Compositions containing a synergistic combination of memantine and vitamin d

Abstract

The invention relates to compositions comprising a synergistic combination of memantine and vitamin D, to the method for preparing same and to the use thereof in the prevention and treatment of neurodegenerative diseases, in particular Alzheimer's disease.

Description

[0001] DESCRIPTION

[0002] Compositions containing a synergistic combination of memantine and vitamin D

[0003] The present invention relates to compositions comprising a synergistic combination of memantine and vitamin D, their method of preparation and their use in the prevention and treatment of neurodegenerative diseases, in particular Alzheimer's disease.

[0004] Glutamate excitotoxicity is implicated in acute neurological damage caused by ischemia and traumatic brain injury, as well as in chronic neurodegeneration in Alzheimer's disease and Huntington's disease. Excitotoxic neuronal death through overactivation of the N-methyl-D-aspartic acid receptor (NMDA receptor or NMDAR) contributes to an excessive and continuous influx of calcium (Ca 2+) in the cell. This triggers a whole series of reactions leading to cell death, including increased oxidative stress, inappropriate activation of proteases such as calpain, dysregulation of Ca-related pathways 2+ , mitochondrial damage and an apoptotic cascade.

[0005] Memantine (MEM) is a low-affinity, voltage-dependent, non-competitive NMDA receptor antagonist. It is currently prescribed for the treatment of Alzheimer's disease in conjunction with acetylcholinesterase inhibitors such as galantamine, donepezil, and rivastigmine (Allgaier et al., Frontiers in Bioscience, 2014, vol. 19, pp. 1345-1354). As a low-affinity antagonist, it blocks NMDA receptors, but is rapidly displaced from them, avoiding the prolonged blockade of the NMDA receptor channel and the negative side effects on learning and memory that have been observed with high-affinity NMDAR antagonists (dissociative anesthetics, ketamine, and MK-801).Another advantage of memantine is that it only interacts with the NMDA receptor channel when it is pathologically activated under excessive glutamate concentration in the synaptic cleft, as is the case in Alzheimer's disease (Masters et al., Nature Reviews / Disease primers, 2015, 1, pp. 1-18).

[0006] It is generally accepted that cholecalciferol is a form of vitamin D, also known as vitamin D3. Cholecalciferol is an essential substance required for calcium and phosphorus homeostasis in the human body. In addition, cholecalciferol receptors (VDRs) and cholecalciferol-activating enzymes have been found in various brain structures, including the hippocampus, prefrontal cortex, and amygdala (Adam et al., Clin Endocrinol Metab, February 2010, 95(2), pp. 471-478). Cholecalciferol may modulate various physiological processes in the brain, such as the regulation of brain-derived neurotrophic factor (BDNF) and other neurotrophic factors, neurogenesis, neuroplasticity, neuroprotection, and neuroimmunomodulation (DeLuca et al., Neuropathology and Applied Neurobiology, 2013, 39(5), pp. 458-484; Koshkina et al., Nutrients, 2019, 11, p. 1726).Used alone, cholecalciferol has no proven efficacy in the treatment of neurodegenerative diseases, particularly Alzheimer's disease.

[0007] In this perspective, early pharmacological treatment with substances reducing glutamate excitotoxicity could represent a very good option to improve neuronal survival (and cognitive function), and could represent an interesting therapeutic strategy for patients diagnosed with Alzheimer's disease and more generally suffering from a neurodegenerative disease.

[0008] Thus, patent FR. 2 965 178 B1 describes pharmaceutical compositions comprising a combination of 5 to 20 mg of memantine hydrochloride and 800 to 2000 IU of cholecalciferol which could be useful in the treatment of Alzheimer's disease. This patent suggests, without however demonstrating it, that there could be a potentiating effect between the two active ingredients, in particular a potentiating effect of vitamin D on memantine.

[0009] Patent application EP 2 363 119 A1 describes pharmaceutical compositions comprising from 1 to 40 mg of memantine and from 200 to 10,000 IU of vitamin D, i.e. from 5 pg to 250 pg of vitamin D (40 IU of vitamin D corresponds to the biological equivalent of 1 pg of vitamin D). These compositions are described as being useful in the treatment of Alzheimer's disease, but no results proving this effect are given.

[0010] Anweiler C. et al. (Neurobiology of Aging, 2014, 35, pp. 331-335) describe a neuroprotective effect of a combination comprising 1 pM of memantine and 100 nM of vitamin D in an in vitro model of glutamate toxicity on cortical cells. D. Charier et al. (Annales Françaises d'Anesthésie et de Réanimation 33S, 2014, A168- A173) describe a synergistic effect of memantine at a concentration of 1 mM and vitamin D at a concentration of 100 nM on the prevention of neuronal death in a model of mouse neurons cultured on microfluidic medium and subjected to blood insult.

[0011] Although the memantine / vitamin D combination and its potential application in the treatment of Alzheimer's disease have already been described, these active ingredients are used at high doses that could have harmful side effects. Indeed, it appears from the various publications of the prior art that memantine, when administered alone, is effective in the treatment of Alzheimer's disease at a dose greater than 5 mg. For example, memantine-based drugs prescribed for the treatment of moderate to severe forms of Alzheimer's disease contain, regardless of the manufacturer, doses of 10 mg or 20 mg of memantine. At such doses, memantine frequently causes adverse effects such as drowsiness, dizziness, balance disorders, shortness of breath, headaches, constipation, high blood pressure and increased transaminases...

[0012] It has also been recognized that the receptors on which memantine is active, namely the NMDA receptors, can become internalized, reducing the effectiveness of memantine and thus creating a therapeutic escape after a certain duration of treatment (Katherine W. Roche et al., Nature Neuroscience, 2001, volume 4, pages 794-802).

[0013] There is therefore a need for pharmaceutical compositions that can overcome these drawbacks in order to prevent and / or treat neurodegenerative diseases, in particular Alzheimer's disease, effectively while limiting the harmful side effects usually observed or likely to be observed when a dose of 5 mg or more of memantine is used and reducing or eliminating therapeutic escape.

[0014] The present invention aims to satisfy this need.

[0015] According to a first aspect, the subject of the invention is a pharmaceutical composition comprising a synergistic combination of memantine and vitamin D and at least one pharmaceutically acceptable excipient, said composition being characterized in that the unit dose of memantine present within said composition is less than 0.5 mg and in that the unit dose of vitamin D present within said composition is less than 0.08 mg.

[0016] According to a second aspect, the invention relates to said pharmaceutical composition for its use in the prevention and / or treatment of neurodegenerative diseases, and in particular Alzheimer's disease.

[0017] According to a third aspect, the invention relates to a combined preparation comprising at least two individual components, said preparation being characterized in that a first component contains a unit dose of memantine in an amount of less than 0.5 mg and in that a second component contains a unit dose of vitamin D in an amount of less than 0.08 mg for simultaneous, separate or sequential use in the prevention and / or treatment of neurodegenerative diseases, and in particular Alzheimer's disease.

[0018] Finally, according to a fourth aspect, the invention relates to a method for preventing, inhibiting, delaying or treating neuronal degeneration, in particular that linked to Alzheimer's disease in a subject who needs it, said method being characterized in that it comprises the administration of a pharmaceutical composition as defined according to the first aspect of the invention or of a combined preparation as defined according to the third aspect of the invention to said subject.

[0019] Indeed, and as demonstrated in the examples which follow, the inventors have shown that the combination of memantine and vitamin D makes it possible to significantly reduce the effective unit dose of memantine usually used in the treatment of Alzheimer's disease. In particular, the inventors have been able to demonstrate that when memantine is used in combination with vitamin D, it is possible to reduce the effective unit dose of memantine by a factor of at least 100.

[0020] The invention therefore has as its first subject a pharmaceutical composition comprising a synergistic combination of memantine and vitamin D, said composition being characterized in that the unit dose of memantine present in said composition is less than 0.5 mg and in that the unit dose of vitamin D present in said composition is less than 0.08 mg. At such unit doses, each of these two compounds has no activity on neuronal survival and protection of the neuritic network.

[0021] In an advantageous embodiment of the invention, memantine is in the form of a salt, in particular hydrochloride.

[0022] According to the invention, vitamin D may be chosen from the group comprising vitamin D2 or ergocalciferol, vitamin D3 or cholecalciferol, vitamin D4 or 22-dihydroergocalciferol, vitamin Ds or sitocalciferol, their derivatives such as, for example, vitamin D ethyl derivative of vitamin D4 or vitamin D7, 24R-methyl derivative of vitamin D3, as well as their salts.

[0023] In another particularly advantageous embodiment of the invention, the vitamin D is cholecalciferol.

[0024] According to a preferred embodiment of the invention, the unit dose of memantine is from about 5 pg to 0.1 mg and the unit dose of vitamin D is from about 0.8 pg to 8 pg.

[0025] Also according to a preferred embodiment of the invention, the memantine / vitamin D molar ratio within said synergistic association is from 1:2 to 1:100 and even more preferably from 1:3.5 to 1:100.

[0026] In accordance with the invention, the term "pharmaceutically acceptable excipient" means any substance other than the active substance(s), intended to provide a consistency, taste or color to a drug, while avoiding any interaction with the active substance(s). The pharmaceutically acceptable excipient according to the invention will be chosen according to the pharmaceutical form and the desired method of administration, from among the usual excipients which are known to those skilled in the art, with a view to being administered to humans or animals.

[0027] The pharmaceutical composition according to the present invention may be in any pharmaceutical form suitable for local, regional, systemic or continuous administration. Examples of routes of administration include oral, sublingual, subcutaneous, parenteral, intramuscular, intravenous, intranasal, or rectal or any other route of administration. Suitable administration forms include oral forms such as tablets, soft or hard capsules, powders, granules and oral solutions or suspensions, sublingual, buccal, intranasal, inhalation forms, subcutaneous, intramuscular or intravenous forms, rectal forms and implants.

[0028] According to a preferred embodiment of the invention, the pharmaceutical composition is formulated for oral administration.

[0029] The pharmaceutical composition in accordance with the present invention can be prepared by any techniques known to those skilled in the art, in particular by mixing memantine and cholecalciferol with at least one pharmaceutically acceptable excipient.

[0030] As previously indicated, the synergistic combination present in the pharmaceutical composition in accordance with the present invention exhibits a neuroprotective effect at a very low dose of memantine which is completely inactive when used alone. Also, the synergistic combination of memantine and vitamin D usable according to the invention exhibits a better therapeutic index than memantine alone.

[0031] The invention also relates to the pharmaceutical composition comprising a synergistic combination of memantine and vitamin D and at least one pharmaceutically acceptable excipient as defined according to the first subject of the invention, for its use in the prevention and / or treatment of degenerative diseases.

[0032] Neurodegenerative diseases include Alzheimer's disease, dementias such as Lewy body dementia, frontotemporal dementia (FTD) and mixed dementia, and other neurodegenerative diseases such as Lou Gehrig's disease (or amyotrophic lateral sclerosis or ALS) or progressive supranuclear palsy (PSP). Among these neurodegenerative diseases, the invention is particularly aimed at the treatment of Alzheimer's disease.

[0033] Memantine and cholecalciferol may be formulated together or may also be mixed just before administration. In this case, they may be in a form suitable for combined administration for use in the prevention and / or treatment of neurodegenerative diseases, and in particular Alzheimer's disease. Thus, the third subject of the invention is a combined preparation comprising at least two individual components, said preparation being characterized in that a first component contains a unit dose of memantine in an amount of less than 0.5 mg and in that a second component contains a unit dose of vitamin D in an amount of less than 0.08 mg, for simultaneous, separate or sequential use in the prevention and / or treatment of neurodegenerative diseases, and in particular Alzheimer's disease.

[0034] Finally, the fourth subject of the invention is a method for preventing, inhibiting, delaying or treating neuronal degeneration, in particular that linked to Alzheimer's disease in a subject in need thereof, in which the method comprises the administration of an effective amount of the pharmaceutical composition as defined according to the first subject of the invention or of a combined preparation according to the third subject of the invention.

[0035] According to this fourth object, the usual dosage for an adult of approximately 70 kg comprises the administration of a pharmaceutical composition as defined according to the first object of the invention or of a combined preparation as defined according to the third object of the invention, once or twice a day.

[0036] According to a particular embodiment of the invention and when said method comprises the administration of a combined preparation, then the unit dose of memantine and the unit dose of vitamin D are preferably administered simultaneously, even more preferably, simultaneously and orally.

[0037] Brief description of the figures

[0038] Figures 1 to 4 and the following example illustrate the invention:

[0039] [Fig.l] Figure 1A represents the effect of memantine alone on the survival of MAP-2 neurons in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant. Figure 1B represents the effect of memantine alone on the protection of the total neuronal network in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs 20 pM glutamate is considered significant.

[0040] [Fig.2] Figure 2A represents the effect of a combination of memantine (at 500 pM in 180 pL of medium) and cholecalciferol (at doses varying from 5 nM to 80 nM in 180 pL of medium) on the survival of MAP-2 neurons in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control, mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant. Figure 2B represents the effect of a combination of memantine (at 500 pM in 180 pL of medium) and cholecalciferol (at doses ranging from 5 nM to 80 nM in 180 pL of medium) on the protection of the total neuronal network in a primary culture of cortical neurons injured by glutamate according to the example.Results are expressed as a percentage relative to the control mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant.

[0041] [Fig.3] Figure 3A represents the effect of a combination of memantine (at 1 nM in 180 pL of medium) and cholecalciferol (at doses varying from 5 nM to 80 nM in 180 pL of medium) on the survival of MAP-2 neurons in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control, mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant. Figure 3B represents the effect of a combination of memantine (at 1 nM in 180 pL of medium) and cholecalciferol (at doses ranging from 5 nM to 80 nM in 180 pL of medium) on the protection of the total neuronal network in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control mean ± standard deviation (n=4-6 / group).Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant. [Fig.4] Figure 4A represents the effect of a combination of memantine (at 10 nM in 180 pL of medium) and cholecalciferol (at doses varying from 5 nM to 80 nM in 180 pL of medium) on the survival of MAP-2 neurons in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as a percentage relative to the control, mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant.Figure 4B represents the effect of a combination of memantine (at 10 nM in 180 pL of medium) and cholecalciferol (at doses varying from 5 nM to 80 nM in 180 pL of medium) on the protection of the total neuronal network in a primary culture of cortical neurons injured by glutamate according to the example. The results are expressed as percentages relative to the control, mean ± standard deviation (n=4-6 / group). Control = 100% corresponds to medium without glutamate. One-way ANOVA followed by an LSD Fisher's test. *p< 0.05 vs. 20 pM glutamate is considered significant.

[0042] EXAMPLE: PREVENTION OF GLUTAMATE TOXICITY OF NEURONAL CELLS BY DIFFERENT CONCENTRATIONS OF MEMANTINE, CHOLECALCIFEROL AND A SYNERGISTIC COMPOSITION ACCORDING TO THE INVENTION

[0043] Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders, including neurodegenerative diseases. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The inventors tested memantine alone for its ability to prevent or reduce the toxic effects of glutamate on glutamate-injured primary cortical neurons.

[0044] 1.1 Materials and methods

[0045] 1.1.1 Primary cultures of cortical neurons

[0046] All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed current European Union regulations (Directive 2010 / 63 / EU). Grant number: B1301310.

[0047] Rat cortical neurons were cultured as described by Callizot et al. (J. Neurosc. Res. (2013), 91(5), 706-716). 15-day-old pregnant rats (Wistar) were killed using deep anesthesia with a CO2 chamber and cervical dislocation. Briefly, fetuses were collected and immediately placed in ice-cold Leibovitz L15 medium with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA) solution. Cortices were treated for 20 minutes at 37°C with a trypsin-EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA. Dissociation was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g / L glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS).Cells were mechanically disrupted by three forced passages through the tip of a 10 mL pipette. Cells were then centrifuged at 515 x g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in defined culture medium consisting of neurobasal medium with 2% B27 supplement, 2 mmol / L L-glutamine, 2% PS solution, and 10 ng / mL brain-derived neurotrophic factor (BDNF). Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion assay. Cells were seeded at a density of 25,000 cells per well in poly-L-lysine-precoated 96-well plates and grown at 37°C in an air (95%) - CO2 (5%) incubator.

[0048] To avoid any side effects, the first and last columns as well as the first and last rows of the culture plates were not used in the study. Empty wells were filled with water. The medium was renewed every 2 days.

[0049] 1.1.2 Compounds tested and glutamate exposure

[0050] Pre-incubation: at I3 ème day of culture, memantine (memantine hydrochloride SIGMA ALDRICH) and cholecalciferol (SIGMA ALDRICH), were diluted in 180 pL of culture medium as defined in point 1.1.1. up to a final concentration of 1x and preincubated with primary cortical neurons for 1 h, before exposure to glutamate.

[0051] Glutamate treatment: I3 èmeOn the day of culture, glutamate was added at a final concentration of 20 pM diluted in the 180 pL of culture medium still in the presence of the compounds (alone or in combination) for 20 minutes. After 20 minutes, the glutamate was washed off and some fresh culture medium with the test compounds at the same 1x concentration was added for an additional 48 hours.

[0052] 1.1.3 Immunostaining: MAP-2 (survival and neural network)

[0053] MAP-2 is a neuronal marker present in the cell bodies and neurites of neurons, and which allows the study of neuron survival and the length of the neuritic network.

[0054] 48 hours after glutamate application, the cell culture supernatant was removed. Cells were fixed with a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20 °C. Cells were washed twice in phosphate-buffered saline (PBS). Cells were permeabilized and nonspecific attachment sites blocked with a PBS solution containing 0.1% saponin and 1% fetal bovine serum for 15 minutes at room temperature.

[0055] The cultures were then incubated for 2 hours with a mouse monoclonal anti-microtubule-associated protein 2 (MAP-2) antibody, at a 1:400 dilution, in PBS containing 1% fetal bovine serum and 0.1% saponin. This antibody was revealed by goat anti-microtubule IgG Alexa Fluor 488 (Molecular Probes) (1:400 dilution) in PBS containing 1% fetal bovine serum, 0.1% saponin, for 1 hour at room temperature.

[0056] 1.1.4 Automatic computer analysis

[0057] For each condition, 30 images (representative of the entire well area) per well were taken using ImageXpress® (Molecular Devices) at 20x magnification. All images were generated using the same acquisition parameters. From the images, analyses were automatically performed by MetaXpress® (Molecular Devices).

[0058] The following data were examined:

[0059] - total neuron survival (number of MAP-2 positive neurons); data are expressed as the average number of neurons from 30 images per well.

[0060] - the total neural network (MAP-2 in pm); the data are expressed as the average length of the neural network of 30 images per well. 1.1.5 Statistical analysis

[0061] All values ​​were expressed as mean + / - SEM (standard deviation of the mean). Statistical analysis was performed by one-way ANOVA, followed by Dunnett's or Fisher's LSD test. p<0.05 was considered significant.

[0062] 1.1.6 Results

[0063] * Effects of memantine alone on glutamate-damaged cortical neurons

[0064] They are given in Figures IA and IB.

[0065] Glutamatergic stress induces a loss of cortical neurons (Fig. 1A) and a shrinkage of the total neuritic network (Fig. 1B).

[0066] Memantine used alone shows neuroprotective effects, as it strongly improves neuronal survival at all tested concentrations between 50 nM and 10 pM (in 180 pL of medium, corresponding to doses between 0.009 nmol and 0.0018 pmol of memantine per well). In addition, a significant protective effect on the neuritic network was observed for concentrations of 100 nM to 10 pM (in 180 pL of medium, corresponding to doses of 0.018 nmol to 0.0018 pmol per well). Only the two lowest concentrations (500 pM and 10 nM, in 180 pL of medium and corresponding to doses of 0.09 pmol and 0.0018 nmol per well) are inactive.

[0067] When memantine is used alone, a dose-dependent neuroprotective effect (survival and neuritic network) is observed.

[0068] * Effects of a synergistic combination of memantine and cholecalciferol on glutamate-damaged cortical neurons

[0069] They are given in Figures 2A, 2B, 3A, 3B, 4A and 4C.

[0070] The first 3 inactive concentrations for memantine were selected, namely concentrations of 500 pM, 1 nM and 10 nM (in 180 pL of medium and corresponding respectively to doses of 0.09 pmol, 0.00018 nmol and 0.0018 nmol per well). Each of these doses of memantine was tested in association with cholecalciferol at concentrations ranging from 5 nM to 80 nM (in 180 pL of medium), corresponding to also very low doses of vitamin D ranging from 0.0009 nmol to 0.0144 nmol per well. Glutamatergic stress induces a loss of cortical neurons (Fig. 2A, 3A and 4A) and a shrinkage of the total neuritic network (Fig. 2B, 3B and 4B).

[0071] These results show:

[0072] - for a memantine concentration of 500 pM (i.e. 0.09 pmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination is observed both on the survival of neurons and on the neuritic network for cholecalciferol concentrations of 35 nM (i.e. 0.0063 nmol / well) and 50 nM (i.e. 0.009 nmol / well). It is interesting to note that when the cholecalciferol concentration is lower than 35 nM or higher than 50 nM, this effect is not observed.

[0073] - for a memantine concentration of 1 nM (i.e. 0.00018 nmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination on neuron survival is observed for cholecalciferol concentrations of 20 nM (i.e. 0.0036 nmol / well) and 35 nM (i.e. 0.0063 nmol / well). It is interesting to note that when the cholecalciferol concentration is lower than 35 nM (i.e. 0.0063 nmol / well) or higher than 50 nM (i.e. 0.009 nmol / well), this effect is not observed.

[0074] - for a memantine concentration of 1 nM (i.e. 0.00018 nmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination on the neuritic network is observed for cholecalciferol concentrations of 20 nM (i.e. 0.0036 nmol / well), 35 nM (i.e. 0.0063 nmol / well) and 50 nM (i.e. 0.009 nmol / well). It is interesting to note that when the cholecalciferol concentration is lower than 20 nM (i.e. 0.0036 nmol / well) or higher than 50 nM (i.e. 0.009 nmol / well), this effect is not observed.

[0075] - for a memantine concentration of 10 nM (i.e. 0.0018 nmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination is observed both on the survival of neurons and on the neuritic network for cholecalciferol concentrations ranging from 20 nM (i.e. 0.0036 nmol / well) to 80 nM (i.e. 0.0144 nmol / well). It is interesting to note that when the cholecalciferol concentration is lower than 20 nM (i.e. 0.0036 nmol / well) or higher than 80 nM (i.e. 0.0144 nmol / well), this effect is not observed.

[0076] Thus, whatever the concentration of memantine tested (500 pM, 1 nM, or 10 nM which are all three concentrations at which memantine alone is inactive with respect to neuronal protection (corresponding respectively to doses of memantine of 0.09 pmol / well, 0.00018 nmol / well and 0.0018 nmol / well), the association with cholecalciferol at a very low concentration also, of 35 nM (i.e. 0.0063 nmol / well), makes it possible to obtain a synergistic association having a significant neuroprotective effect.

[0077] All of these results demonstrate that the combination of memantine with vitamin D makes it possible to reduce by a factor of at least 100 the effective doses of memantine usually used alone in the treatment of Alzheimer's disease. Without wishing to be bound by any theory, the Inventors believe that these results are due to new mechanisms of action among which one can envisage, among others, the stimulation of another subunit of the NMDA receptor than that usually implemented with higher doses of memantine, or a morphological modification of this receptor itself, and / or a better use of calcium ions.

Claims

CLAIMS 1. Pharmaceutical composition comprising a synergistic combination of memantine and vitamin D and at least one pharmaceutically acceptable excipient, said composition being characterized in that the unit dose of memantine present in said composition is less than 0.5 mg and in that the unit dose of vitamin D present in said composition is less than or equal to 0.08 mg.

2. Composition according to claim 1, characterized in that the memantine is in the form of a salt, in particular hydrochloride.

3. Composition according to claim 1 or 2, characterized in that the unit dose of memantine is from 5 pg to 0.1 mg and the unit dose of vitamin D is from 0.8 pg to 8 pg.

4. Composition according to any one of claims 1 to 3, characterized in that the memantine / vitamin D molar ratio within said synergistic association is from 1:2 to 1:

100.

5. Composition according to any one of claims 1 to 4, characterized in that the vitamin D is cholecalciferol.

6. Pharmaceutical composition according to any one of claims 1 to 5 for its use in the prevention or treatment of neurodegenerative diseases.

7. Pharmaceutical composition for its use according to claim 6, characterized in that the neurodegenerative disease is Alzheimer's disease.

8. Combined preparation comprising at least two individual components, said preparation being characterized in that a first component contains a unit dose of memantine in an amount of less than 0.5 mg and in that a second component contains a unit dose of vitamin D in an amount of less than 0.08 mg, for simultaneous, separate or sequential use in the prevention and / or treatment of neurodegenerative diseases, in particular Alzheimer's disease.

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