V) in the same solution.

[0363] For each condition, 20 pictures per well were taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. Images of each culture well were taken in same condition. The number of dopaminergic neurons (TH) and the neurite length of dopaminergic neurons are automatically evaluated with Developer system analysis (GE Healthcare). A total of 6 data per experimental condition were provided.

[0364] Statistics

[0365] The data were expressed as mean + s.e.mean (of 6 data per condition, 1 culture). A global analysis of the data was performed using a one-way analysis of variance (ANOVA) following by Dunnett’s test. The level of significance is set at p<0.05.

[0366] Results

[0367] Neurite length

[0368] Figure 12 presents the effect of metformin (CXS001 ) and glibenclamide (CXS002) after 6-OHDA injury on neurites length of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; # N / A (reference), * p<0.05, *** p<0.001 , **** p<0.0001 ; stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0369] Cell viability

[0370] Figure 13 presents the effect of Ambroxol, lixisenatide, metformin (CXS001) and glibenclamide (CXS002) after 6-OHDA injury on neuron survival of rat primary dopaminergic neurons expressed in percentage of control. Protective protocol (mean ± s.e.m; # N / A (reference), * p<0.05, *** p<0.001 , **** p<0.0001 , stats vs 6-OHDA 20pM; one-way ANOVA followed by Dunnett’s test).

[0371] These results presents concentrations in vitro in rat dopaminergic neurons intoxicated by 6OHDA, to reach a target concentration behind the blood-brain barrier.

[0372] To reach these concentrations, the drugs must penetrate the blood brain barrier, and the equilibrium concentration range upon daily oral administration must ensure a certain plasma concentration range.[03731 Example 5: Metabolic profiling of ALS patient primary fibroblasts:

[0374] EXPERIMENTAL PROTOCOL

[0375] We used ALS patient primary fibroblasts, with no identified ALS-causing genetic mutation. ALS patient primary fibroblasts are cultured in standard high- glucose Dulbecco's Modified Eagle medium complemented with 10% fetal bovine serum before challenge.

[0376] Cells then undergo a metabolic challenge, caused by a change of medium.

[0377] High-glucose Dulbecco's Modified Eagle medium is replaced by no-glucose Dulbecco's Modified Eagle medium containing 10% fetal bovine serum before challenge and 10mM galactose to decrease glycolysis and increase ATP synthesis for 16 hours before analysis.

[0378] Mitochondrial respiration, glycolysis and ATP respiration are then measured using a Seahorse XF apparatus.

[0379] Fibroblasts are treated upon medium replacement by 10pM of metformin and 1 pm glibenclamide.

[0380] RESULTS

[0381] Quantitative results regarding glycolysis, ATP synthesis and mitochondrial metabolic profile are compared to those obtained with non-ALS patients.

[0382] These results show an improvement regarding mitochondrial metabolic profile for said cultured ALS patients primary fibroblasts treated upon medium replacement by 10pM of metformin and 1 m glibenclamide as compared to healthy primary fibroblasts. The metabolic profile of ALS patient fibroblasts shifts towards that of non-ALS patients. This means that the combination of glibenclamide and low-dose metformin has the potential to normalize the metabolic profile of ALS patient primary fibroblasts, independent on any ALS- related genetic mutation.

[0383] Example 6: Pharmacokinetics of the composition according to the invention in humans

[0384] The pharmacokinetic profile of this low dose formulation has been studied in human.

[0385] Briefly, after a single dose of the combination, ten dosing points were sampled at TO, T0+1 h, +2h, +5h, +7h, +10h, +12h, +18h, +20h, +24h.

[0386] The sampling was conducted using a 17G needle and a vacutainer, the plasma was stored at -80°C until analysis.

[0387] The dosing was performed ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS), and the results were compared to a calibration curve, ranging from 0.1 to 100ng / mL for both compounds.

[0388] The results were as follows (table 1 and Figure 14) :[Table 1]Time (h) Glibenclamide Metformin(ng / mL) (ng / mL)0 0 01 6 82 13 245 30 367 18 3810 10 3812 7 3218 6 1220 5 824 3 4[03891 Example 7: Neuroprotection effect of qlibenclamide and metformin at 2 concentrations alone or in combination on primary rat cortical neurons after 8 amyloid oligomer intoxication - Model of Alzheimer Disease.

[0390] The aim of this study is to assess neuroprotective effect of the association of glibenclamide and metformin at 2 concentrations alone or in combination on rat primary culture of cortical neurons after intoxication amyloid- peptides at 10 pM (high toxicity test, 50% of cortical neuron death) and at 5 pM (low toxicity test, 25% of cortical neuron death).

[0391] Neuron cell bodies count and neurite length in neuronal nuclei will be studied.

[0392] Experimental protocol

[0393] 1 . Rat primary culture of cortical neurons

[0394] Rat cortical neurons will be cultured as described by Verleye (2016). Briefly pregnant female rats of 15 days gestation will be killed by cervical dislocation and the fetuses will be removed from the uterus. The cortex will be removed and placed in ice-cold medium of Leibovitz; Penicillin and Streptomycin (PS) and bovine serum albumin (BSA). Cortex will be dissociated by trypsinisation.Reaction will be stopped by the addition of Dulbecco's modified Eagle’s medium (DMEM) containing DNAase I grade II and foetal calf serum (FCS).

[0395] Cells will be then centrifuged and re-suspended in a defined culture medium consisting of Neurobasal supplemented with B27 supplement, L-glutamine, PS and 10 ng / ml of BDNF.

[0396] The cells will be seeded at a density of 30 000 cells / well in 96 well-plates precoated with poly-D-lysine and will be cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere.

[0397] Half of the medium will be changed every 2 days with fresh medium.

[0398] 2. p-amyloid intoxication and drug treatment on cortical neurons.

[0399] Briefly, 1-42 A O will be prepared according to Neuron Experts original protocol. Human P-Amyloid 1-42 peptide (Bachem; ref: H-1368) will be dissolved in 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP) and evaporated to obtain a dried film. Then peptide will be resuspended in DMSO to prepare a 5 mM stock. This solution will be sonicated for 10 min and oligomers will be prepared by diluting stock solution to 100 pM with culture medium without phenol red and left for 24h at 4°C prior to use.

[0400] On day 11 , two test compounds (2 concentrations) or Mix of 2 compounds (2 concentrations) or BDNF internal reference compound (50ng / ml) will be preincubated with hippocampal neurons for 1 h before Apo application. Then, Apo (at 10pM and 5pM, for 48H) will be added in the culture medium. The test compounds will be let during Apo intoxication.

[0401] The following experimental conditions will be assessed:

[0402] Plate 1 , 10 pM of ApO for 48 h:• Control medium (2 days)• Control medium intoxicated with ApO (10 pM, 48h)• Control medium + CX001 at 10 pM intoxicated with ApO (10 pM, 48h)• Control medium + CX001 at 1 pM intoxicated with ApO (10 pM, 48h)• Control medium + CX002 at 1 pM intoxicated with ApO (10 pM, 48h)• Control medium + CX002 at 0.1 pM intoxicated with A O (10 pM, 48h)• Control medium + CX001 at 10pM + CX002 at 1 pM intoxicated with A O (10 pM,48h).• Control medium + CX001 at 1 pM + CX002 at 0.1 pM intoxicated with A O (10 pM, 48h).Control medium with BDNF (50ng / mL) intoxicated with A O (10 pM, 48h)

[0403] Plate 2, 5 pM of ApO for 48h:• Control medium (2 days)• Control medium intoxicated with A O (5 UM, 48h)• Control medium + CX001 at 10 pM intoxicated with ApO (5 pM, 48h)• Control medium + CX001 at 1 pM intoxicated with ApO (5 pM, 48h)• Control medium + CX002 at 1 pM intoxicated with ApO (5 pM, 48h)• Control medium + CX002 at 0.1 pM intoxicated with ApO (5 pM, 48h)• Control medium + CX001 at 10pM + CX002 at 1 pM intoxicated with ApO(5 pM, 48h).• Control medium + CX001 at 1 pM + CX002 at 0.1 pM intoxicated with ApO (5 pM, 48h).• Control medium with BDNF (50ng / mL) intoxicated with ApO (5 pM, 48h)

[0404] Six wells per condition (1 culture) will be done.

[0405] 3. Cell survival evaluation after AO injury: MAP-2 immunostaining.

[0406] After 48h of intoxication, cells will be fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min. Then, cells will be permeabilized and non-specific sites will be blocked with a solution of PBS containing saponin and FCS. Cell will be then incubated with mouse monoclonal primary antibody MAP-2. This antibody will be revealed with Alexa Fluor 488 goat anti-mouse. Nuclei of cells will be labelled by a fluorescent marker (Hoechst solution).

[0407] For each well of culture, 20 pictures per well will be taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. All the images will betaken in the same conditions. The number of neurons will be automatically evaluated with Developer system analysis (GE Healthcare). All values will be expressed as mean + / - s.e.mean.

[0408] The total neuronal survival (number of MAP-2 positive neuronal cell bodies) and the MAP-2 neurite length will be evaluated.

[0409] Statistical analysis will be done on different conditions (ANOVA followed by Dunnett's test).

[0410] Six wells will be analyzed per culture condition.

[0411] Results

[0412] Evaluation of number and length of cortical neurons in cellular model of Alzheimer disease based on A O intoxication.

[0413] Cortical neurons survival

[0414] As shown in figure 15A, the uncombined use of the molecules shows significant efficacy against A O intoxication only in the case of 10pM metformin. This effect is largely increased by the combined use of 10pM metformin and 1 pM glibenclamide. BDNF, the internal positive control, shows in this case a maximal protective effect compared to the other experimental conditions.

[0415] Cortical neurons length

[0416] As shown in figure 15B, the uncombined use of metformin and glibenclamide does not exert a significant effect on the improvement of neuron length after 10pM ABO intoxication. This effect is much more marked when the two molecules are used together (metformin 10pM + glibenclamide 1 pM). Interestingly, this effect is as effective as BDNF, the internal positive control.

[0417] In conclusion, the simultaneous use of metformin and glibenclamide exerts a beneficial synergistic effect on the survival and the length of cortical neurons in this cellular model of Alzheimer's disease based on A O intoxication.

[0418] General conclusion regarding the results :

[0419] The study investigates the effects of metformin and glibenclamide on cortical neurons exposed to A O, a cellular model of Alzheimer’s disease.

[0420] Neuron survival: Metformin alone (10 pM) shows some protective effect, which is strongly enhanced when combined with glibenclamide (1 pM). BDNF, used as a positive control, provides the strongest protection.

[0421] Neuron length: Neither drug alone improves neurite length, but their combination produces a marked effect, comparable to BDNF.

[0422] Conclusion: The combined use of metformin and glibenclamide has a synergistic beneficial effect on both neuron survival and neurite growth in this Alzheimer’s disease model.[04231 Example 8: Neuroprotection effect of glibenclamide and metformin at 2 concentrations alone or in combination on primary rat glutamatergic neurons after glutamate intoxication - Model of Huntington Disease.

[0424] The aim of this study will be to assess neuroprotective effect of one mix of 2 compounds at 2 concentrations and 2 compounds at 2 concentrations on rat primary culture of medium spiny neurons (MSNSs) of striatum after glutamate injury, an in vitro model of Huntington's disease.

[0425] MK 801 , an antagonist of NMDA receptor, will be used as reference compound.

[0426] 1 . Rat primary cultures of MSN neurons

[0427] Rat MSN of striatum will be cultured as described by Ivkovic et al., 1999.Briefly pregnant female rats of 15 days gestation will be killed by cervical dislocation (Rats Wistar; Janvier) and the foetuses will be removed from the uterus and placed in ice-cold medium of Leibovitz containing Penicillin and Streptomycin (PS). The embryonic midbrains will be removed. Only the ventral portions of the mesencephalic flexure will be used for the cell preparations as this is the region of the developing brain rich in dopaminergic neurons.

[0428] The midbrains will be dissociated by trypsinization. Cells will be then centrifuged and re-suspended in a defined culture medium consisting of Neurobasal supplemented with B27 supplement. The cells will be seeded in 96 well- and will be cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium will be changed every 2 days with fresh medium.In these conditions, after 6 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation.

[0429] Half of the medium will be changed every 2 days with fresh medium.

[0430] 2. Glutamate intoxication and drug treatment.

[0431] Briefly, after 11 days of culture, the medium will be removed and fresh medium with test compounds at 3 concentrations will be added 1 hour before the intoxication. Then glutamate (40 pM, during 20 min) will be added, with test compound. After 20 min of intoxication, the supernatant will be changed with culture medium without glutamate and with test compounds during the next 48 hours after glutamate intoxication. The following conditions will be done:

[0432] The following experimental conditions will be assessed:- Control medium- Control medium intoxicated with Glutamate (40 pM, 20min)- Control medium + CX001 at 10 pM intoxicated with Glutamate (40pM, 20 min)- Control medium + CX001 at 1 pM intoxicated with Glutamate (40pM, 20 min)- Control medium + CX002 at 1 pM intoxicated with Glutamate (40pM, 20 min)- Control medium + CX002 at 0.1 pM intoxicated with Glutamate (40pM, 20 min)- Control medium + CX001 at 10pM + CX002 at 1 pM intoxicated with Glutamate (40pM, 20 min)- Control medium + CX001 at 1 pM + CX002 at 0.1 pM intoxicated with Glutamate (40pM, 20 min)- MK 801 at 1 concentration intoxicated with Glutamate (40 pM, 20 min)

[0433] Six wells per condition and one 96 wells culture plate will be done

[0434] 3. Endpoint evaluation: Neuron survival evaluation.

[0435] After 48 hs of intoxication, cells will be fixed by a solution of paraformaldehyde. Then, cells will be permeabilized and non-specific sites will be blocked with a solution of PBS containing saponin and fetal calf serum (FCS). Then, cells will be incubated with a rabbit polyclonal primary antibody anti- DARPP32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) and with a mouse monoclonal primary antibody anti-MAP2 respectively revealed with an Alexa Fluor 568 goat anti-rabbit and an Alexa Fluor 488 goat anti-mouse. Nuclei of cells will be labelled by a fluorescent marker (Hoechst solution).

[0436] The total neuronal survival medium spiny neurons (total number of neuronal cell bodies stained with DARPP32 and MAP2) will be quantified. For each well of culture, 20 pictures per well will be taken using InCell AnalyzerTM 2200 (GE Healthcare) with 20x magnification. All the images will be taken in the same conditions. The number of neurons will be automatically evaluated with Developer system analysis (GE Healthcare).

[0437] All values will be expressed as mean + / - s.e.mean. Statistical analyses will be done on the different conditions (ANOVA followed by Dunnett's test).

[0438] Results

[0439] Mesencephalic neurons survival

[0440] As shown in figure 17A, 10pM metformin and 1 pM glibenclamide, used independently, do not induce a protective effect on glutamate-induced toxicity. When using both compounds simultaneously, the protective effect is observed. MK801 , a glutamate receptor inhibitor and positive control of the experiment, allows to suppress all glutamate-induced toxicity.

[0441] GABAergic neurons survival

[0442] As shown in figure 17B, 10pM metformin and 1 pM glibenclamide, used alone, exert a significant protective effect against glutamate-induced toxicity. The combined use of these molecules seems to exerts a stronger protective effect against the toxic. Once again MK801 allows to suppress all glutamate-induced toxicity.

[0443] In conclusion, the combined use of 10pM metformin and 1 pM glibenclamide is associated with improved survival and total length of mesencephalic neurons andimproved survival of GABAergics neurons in an in vitro model of Huntington's disease based on glutamate intoxication.

[0444] General conclusion regarding the results

[0445] The study evaluates the effects of metformin (10 pM) and glibenclamide (1 pM), alone or in combination, on glutamate-induced toxicity in an in vitro neuronal model.

[0446] 1 . Mesencephalic neurons: No protection is observed with either drug alone, contrary to protective effect induced when metformin and glibenclamide are combined.

[0447] 2. GABAergic neurons: Both drugs alone show significant protection; combined treatment provides even stronger protection.

[0448] Conclusion: The combined use of metformin and glibenclamide enhances survival and neurite length in mesencephalic neurons and improves GABAergic neuron survival in this glutamate-induced in vitro Huntington’s disease model.

[0449] Example 9: Study of the potential effect of glibenclamide and Metformin alone or in combination in a rat model of Parkinson’s disease: 6-OHDA induced akinesia.

[0450] 1 . Objective of the study

[0451] The aim of the project is to assess the potential efficacy of Metformin (CXS001) and glibenclamide (CXS001 ) alone or in combination (CXS003), on several behavioral tests assessing akinesia in the 6-OHDA rat model of Parkinson’s disease. First, animals will be subjected to stereotaxic injection with 6-OHDA (or its vehicle) to induce the dopaminergic lesion on DO. Forty-eight hours (48 h) after stereotaxic injection, animals will be daily treated during 5 weeks with the CXS001 , CXS002 or CXS003. On D35, rats will be subjected to several behavioral tests that is the initiation time test, and the stepping test. After behavioral measurements, terminal CSF or brain collections will be carried out on all animals for further molecular and / or immunohistological analysis.

[0452] 2. Materials and methods

[0453] The experimental procedures will be carried out in accordance to European guidelines for the care and use of laboratory animals (Directive 2010 / 63 / UE). Theprotocol that will be used both to induce and evaluate akinesia was approved by an Animal Ethical Committee (French National Committee N°71) and by the Higher Education and Research Ministry (APAFIS # 2020020715441357_v1).Every effort will be done to minimize animal suffering and reduce the number of animals used in the experiments. The animals, which will be included in these experiments, will be naive to previous administration of drugs.

[0454] 2.1 . Test items and reference compounds

[0455] 2.1.1. Reference compound

[0456] Rats from the group 3 (see Table 2) will be administered with a combination of L-DOPA and benserazide, a peripheric DOPA decarboxylase inhibitor 1 h30 before the behavioural tests carried out on D35.[Table 2]Reference compoundsNature Dose (mg / kg) Vehicle Route of Volume ofSchedule administration administration (ml. kg1)L-DOPA 10 mg. kg1Once on the day ofNaCI°’9%akinesia tests containing0,1 % i.p. 1Benserazide 12,5 mga. kga 1ascor .b .icOnce on the day of acid akinesia tests

[0457] Preparation of reference compound

[0458] The appropriate amounts of L-DOPA and benserazide will be weighed separately as detailed in Table 1 . Each of the two compounds will be dissolved in the appropriate volumes of sterile 0.9% NaCI containing 0.1 % ascorbic acid to reach the final expected concentrations. The final preparation will be freshly prepared by mixing equal volumes of solutions of L-DOPA and benserazide.

[0459] 2.1 .2. Candidate compounds

[0460] Candidate compounds details

[0461] Details regarding the test items to be tested within the scope of the present study are presented in Table 3. Metformin (CXS001) and glibenclamide (CXS002) alone will be tested at one dose. The CXS003, a mixture of CXS001 and CXS002 at the same dose for each, will also be tested.[Table 3]

[0462] On CXS Therapeutics request, the two compounds to be tested have been ordered by SYNCROSOME directly from the supplier: CXS001 (1 ,1- dimethylbiguanide hydrochloride; Metformin, D150959) and CXS002 (Glibenclamide, PHR1287) will be purchased respectively from Sigma Aldrich.

[0463] Determination of doses

[0464] Information regarding the respective doses of CXS001 , CXS002 and CXS003 to be tested within the scope of the present study were transmitted by email by CXS Therapeutics to SYNCROSOME on March 12th, 2025. Details of the doses that will be tested are described in Table 3.

[0465] 2.1 .3. Preparation of the 3 candidate compounds

[0466] Vehicle preparation of each candidate compound

[0467] CXS001 : this compound will be solubilized in Sterile water.

[0468] CXS002: this compound will be solubilized in Sterile water containing 10% of Dimethyl sulfoxide (DMSO). For a final vehicle solution of 100 ml: a 10% DMSO (i.e. , 10 ml) will be first diluted in a volume of sterile water (e.g., 10 ml in 50 ml of sterile water) by stirring the mix. Finally, the final volume will be completed byadding sterile water until reaching 100 ml under magnetic stirring. The vehicle solution will be prepared once a week and will be stored at +4°C until the day of treatment.

[0469] NB: The vehicle formulation administered to groups 1 , 2 and 3 will contain 5% DMSO in sterile water as detailed in the following paragraph.

[0470] Final CXS001, CXS002 and CXS003 formulation preparations

[0471] Briefly, the required amount of CXS001 and CXS002 will be accurately weighed separately (1.4 mg.ml-1 and 14 pg.ml-1 , respectively). Each of the two compounds will be dissolved in the appropriate volume of sterile water or in sterile water containing 10% DMSO to reach the final expected concentrations. For CXS003, the required amount of CXS001 and CXS002 will be accurately weighed separately (2.8 mg.ml-1 and 28 pg.ml-1 , twice as concentrated as the final administrated solution) and dissolved in the appropriate volume of sterile water or in sterile water containing 10% DMSO to reach the final expected concentrations. Then, the final formulation will be prepared by mixing equal volumes of each solution. The final formulation of and will be prepared freshly once a day. As CXS001 and CXS002 will be respectively dissolved in sterile water and 10% DMSO, the final formulation (CXS003) will contain 5% DMSO in sterile water.

[0472] 2.2. Test system

[0473] Eighty-four (84) adult male Wistar rats (7 weeks-old at the beginning of the study, JANVIER, France) will be used in the present study.

[0474] 2.2.1 . Animal housing

[0475] The acclimatisation of the animals will last at least 5 days. At receipt, animals will be collectively housed in cages at SYNCROSOME’s facilities. Food (RM1 , SDS Dietex) and water will be daily distributed in food and water dispensers.

[0476] 2.2.2. Experimental groups

[0477] The experiments will be performed on 6 groups of 14 animals as described in Table 4.

[0478] [Table 4]Groups / treatment Number Induction Schedule Treatment BehavioralProtocol of . . . , . testsVolume / route of treatment . . . . .. administration1 14 Vehicle on Do From D2 % DMSO with( ,s .ham / ,ve .h .) to D35 sterile waterOn D35 5 ml. kg-1 / p.o.0,9% NaCI1 ml. kg-1 / i.p.2 14 6-0 HD A on From D2% DMSO with(6-OHDA / veh)Do to°35sterile waterOn D35 5 ml. kg-1 / p.o.0,9% NaCI1 ml. kg-1 / i.p.3 14 6-OHDA on From D2% DMSO with(6-OHDA / L-DOPA)Do to°35sterile waterOn D35 5 ml. kg-1 / p.o.L-DOPA + Benserazide1 ml. kg-1 / i.p.4 14 6-OHDA on From D2CXS001(6-OHDA / CXS001)Do t0°355 ml. kg1 / p.o.On D350,9% NaCI1 ml. kg-1 / i.p.5 14 6-OHDA on From D2CXS002(6-OHDA / CXS002)Do t0°355 ml. kg1 / p.o.On D350,9% NaCI1 ml. kg-1 / i.p.3 14 6-OHDA on From D2CXS003(6-OHDA / CXS003)Do t0°355 ml. kg1 / p.o.On D350,9% NaCI1 ml. kg-1 / i.p.6-OHDA : 6 hydroxydopamine; D: Day; i.p. intraperitoneal; L-DOPA : L-3,4- dihydroxyphenylalanine ; p.o. : per os ; i.p. intraperitoneal

[0479] The acclimatisation of the animals will last at least 5 days. At receipt, animals will be collectively housed in cages at SYNCROSOME’s facilities. Food (RM1 , SDS Dietex) and water will be daily distributed in food and water dispensers.

[0480] 2.3. Work plan

[0481] The Experimental procedures include intracerebral administration of 6-OHDA, chronic oral administrations of the molecules, akinesia tests and CSF sampling and brain collections.

[0482] 2.4. Stereotaxic 6-OHDA lesion

[0483] For animals of groups 1 to 6, surgery will be performed on DO under isoflurane (4% induction 12-3% for maintain with a mix of oxygen and air). Animals will receive an unilateral stereotaxic injection with a micro syringe pump (SYS-Micro4, WPI) of 12 pg of 6-OHDA (sigma Aldrich) dissolved in 6 pl of 0.9% sterile NaCI containing 0.1 % ascorbic acid (sigma Aldrich) at the flow rate of 1 pl.min-1 , in the left substantia nigra pars compacta. Animal from Sham group (G1 ) will be injected with the 6-OHDA vehicle. Before the surgery, systemic (30 min) and local (5 min) analgesia will be achieved using subcutaneous (s.c) injections of buprenorphine (0.03 mg.kg-1) and lidocaine (4 mg.kg-1 ), respectively. The stereotaxic coordinates of the injection site will be as follows from the bregma: anteroposterior -5.3 mm, lateral +2.1 mm, dorsoventral -7.6 mm, with the incisor bar at -3.3 mm below the interaural plane, according to the rat stereotaxic atlas by Paxinos & Watson, 2007. At the end of surgery, animals will be treated with Carprofen (5 mg.kg-1 , s.c).

[0484] 2.5. Oral and intraperitoneal administrations

[0485] Animals from groups 1 to 3 will be daily treated with the vehicle while animals from group 4, 5 and 6 will be treated with CXS001 , CXS002 and CXS003 respectively over a 5-week period (from D2 to D35 included). CXS001 , CXS002 and CXS003 will be prepared as detailed in §2.1 and T able 3. The first administration of the candidate compounds or their vehicle will start 48 hours after stereotaxic 6-OHDA lesion, and the treatment will be administered until D35. During the study and for each animal, the volume of p.o. administrations will bedetermined based on the individual body weight of the animals. Body weights will be determined twice a week, and the volume of administration will thus be adjusted accordingly.

[0486] The day of behavioral tests (D35), at the same time of p.o. administration, animals from group 3 will be treated with the combination of L-DOPA I benserazide (10 mg.kg-1 1 12.5 mg.kg-1 ) (Table 4) while animals from groups 1 , 2, 4, 5 and 6 will be treated with 0.9% NaCI. Animals will be treated around 1 h-1 h30 before the behavioural tests (Table 4). For each animal, the volume of p.o. and i.p. administrations on D35 will be determined based on its own body weight.

[0487] The p.o. and i.p. administrations will be performed in accordance with standard operating procedure in force at SYNCROSOME.

[0488] 2.6. Behavioural tests

[0489] As depicted in §2.3, the behavioral tests will be carried out on D35. For that, rats will be accustomed to experimental rooms one hour prior to test sessions. To assess the test validity, animals of the group 3 will be administered with the reference compound, i.e. , L-DOPA, that is known to be efficient for behavioral recovery 1 h-1 h30 before the first test. Behavioral tests will be performed in a single blind manner.

[0490] 2.6.1 . Initiation time test (IT test)

[0491] On D35, the rat is held by its torso with its hindlimbs and one forelimb lifted above the surface of a table so that the weight of the animal's body is supported by one forelimb alone only one of the two forelimbs will be left free to move and the time that is necessary to initiate the movement toward a plane surface will be recorded, using 90 sec as break-off point. The test will be performed 8 times for each limb and for each animal.

[0492] 2.6.2. Stepping test (ST)

[0493] On D35, the rat is held by the experimenter and only one of the two forelimbs left free to move above a plane surface. The other hand fixed the forelimb not to be monitored with one paw touching the table. Then, Animal is moved slowly forward by the experimenter over a 90 cm distance. Each forelimb will be tested 6times. The performance of each forelimb will be evaluated and the mean number of adjusting steps for each forepaw will be calculated.

[0494] 2.7. Collection of cerebrospinal fluid

[0495] On D35, soon after the behavioral tests, rats of groups 1 to 6 (see Table 4) will be subcutaneously injected with 0.05 mg.kg-1 of buprenorphine. Then, animals will be deeply anesthetized with an i.p. combination of ketamine (50 mg.kg-1) I medetomidine (0.5 mg.kg-1) approximatively 15-20 minutes before CSF sampling. Around 50 pl of CSF will be collected by direct puncture of the cisterna magna for all groups. CSF samples will be then transferred into an Eppendorf tube and snap frozen in dry ice.

[0496] Samples identification will include the following information:- SYNCROSOME (SYN 94813)- Animal identification + group- Nature of the sample: CSF- Sampling time point: D35

[0497] CSF samples will be stored at -80°C until shipment to a dedicated partner (to be determined by the sponsor).

[0498] 2.8. Terminal brain collection

[0499] After CSF sampling, animals will be euthanized with pentobarbital sodium (140 mg.ml-1 ). Half of the animals of each group will be transcardially perfused with a successive solution of PBS (5 min), PBS + 5000 III of heparin per liter (3 min) followed by 4% PFA (5 min), at a flow rate of 14 ml.min-1 , to fix the brain tissue. The whole brain will then be removed, left 24 h in 4% PFA, transferred to successive bath of 10, 20 and 30% sucrose before being snap frozen in frozen isopentane.

[0500] The brain of the other half of animals of each group will be directly collected after euthanasia and brain will be snap frozen on dry ice before being stored at - 80°C.

[0501] Brain samples will be stored at -80°C until shipment.

[0502] 2.9. Statistical analysis

[0503] The statistical analysis will be performed by SYNCROSOME. If samples will be drawn from normally distributed populations with equal variances, differences in the mean values from behavioral tests at each time point in the different groups will be assessed with an analysis of variance (one way ANOVA followed by appropriate correction test). If samples will be drawn from non-normal populations, a Kruskal-Wallis ANOVA on ranks will be performed followed by appropriate correction test. A level of probability of 0.05 or less will be accepted as significant.

[0504] Results

[0505] Initiation time test results (Figure 18A and 18B)

[0506] 6-OHDA rats treated with vehicle show a significant increase of the time to initiate the movement of the RF (contra lesional forepaw) compared to Sham rats.

[0507] The reference compound, L-DOPA (10 mg / kg), administered in 6-OHDA significantly reduced the time to initiate the movement of the RF compared to 6- ODHA / vehicle.

[0508] CXS001 and CXS002 administered in 6-OHDA animals did not reduce the time to initiate the movement of the RF compared to 6-OHDA / vehicle.

[0509] CXS003 (combination of metformin and glibenclamide) administered in 6- OHDA animals significantly reduced the time to initiate the movement of the RF compared to 6-OHDA / vehicle.

[0510] Stepping test results (Figure 19A and 19B)

[0511] 6-OHDA rats treated with vehicle show a significant decrease of the number of RF steps compared to Sham rats.

[0512] The reference compound, L-DOPA (10 mg / kg), administered in 6-OHDA significantly increased the number of RF steps compared to 6-ODHA / vehicle.

[0513] CXS001 , CXS002 administered in 6-OHDA animals did not increase the number of RF steps compared to 6-OHDA / vehicle.

[0514] CXS003 (combination of metformin and glibenclamide) administered in 6- OHDA animals significantly increased the number of RF steps compared to 6-OHDA / vehicle (after exclusion test performed on 1 / 14 animal in 6-OHDA + vehicle group).

[0515] Conclusion

[0516] Regarding the issue of CXS001 / CXS002 synergy

[0517] As shown in the various analyses, CXS001 and CXS002 administered in 6- OHDA animals did not reduce the time to initiate the movement of the RF and the number of RF steps compared to 6-0 HDA / ve hide.

[0518] This data is fundamental since it also demonstrates in vivo the necessity of the two molecules in presence to obtain a synergistic biological effect, and even a beneficial effect outright.

[0519] It also suggests two closely linked and dependent molecular mechanisms.

[0520] Regarding the responses provided by the types of behavioral tests conducted

[0521] The Stepping Test has been shown to be one of the simplest and most sensitive behavioral measures of innate motor behavior, which is also dependent on the degree of DA lesion (Tseng et al., 2005).

[0522] Furthermore, the initiation time (IT) is considered the most sensitive parameter in the stepping test procedure.

[0523] The protocol used therefore allowed us to reliably and accurately measure the degree of motor impairment based on the different treatments used.

[0524] Regarding the non-preventive nature of the administration of the treatment

[0525] The first protocol used when conducting behavioral tests on the 6-OHDA rat model included preventive treatment with CXS003 before the induction of stereotaxic lesions with 6-OHDA.

[0526] This first protocol had already demonstrated the efficacy of the molecule on stepping test measurement.

[0527] In order to bring us closer to the patient's reality, the second in vivo protocol did not include preventive treatment, and we waited for disease induction protocol.

[0528] It should be noted that the duration of post-lesion treatment was this time longer, increasing from 2 weeks to 5 weeks.

[0529] Even without preventive administration, CXS003 conserves its beneficial effects against motor impairment, and duration treatment extending also increases its effectiveness.

[0530] Regarding the model that we used (6-OHDA rat model of Parkinson’s disease)

[0531] Akinesia is a major manifestation of Parkinson's disease related to difficulties or failures of willed movement to occur.

[0532] OHDA-lesioned rats do exhibit motor deficits that share essential functional similarities with parkinsonian akinesia or dyskinesia.

[0533] Toxin-induced models, such as those using 6-OHDA or MPTP, are celebrated for their reproducibility and capacity to produce clear, quantifiable motor deficits within a relatively short period.

[0534] They enabled the focused study of oxidative stress, mitochondrial dysfunction, and acute neurodegenerative processes.

[0535] Toxins like 6-OHDA and MPTP cause a rapid loss of dopaminergic neurons and produce significant motor deficits, closely mimicking the dopaminergic depletion seen in PD patients.

[0536] The unilateral 6-OHDA lesion model provides an internal control that is highly valuable for evaluating the efficacy of neuroprotective agents and symptomatic therapies.

[0537] Oral administration issue

[0538] This study finally shows that the selected route of administration (oral administration) is adapted to a satisfactory therapeutic effect and the synergy of the molecules combined into CXS003 treatment.

[0539] Example 10 : Neuroprotection effect of one mix compounds at 2 concentrations and 2 compounds alone at 2 concentrations on primary mouse motor neurons overexpressing TDP-43. Model of Amyotrophic Lateral Sclerosis

[0540] Study protocol

[0541] Mouse primary culture of motor neurons, transfection and drug treatment.

[0542] Mouse motor neurons have been cultured as described by Camu et al., 1994. After 5 days of culture, astrocytes are present in the culture and release growth factor allowing neurons differentiation. After day 7, cells have been transfected using Lipofectamine 3000. For each well of a 96-well plate, lipofectamine and plasmid have been mixed together and incubate for 10 min at RT before adding to the cells. The medium has been changed after 6h of transfection. One day after, the conditions described in the following graphics have been performed.

[0543] Endpoint evaluation: neuron survival evaluation

[0544] After 10 days of incubation with compounds (8 days post transfection), cells have been fixed by a solution of 4% PFA for 20 min at RT. The cells have been the permeabilized and non-specific sites blocked with a solution of PBS with saponin and FCS for 15 min at RT. Cells have been incubated with a chicken anti-200Kd NF, a rabbit anti-phospho (S409 / 410) TDP-43 and a mouse monoclonal anti-lslet antibodies. These antibodies have been revealed with different Alexa Fluor goat anti-mouse IgG, goat anti-rabbit IgG and goat antichicken IgG. Nuclei of cells have been labelled by a fluorescent marker in the same solution (Hoechst). For each well of culture, 20 pictures per well have been taken using InCell AnalyzerTM 2200. All images will be taken in the same conditions. The number of motor neurons, the NF neurite length, the number of motor neurons with TDP-43 aggregation signal have been automatically evaluated with Developer system analysis. All value are expressed as mean ± s.e. mean. Statistical analyses have been done on the different conditions (ANOVA followed by Dunnett’s test).

[0545] Results

[0546] Motor neurons survival

[0547] As shown in figure 16A, the uncombined use of 10 pM metformin and 1 pM glibenclamide is associated with a significant improvement in the survival of motor neurons transfected with pTDP-43. The combination of 10 pM metformin with 1 pM glibenclamide further improves neuron survival, indicating a synergistic effect of the two molecules at these doses.

[0548] Motor neurons length

[0549] As shown in figure 16B, the uncombined use of metformin and glibenclamide did not induce a significant change in neurite length of pTDP-43 motor neurons. Interestingly, combination of metformin and glibenclamide exerted a positive effect on the neurite length phenotype in an exclusively synergistic manner.

[0550] Motor neurons nuclear and cytoplasmic inclusion of pTDP-43

[0551] As shown in figure 16C, the uncombined use of metformin and glibenclamide exerts a slight significant effect on the reduction of the number of pTDP-43 nuclear inclusions in motor neurons. This effect is very clearly amplified when using 10 pM metformin and 1 pM glibenclamide in combination.

[0552] As shown in figure 16D, the uncombined use of metformin and glibenclamide exerts a significant effect on the reduction of the number of pTDP-43 cytoplasmic inclusions in motor neurons. A robust synergy is observed when using both 10pM metformin + 1 pM glibenclamide.

[0553] In conclusion, the simultaneous use of metformin and glibenclamide exerts a beneficial synergistic effect on survival, length and pTDP-43 inclusions in motor neurons in this cellular model of Alzheimer's disease based on A O intoxication.

[0554] General conclusion regarding the results

[0555] The study investigates the effects of metformin and glibenclamide, alone or combined, on motor neurons overexpressing pTDP-43 (an in vitro model of Charcot disease).

[0556] 1. Neuron survival: Each drug improves survival, but their combination produces a stronger synergistic effect.

[0557] 2. Neurite length: Neither drug alone significantly affects neurite length, while their combination promotes neurite elongation through synergy.

[0558] 3. pTDP-43 nuclear and cytoplasmic inclusions: Each treatment slightly reduces inclusions, but the combined use markedly enhances this effect at 10 pM metformin + 1 pM glibenclamide.

[0559] Conclusion: The combined treatment with metformin and glibenclamide shows a beneficial synergistic effect on neuron survival, neurite growth, and reduction of pTDP-43 inclusions, highlighting potential therapeutic value in this neurodegenerative disease model. |

Claims

Claims

1. A Pharmaceutical composition for use in the prevention and / or treatment of neurodegenerative diseases comprising metformin and glibenclamide.

2. The pharmaceutical composition for use according to the preceding claim, wherein said neurodegenerative disease is chosen among: Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia, Huntington’s disease or Alzheimer’s disease.

3. The pharmaceutical composition for use according to any one of the preceding claims, wherein metformin is present in an amount providing an effective dose of between 0.025 mg / day and 250 mg / day.

4. The pharmaceutical composition for use according to the preceding claim, wherein said metformin is present in an amount providing an effective dose of about 50 mg / day.

5. The pharmaceutical composition for use according to any one of the preceding claims, wherein glibenclamide is present in an amount providing an effective dose of between 0,001 mg / day and 5 mg / day.

6. The pharmaceutical composition for use according to the preceding claim, wherein said glibenclamide is present in an amount providing an effective dose of 2 mg / day.

7. The pharmaceutical composition for use according to claim 5, wherein said glibenclamide is present in an amount providing an effective dose of 0.5 mg / day.

8. The pharmaceutical composition for use according to any one of the preceding claims, wherein metformin and glibenclamide are administered concomitantly.

9. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein metformin and glibenclamide are administered sequentially.

10. The pharmaceutical composition for use according to any one of the preceding claims, further comprising at least one other acceptable activepharmaceutical ingredient, and / or at least one acceptable pharmaceutical excipient or carrier.

11. The pharmaceutical composition for use according to the preceding claim, wherein said active pharmaceutical ingredient is another agent for preventing or treating a neurodegenerative disease.

12. The pharmaceutical composition for use according to the preceding claim, wherein said agent is for preventing or treating Parkinson’s disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy body dementia and / or Alzheimer’s disease.

13. The pharmaceutical composition for use according to any one of the preceding claims, wherein the composition is formulated to be suitable for oral, nasal, transethmoidal, rectal, topical, intramuscular, intravenous or subcutaneous administration.

14. The pharmaceutical composition for use according the preceding claim, wherein the composition is formulated to be suitable for oral administration.

15. The pharmaceutical composition for use according to the preceding claim, wherein the oral administration is made by gelatin capsules, capsules, tablets, caplet, pill, compressed tablet, powders, granules, oral solutions, microgranules, multilayer tablets, gastroresistant tablets, extended release insoluble matrix tablets, or suspensions.

16. The pharmaceutical composition according to any one of the preceding claims, which is formulated for a delayed-release administration, preferably a combined administration of an immediate release form of metformin, an extended-release form of metformin, and glibenclamide

17. The pharmaceutical composition for use according to any one of the preceding claims, comprising a core containing coated particles of metformin and uncoated particles of glibenclamide.

18. The pharmaceutical composition for use according to the preceding claim, wherein said core is surrounded by an outer layer containing uncoated particles of metformin and uncoated particles of glibenclamide.

19. The pharmaceutical composition for use according to any one of claims 1 to 16, comprising a core containing coated particles of metformin.

20. The pharmaceutical composition for use according to the preceding claim, wherein said core is surrounded by an outer layer containing uncoated particles of metformin and uncoated particles of glibenclamide.

21. The pharmaceutical composition for use according to any one of claims 15 to 20, wherein glibenclamide is released over a period of at least 6 hours, preferably at least 12 hours, more preferably, at least 24 hours.

22. The pharmaceutical composition for use according to any one of claims 15 to 21 , wherein metformin is released over a period of at least 6 hours, preferably at least 12 hours, more preferably, at least 24 hours. |

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