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

Compositions containing a synergistic combination of memantine and vitamin D The present 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. Glutamate excitotoxicity is implicated in acute neurological damage caused by ischemia and traumatic brain injury, as well as in the chronic neurodegeneration associated with Alzheimer’s disease and Huntington’s disease. Excitotoxic neuronal death caused by overactivation of the N-methyl-D-aspartate receptor (NMDA receptors or NMDARs) contributes to an excessive and continuous influx of calcium (Ca2+) into the cell. This triggers a whole series of reactions leading to cell death, such as, in particular, increased oxidative stress, inappropriate activation of proteases such as calpain, dysregulation of Ca2+-related pathways, mitochondrial damage, and an apoptotic cascade. 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 combination with acetylcholinesterase inhibitors such as galantamine, donepezil and rivastigmine (Allgaier et al., Frontiers in Bioscience, 2014, vol. 19, pages 13451354). Because it is a low-affinity antagonist, it blocks the NMDA receptors but is rapidly displaced therefrom, thereby preventing prolonged blockage of the NMDA receptor channel and the adverse effects on learning and memory that have been observed in the case of high-affinity NMDAR antagonists (dissociative anesthetics, ketamine, and MK-801). Another advantage of memantine is that it interacts with the NMDA receptor channel only when it is pathologically activated by excessive glutamate concentrations in the synaptic cleft, as is the case in Alzheimer’s disease (Masters et al., Nature Reviews / Disease primers, 2015, 1, pages 1-18). It is generally accepted that cholecalciferol is a form of vitamin D, also referred to 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, in particular the hippocampus, the prefrontal cortex, and the amygdala (Adam et al., Clin Endocrinol Metab, February 2010, 95(2), pages 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), pages 458-484; Koshkina et al., Nutrients, 2019, 11, page 1726). When used alone, cholecalciferol has no demonstrable efficacy in the treatment of neurodegenerative diseases, and in particular Alzheimer’s disease. With this in mind, early pharmacological treatment with substances that reduce glutamate excitotoxicity could be a very good option for improving neuron survival (and cognitive function) and could represent a promising therapeutic strategy for patients diagnosed with Alzheimer’s disease and, more generally, those suffering from neurodegenerative diseases. For instance, patent FR2965178B1 describes pharmaceutical compositions comprising a combination of 5 to 20 mg of memantine hydrochloride and 800 to 2000 IU of cholecalciferol that may be useful in the treatment of Alzheimer’s disease. Said patent suggests, though does not demonstrate, that there may be a potentiating effect between the two active ingredients, in particular a potentiating effect of vitamin D on memantine. Patent application EP2363119A1 describes pharmaceutical compositions comprising 1 to 40 mg of memantine and 200 to 10,000 IU of vitamin D, i.e., 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 potentially useful in the treatment of Alzheimer’s disease, but no results proving this effect are given. Anweiler C. et al. (Neurobiology of Aging, 2014, 35, pages 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 in cortical cells. D. Charier et al. (Annales Francises d’Anesthesie et de Reanimation 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 in preventing neuronal death in a model of mouse neurons cultured in a microfluidic environment and exposed to blood- induced stress. Although the combination of memantine and vitamin D and the potential use thereof in the treatment of Alzheimer’s disease have already been described, these active ingredients are used at high doses that could cause adverse side effects. Indeed, it is evident from various prior art publications that memantine, when administered alone, is effective in treating Alzheimer’s disease at a dose greater than 5 mg. By way of 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 problems, shortness of breath, headaches, constipation, high blood pressure, and elevated transaminase levels. It has also been recognized that the receptors on which memantine acts, namely NMDA receptors, may undergo internalization, thereby reducing the efficacy of memantine and leading to a loss of therapeutic response after a certain period of treatment (Katherine W. Roche et al., Nature Neuroscience, 2001, volume 4, pages 794-802). There is therefore a need for pharmaceutical compositions that can overcome these drawbacks in order to effectively prevent and / or treat neurodegenerative diseases, in particular Alzheimer’s disease, while limiting the adverse side effects typically observed or likely to be observed when using a memantine dose of 5 mg or more, and while reducing or eliminating the loss of therapeutic response. The aim of the present invention is to satisfy this need. According to a first aspect, the invention relates to 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 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. According to a second aspect, the invention relates to said pharmaceutical composition for use in the prevention and / or treatment of neurodegenerative diseases, and in particular Alzheimer’s disease. According to a third aspect, the invention relates to a combination 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. Finally, according to a fourth aspect, the invention relates to a method for preventing, inhibiting, slowing or treating neuronal degeneration, in particular that associated with Alzheimer’s disease, in a subject in need thereof, said method being characterized in that it comprises administering to said subject a pharmaceutical composition as defined according to the first aspect of the invention or a combination preparation as defined according to the third aspect of the invention. Indeed, and as demonstrated in the examples that 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 typically used in the treatment of Alzheimer’s disease. In particular, the inventors were 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. Therefore, a first aspect of the invention is 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. Each of these two compounds, at such unit doses, does not exhibit any effect on neuron survival or protection of the neurite network. In one advantageous embodiment of the invention, the memantine is in the form of a salt, in particular hydrochloride. According to the invention, the vitamin D may be selected from the group comprising vitamin D2 or ergocalciferol, vitamin D3 or cholecalciferol, vitamin D4 or 22-dihydroergocalciferol, vitamin D5 or sitocalciferol, derivatives thereof, such as vitamin D6, the ethyl derivative of vitamin D4, or vitamin D7, the 24R-methyl derivative of vitamin D3, and salts thereof. In another particularly advantageous embodiment of the invention, the vitamin D is cholecalciferol. According to one preferred embodiment of the invention, the unit dose of memantine is approximately 5 pg to 0.1 mg, and the unit dose of vitamin D is approximately 0.8 pg to 8 pg. Also according to one preferred embodiment of the invention, the molar ratio of memantine to vitamin D in said synergistic combination is 1:2 to 1:100, and even more preferentially 1:3.5 to 1:100. According to the invention, the term “pharmaceutically acceptable excipient” will be understood to mean any substance other than the active ingredient(s) that is intended to confer consistency, taste or color on a drug, while avoiding any interaction with the active ingredient(s). The pharmaceutically acceptable excipient according to the invention will be selected, depending on the desired pharmaceutical form and route of administration, from among the conventional excipients known to a person skilled in the art, for administration to humans or animals. The pharmaceutical composition according to the present invention may be in any pharmaceutical form suitable for local, regional, systemic or continuous administration. The following routes of administration may be mentioned by way of example: oral, sublingual, subcutaneous, parenteral, intramuscular, intravenous, intranasal, rectal, or any other route of administration. Suitable forms of administration include oral forms, such as tablets, soft or hard capsules, powders, granules, and oral solutions or suspensions; sublingual, buccal, intranasal and inhalation forms; subcutaneous, intramuscular or intravenous forms; rectal forms; and implants. According to one preferred embodiment of the invention, the pharmaceutical composition is formulated for oral administration. The pharmaceutical composition according to the present invention may be prepared by any technique known to a person skilled in the art, in particular by mixing memantine and cholecalciferol with at least one pharmaceutically acceptable excipient. As indicated above, the synergistic combination present in the pharmaceutical composition according to the present invention exhibits a neuroprotective effect at a very low dose of memantine that is completely inactive when used alone. The synergistic combination of memantine and vitamin D that can be used according to the invention also has a better therapeutic index than memantine alone. A further aspect of the invention is 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 aspect of the invention, for use in the prevention and / or treatment of degenerative diseases. Among neurodegenerative diseases, mention may be made in particular of Alzheimer’s disease, dementias such as Lewy body dementia, frontotemporal dementia (FTD), and mixed dementia, as well as other neurodegenerative diseases such as Charcot’s disease (also known as amyotrophic lateral sclerosis, or ALS) and progressive supranuclear palsy (PSP). Among these neurodegenerative diseases, the invention is directed more particularly to the treatment of Alzheimer’s disease. Memantine and cholecalciferol may be jointly formulated or else may be mixed just prior to 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, a third aspect of the invention is a combination 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. Finally, a fourth aspect of the invention is a method for preventing, inhibiting, slowing or treating neuronal degeneration, in particular that associated with Alzheimer’s disease, in a subject in need thereof, wherein the method comprises administering an effective amount of the pharmaceutical composition as defined according to the first aspect of the invention or a combination preparation as defined according to the third aspect of the invention. According to this fourth aspect, the typical dosage regimen for an adult weighing approximately 70 kg comprises administering, once or twice daily, a pharmaceutical composition as defined according to the first aspect of the invention or a combination preparation as defined according to the third aspect of the invention. According to one particular embodiment of the invention, and when said method comprises administering a combination preparation, the unit dose of memantine and the unit dose of vitamin D are preferably administered simultaneously, even more preferentially simultaneously and orally. Brief description of the figures The following figures 1 to 4 and the example illustrate the invention: [Fig. 1] Figure 1A shows the effect of memantine alone on the survival of MAP-2 neurons in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. Figure 1B shows the effect of memantine alone on the protection of the total neuron network in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. [Fig. 2] Figure 2A shows 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 survival of MAP-2 neurons in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. Figure 2B shows 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 neuron network in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. [Fig. 3] Figure 3A shows 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 survival of MAP-2 neurons in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. Figure 3B shows 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 neuron network in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. [Fig. 4] Figure 4A shows the effect of a combination of memantine (at 10 nM in 180 pL of medium) and cholecalciferol (at doses ranging from 5 nM to 80 nM in 180 pL of medium) on the survival of MAP-2 neurons in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. Figure 4B shows the effect of a combination of memantine (at 10 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 neuron network in a primary culture of glutamate-damaged cortical neurons, in accordance with the example. The results are expressed as a percentage relative to the control; mean ± standard deviation (n = 4-6 per group). Control = 100% corresponds to a glutamate-free medium. One-way ANOVA followed by a Fisher’s LSD test. *p < 0.05 vs 20 pM glutamate is considered significant. EXAMPLE: PREVENTION OF GLUTAMATE TOXICITY IN NEURONAL CELLS USING   DIFFERENT   CONCENTRATIONS   OF   MEMANTINE,   OF CHOLECALCIFEROL, AND OF A SYNERGISTIC COMPOSITION ACCORDING TO THE INVENTION Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders, including neurodegenerative diseases. The 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-damaged primary cortical neurons. 1.1 Materials and methods 1.1.1 Primary cultures of cortical neurons All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with current European Union regulations (Directive 2010 / 63 / EU). Agreement Number: B1301310. Cortical neurons from rats were cultured as described by Callizot et al. (J. Neurosc. Res. (2013), 91(5), 706-716). Pregnant (Wistar) rats at 15 days of gestation were euthanized using deep anesthesia in a CO2 chamber followed by cervical dislocation. In short, the fetuses were harvested and immediately placed in ice-cold Leibovitz L15 medium with a 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) (PS) solution and a 1% bovine serum albumin (BSA) solution. The cortices were treated for 20 minutes at 37°C with a trypsin and EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA. Dissociation was stopped by adding 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). The cells were mechanically dissociated by forcing them through the tip of a 10 mL pipette three times. The cells were then centrifuged at 515 x g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of neurobasal medium with a 2% solution of B27 supplement, 2 mmol / L of L-glutamine, 2% PS solution, and 10 ng / mL of brain-derived neurotrophic factor (BDNF). Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion assay. The cells were seeded at a density of 25,000 cells per well into 96-well plates pre-coated with poly-L-lysine and were cultured at 37°C in an air (95%) / CO2 (5%) incubator. To avoid any edge effects, the first and last columns and the first and last rows of the culture plates were not used in the study. The empty wells were filled with water. The medium was renewed every two days. 1.1.2 Compounds tested, and exposure to glutamate Pre-incubation: On day 13 of culture, memantine (memantine hydrochloride, SIGMA ALDRICH) and cholecalciferol (SIGMA ALDRICH) were diluted in 180 pL of culture medium, as defined in section 1.1.1, to a final concentration of 1x and pre-incubated with primary cortical neurons for 1 hour before being exposed to glutamate. Treatment with glutamate: On day 13 of culture, glutamate was added to a final concentration of 20 pM diluted in 180 pL of culture medium, while the compounds (alone or in combination) were still present, for 20 minutes. After 20 minutes, the glutamate was removed by washing, and a small amount of fresh culture medium containing the test compounds at the same 1x concentration was added for an additional 48 hours. 1.1.3 Immunostaining: MAP-2 (neuron survival and network) MAP-2 is a neuronal marker which is present in the cell bodies and neurites of neurons and which makes it possible to study neuron survival and the length of the neurite network. 48 hours after the glutamate was added, the cell culture supernatant was removed. The cells were fixed using a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20°C. The cells were washed twice in phosphate-buffered saline (PBS). The cells were permeabilized, and non-specific binding sites were blocked using a PBS solution containing 0.1% saponin and 1% fetal calf serum for 15 minutes at room temperature. The cultures were then incubated for 2 hours with a mouse monoclonal anti microtubule-associated protein-2 (MAP-2) antibody diluted 1:400 in PBS containing 1% fetal calf serum and 0.1% saponin. This antibody was detected using an Alexa Fluor 488 goat anti-microtubule IgG (Molecular Probes) (diluted 1:400) in PBS containing 1% fetal calf serum and 0.1% saponin for 1 hour at room temperature. 1.1.4 Automated computer analysis For each condition, 30 photos (representative of the entire well area) per well were captured using ImageXpress® (Molecular Devices) at 20x magnification. All the images were generated using the same acquisition parameters. Based on the images, analyses were automatically performed using MetaXpress® (Molecular Devices). The following data were examined: - total neuron survival (number of MAP-2-positive neurons); the data are expressed as the mean number of neurons from 30 images per well. - total neuron network (MAP-2, in pm); the data are expressed as the mean neuron network length from 30 images per well. 1.1.5 Statistical analysis All values are expressed as a mean + / - SEM (standard error of the mean). Statistical analysis was performed using one-way ANOVA, followed by a Dunnett or Fisher’s LSD test. A p-value of <0.05 was considered significant. 1.1.6 Results * Effects of memantine alone on glutamate-damaged cortical neurons. These are shown in figures 1A and 1B. Glutamatergic stress induces a loss of cortical neurons (Fig. 1A) and a shrinkage of the total neurite network (Fig. 1B). Memantine used alone exhibits neuroprotective effects since it significantly improves neuron 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 neurite network was observed at 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, corresponding to doses of 0.09 pmol and 0.0018 nmol per well) are inactive. When memantine is used alone, a dose-dependent neuroprotective effect (survival and neurite network) is observed. * Effects of a synergistic combination of memantine and cholecalciferol on glutamate-damaged cortical neurons These are shown in figures 2A, 2B, 3A, 3B, 4A and 4C. The first three inactive concentrations of memantine were selected, namely concentrations of 500 pM, 1 nM and 10 nM (in 180 pL of medium, corresponding to doses of 0.09 pmol, 0.00018 nmol and 0.0018 nmol per well, respectively). Each of these doses of memantine was tested in combination with cholecalciferol at concentrations ranging from 5 nM to 80 nM (in 180 pL of medium), corresponding to equally 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 (Figs. 2A, 3A and 4A) and a shrinkage of the total neurite network (Figs. 2B, 3B and 4B). These results show: - at a memantine concentration of 500 pM (i.e., 0.09 pmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination both on neuron survival and on the neurite network is observed at 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 this effect is not observed when the cholecalciferol concentration is less than 35 nM or greater than 50 nM. - at 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 at 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 this effect is not observed when the cholecalciferol concentration is less than 35 nM (i.e., 0.0063 nmol / well) or greater than 50 nM (i.e., 0.009 nmol / well). - at a memantine concentration of 1 nM (i.e., 0.00018 nmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination on the neurite network is observed at 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 this effect is not observed when the cholecalciferol concentration is less than 20 nM (i.e., 0.0036 nmol / well) or greater than 50 nM (i.e., 0.009 nmol / well). - at a memantine concentration of 10 nM (i.e., 0.0018 nmol / well), a significant neuroprotective effect of the memantine + cholecalciferol combination both on neuron survival and on the neurite network is observed at 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 this effect is not observed when the cholecalciferol concentration is less than 20 nM (i.e., 0.0036 nmol / well) or greater than 80 nM (i.e., 0.0144 nmol / well). Thus, regardless of the memantine concentration tested (500 pM, 1 nM or 10 nM, all three of which are concentrations at which memantine alone is inactive in terms of neuron protection (corresponding to memantine doses of 0.09 pmol / well, 0.00018 nmol / well and 0.0018 nmol / well, respectively), the combination with cholecalciferol also at a very low concentration, namely 35 nM (i.e., 0.0063 nmol / well), makes it possible to obtain a synergistic combination that has a significant neuroprotective effect. This set of results demonstrates that combining memantine with vitamin D makes it possible to lower by a factor of at least 100 the effective doses of memantine typically used alone in the treatment of Alzheimer’s disease. Without wishing to be bound by any particular theory, the inventors believe that these results are due to novel mechanisms of action, which may include, inter alia, the stimulation of an NMDA receptor subunit other than the one typically activated by higher doses of memantine, or a morphological modification of this receptor itself, and / or better utilization of calcium ions.

Claims

1. A pharmaceutical composition comprising a synergistic combination ofmemantine 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. The composition according to claim 1, characterized in that the memantine isin the form of a salt, in particular hydrochloride.

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

4. The composition according to any one of claims 1 to 3, characterized in that themolar ratio of memantine to vitamin D in said synergistic combination is 1:2 to 1:100.

5. The composition according to any one of claims 1 to 4, characterized in thatthe vitamin D is cholecalciferol.

6. A pharmaceutical composition according to any one of claims 1 to 5 for use inthe prevention or treatment of neurodegenerative diseases.

7. The pharmaceutical composition for use according to claim 6, characterizedin that the neurodegenerative disease is Alzheimer’s disease.

8. A combination 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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