The combination of metformin and glibenclamide in the treatment of Parkinson's disease

Abstract

The present invention provides a pharmaceutical composition comprising metformin and glibenclamide for use in the treatment of Parkinson's disease. The present invention also includes the combined administration of metformin and glibenclamide. In preferred embodiments, administration is carried out by oral route.

Description

Technical Field

[0001] The present invention provides a pharmaceutical composition containing metformin and glibencamide for use in the treatment of Parkinson's disease. The present invention also includes the combined administration of metformin and glibencamide. In a preferred embodiment, the administration is carried out by the oral route.

[0002] (Field of the Invention) The technical field of the present invention is the treatment of Parkinson's disease.

Background Art

[0003] Parkinson's disease (PD) has a considerable impact on society, with approximately 6.1 million patients worldwide in 2016. For reasons that are not yet fully understood, the incidence and prevalence of this disease have been rapidly increasing over the past 20 years. The consequences of Parkinson's disease for an individual are severe, the duration of the disease can span decades, and the patient accumulates disabilities. Parkinson's disease also has serious consequences for caregivers, with most caregivers being exposed to excessive stress.

[0004] Parkinson's disease is a complex multifactorial neurodegenerative disease mainly associated with elderly patients. Modifying proteins aggregate in the dopaminergic neurons of the substantia nigra to form and accumulate Lewy bodies, resulting in a progressive decrease in these neurons. The cause of the aggregation and accumulation of proteins in these specific cells has not been elucidated, but the progressive loss of dopaminergic neurons causes the three main symptoms of the syndrome associated with Parkinson's disease and Parkinson's syndrome: rigidity, akinesia, and tremor (Bloem, Okun, et Klein 2021). Although several genetic polymorphisms are known to be risk factors, they have not been systematically elucidated, and some associations with physical trauma or long-term exposure to certain toxic substances have been identified.

[0005] Protein aggregation occurs after cellular stress involving reactive oxygen species and mitochondrial metabolism. More recently, glucose metabolism has been suggested to be a source of reactive oxygen species, which react with intracellular proteins and cause the accumulation of Lewy bodies. Glycolysis produces reactive metabolites such as 1,3-bisphosphoglycerate, which are sources of reactive oxygen species (Heremans et al. 2022a). However, the specific sensitivity of motor neurons in the substantia nigra remains unclear (Heremans et al. 2022b).

[0006] To date, there are no drugs that can slow the progression of the disease, that is, the neurodegeneration of dopaminergic neurons in the substantia nigra, which progresses over decades or months before and after the onset of symptoms and can be either rapid or slow. Approved drugs work by increasing dopamine release from remaining neurons or by acting as dopamine substitutes. They help maintain patient autonomy when combined with physical activity, but they do not slow neurodegeneration (Radhakrishnan et Goyal 2018).

[0007] Glucose metabolism has been identified as a promising target for disease-modifying therapies. Indeed, the Mediterranean diet, consisting of a variety of unprocessed fruits, vegetables, vegetable oils, fish, and generally small amounts of fast-acting carbohydrates, has been shown to be associated with the slight maintenance of motor function in patients with Parkinson's disease (Paknahad et al. 2020). This diet is associated with a metabolic shift to ketosis.

[0008] The efficacy of metformin, an antihyperglycemic antidiabetic drug, in preclinical models of Parkinson's disease, as well as in the protection of non-dementia vascular cognitive impairment and glucose metabolism disorders, supports the metabolic switch from glycolysis to ketosis (Mor et al. 2020b). However, at present, in Parkinson's disease, it has been observed to result in only slight improvement of the disease, or in some cases, even worsening (Mohamed Alrouji et al. 2023).

[0009] Metformin exerts a triple neuroprotective effect. First, it reduces serum glucose availability by lowering glucose levels, which are then converted to cyclic 1,3-bisphosphoglycerate during glycolysis. Second, metformin excretes glycerol, thus detoxifying intermediate metabolites involved in the accumulation of cyclic 1,3-bisphosphoglycerate. Finally, it promotes neurogenesis (Mor et al. 2020b; Markowicz-Piasecka et al. 2017; Wang et al. 2012).

[0010] Therefore, it is not surprising that metformin is attracting attention for the treatment of Parkinson's disease. The main drawback of this molecule lies in its tolerability. Metformin is mainly removed by ultrafiltration in the kidneys. If renal function is insufficient, metformin can accumulate, increasing serum lactate to life-threatening levels and leading to lactic acidosis. For this reason, the use of this molecule is contraindicated in patients over 65 years of age whose renal function, as confirmed by blood clearance, has decreased to less than 30 mL / min.

[0011] However, half of patients suspected of having Parkinson's disease are over 75 years old and tend to have cachexia, which carries risks for the use of metformin (Barichella, Cereda, et Pezzoli 2009).

[0012] Based on the hypothesis that neuroprotection is achieved by inhibiting the accumulation of cyclic 1,3-bisphosphoglycerate within dopaminergic neurons in the substantia nigra, the ideal treatment would be one that possesses the properties of metformin without the side effects that endanger the target population.

[0013] Considering the sustained need for medication to slow the progression of Parkinson's disease, the applicant has remarkably identified a synergistic and ingenious combination of molecules—specific doses of metformin and glibenclamide—that combines efficacy with minimal side effects, enabling safe long-term treatment for the majority of patients. While each compound alone may be ineffective or even toxic to dopaminergic neurons, their combination at low doses increases cellular health, characterized by neurite length, and prevents neurodegeneration. [Overview of the project]

[0014] The present invention relates to a pharmaceutical composition comprising metformin and glibenclamide for use in the treatment of Parkinson's disease.

[0015] The above pharmaceutical composition contains therapeutically effective amounts of metformin and glibenclamide.

[0016] In one embodiment, the metformin is present in an amount that provides an effective dose of 0.025 mg / day to 450 mg / day.

[0017] In one embodiment, the metformin is present in an amount that provides an effective dose of 0.025 mg / day to 250 mg / day.

[0018] Preferably, the metformin is administered in doses of 0.025 mg / day, 0.050 mg / day, 0.075 mg / day, 0.1 mg / day, 0.125 mg / day, 0.150 mg / day, 0.175 mg / day, 0.200 mg / day, 0.225 mg / day, 0.250 mg / day, 0.275 mg / day, 0.300 mg / day, 0.325 mg / day, 0.350 mg / day, 0.375 mg / day, or 0.400 mg / day, or 0 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 0.85 0 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 / 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.It is available in quantities that provide effective doses of 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.

[0019] More preferably, the metformin is present in an amount that provides an effective dose of about 50 mg / day.

[0020] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.001 mg / day to 2 mg / day.

[0021] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.001 mg / day to 1 mg / day.

[0022] Preferably, the above glibenclamide is administered in doses of 0.001 mg / day, or 0.005 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 It exists in amounts that provide an effective dose of 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.

[0023] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.5 mg / day.

[0024] In one embodiment, the pharmaceutical composition according to the present invention further comprises at least one other acceptable active pharmaceutical ingredient and / or at least one acceptable pharmaceutical excipient or pharmaceutical carrier.

[0025] In a preferred embodiment, the active pharmaceutical ingredient is another antiparkinson's disease agent.

[0026] Definition "Pharmaceutical composition" or "pharmaceutical preparation" refers to the formation of a pharmaceutical product in which different chemical substances, including the active drug, are combined to produce the final pharmaceutical product.

[0027] "Combination" in the present invention refers to the relationship between two compounds administered to the same subject, for example, metformin and glibenclamide. According to the present invention, the above two compounds can be administered simultaneously or sequentially. Therefore, according to the present invention, the administration of the first compound does not necessarily overlap with the administration of the second compound.

[0028] "Simultaneously" means administering metformin and glibenclamide at the same time, and the administration of the first compound overlaps with the administration of the second compound.

[0029] "Sequentially" means the administration of metformin and glibenclamide that is not carried out simultaneously. 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.

[0030] "Subject", "individual" or "patient" refers to a mammal, preferably a human. In one embodiment, the subject is diagnosed with Parkinson's disease. In one embodiment, the subject is waiting to receive medical treatment, is receiving medical treatment, or has been / will be / is the subject of a medical procedure, or a patient whose onset or progression of Parkinson's disease is being monitored, preferably a human patient. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject is an adult. In another embodiment, the subject is a child. <​​​

[0032] "To treat," "to treat," or "treatment" refers to a therapeutic measure, a prophylactic or preventative measure, or both, whose purpose is to prevent Parkinson's disease or to slow the progression of the disease. If, after administration of a therapeutic dose of metformin and glibenclamide in accordance with the present invention, the subject shows an observable and / or measurable reduction of the consequences of Parkinson's disease, as indicated by a reduction in symptoms, a restoration of the effectiveness of previously lost or impaired symptomatic treatment, the subject is successfully "treated."

[0033] The word "approximately" preceding a number includes a range of up to plus or minus 10% of the given number. It should be understood that the value referred to by the term "approximately" is itself specifically disclosed.

[0034] Idiopathic Parkinson's disease (IPD) is a multisystem synucleinopathy of the human nervous system with functional consequences and diagnostic applicability extending beyond the substantia nigra-striatal system. In the brain, inclusion body lesions manifest in the form of Lewy processes, Lewy bodies, and Lewy plaques in a small number of predisposed types of nerve cells. Melanoneurons or other projection neurons that form long axons and are unmyelinate or sparsely myelinate are particularly susceptible. This selective vulnerability in specific neuronal populations and anatomically and functionally interrelated brain regions results in a characteristic local distribution pattern of brain lesions that is observed almost consistently across autopsy cases, making it possible to predict and neuropathologically grade the brain progression of IPD. In Stage 1, lesions are limited to predisposed triggering sites, namely the dorsal visceral motor nuclei of the vagus nerve, the intermediate reticular zone, and / or the telencephalonal bulb. In stage 2 cases, inclusion bodies begin to appear in the caudal raphe nuclei (particularly the great raphe and uncertain raphe nuclei), the megacellular reticular nucleus, and the locus coeruleus / subcoeruleus complex nucleus. The severity of the lesions in stage 1 typically increases in stage 2 cases, and the pathology in the brainstem not only worsens throughout the subsequent stages but also essentially moves upward, toward the cerebral cortex. In stage 3, neuronal damage begins in the substantia nigra pars compacta of the midbrain, accompanied by changes in the pedunculopontine tegmental nucleus and forebrain (central subnucleus and basal complex of the amygdala, megacellular nuclei of the forebrain base, and hypothalamic tuberomammillary nucleus). In stage 4, the disease process reaches the cerebral cortex (anteromedial temporal mesocortex) for the first time. In stage 4 and beyond, IPD progresses primarily to the transitional entorhinal area, hippocampus, anterior cingulate cortex (all limbic loop structures), and further telencephalic regions, including the insula and subgenital regions of the intercortex (cortical components of the autonomic loop). Pathogenesis in these and other non-somatic motor structures almost certainly results in detectable olfactory dysfunction, lack of response to emotional stimuli, visceromotor and endocrine dysfunction, and, most likely, cognitive decline, including dementia in some individuals. Reduced influence of higher limbic centers on both the cerebral cortex and brainstem reticular formation is thought to contribute to the voluntary and emotional motor deficits that typically become apparent during the progression of IPD.In the final stages, 5 and 6, the disease progresses to encompass a wide area of ​​the neocortex, starting in the higher-order sensory association areas and prefrontal cortex, then the primary somatic cortex and premotor cortex, and finally reaching the primary somatic cortex and primary motor cortex of the mature neocortex. (Kelly del Tredici and Heiko Braak, Madame Curie Bioscience Database).

[0035] Early-onset Parkinson's disease (YOPD) refers to a type of Parkinson's disease diagnosed in individuals between the ages of 21 and 50. While the general symptoms of Parkinson's disease can be similar regardless of age, there are often differences in progression. For example, in younger individuals, involuntary movement problems are often exacerbated by levodopa, the most commonly prescribed medication for Parkinson's disease. Younger individuals with this disorder tend to experience fewer of the other Parkinson's disease-related problems, such as memory loss, confusion, and balance disorders.

[0036] "Parkinson's disease associated with splicing abnormalities" refers to Parkinson's disease that includes splicing abnormalities. In PD patients, at least PARK2, SNCAIP, LRRK2, SNCA, SRRM2, and MAPT are involved in abnormal AS events (FU, Ru-Huei, et al. 2013).

[0037] Mutations in PARK2 are associated with imbalances in programmed cell death systems, where non-apoptotic molecular mechanisms play a major role (Konovalova et al. 2015). Nearly half of cases of early-onset autosomal recessive Parkinson's disease are caused by the PARK2 gene (Olga Corti 2014).

[0038] Mutations in the MAPT gene, which encodes the microtubule-associated protein tau, result in the clinical phenotype of frontotemporal dementia with parkinsonism. Genome-wide association studies have linked MAPT H1 as a significant risk factor for Parkinson's disease (PD), while preliminary subhaplotype analyses suggest that different gene variants within the MAPT H1 haplotype are associated with each of these Parkinson's disease-like disorders (Ross Owen 2012).

[0039] Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common genetic cause of Parkinson's disease (PD) and one of the strongest genetic risk factors in sporadic PD. The LRRK2 protein contains a GTPase domain, a kinase domain, and several protein-protein interaction domains (Yulan Xiong et al. 2017).

[0040] Symphyrin-1 (SNCAIP) is a presynaptic protein that associates with synaptic vesicles (Ribeiro et al. 2002). This protein is an endogenous component of Lewy bodies (Wakabayashi et al. 2000), and mutations in the SNCAIP gene have been identified in some PD patients (Marx et al. 2003). Since the accumulation of SNCAIP and its interaction with SNCA may be related to Lewy body formation in PD, it is considered to be associated with Parkinson's disease (PD). SNCAIP is ubiquitinated by several different E3 ubiquitin ligases, including parkin (PARK2).

[0041] The alpha-synuclein gene (SNCA) is considered to be the first pathogenic gene responsible for autosomal dominant Parkinson's disease (PD), supported by the fact that its protein aggregation is a major pathological feature in patients, although only minor mutations have been identified (Polymeropoulos et al., 1997; Fields et al., 2019).

[0042] SRRM2 is an RNA splicing factor that has been reported to be consistently dysregulated in different PD neuronal sources (Shehadeh et al. 2010).

[0043] "Traumatic Parkinson's disease" refers to Parkinson's disease associated with traumatic brain injury. Traumatic brain injury (TBI) is considered a risk factor for PD. This includes mild TBI (mTBI), which is responsible for the 56% higher risk of developing PD among US veterans, and this risk is known to increase with the severity of the injury (Delic et al. 2020).

[0044] Vascular Parkinson's disease refers to Parkinson's disease associated with vascular injury or pseudovascular injury. This name includes progressive gait disturbance and abnormal white matter (WM) signaling visible on neuroimaging.

[0045] "Drug-induced Parkinson's disease" refers to Parkinson's disease that develops after taking a drug. This subtype is often reversible after discontinuation of the causative drug. All known causative drugs were prescribed outside of neurology, with more than half prescribed outside of psychiatry, and the majority prescribed to treat depression or abdominal discomfort (Shiraiwa et al. 2018).

[0046] "Parkinson's disease in the elderly" refers to Parkinson's disease in patients over 65 years of age.

[0047] "Acceptable pharmaceutical excipients or carriers" refer to excipients or carriers that, when administered to mammals, preferably humans, do not cause adverse reactions, allergic reactions, or other adverse reactions. These include any solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent, and absorption retarder. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation aid. For administration to humans, preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the FDA or EMA.

[0048] "Antiparkinson's disease agents" refer to molecules known to prevent, treat, or slow the progression of Parkinson's disease and / or its consequences.

[0049] "Administration" refers to the ingestion of a drug for it to pass through a target site in the patient's body, regardless of the route taken.

[0050] "Oral administration" refers to a route of administration in which a pharmaceutical composition is ingested orally, swallowed, and then processed by the digestive system.

[0051] "Rectal administration" refers to a route of administration that uses the rectum. The pharmaceutical composition is absorbed by the blood vessels in the rectum, enters the body's circulatory system, and distributes the drug to the target site.

[0052] "Intramuscular administration" refers to the injection of a substance into the muscle. Muscles have a larger and greater number of blood vessels than subcutaneous tissue, resulting in faster absorption than subcutaneous or intradermal injections.

[0053] "Intravenous administration" refers to administering a drug directly into a human vein.

[0054] "Subcutaneous administration" refers to the insertion of a drug under the skin by either injection or infusion. [Modes for carrying out the invention]

[0055] Therefore, the present invention relates to a pharmaceutical composition comprising metformin and glibenclamide for use in the treatment of Parkinson's disease.

[0056] Parkinson's disease is a brain disorder that causes involuntary or uncontrollable movements such as tremors, rigidity, and difficulty with balance and coordination. The pathophysiology of Parkinson's disease is thought to be due to a complex interaction of abnormal alpha-synuclein aggregation, dysfunction of mitochondrial, lysosome, or vesicular transport, problems with synaptic transport, and neuroinflammation.

[0057] While these pathological mechanisms primarily accelerate the death of dopaminergic neurons, numerous other motor and non-motor circuits are also involved in the neuropathology.

[0058] Loss of dopaminergic cells in the substantia nigra and striatum leads to a gradient of striatal dopamine depletion, creating an imbalance between direct (facilitative) and indirect (inhibitory) pathways through the basal ganglia, resulting in motor laxity.

[0059] Neurophysiological recordings conceptualized the imbalance between different oscillatory rhythms, namely, an excess of beta activity (anti-motor activity) and a deficiency of gamma activity (motor-promoting activity). More precisely, beta oscillations are associated with a dopaminergic inactivation state and are abolished by dopaminergic drugs or deep brain stimulation.

[0060] A relatively recent finding suggests that these pathological changes are accompanied by compensatory changes in brain activity in areas not initially affected by the pathogenesis of Parkinson's disease, such as a shift to more anterior corticostriatal circuits and recruitment of cortical regions not so closely connected to the basal ganglia (Bastiaan R Bloem et al., Parkinson's disease, Lancet 2021).

[0061] Metformin is the international generic name (INN) for the substance 1,1-dimethyl biguanide (CAS 657-24-9). Metformin is a widely used drug for the treatment of type 2 diabetes, particularly its hydrochloride C4H 11 N 5 It is used in the form of HCl. Classically, its role is to reduce insulin resistance in carbohydrate-intolerant organisms and to reduce hepatic nascent glucose production. Metformin can be prepared by known methods or is available from commercial sources (e.g., GLUCOPHAGE®, STAGID®, GLUMETZA®, and FORTAMET®). Metformin has the structure shown below:

[0062] [ka] It has.

[0063] As used herein, the term “metformin” includes any of its prodrugs, pharmaceutically acceptable salts, hydrates, and solvates. In particular, the term “metformin” includes its hydrochloride and emponates, as well as its hydrochloride and monohydrochloride, for example, metformin hydrochloride and metformin monohydrochloride. The term “metformin” also includes the crystalline forms of the above compounds.

[0064] Glibenclamide, also known as glibride, is an antidiabetic drug belonging to a class of drugs known as sulfonylureas, closely related to sulfonamides. This molecule exhibits a blood glucose-lowering effect, a favorable tolerability profile, compatibility with combination formulations, and synergistic effects that enable low doses for frequent polypharmacy in the target population. Glibenclamide can be prepared by known methods or is available from commercial sources (e.g., DAONIL®). The structure of glibenclamide is shown below:

[0065] [ka] It has.

[0066] The applicant has surprisingly demonstrated that the combined administration of metformin and glibenclamide provides significant and synergistic neuroprotection in the context of Parkinson's disease by modulating blood-brain barrier integrity and promoting neuronal survival in situ.

[0067] Furthermore, using a low dose of metformin makes it possible to prevent the two main side effects of this molecule: lactic acidosis and vitamin B12 deficiency.

[0068] This combination is beneficial for Parkinson's disease patients, especially elderly patients, as it reduces metformin-related toxicity while retaining its inherent neuroprotective and antioxidant effects.

[0069] This combination of molecules acts through a novel mechanism of action characterized by the applicant.

[0070] Increasingly, studies have shown a strong association between metformin and the regulation of AMPK, primarily mediated by the inhibition of respiratory chain complex I and glycerol-3-phosphate dehydrogenase at the mitochondrial level (Francesco Agostini et al., Int.J.Mol.Sci., 2022 and Vial et al., Front.Endocrinol., 07 May 2019). One of its major transporters, OCT1, is expressed on the surface of numerous cell types, including neurons and endothelial cells. These mechanisms, applicable to the treatment of Parkinson's disease, therefore deserve the most attention and study.

[0071] Next, metformin is likely to exert its neuroprotective role primarily by regulating mitochondrial function, namely, This is achieved by inhibiting glycerol-3-phosphate dehydrogenase activity, reducing gluconeogenesis, and ultimately suppressing the accumulation of toxic protein aggregates through abnormal protein glycation events. - By suppressing protein misfolding, oxidative stress, and protein synthesis, and by promoting the autophagy process (regulation of mitochondrial AMPK levels).

[0072] The NLRP3 inflammasome complex is known to be associated with blood-brain barrier (BBB) ​​permeability and neuroinflammation (Lawrence et al. 2022). The NLRP3-inflammasome is an intracellular protein complex and a key mediator of inflammation in numerous pathological conditions. Its activation by various effectors leads to the release of pro-inflammatory cytokines IL-1B and IL-18, as well as pyroptosis, a type of programmed cell death characterized by plasma membrane permeabilization by family member gasdermin proteins (Rebecca C. Coll et al., Trends in Pharmacological Sciences. 2022). Recent studies have shown that the use of glibenclamide can improve BBB integrity by reducing NLRP3 activity (a mechanism explained by decreased cellular potassium efflux) and pyroptosis at the endothelial cell level (Fulin Xu et al., Brain Behav. 2019).

[0073] Subsequently, glibenclamide primarily mediates its neuroprotective effects by inhibiting NLRP3 inflammasome activity and ultimately inhibiting programmed cell death induced by pyroptosis. Two different sites of action can constitute targets for this mechanism, namely, - Helps maintain barrier integrity within the BBB. - In neurons, it is in situ and suppresses the decrease in cell population.

[0074] The combined use of the two molecules (metformin and glibenclamide) and targeting of the aforementioned mechanisms appears to provide neuroprotection in situ, in the context of Parkinson's disease, by modulating blood-brain barrier integrity and promoting neuronal survival.

[0075] Therefore, the present invention relates to a combination comprising the above-mentioned metformin and the above-mentioned glibenclamide for use in the treatment of Parkinson's disease.

[0076] A second object of the present invention relates to a method for treating Parkinson's disease, comprising the administration of metformin and glibenclamide.

[0077] According to a third object, the present invention relates to the combined administration of metformin and glibenclamide for use in the treatment of Parkinson's disease.

[0078] Another object of the present invention is a kit-of-parts for use in the treatment of Parkinson's disease, comprising a first part containing metformin and a second part containing glibenclamide.

[0079] Another object of the present invention is the use of metformin and glibenclamide, or a combination thereof, for the manufacture of pharmaceuticals for the treatment of Parkinson's disease.

[0080] Another object of the present invention is a pharmaceutical combination comprising, or consisting of, the above-mentioned metformin and glibenclamide and at least one pharmaceutically acceptable excipient, for use in the treatment of Parkinson's disease.

[0081] Another object of the present invention is a pharmaceutical product comprising or consisting of the above-mentioned combination of metformin and glibenclamide, or the above-mentioned pharmaceutical combination, or the above-mentioned kit-of-parts, for use in the treatment of Parkinson's disease.

[0082] In one embodiment, Parkinson's disease is early-onset Parkinson's disease, idiopathic Parkinson's disease, Parkinson's disease associated with splicing abnormalities, traumatic Parkinson's disease, vascular Parkinson's disease, drug-induced Parkinson's disease, or Parkinson's disease in elderly adults.

[0083] It should be understood that the total daily dose of metformin and glibenclamide combined according to the present invention will be determined by the attending physician within the bounds of reasonable medical judgment.

[0084] The above pharmaceutical composition contains therapeutically effective amounts of metformin and glibenclamide.

[0085] In one embodiment, the metformin is present in an amount that provides an effective dose of 0.025 mg / day to 450 mg / day.

[0086] In one embodiment, the metformin is present in an amount that provides an effective dose of 0.025 mg / day to 250 mg / day.

[0087] Preferably, the metformin is administered in doses of 0.025 mg / day, 0.050 mg / day, 0.075 mg / day, 0.1 mg / day, 0.125 mg / day, 0.150 mg / day, 0.175 mg / day, 0.200 mg / day, 0.225 mg / day, 0.250 mg / day, 0.275 mg / day, 0.300 mg / day, 0.325 mg / day, 0.350 mg / day, 0.375 mg / day, or 0.400 mg / day, or 0 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 0.85 0 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 / 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.It is available in quantities that provide effective doses of 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.

[0088] More preferably, the metformin is present in an amount that provides an effective dose of about 50 mg / day.

[0089] In one embodiment, the subject is a mammal, preferably a human, and the therapeutically effective dose of metformin is a daily dose administered in a single oral or intravenous injection.

[0090] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.001 mg / day to 2 mg / day.

[0091] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.001 mg / day to 1 mg / day.

[0092] Preferably, the above glibenclamide is administered in doses of 0.001 mg / day, or 0.005 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 g / 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 It exists in amounts that provide an effective dose of g / 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 1.6 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.

[0093] In one embodiment, the glibenclamide is present in an amount that provides an effective dose of 0.5 mg / day.

[0094] In one embodiment, the subject is a mammal, preferably a human, and the therapeutically effective dose of glibenclamide is a daily dose administered in a single oral or intravenous injection.

[0095] According to the present invention, metformin and glibenclamide in the combination of the present invention can be administered simultaneously, separately, or sequentially.

[0096] According to one embodiment, the method for treating Parkinson's disease in a subject of interest comprises administering metformin and glibenclamide, or a combination thereof, to the subject, wherein a therapeutically effective dose of metformin is administered once to the subject, and then, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the administration of the therapeutically effective dose of metformin, a therapeutically effective dose of glibenclamide is administered to the subject.

[0097] According to one embodiment, the method for treating Parkinson's disease in a subject of interest comprises administering metformin and glibenclamide, or a combination thereof, to the subject, wherein a therapeutically effective dose of meglibenclamide is administered once to the subject, and then, 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, a therapeutically effective dose of metformin is administered to the subject.

[0098] In one embodiment, the pharmaceutical composition according to the present invention further comprises at least one other acceptable active pharmaceutical ingredient and / or at least one acceptable pharmaceutical excipient or pharmaceutical carrier.

[0099] In a preferred embodiment, the active pharmaceutical ingredient is another antiparkinson's disease agent.

[0100] In a more preferred embodiment, the antiparkinson'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 anticholinergic agents.

[0101] Levodopa (or L-dopa) is converted to dopamine in the brain. This is the standard treatment for Parkinson's disease, offering the best efficacy-to-side-effect ratio. To prolong its effects, all drugs containing levodopa also contain substances that inhibit the body's breakdown of levodopa.

[0102] Dopamine agonists are used in patients with Parkinson's disease. Dopamine agonists such as ropinirole are the first-line treatment for restless limb syndrome, and bromocriptine is administered for neuroleptic malignant syndrome. Dopamine agonists are also prescribed to counteract dopamine antagonist-induced hyperprolactinemia.

[0103] MAO-B inhibitors allow the brain to utilize more dopamine. They can be useful as early monotherapy (drugs used alone) or as add-ons to other medications, including levodopa. When used with other medications, MAO-B inhibitors can reduce motor variability (periods in which symptom control weakens as the levodopa dose decreases), minimizing "off" time and extending "on" time.

[0104] Amantadine (brand names Gocovri, Symadine, and Symmetrel) is a drug used to treat dyskinesia associated with Parkinson's disease. Amantadine is the organic compound 1-adamantylamine or 1-aminoadamantane, which consists of an adamantane skeleton with an amino group substituted on one of the four tertiary carbon atoms. Rimantadine is a closely related adamantane derivative with similar biological properties.

[0105] Carbidopa / levodopa, also known as levocarb and ko-caredopa, is a combination of two drugs, carbidopa and levodopa. It is primarily used to manage the symptoms of Parkinson's disease, but it does not slow the progression of the disease or prevent its worsening. It is administered via a mouthdopamine agonist. Foslevodopa-foscarbidopa is a soluble formulation of levodopa / carbidopa that has solubility to allow subcutaneous injection for the treatment of motor complications in patients with Parkinson's disease.

[0106] Istradefylline, marketed under the brand name Nourianz, is a medication used as an add-on to levodopa / carbidopa treatment for adults with Parkinson's disease (PD) experiencing "off" episodes. Istradefylline shortens the "off" period that results from long-term treatment with the antiparkinsonian drug levodopa. An "off" episode is a period when the patient's medication is not working effectively, leading to an increase in PD symptoms such as tremors and difficulty walking.

[0107] Inosine is a nucleoside formed when hypoxanthine is bonded to a ribose ring (also known as ribofuranose) via a β-N9 glycosidic bond. Previous studies have suggested that patients with the highest serum uric acid levels experience slower progression of Parkinson's disease symptoms. This study uses inosine to raise uric acid levels in individuals whose uric acid levels are below the population mean (6 mg / dL).

[0108] Isradipine (brand names DynaCirc, Prescal) is a calcium channel blocker in the dihydropyridine class.

[0109] In particular, apomorphine, marketed under the trade name Apokyn, is a type of apomorphine that acts as a non-selective dopamine agonist, activating both D2-like receptors and, to a much lesser extent, D1-like receptors. Apomorphine also acts with high affinity as an antagonist of 5-HT2 receptors and α-adrenergic receptors. Apomorphine is used for intermittent hypokinesia ("off" episodes) in advanced Parkinson's disease, where a reduced response to antiparkinsonian drugs such as L-dopa leads to muscle rigidity and loss of muscle control.

[0110] Donepezil, particularly marketed under the brand name Aricept, is a medication used to treat dementia associated with Parkinson's disease, and some evidence suggests that donepezil may improve cognitive, executive function, and overall condition in Parkinson's dementia.

[0111] In particular, varenicline, marketed under the brand names Chantix and Champix, is a nicotinic receptor partial agonist and cholinergic agonist. This molecule is thought to potentially affect balance and cognition in patients with Parkinson's disease.

[0112] Anticholinergics are substances that block the action of a neurotransmitter called acetylcholine (ACh) at synapses in the central and peripheral nervous systems. These drugs suppress the parasympathetic nervous system by selectively blocking the binding of ACh to its receptors in nerve cells. The nerve fibers of the parasympathetic nervous system are involved in the involuntary movements of smooth muscles in the gastrointestinal tract, urinary tract, lungs, sweat glands, and many other parts of the body. In a broad sense, anticholinergics are divided into two categories, antimuscarinic and antinicotinic (ganglionic and neuromuscular) drugs, according to their specific targets in the central and peripheral nervous systems and neuromuscular junctions.

[0113] In another embodiment, the active pharmaceutical ingredient is preferably an antidiabetic agent selected from repaglinide, gliclazide, glimepiride, and glipizide.

[0114] According to one embodiment, metformin and glibenclamide, combinations thereof, pharmaceutical combinations, pharmaceuticals, or kit-of-parts according to the present invention are formulated for administration to a subject.

[0115] In one embodiment, the pharmaceutical composition is formulated to be suitable for oral, rectal, topical, intramuscular, intravenous, or subcutaneous administration of the compound.

[0116] In preferred embodiments, the pharmaceutical composition is formulated to be suitable for oral administration.

[0117] In one embodiment, oral administration is carried out by gelatin capsules, capsules, tablets, powders, granules, oral solutions, or oral suspensions.

[0118] In one embodiment, the metformin described above is in a form suitable for oral administration. In another embodiment, the metformin described above is in a form suitable for injection. Thus, in one embodiment, the metformin described above can be injected into the target by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion. In one embodiment, the glibenclamide described above is in a form suitable for oral administration.

[0119] Therefore, in one embodiment, the above-mentioned glibenclamide can be administered orally to a subject, for example, in the form of a capsule or tablet. In another embodiment, the above-mentioned glibenclamide is in a form suitable for injection. Therefore, in another embodiment, the above-mentioned glibenclamide can be injected by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion, preferably by intravenous injection.

[0120] In one embodiment, the combination, pharmaceutical combination, pharmaceutical, or kit-of-parts according to the present invention is in a form suitable for oral administration. In other words, the combination, pharmaceutical combination, pharmaceutical, or kit-of-parts according to the present invention comprises metformin and glibenclamide, both of which are in a form suitable for oral administration. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical, or kit-of-parts according to the present invention comprises metformin and glibenclamide, which can be administered orally to a subject.

[0121] Examples of forms suitable for oral administration include, but are not limited to, liquid, paste, or solid compositions, more specifically tablets, tablets formulated for sustained release or extended release, capsules, pills, sugar-coated tablets, liquids, gels, syrups, slurries, and suspensions. In one embodiment, the combination, pharmaceutical combination, drug, or kit-of-parts according to the present invention is in a form suitable for parenteral administration. In other words, the combination, pharmaceutical combination, drug, or kit-of-parts according to the present invention comprises metformin and glibenclamide, both of which are in a form suitable for parenteral administration. Therefore, in one embodiment, the combination, pharmaceutical combination, drug, or kit-of-parts according to the present invention comprises metformin and glibenclamide, which can be administered parenterally to a subject.

[0122] In one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention is in a form suitable for injection, such as intravenous, subcutaneous, intramuscular, intradermal, or transdermal injection or infusion. In other words, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, both of which are in a form suitable for injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, which can be administered to a subject by injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion.

[0123] The metformin and glibenclamide, combination thereof, or pharmaceutical combination, or pharmaceutical according to the present invention in sterile injectable form, may be a solution, an aqueous suspension, or an oily suspension. These suspensions may be formulated according to techniques known in the art using appropriate dispersants or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic, pharmaceutically acceptable diluent or solvent. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixative oils have conventionally been used as solvents or suspension media. For this purpose, any non-irritating fixative oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful for the preparation of injectable preparations, as are natural, pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Spans, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes. In one embodiment, the combination, pharmaceutical combination, drug, or kit-of-parts according to the present invention comprises metformin in a form suitable for oral administration and glibenclamide in a form suitable for injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical, or kit-of-parts according to the present invention comprises metformin, which can be administered orally, and glibenclamide, which can be administered to a subject by injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion.In one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin in a form suitable for injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion, and glibenclamide in a form suitable for oral administration. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin that can be administered to a subject by injection, such as intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, or transdermal injection or infusion, and glibenclamide that can be administered orally.

[0124] In another embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention is in a form suitable for topical administration. In other words, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, both of which are in a form suitable for topical administration. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, which can be administered topically to a subject.

[0125] Examples of forms suitable for topical administration include, but are not limited to, liquid, paste, or solid compositions, more specifically aqueous solutions, droplets, dispersions, sprays, microcapsules, microparticles or nanoparticles, polymer patches, or controlled-release patches.

[0126] In another embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention is in a form suitable for rectal administration. In other words, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, both of which are in a form suitable for rectal administration. Therefore, in one embodiment, the combination, pharmaceutical combination, pharmaceutical or kit-of-parts according to the present invention comprises metformin and glibenclamide, which can be administered rectally to a subject.

[0127] Examples of forms suitable for rectal administration include, but are not limited to, suppositories, microenemas, enemas, gels, enema foams, creams, and ointments.

[0128] In one embodiment, metformin may be administered after glibenclamide. In one embodiment, metformin may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, or 10 days after glibenclamide.

[0129] In this specification and the following embodiments, unless otherwise specified, the range of values ​​expressed as "...~...(between...and...)" includes the specified lower and upper limits. [Brief explanation of the drawing]

[0130] [Figure 1] Histogram showing the effects of metformin (CXS001) and glibenclamide (CXS002) on dopaminergic neuron survival after 6-OHDA injury. Metformin (CXS001) alone and glibenclamide (CXS002) alone did not improve dopaminergic neuron survival in 6-OHDA toxicity compared to brain-derived neurotrophic factor. High concentrations of CXS002 appear to reduce neuronal viability. [Figure 2]Histogram showing the effects of metformin (CXS001) and glibenclamide (CXS002) on neurite length of dopaminergic neurons after 6-OHDA injury. The results show that metformin (CXS001) alone and glibenclamide (CXS002) alone did not improve the health (neurite length) of dopaminergic neurons in 6OHDA toxicity compared to brain-derived neurotrophic factor. [Figure 3] Histogram showing the effects of metformin (CXS001) and glibenclamide (CXS002) on dopaminergic neuron survival after 6-OHDA injury. Low doses of metformin (CXS001) and glibenclamide (CXS002) have been shown to improve the survival rate of dopaminergic neurons in 6-OHDA toxicity. With metformin 10 μM + glibenclamide 10 μM, a certain trend appears to be observed, although it is not statistically significant at this dose. [Figure 4] Histogram showing the effects of metformin (CXS001) and glibenclamide (CXS002) on neurite length of dopaminergic neurons after 6-OHDA injury. Low doses of metformin (CXS001) and glibenclamide (CXS002) have been shown to improve the health of dopaminergic neurons, as estimated by mean neurite length, in 6OHDA toxicity. [Figure 5] Representative photographs of primary neuron cell cultures under standard conditions (A) or under 6OHDA toxicity (B, C, D). Arrows point to surviving dopaminergic neurons that are differentially stained (in a tyrosine hydroxylase expression-dependent manner). Four conditions are shown: standard conditions (A), 6OHDA toxicity (B), 6OHDA toxicity after 48 hours of pretreatment with metformin and glibenclamide (C), and 6OHDA toxicity after 48 hours of pretreatment with brain-derived neurotrophic factor (D). [Figure 6]Effects of metformin (CXS001) and glibenclamide (CXS002) on the survival of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage compared to the control. Protective protocol (mean ± sem; *p<0.05, ****p<0.0001, statistics against 6-OHDA 20 μM; one-way ANOVA, followed by Dunnett's test). [Figure 7] The effect of the combination of metformin (CXS001) and glibenclamide (CXS002) on the survival of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage relative to the control. Protective protocol (mean ± sem; *p<0.05, ****p<0.0001, statistics relative to 6-OHDA 20 μM; one-way ANOVA, followed by Dunnett's test). [Figure 8] The effect of the combination of metformin (CXS001) and glibenclamide (CXS002) on the survival of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage compared to the control. Protective protocol (mean ± sem; *p<0.05, ***p<0.001, statistics relative to 6-OHDA 20 μM; one-way ANOVA, followed by Dunnett's test). [Figure 9] Effects of metformin (CXS001) and glibenclamide (CXS002) on neurite length of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage relative to control. Protective protocol (mean ± sem; *p<0.05, ***p<0.001, statistics relative to 20 μM 6-OHDA; one-way ANOVA, followed by Dunnett's test). [Figure 10] The effect of the combination of metformin (CXS001) and glibenclamide (CXS002) on neurite length of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage relative to the control. Protective protocol (mean ± sem; *p<0.05, ****p<0.0001, statistics relative to 20uM 6-OHDA; one-way ANOVA, followed by Dunnett's test). [Figure 11]Effects of the combination of metformin (CXS001) and glibenclamide (CXS002) on neurite length of primary dopaminergic neurons in rats after 6-OHDA injury, expressed as a percentage compared to the control. Protective protocol (mean ± sem; *p<0.05, ***p<0.001, statistics relative to 20uM 6-OHDA; one-way ANOVA, followed by Dunnett's test). [Examples]

[0131] Example 1: Testing the potential neuroprotective effects of two test compounds and a mixture of these two compounds on the survival of primary dopaminergic neurons in rats after 6OH-DA injury: Analysis of the number of dopaminergic neurons: 6-hydroxydopamine (6OH-DA) is a selective catecholamine-agonist neurotoxin used as a pharmacological agent capable of inducing PD-like symptoms (Sauer and Ortel, 1994; Cass et al., 2002). Furthermore, it is highly likely to correspond to a naturally occurring dopaminergic catabolic metabolite that accumulates in the brains of Parkinson's disease patients and is thought 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, which induces neurodegeneration of dopaminergic neurons in vitro, provides a useful model for Parkinson's disease.

[0132] This study will investigate the effects of two compounds and a mixture of these two compounds at three different concentrations on primary rat midbrain cultures damaged by 6OH-DA, an in vitro model of Parkinson's disease. For Parkinson's disease, the total number of dopaminergic neurons will be used. BDNF will be used as a positive control in this study.

[0133] To determine the maximum non-toxic concentration to be used for the remainder of the experiment, initial cultures of dopaminergic neurons are performed to determine the toxic concentrations of the compound alone (4 concentrations) and the compound mixture (12 concentrations).

[0134] Second and third cultures of dopaminergic neurons were prepared, and neuronal protection in the presence of the compound was tested after poisoning with 6OHDA.

[0135] Experimental protocol 1. Cell culture of dopaminergic neurons Rat dopaminergic neurons are cultured as described by Schinelli et al. (1988). Briefly, pregnant female rats on day 15 of gestation are sacrificed by cervical dislocation (Janvier rat), and the fetuses are removed from the uterus. The embryonic midbrain is removed and placed in ice-cold medium. Of the developing brain, only the ventral portion of the midbrain curvature is used for the cell preparation because it is a region rich in dopaminergic neurons. The midbrain is dissociated by trypsin treatment. The dissociated cells are resuspended in the prescribed culture medium, seeded in a 96-well plate, and cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere.

[0136] Half of the culture medium is replaced with fresh medium every two days. Under these conditions, after 5 days of culture, astrocytes are present in the culture, releasing growth factors that enable neuronal differentiation. 3-6% of the neuronal population are dopaminergic neurons.

[0137] After 6 days of incubation, the culture medium is replaced with a medium that does not contain growth factors.

[0138] 2. Cytotoxicity of the compound The initial cultures of dopaminergic neurons are prepared for cytotoxicity testing. Briefly, on day 7 of culture, the cells are incubated with four concentrations of the compound and 16 concentrations of a mixture of these two compounds for 48 hours.

[0139] The following conditions must be met: -Control medium (standard vehicle, 48 hours). -Control medium + 1 mM, 100 μM, 10 μM, and 1 μM of CX001. -Control medium + CX002 at concentrations of 100 μM, 10 μM, 1 μM, and 0.1 μM. -Control medium + 1 mM CX001 + 100 μM, 10 μM, 1 μM and 0.1 μM CX002. -Control medium + 100 μM CX001 + 100 μM, 10 μM, 1 μM and 0.1 μM CX002. -Control medium + 10 μM CX001 + 100 μM, 10 μM, 1 μM and 0.1 μM CX002. -Control medium + 1 μM CX001 + 100 μM, 10 μM, 1 μM and 0.1 μM CX002.

[0140] One culture is performed using 6 wells and 3 96-well plates per condition.

[0141] 3. Endpoint evaluation: Measurement of the total number of TH-positive neurons. After a two-day incubation, the cells were fixed in a 4% paraformaldehyde solution at room temperature for 20 minutes, and the control cells were fixed using the same procedure. The cells were then permeabilized, and nonspecific sites were blocked at room temperature for 15 minutes with a solution of phosphate-buffered saline (PBS) containing saponin and FCS. The cells were incubated overnight at 4°C with mouse monoclonal anti-tyrosine hydroxylase antibody (anti-TH, Sigma) in PBS containing saponin and FCS. This antibody was visualized at room temperature for 1 hour using Alexa Fluor 488 goat anti-mouse IgG in PBS containing 1% FCS and 0.1% saponin. The cell nuclei were labeled with a fluorescent marker (Hoechst solution) in the same solution.

[0142] For each well of the culture, 20 images per well were taken at 20x magnification using an InCell Analyzer® 2200 (GE Healthcare). All images were taken under the same conditions. Cell bodies of TH-positive neurons were analyzed using Developer software (GE Healthcare). A total of six data points were obtained per experimental condition.

[0143] All values ​​are expressed as mean ± standard error of the mean. Statistical analysis is performed under different conditions (ANOVA, followed by Dunnett's test).

[0144] For the following tests, the maximum non-toxic concentration of the mixture will be selected by the test lab.

[0145] 4.6 OH-DA exposure and drug handling: protective protocols Second and third initial cultures of dopaminergic neurons are prepared for 6-OHDA exposure. On day 7 of culture, cells are pretreated with the test compound or reference compound for 1 hour, and then poisoned with 6-OHDA (20 μM) for 48 hours.

[0146] The following conditions must be met: The following conditions must be met: -Control medium (standard vehicle, 48 hours). -Control medium + 6OHDA (20 μM, 48 hours). -Control medium + CX001 + 6OHDA (20 μM, 48 hours) for C1, C2, and C3. -Control medium + CX002 + 6OHDA (20 μM, 48 hours) for C1, C2, and C3. -Control medium + CX001 in C1 + CX002 in C1, C2 and C3 + 6OHDA (20 μM, 48 hours). -Control medium + CX001 for C2 + CX002 for C1, C2 and C3 + 6OHDA (20 μM, 48 hours). -Control medium + CX001 in C3 + CX002 in C1, C2 and C3 + 6OHDA (20 μM, 48 hours). -BDNF (50 ng / mL); +6OHDA (20 μM, 48 hours) as reference compound.

[0147] Two cultures are performed using 96-well plates with 6 wells per condition and 2 wells per culture.

[0148] 5. Endpoint evaluation: Measurement of the total number of TH-positive neurons after 6OH-DA poisoning.

[0149] After a two-day incubation, the cells were fixed in a 4% paraformaldehyde solution at room temperature for 20 minutes, and the control cells were fixed using the same procedure. The cells were then permeabilized, and nonspecific sites were blocked at room temperature for 15 minutes with a solution of phosphate-buffered saline (PBS) containing saponin and FCS. The cells were incubated overnight at 4°C with mouse monoclonal anti-tyrosine hydroxylase antibody (anti-TH, Sigma) in PBS containing saponin and FCS. This antibody was visualized at room temperature for 1 hour using Alexa Fluor 488 goat anti-mouse IgG in PBS containing 1% FCS and 0.1% saponin. The cell nuclei were labeled with a fluorescent marker (Hoechst solution) in the same solution.

[0150] For each well of the culture, 20 images per well were taken at 20x magnification using an InCell Analyzer® 2200 (GE Healthcare). All images were taken under the same conditions. Cell bodies of TH-positive neurons were analyzed using Developer software (GE Healthcare). A total of six data points were obtained per experimental condition.

[0151] All values ​​are expressed as mean ± standard error of the mean. Statistical analysis is performed under different conditions (ANOVA, followed by Dunnett's test).

[0152] result In vitro, primary cultures of rat neurons containing approximately 5% dopaminergic neurons (identified by tyrosine hydroxylase expression) were incubated with metformin (CXS001) and glibenclamide (CXS002) both individually and in combination. The cultures, evaluated for vitality (number of living dopaminergic neurons) and health (neurite length), showed the results shown in Figures 1-5: Metformin (CXS001) alone and glibenclamide (CXS002) alone did not improve the survival of dopaminergic neurons in 6OHDA toxicity compared to brain-derived neurotrophic factor. High concentrations of CXS002 appeared to decrease neuronal viability. - Metformin (CXS001) alone and glibenclamide (CXS002) alone did not improve the health of dopaminergic neurons (neurite length) in 6OHDA toxicity compared to brain-derived neurotrophic factor. Low doses of metformin (CXS001) and glibenclamide (CXS002) improve the survival rate of dopaminergic neurons in 6OHDA toxicity. A trend appears to be observed with CXS001 10 μM + CXS002 10 μM, although this is not statistically significant at this dose. - Low doses of metformin (CXS001) and glibenclamide (CXS002) improve the health of dopaminergic neurons, as estimated by mean neurite length, in 6OHDA toxicity.

[0153] Consideration Metformin treatment has been shown in large-scale trials to correlate with an increased risk of developing Parkinson's disease, and glibenclamide is known to potentially exhibit neurotoxicity through its excessive hypoglycemic effect. This example demonstrates that treatment including low doses of metformin and glibenclamide can protect dopaminergic neurons from 6OHDA toxicity. In healthy subjects, metformin reaches the brain and is transported across the blood-brain barrier (BBB) ​​by OCT family transporters, while glibenclamide remains outside. However, in PD, the BBB is more susceptible to leakage, and this mechanism contributes to disease progression. Glibenclamide inhibits the activation of the NLRP3 / inflammasome complex in brain compartments by inhibiting the NLRP3 / inflammasome complex on astrocytes in cases of BBB hyperpermeability. Glibenclamide enhances the BBB by inhibiting the NLRP3 / inflammasome on the endothelial cells of the BBB itself, thus preventing further leakage. This demonstrates the validity of discontinuous administration of glibenclamide in combination therapy, including metformin.

[0154] Example 2: A study to investigate the potential neuroprotective effects of the combination alone or in combination according to the present invention on the survival of primary dopaminergic neurons in rats after 6-OHDA injury. (Parkinson's disease model) The objective of this study was to investigate the neuroprotective effects of metformin (CXS001) and glibenclamide (CXS002), either alone or in combination of three different concentrations, on the survival of primary rat dopaminergic cultures damaged by 6-hydroxydopamine (6-OHDA), an in vitro model of Parkinson's disease. The total number of dopaminergic neurons and neurite length were evaluated. Brain-derived neurotrophic factor (BDNF) was used as a reference compound.

[0155] Experimental protocol Cell culture of dopaminergic neurons Rat midbrain neurons were cultured as described by Schinelli et al. (1988). Briefly, pregnant female rats on day 15 of gestation were sacrificed by cervical dislocation (Wistar rats; Janvier Lab), and the fetuses were removed from the uterus. The midbrain of the embryos was removed and placed in ice-cold Leibovitz 15 medium (L15; PanBiotech, reference number P04-27055, batch number 1260323) containing 2% penicillin-streptomycin (PS; PanBiotech, reference no. P06-07100, batch no. 2585615) and 1% bovine serum albumin (BSA; Sigma, reference no. 810533, batch no. 73). Only the ventral portion of the midbrain curvature was used for the cell preparation because it is a region rich in dopaminergic neurons. The midbrain was dissociated by 31-rypsinization at 37°C for 20 minutes (Trypsin EDTA 1×; PanBiotech, reference number: P10-023100, batch number: 8290223). The reaction was stopped by adding Dulbecco's modified Eagle medium (DMEM; PanBiotech, reference number: P04-03600, batch number: 6041222) containing DNase Grade I II (0.1 mg / mL; PanBiotech, reference number: P60-37780100, batch number: H210916) and 10% fetal bovine serum (FCS; Invitrogen, reference number: 10270106, batch number: 2534381). The cells were then mechanically dissociated by passing them through a 10 mL pipette three times. Next, the cells were centrifuged at 180×g for 10 minutes at +4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded. Cell pellets were resuspended in a standard medium consisting of Neurobasal Plus (Gibco, reference number: A3582901, batch number: 2537248) supplemented with 2% B27 Plus (Gibco, reference number: A3582801, batch number: 2722550), L-glutamine (2 mM; PanBiotech, reference number: P04-80100, batch number: 7061121), 2% PS, 10 ng / mL BDNF (PeproTech, reference number: 450-02, batch number: 092361), and 1 ng / mL glial neurotrophic factor (GDNF; PeproTech, reference number: 450-10, batch number: 012364). Viable cells were then counted using a Neubauer cytometer with a trypan blue exclusion test. Cells were seeded at a density of 40,000 cells / well in 96-well plates coated with poly-L-ornithine (Sigma, reference number: P4957, batch number: RNBL6953) and laminin (Sigma, reference number: L2020, batch number: 0000216258) and cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the culture medium was replaced with fresh medium every two days. Under these conditions, after 5 days of culture, astrocytes were present in the culture, releasing growth factors and enabling neuronal differentiation. Under these conditions, 2-5% of the neurons were dopaminergic neurons.

[0156] 6-OHDA preparation, exposure, and drug-treated dopaminergic neuron cell culture In short, 6-OHDA (Sigma, reference number: H4381, batch number: MKCQ5002) was reconstituted at 40 μM in the specified culture medium (stock solution). A control medium was prepared under the same conditions. After 7 days of incubation, primary midbrain neurons were pretreated with the test compound or reference compound (BDNF, 50 ng / mL) for 1 hour, and then poisoned with 6-OHDA at a final concentration of 20 μM over a 2-day incubation period to induce approximately 40% neuronal cell death. The following conditions were followed: Plate 1: - Control (0.05% DMSO) -Control + 6-OHDA (20 μM, 2 days) -10 pM, 3 μM, and 1 μM CXS001+6-OHDA (20 μM, 2 days) -10uM, 3uM, and 1uM CXS002+6-OHDA (20uM, 2 days) -50 ng / mL BDNF + 6-OHDA (20 μM, 2 days) Plate 2: - Control (0.05% DMSO) -Control + 6-OHDA (20 μM, 2 days) -10uM CXS001 + 10uM, 3uM, and 1uM CXS002 + 6-OHDA (20uM, 2 days) -3uM CXS001 + 10uM, 3uM and 1uM CXS002 + 6-OHDA (20uM, 2 days) -50 ng / mL BDNF + 6-OHDA (20 μM, 2 days) Plate 3: - Control (0.05% DMSO) -Control + 6-OHDA (20 μM, 2 days) - TUM's CXS001+10uM, 3uM, and 1uM CXS002+6-OHDA (20uM, 2 days) -50 ng / mL BDNF + 6-OHDA (20 μM, 2 days)

[0157] Two cultures were performed in six wells per condition.

[0158] Endpoint evaluation: Measurement of the total number of TH-positive neurons and the length of TH-positive neurites. After poisoning for two days, the cells were fixed at room temperature for 20 minutes with a solution of 4% paraformaldehyde (Alpha Aesar, reference number J19943, batch number: 211457), and the control cells were fixed using the same procedure. The cells were then permeabilized, and nonspecific sites were blocked at room temperature for 15 minutes with a solution of phosphate-buffered saline (PBS; VWR; reference number: L0615-500, batch number: MS01MB) containing 0.1% saponin (Sigma; reference number: S7900, batch number: BCBL8667V) and 1% FCS. The cells were incubated overnight at 4°C with rabbit polyclonal anti-tyrosine hydroxylase antibody (TH, 1 / 1000, Sigma, reference number: AB152, batch number: 3870479) in PBS solution.

[0159] Staining was visualized by adding Alexa Fluor 568 goat anti-rabbit IgG (1 / 400, Molecular probe, reference number: A11011, batch number: 2500544) to PBS containing 1% FCS and 0.1% saponin at room temperature for 1 hour. Cell nuclei were labeled with a fluorescent marker (Hoechst, Sigma; reference number: B1155, batch number: 046M4048V) in the same solution.

[0160] For each condition, 20 images were taken per well at 20x magnification using the InCell Analyzer® 2200 (GE Healthcare). Images of each culture well were taken under the same conditions. The number of dopaminergic neurons (THs) and the length of their neurites were automatically evaluated using Developer System Analysis (GE Healthcare). A total of six data points were obtained per experimental condition.

[0161] statistics Data were expressed as the mean value plus the standard error of the mean (6 data points and 1 culture per condition). A one-way analysis of variance (ANOVA), followed by Dunnett's test, was performed to analyze the overall data. The significance level was set to p<0.05.

[0162] result Effects of CXS001 and CXS002 on dopaminergic neuron survival after 6-OHDA injury (Plate 1) As observed in Figure 6, treatment with 20 μM 6-OHDA for 48 hours resulted in a significant decrease in dopaminergic neuron survival (54% cell death, p<0.0001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurons from cell death (81% compared to control, p<0.05). These results allow for the validation of culture conditions.

[0163] Pretreatment with 10 μM, 3 μM, and 1 μM CXS001 for 1 hour did not significantly rescue dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure (51%, 44%, and 43% cell death, respectively).

[0164] Furthermore, pretreatment with 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure (47%, 57%, and 58% cell death, respectively).

[0165] Effects of CXS001 and CXS002 on dopaminergic neuron survival after 6-OHDA injury (Plate 2) As observed in Figure 7, treatment with 20 μM 5-OHDA for 48 hours resulted in a significant decrease in dopaminergic neuron survival (55% cell death, p<0.0001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurons from cell death (87% compared to control, p<0.0001). These results allow for the validation of culture conditions.

[0166] Pretreatment with 10 μM CXS001 and a combination of 10 μM and 3 μM CXS002 for 1 hour did not significantly rescue dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure (45% and 37%, respectively). However, simultaneous treatment with 10 μM CXS001 and 1 μM CXS002 significantly rescued dopaminergic neurons from cell death (72% compared to control, p<0.05).

[0167] Furthermore, pretreatment with a combination of 3 μM CXS001 and 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure (45%, 45%, and 47% cell death, respectively).

[0168] Effects of CXS001 and CXS002 on dopaminergic neuron survival after 6-OHDA injury (Plate 3) As observed in Figure 8, treatment with 20 μM 6-OHDA for 48 hours resulted in a significant decrease in dopaminergic neuron survival (46% cell death, p<0.001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurons from cell death (83% compared to control, p<0.05). These results allow for the validation of culture conditions.

[0169] Pretreatment with 1 μM CXS001 combined with 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure (41%, 39%, and 47% cell death, respectively).

[0170] Effects of CXS001 and CXS002 on neurite length of dopaminergic neurons after 6-OHDA injury (Plate 1) As observed in Figure 9, treatment with 20 μM 6-OHDA for 48 hours induced a significant reduction in neurite length (54% neurite loss, p<0.001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurite length (79% compared to control, p<0.05). These results allow for the validation of culture conditions.

[0171] Pretreatment with 10 μM, 3 μM, and 1 μM CXS001 for 1 hour did not significantly rescue neurite length in dopaminergic neurons after 48 hours of 6-OHDA exposure (neurite loss of 55%, 43%, and 38%, respectively).

[0172] Furthermore, pretreatment with 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue the neurite length of dopaminergic neurons after 48 hours of 6-OHDA exposure (neurite loss of 47%, 57%, and 58%, respectively).

[0173] Effects of CXS001 and CXS002 on neurite length of dopaminergic neurons after 6-OHDA injury (Plate 2) As observed in Figure 10, treatment with 20 μM 6-OHDA for 48 hours induced a significant reduction in neurite length (56% neurite loss, p<0.0001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurite length (76% compared to control, p<0.05). These results allow for the validation of culture conditions.

[0174] Pretreatment with 10 μM CXS001 and 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue the neurite length of dopaminergic neurons after 48 hours of 5-OHDA exposure (neurite loss of 47%, 42%, and 42%, respectively).

[0175] Furthermore, pretreatment with a combination of 3 μM CXS001 and 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly rescue the neurite length of dopaminergic neurons after 48 hours of 6-OHDA exposure (neurite loss of 52%, 52%, and 41%, respectively).

[0176] Effects of CXS001 and CXS002 on neurite length of dopaminergic neurons after 6-OHDA injury (Plate 3) As observed in Figure 11, treatment with 20 μM 6-OHDA for 48 hours induced a significant reduction in neurite length (45% neurite loss, p<0.001). As expected, application of the reference molecule BDNF at 50 ng / mL for 48 hours significantly rescued neurite length (85% compared to control, p<0.05). These results allow for the validation of culture conditions.

[0177] Pretreatment with 1 μM CXS001 combined with 10 μM, 3 μM, and 1 μM CXS002 for 1 hour did not significantly save the neurite length of dopaminergic neurons after 48 hours of 5-OHDA exposure (40%, 46%, and 43% cell death, respectively).

[0178] conclusion Model validation As expected, application of 6-OHDA at 20 μM over two days induces significant loss of dopaminergic neurons and their neurites. Furthermore, BDNF at 50 ng / mL can significantly rescue these neurons from cell death.

[0179] Metformin (CXS001) and glibenclamide (CXS002) Pretreatment with CXS001 or CXS002 alone for one hour does not protect dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure, nor does it protect neurites from damage induced by 48 hours of 6-OHDA exposure.

[0180] Pretreatment with a combination of CXS002 and CXS001 for 1 hour did not significantly protect dopaminergic neurons from cell death induced by 48 hours of 6-OHDA exposure, nor did it significantly protect neurites from degradation induced by 48 hours of 6-OHDA exposure. However, a combination of 10 μM CXS001 and 1 μM CXS002 partially and significantly protected dopaminergic neurons after 48 hours of 6-OHDA exposure.

Claims

1. A pharmaceutical composition containing metformin and glibenclamide for use in the treatment of Parkinson's disease.

2. The pharmaceutical composition for use according to claim 1, wherein the Parkinson's disease is early-onset Parkinson's disease, idiopathic Parkinson's disease, Parkinson's disease associated with splicing abnormalities, traumatic Parkinson's disease, vascular Parkinson's disease, drug-induced Parkinson's disease, or Parkinson's disease in elderly adults.

3. A pharmaceutical composition for use according to any one of claims 1 or 2, wherein metformin is present in an amount that provides an effective dose of 0.025 mg / day to 250 mg / day.

4. The pharmaceutical composition for use according to claim 3, wherein the metformin is present in an amount that provides an effective dose of about 50 mg / day.

5. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein glibenclamide is present in an amount that provides an effective dose of 0.001 mg / day to 2 mg / day.

6. The pharmaceutical composition for use according to claim 5, wherein the glibenclamide is present in an amount that provides an effective dose of 2 mg / day.

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

8. A pharmaceutical composition for use according to any one of claims 1 to 7, wherein metformin and glibenclamide are administered simultaneously.

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

10. A pharmaceutical composition for use according to any one of claims 1 to 9, further comprising at least one other acceptable active pharmaceutical ingredient and / or at least one acceptable pharmaceutical excipient or pharmaceutical carrier.

11. The pharmaceutical composition for use according to claim 10, wherein the active pharmaceutical component is another anti-Parkinson's disease agent.

12. The pharmaceutical composition for use according to claim 11, wherein the antiparkinson'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 anticholinergic agents.

13. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the composition is formulated to be suitable for oral administration, rectal administration, topical administration, intramuscular administration, intravenous administration or subcutaneous administration.

14. The pharmaceutical composition for use according to claim 13, wherein the composition is formulated to be suitable for oral administration.

15. The pharmaceutical composition for use according to claim 14, wherein the oral administration is carried out by a gelatin capsule, capsule, tablet, powder, granule, oral solution, or oral suspension.

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