Pharmaceutical composition formulated to be administered by intranasal route and suitable for use in the treatment of a neurodegenerative disease

Abstract

The present invention relates to a pharmaceutical composition formulated to be administered by intranasal route comprising, as active compound(s), at least one compound chosen from the compound of the following formula (I), the fumarate salt of the compound of the following formula (I): (I) and mixtures thereof, at least one pharmaceutically acceptable excipient, said composition being in the form of a gel free of hydroxypropylmethylcellulose.

Description

[0001] PHARMACEUTICAL COMPOSITION FORMULATED FOR ADMINISTRATION BY INTRANASAL ROUTE AND USABLE FOR THE TREATMENT OF A NEURODEGENERATIVE DISEASE

[0002] technical field

[0003] The present invention relates to a pharmaceutical composition formulated for intranasal administration and usable for the treatment of a neurodegenerative disease, selected in particular from Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis.

[0004] Previous art

[0005] Document W02020 / 065028 A1 describes several compounds for use in the preventive treatment of a selected neurodegenerative disease, including Alzheimer's and Parkinson's diseases. This document indicates that the compound with the following formula(l) exhibits an in vitro neuroprotective effect on neurons, including hippocampal and mesencephalic neurons expressing tyrosine hydroxylase, microtubule-associated protein MAP2-expressing neurons subjected to glutamate, and spinal neurons subjected to glutamate.

[0006] Document EP3 004 058 B1 describes a fluorinated compound similar to the compound of formula (I) below. This compound differs from the compound of formula (I) in that the cycloheptyl of formula (I) is replaced by a cyclohexyl.

[0007] The publication by Adnet et al., entitled "Pharmacotechnical development of a nasal drug delivery composite nanosystem intended for Alzheimer's disease treatment," published in the journal Pharmaceutics (vol. 12, no. 3, page 251) on March 11, 2020, describes a thermosensitive intranasal gel containing an unidentified AChE and BuChE inhibitor. This inhibitor is contained within liposomes. In addition to the liposomes, the gel also contains two poloxamers. The publication indicates that the interactions between the poloxamers and the liposomes increase the gel's adhesion to the nasal mucosa.

[0008] The publication entitled "A Mucoadhesive, thermoreversible in situ nasal gel of Geniposide for neurodegenerative diseases," published in the journal PLOS ONE on December 4, 2017 (volume 12, no. 12), describes a thermosensitive gel containing geniposide as its active ingredient. The geniposide is not contained in liposomes. The gel contains hydroxypropylmethylcellulose to ensure good adhesion to the nasal mucosa.

[0009] Technical problem

[0010] One object of the present invention is to provide a pharmaceutical composition containing, as an active agent, at least one compound selected from compound of formula (I), the addition salt of fumaric acid with this compound, and mixtures thereof, and which allows for the efficient delivery of this active agent to the brain. Another object of the invention is to provide a pharmaceutical composition that increases the bioavailability of the aforementioned active agent(s).

[0011] Another objective of the present invention is to propose a pharmaceutical composition which makes it possible to reduce the concentration of at least one of the aforementioned compounds in the gastrointestinal tract and more particularly in the small intestine and / or the large intestine.

[0012] Another objective of the present invention is to propose a pharmaceutical composition that takes into account the diffusion kinetics of the active compound(s) in the body and in particular in the blood system.

[0013] Another objective of the present invention is to provide a pharmaceutical composition that is stable over time.

[0014] Another objective of the present invention is to provide a pharmaceutical composition that causes little or no irritation of the intranasal mucosa.

[0015] Another objective of the present invention is to provide a pharmaceutical composition which exhibits sufficient adhesion to the nasal wall to prevent it from being swept away by cilia and intranasal mucus.

[0016] Another objective of the present invention is to provide a pharmaceutical composition which adheres for at least 1 min, preferably at least 5 minutes and more particularly at least 10 minutes to the intranasal mucosa of a mammal, in particular a human.

[0017] Another objective of the present invention is to provide a pharmaceutical composition which allows, by spraying, in particular, the formation of droplets having a size equal to or greater than 30pm and less than or equal to 800pm.

[0018] Summary of the invention

[0019] One object of the present invention is to solve at least one of the aforementioned technical problems.

[0020] According to a first aspect, the present invention relates to a pharmaceutical composition formulated for intranasal administration comprising, as active compound(s), at least one compound selected from the following compound of formula (I), the fumarate salt of the compound of formula (I), and mixtures thereof: less one pharmaceutically acceptable excipient, the composition is in the form of a gel and is free of hydroxypropylmethylcellulose.

[0021] The inventors deserve credit for demonstrating that the compound of formula (I) or its fumarate salt can form an intranasal gel without the addition of hydroxypropyl methylcellulose. Hydroxypropyl methylcellulose is suspected of containing mutagenic propylene chlorohydrin impurities. The fact that the gel of the invention does not contain these impurities makes it safer.

[0022] Detailed description

[0023] Advantageously, the active compound is chosen from the fumarate salt of the compound of formula (I) and mixtures of this salt with the compound of formula (I).

[0024] Preferably, the active compound is the fumarate salt of the compound of formula (I).

[0025] The fumarate salt of the compound of formula (I), hereinafter referred to as the fumarate salt of Neocopride, contains in moles a Neocopride: fumaric acid ratio equal to 1.0:1.0. This is the addition salt of fumaric acid with Neocopride.

[0026] According to a particular embodiment, the composition of the invention comprises, as excipients, at least one pharmaceutically acceptable polymer and at least one pharmaceutically acceptable liquid excipient. The pharmaceutical composition of the invention may be in the form of a powder, a solution, a suspension, a colloidal suspension, an emulsion (cream), or a more or less viscous solution, such as an ointment, or a gel.

[0027] It can also, for example, consist of the fumarate salt of the compound of formula (I) alone, possibly with a solid excipient in powder form, and be presented in powder form.

[0028] According to a preferred embodiment, the composition is in the form of a gel, and preferably in the form of a thermosensitive gel. This gel may be chemical or physical.

[0029] Advantageously, the gel is a physical gel that can, depending on a parameter, reversibly transition from a solution state to a gel state. Advantageously, the pharmaceutical composition of the invention is capable of transforming into a gel under the effect of temperature; preferably, it transforms into a gel at a temperature greater than or equal to 31 °C (heat-sensitive gel).

[0030] Preferably, the pharmaceutical composition of the invention is a hydrogel and preferably a thermosensitive hydrogel as mentioned above.

[0031] The pharmaceutically acceptable polymer(s) is / are not limited according to the invention. Preferably, the polymer(s) form a suspension, a colloidal suspension, or a gel with the liquid excipient(s).

[0032] The pharmaceutically acceptable polymer may be selected from polysaccharides, in particular starches, cellulose, chitin, alginates, chitosans, in particular chitosans having a molecular mass less than 150 kDa, hyaluronic acid, gellan gum, galactan, polypeptides, in particular collagen, elastin, reticulin, gluten, albumin, casein, zein, glycinin, B-conglycin, modified celluloses, in particular methyl cellulose, hydroxypropylcellulose, cyclodextrins, synthetic polymers, in particular poly(N-isopropylacrylamide, poly[2-(dimethylamino)ethyl methyl methacrylate, poly(vinylcaprolactam), polyvinyl methyl ether, carboxypolymethylenes, poly(ethylene glycol) copolymers / polyester(s), poly(ethylene glycol) / poly(D,L-lactide-co-glycolide) block copolymers, poloxamers and mixtures of these polymers.Preferably, said polymer is amphiphilic. The inventors have demonstrated that it is possible to form a thermosensitive intranasal gel that exhibits satisfactory adhesion to the intranasal mucosa without the addition of hydroxypropylmethylcellulose, particularly when the polymer is amphiphilic. While the inventors are not affiliated with this explanation, it is likely that the compound of formula (I) or its fumarate salt, which is an ionic compound, interacts with such a polymer(s) to enhance adhesion to the nasal mucosa.

[0033] Regardless of the embodiment, the mass percentage of polymer(s) relative to the quantity of liquid excipient(s) is not limited according to the invention. The pharmaceutical composition of the invention may contain from 0.5% by mass to 30% by mass of polymer(s), and in particular 10%, 15%, 20%, or 30% by mass of polymer(s).

[0034] In a particular embodiment, it contains poloxamers, preferably two poloxamers. Preferably, it contains one or more poloxamers, preferably two poloxamers, excluding any other polymer.

[0035] The inventors demonstrated that it was possible to form a thermosensitive intranasal gel that exhibited satisfactory adhesion to the intranasal mucosa without the addition of hydroxypropylmethylcellulose, particularly when the polymer was a poloxamer or a mixture of poloxamers. While the inventors are not affiliated with this explanation, it is likely that the compound of formula (I) or its fumarate salt, which is an ionic compound, interacts with the poloxamers to maintain the gel's cohesion. Surprisingly, the gel nevertheless remains thermosensitive.

[0036] The pharmaceutically acceptable liquid excipient is not limited according to the invention. The composition of the invention may also contain several pharmaceutically acceptable liquid excipients. Examples include (purified) water, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, methyl tert-butyl ether, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, 2-methyltertrahydrofuran, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, triethylamine and mixtures thereof.

[0037] According to a variant combinable with all the aforementioned embodiments, it contains water as a liquid excipient, and preferably, it contains water as the only liquid excipient to the exclusion of any other liquid excipient.

[0038] According to a particular embodiment, it contains a first poloxamer of the following formula: H(OCH2CH2)x(OCH(CH3)CH2)y(OCH2CH2)xOH in which x=101 and y=56 and / or a second poloxamer of the aforementioned formula in which x is equal to or greater than 75 and equal to or less than 85 and y is equal to or greater than 25 and equal to or less than 30 and preferably x=80 and y=27.

[0039] Such polymers are pharmaceutically acceptable and allow the preparation of a thermosensitive gel that reversibly transitions from a solution (particularly aqueous) to a gel at temperatures of approximately 15°C, 20°C, 29°C, 30°C, 31°C, 32°C, 35°C, or 40°C. This pharmaceutical composition can thus be administered either by simple application when it is in gel form at room temperature (like an ointment or cream), or as a spray, the composition being liquid at room temperature and transforming into a gel upon contact with the intranasal mucosa, which is at a temperature of approximately 32°C or 34°C depending on the depth within the nostril.

[0040] The inventors have demonstrated that, surprisingly, the composition of the invention in the form of a gel or solution which transforms into a gel upon contact with the mucous membrane makes it possible to reduce the effective dose by a factor of 10 compared with oral administration.

[0041] They also highlighted that a gel formulation allows for better control of the diffusion kinetics of the active compound(s), since gel administration reduces the amount of the active compound(s) reaching the small intestine. This is particularly advantageous when the active compound affects intestinal transit. The gel formulation thus helps to avoid or reduce adverse effects.

[0042] Advantageously, the pharmaceutical composition of the invention remains adherent to the intranasal mucosa for at least 1 minute, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes. This time can be measured experimentally.

[0043] Thus, the pharmaceutical composition of the invention limits the spread of Neocopride fumarate salt in the small intestine to a distance less than or equal to 260mm, 20 minutes after administration.

[0044] The mass percentage of the polymer(s), particularly the poloxamer(s), is not limited according to the invention. By way of example, the pharmaceutical composition according to the invention may contain, as a mass percentage relative to the mass of liquid excipient(s), particularly water, contained in the composition of the invention: at least 5% by mass, preferably at least 10% by mass, in particular 15%, or at least 20% by mass of the aforementioned first poloxamer. It contains, as a mass percentage relative to the mass of water contained in the composition of the invention, from 0% to 5% by mass of the aforementioned second poloxamer, in particular, 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3%, or 4% of the aforementioned second poloxamer.

[0045] According to a particular embodiment, it contains 15% by mass of said first poloxamer and 1% by mass of said second poloxamer.

[0046] The selection of the two aforementioned poloxamers yields a heat-sensitive gel containing the fumarate salt of neocoprid, which solidifies at temperatures of 31°C or higher. It is thus present in gel form within the nasal cavities of mammals, which are at temperatures of 31°C or higher. The fact that the composition is liquid below 20°C, and particularly at 4°C, allows for homogeneous mixing before gel formation, and potentially for administration as a spray, with the droplets transforming into a gel upon contact with the mucous membrane.

[0047] Unexpectedly, and without the inventors being involved in this explanation, it appears that the active compound, specifically the fumarate salt of Neocopride, reaches the olfactory bulb via intraneuronal absorption and enters the brain, where it then diffuses. The inventors further observed that, unexpectedly, the fumarate salt of Neocopride diffuses through the mucus layer lining the intranasal wall without being affected by the enzymes it contains. Advantageously, the pharmaceutical composition according to the invention has a viscosity that allows the formation of droplets with a size equal to or greater than 30 µm and less than or equal to 800 µm.

[0048] Surprisingly, it was found that the gel, particularly the heat-sensitive gel, allows for a reduction in the amount of active compound in the composition of the invention. Thus, compared with oral administration, the same in vivo effects are obtained with a quantity of active compound, specifically Neocopride fumarate salt, ten times lower with intranasal administration.

[0049] The simple instillation of the pharmaceutical composition of the invention into the nasal cavities allows the active compound to reach the brain at a therapeutically effective dose. However, the gel form allows for dose optimization. It is likely that the instillation of an aqueous solution, due to its lower viscosity, would not allow for consideration of the contact time between the mucous membrane and the active compound. The gel composition according to the invention allows for consideration of the diffusion kinetics of the active compound to and within the brain.

[0050] The mass concentration of the active compound(s) is not limited according to the invention. Thus, the composition according to the invention may contain, in mass concentration relative to the mass of water contained in the composition of the invention, a content equal to or greater than 0.2 mg / g, equal to or greater than 0.8 mg / g, equal to or greater than 1.2 mg / g, equal to or greater than 1.3 mg / g or equal to or greater than 2.0 or 2.1 or 2.5 mg / g.

[0051] The composition of the invention may also contain at least one agent selected from preservatives, pH buffers in particular saline pH buffers of pH equal to or greater than 3 and less than or equal to 9, and ideally a saline pH buffer of pH between 4.5 and 6.5, and preferably equal to or greater than 5 and equal to or less than 6.5, solutions of sodium chloride, magnesium chloride and potassium chloride (in particular to make the composition of the invention isotonic), humectants, preservatives and lubricants.

[0052] According to a particular embodiment, which can be combined with each of the aforementioned embodiments, the pharmaceutical composition of the invention has an osmolality greater than or equal to 200 mOsm / kg and less than or equal to 600 mOsm / kg, and preferably equal to 200 mOsm / kg and less than or equal to 300 mOsm / kg, and in particular equal to 284 mOsm / kg. The pharmaceutical composition of the invention thus prevents the epithelial mucosa from drying out by osmosis and from generating a flow of mucus or water that could wash it away. The osmolality can be measured by the freezing point method. Preferably, the pharmaceutical composition of the invention is free of liposomes containing the compound of formula (I), its fumarate salt, or mixtures thereof.

[0053] Preferably the composition of the invention is free of liposomes.

[0054] The composition of the invention may be in the form of an ointment in a tube or in the form of doses or be contained in a spray bottle allowing the formation of a spray.

[0055] The present invention also relates to a nasal spray device, which contains the composition of the invention.

[0056] This device is preferably capable of forming droplets with a size equal to or greater than 30pm and less than or equal to 800pm.

[0057] The present invention also relates to a product selected from an ointment, a gel, a solution, a suspension which contains the pharmaceutical composition of the invention.

[0058] The pharmaceutical composition according to the invention has proven useful for the treatment of Alzheimer's disease and Parkinson's disease.

[0059] The present invention also relates to a compound selected from the compound of formula (I), the fumarate salt of this compound and mixtures thereof, said compound preferably being in a form suitable for intranasal administration, for use in the treatment of a neurodegenerative disease, in particular Alzheimer's disease and / or Parkinson's disease.

[0060] Advantageously, said active compound(s) is in a composition administrable by intranasal route and preferably in an intranasal gel or in a thermosensitive gel, in particular a hydrogel as mentioned above.

[0061] The present invention also relates to a method of treating a neurodegenerative disease selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, according to which a therapeutically effective amount of an active compound selected from the compound of formula (I), its fumarate salt and mixtures thereof is administered intranasally to a subject in need of such treatment.

[0062] The active compound(s) is preferably contained in a gel as described above.

[0063] The aforementioned active compound(s) may be administered intrasalinally at a dose greater than or equal to 0.1 mg / kg and less than or equal to 0.6 mg / kg and in particular equal to 0.3 mg / kg.

[0064] The frequency of administration can be from one dose to three doses per day depending on the subject's condition.

[0065] Definitions

[0066] For the purposes of the present invention, the expression "pharmaceutically acceptable" refers to compounds, materials, excipients, compositions or dosage forms which are, within the framework of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problematic complications corresponding to an acceptable benefit-risk balance.

[0067] The terms "treat," "treat," "treated," or "treatment," as used in the context of the invention, refer to a therapeutic treatment whose purpose is to eliminate or alleviate symptoms. Beneficial or desired clinical outcomes include, but are not limited to, the elimination of symptoms, the alleviation of symptoms, the reduction of the extent of the condition, a stabilized (i.e., non-aggravated) health status, and a delay or slowing of the progression of the condition.

[0068] The term "patient" or "subject" refers to a warm-blooded animal such as a mammal, including a human being, male or female, unless otherwise indicated, that is suffering from or is likely to suffer from one or more of the diseases and conditions described herein.

[0069] The term "fumarate salt" refers to the addition salt of the compound in question with fumaric acid.

[0070] The term "gel" in the context of this invention refers to a cross-linked system of long, dilute molecular chains (macromolecules, often polymers) that exhibit no flow when in a stable state. The term "gel" encompasses gels that remain in this state regardless of temperature and temperature-sensitive gels that transition from a solution or colloid state to a gel state depending on the temperature.

[0071] The term "poloxamers" refers to any triblock polymer with the following formula: H(OCH2CH2)x(OCH(CH3)CH2) y(OCH2CH2)xOH in which x is greater than or equal to 2 and less than or equal to 130 and y is greater than or equal to 15 and less than or equal to 67.

[0072] Poloxamer P407 or Kolliphor 407 corresponds to a poloxamer with the following formula: H(OCH2CH2)x(OCH(CH3)CH2)y(OCH2CH2)xOH in which x=101 and y=56.

[0073] Poloxamer P118 or Kolliphor 188 corresponds to a poloxamer with the following formula: H(OCH2CH2)x(OCH(CH3)CH2)y(OCH2CH2)xOH where x=80 and y=27

[0074] Brief description of the drawings

[0075] Other features and advantages of the present invention will become apparent from the description of the specific and non-limiting embodiments of the present invention below, with reference to the attached Figures 1 to 12, in which:

[0076] Fig. 1 shows the exploration time (s) in the new branch of the Y maze for each group (Figure A) and the total distance traveled (cm) in the Y-shaped maze (Figure B); (groups for which the active molecule is not indicated correspond to groups treated with Neocopride fumarate salt).

[0077] Fig. 2 is a graph showing the exploration time in seconds in the area near the platform during the Morris water maze test for each group (groups for which the active molecule is not indicated correspond to groups treated with Neocopride fumarate salt).

[0078] Fig. 3 groups two graphs which respectively represent the survival rate of pyramidal neurons (NeuN(+)) compared to the control group, for each group (graph A) and the survival rate of pyramidal neurons (NeuN(+)) compared to the control group obtained for an intranasal administration of 0.3mg / kg of Neocopride fumarate salt and for an oral administration of 3mg / kg of Neocopride fumarate salt (the groups for which the active molecule is not indicated correspond to the groups treated with Neocopride fumarate salt).

[0079] Fig. 4 is a graph that shows, as a percentage of the control, the area occupied by the hyperphosphorylated tau protein in the CA1 area (the groups for which the active molecule is not indicated correspond to the groups treated with the fumarate salt of Neocopride).

[0080] Fig. 5 groups together two graphs which respectively represent microglial activation (Iba1 (+) cells) relative to the control group for each group (graph A) and microglial activation (Iba1 (+) cells) relative to the control group, obtained for intranasal administration of 0.3mg / kg of Neocopride fumarate salt and for oral administration of 0.3mg / kg of Neocopride fumarate salt (Fig. 5B) (the groups for which the active molecule is not indicated correspond to the groups treated with Neocopride fumarate salt).

[0081] Fig. 6 is a graph showing the number of astrocytes (GFAP(+) cells) relative to the control group for each group (groups for which the active molecule is not indicated correspond to groups treated with Neocopride fumarate salt).

[0082] Fig. 7 groups together two graphs representing respectively the PSD95 / GAPDH ratio relative to the control group for each group (Fig. 7A) and that obtained for an intranasal administration of 0.3mg / kg of Neocopride fumarate salt and for an oral administration of 3mg / kg of Neocopride fumarate salt (Fig. 7B) (the groups for which the active molecule is not indicated correspond to the groups treated with Neocopride fumarate salt).

[0083] Figure 8 shows two graphs representing, respectively, the time taken in seconds to reach the bar for mice in each group (graph A) and the number of false steps for each group (graph B), in the bar test in the mouse model of Parkinson's disease (the group for which the active molecule is not indicated corresponds to the group treated with Neocopride fumarate salt). Figure 9 shows two graphs representing, respectively, the time taken in seconds to reach the bar for mice in each group (graph A) and the number of false steps for each group (graph B), in the ladder test in the mouse model of Parkinson's disease (the group for which the active molecule is not indicated corresponds to the group treated with Neocopride fumarate salt).

[0084] Fig.10 groups three graphs showing respectively the percentage of TH(+) neurons in the SNpc compared to the control group for the group that received a protein injection (Parkinson's disease model) and for mice that received this injection and were then treated with Neocopride fumarate salt (graph A), the aggregation of α-syn in TH neurons, in the SNpc compared to the control group, for mice that received this injection and were treated with Neocopride fumarate salt (graph B) and the percentage of activation of Iba1-positive microglial cells in the SNpc compared to the control group for mice that received this injection and were treated with Neocopride fumarate salt (graph C), (the group for which the active molecule is not indicated corresponds to the group treated with Neocopride fumarate salt).

[0085] Fig. 11 groups together two graphs which respectively represent the concentration of Neocopride fumarate salt in the plasma for each group (graph A) and the concentration of Neocopride fumarate in the hippocampus (graph B).

[0086] Fig. 12 is a graph that represents the distance traveled by Neocopride fumarate salt or Prucalopride from the pylorus of the stomach, (the groups for which the active molecule is not indicated correspond to the groups treated with Neocopride fumarate salt).

[0087] Examples

[0088] Example 1: Synthesis of Neocoprid and its fumarate salt

[0089] Steps 1 and 2 Step 1:

[0090] 4-Amino-2,5-Difluorobenzonitrile (200.0 g, 1.30 mol, 1.0 eq) is introduced into a 4 L double-walled reactor. 2-Methyltetrahydrofuran (3.0 L) is added. A 30% (w / w) sodium methoxide solution in methanol is then added dropwise over 20 min with mechanical stirring under a nitrogen atmosphere. After the addition, the reaction mixture is heated to 75°C and stirred, while being maintained at this temperature for 20 hours. Compound 2 is obtained.

[0091] Characterization of compound 2 (4-amino-2-methoxy-5-fluorobenzonitrile)

[0092] NMR 1 H (DMSO-d6) 5 7.36 (d, J = 12 Hz, 1 H), 6.43 (d, J = 8 Hz, 1 H), 6.28 (s, 2H), 3.78 (s, 3H)

[0093] Step 2:

[0094] The reaction mixture is then cooled to 40°C and 1 L of water is added. The two phases are separated, and the organic phase is washed three times with water (1 L x 3). The resulting aqueous phases are combined, and an extraction is performed with 2-methyltetrahydrofuran (2 x 500 mL). The organic phases are combined and evaporated until a brown solid (m = 208 g) is obtained. The solid is introduced into a clean reactor, and ethylene glycol (830 mL) is added. An aqueous solution of KOH (244.6 g, 4.36 mol, 3.6 eq + 830 mL of water) is added. The reaction mixture is heated to 110°C and stirred for 24 hours. The reaction mixture is then cooled to 23°C, and water (500 mL) is added. The mixture is washed with DCM (dichloromethane) (3 x 8000 mL) and the organic phase is discarded. The aqueous phase is slowly acidified with 6N hydrochloric acid (750 mL) until pH equals 1.A white precipitate forms, 700 mL of water is added, and the suspension is stirred at 23°C for 1 hour. The solid is filtered and washed with 500 mL of water, then resuspended in 3 L of water. The suspension is stirred at 23°C for 18 hours. The solid is filtered and dried under vacuum at 50°C for 7 days to obtain compound 3 as a white solid (206 g, 85% in two steps).

[0095] Characterization of compound 3 (4-amino-2-methoxy-5-fluoro-2-methoxybenzoic acid) 1 H NMR (DMSO-d6) 5 11 , 79 (s, 1 H), 7.37 (d, J = 12 Hz, 1 H), 6.40 (d, J = 8 Hz, 1 H), 5.98 (s, 2 H), 3.71 (s, 3 H).

[0096] Step 3:

[0097] 3. EtOH-water recrystallization

[0098] 4-Amino-5-fluoro-2-methoxybenzoic acid (compound 3) (181.0 g, 0.98 mol, 1 eq) is introduced into a 10 L double-walled reactor equipped with a stirrer. THF (4.5 L) is then added. The suspension is heated to 45°C and becomes a solution. Carbonyldiimidazole (CDI, 190.2 g, 1.17 mol, 1.2 eq) is added in three portions (60 g, 62 g, and 70 g) over one hour. The mixture is stirred at 45°C for one hour. A solid combination of dry MgCl2 (139.6 g, 1.47 mol, 1.5 eq) and potassium ethyl malonate (249.6 g, 1.47 mol, 1.5 eq) (previously stored under vacuum at 45°C) was added in portions over 20 min (slight heat release) and the reaction mixture was stirred for 18 h at 45°C. 3.0 L of THF were removed by evaporation under vacuum and 2-MeTHF (2.0 L) followed by 1 N aqueous HCl (1.5 L) were added to the reaction mixture.The phases were separated at 40°C and the organic layer was washed with 1 N aqueous HCl (500 mL), then with a saturated aqueous solution of NaHCO3 (1.5 L), and finally with water (1.5 L). The organic phase was then evaporated under vacuum until compound 4 was obtained as a brown solid (250 g).

[0099] This solid is then dissolved in ethanol at 80°C. Water (1.2 L) is then added to maintain a temperature above 70°C. The solution is stirred for 30 minutes and then cooled for 18 hours to 23°C. The crystallized solid is filtered, washed with water, and dried under vacuum at 45°C, yielding compound 4 as a pale yellow solid.

[0100] Characterization of compound 4 (3-(-4-amino-5-fluoro-2-methoxyphenyl)-3-ethyl oxopropanoate)

[0101] 1 H NMR (DMSO-d6) 5 7.37 (d, J = 12 Hz, 1 H), 6.39 (d, J = 8 Hz, 1 H), 4.08 (qd, J = 8Hz, 2H), 3.77 (s, 2H), 3.75 (s, 3H), 1.18 (t, J = 8Hz, 3H). Step 4:

[0102] Compound 4 (182.0 g, 0.71 mol, 1.0 eq) was introduced into a 4 L double-walled reactor containing K₂CO₃ (197.1 g, 1.43 mol, 2.0 eq) and DMSO (550 mL). The suspension was heated to 45°C and dissolved. Five portions of N-Boc-4-iodomethylpiperidine (255.0 g, 0.78 mol, 1.1 eq) were added over one hour. The reaction mixture was stirred at 55°C for 4 h. The mixture was filtered to remove potassium carbonate, and the resulting solid was washed with ethanol. This yielded compound 5', which was not isolated or characterized.

[0103] The filtrate is returned to the reactor and water (1.2 L) is added, followed by potassium hydroxide (KOH) (184.0 g, 3.28 mol, 4.6 eq). The reaction mixture is heated to 80°C and stirred for 2 hours, then cooled to 40°C for 18 hours, and then heated again to 80°C. 800 mL of ethanol is removed by distillation. The reaction mixture is cooled to 40°C and 800 mL of EtOAc is added. The two phases are separated, and the aqueous phase is extracted with a 1:1 EtOAc / heptane mixture (3 x 500 mL). The organic phases are combined and washed with water (500 mL) and refluxed. 1.1 L of EtOAc is removed by distillation, and the product crystallizes. The suspension is cooled to 23°C over a period of 5 hours. The solid is collected by filtration and washed with heptane (500 mL) and dried under vacuum at 45°C for 18 hours. Compound 6 is obtained as a pale yellow solid.

[0104] Characterization of compound 6: 4-(3-(-4-amino-5-fluoro-2-methoxyphenyl)-3-oxopropyl)piperidine-1-tert-butyl carboxylate

[0105] NMR 1 H (DMSO-d6) 5 7.32 (d, J = 12 Hz, 1 H), 6.41 (d, J = 8 Hz, 1 H), 6.09 (s, 2H), 3.91 (m, 2H), 3.80 (s, 3H), 2.83 (t, J = 8 Hz, 2H), 2.67 (m, 2H), 1.63 (m, 2H), 1.46 (m, 2H), 1.43 (s, 9H), 1.41 (m, 1 H), 0.96 (m, 2H) Step 5:

[0106] Compound 6 (280.0 g, 0.73 mol, 1.0 eq) is introduced with 1.4 L of iProH into a 4 L double-walled reactor. The solid dissolves at 50°C. A mixture of 1.4 L of 6N hydrochloric acid in iProH (7.36 mol, 10.0 eq) is added, and the mixture is heated to 50°C with stirring for one hour. The mixture is cooled to 23°C, and the resulting solid is filtered and washed with diisopropyl ether (500 mL) and dried under vacuum at 45°C for 18 hours. Compound 7 is obtained as a white solid.

[0107] Characterization of compound 7 (4-(4-amino-5-fluoro-2-methoxyphenyl)-3-piperidin-4-yl)propan-1-one dihydrochloride)

[0108] NMR 1 H (DMSO-d6) 5 9.10 (s, 1 H), 8.81 (s, 1 H), 7.33 (d, J = 12 Hz, 1 H), 6.44 (d, J = 8 Hz, 1 H), 3.80 (s, 3H), 3.20 (m, 2H), 2.85 - 2.77 (m, 4H), 1 .77 (m, 2H), 1.51 - 1.48 (m, 3H), 1.34 (m, 2H)

[0109] Step 6:

[0110] Cycloheptane carboxylic acid (200.0 g, 1.41 mol, 1.0 eq) is introduced into a 500 mL round-bottom flask, 1.3 L of ethanol and 20 mL of sulfuric acid are added. The solution is heated to 80°C and stirred for 3 hours.

[0111] The reaction mixture is evaporated under reduced pressure until a volume of 300 mL is obtained. 500 mL of water and 500 mL of dichloromethane are added. The phases are separated, and the aqueous phase is extracted with 2 x 250 mL of dichloromethane. The organic phases are combined and washed with a saturated NaHCO3 solution (2 x 200 mL), water (200 mL), then dried over magnesium sulfate and evaporated until a colorless oil is obtained. The resulting oil is introduced into a 4 L double-walled reactor equipped with a stirrer. Toluene (2.5 L) is added. Sodium bis(2-methoxyethoxy)aluminohydride (460 µL of a 60% solution in 1.41 mol toluene, 1.06 eq) was heated at 23°C for 2 hours, maintaining the temperature below 35°C. The mixture was then heated to 90°C and stirred for 2 hours. The mixture was cooled to 50°C, and 70 mL of a 2N sodium hydroxide solution was added dropwise.The phases are separated, and the aqueous phase is extracted with 300 mL of toluene. The organic phases are combined and washed with 500 mL of 1 N sodium hydroxide solution, then with water. The organic phases are filtered and evaporated until a pale yellow liquid is obtained. The liquid is purified by distillation under reduced pressure (0.2 mbar, 70°C at the column head). Compound 11 (145.5 g, 85%) is obtained as a colorless oil.

[0112] Characterization of compound 11 (cycloheptane methanol)

[0113] NMR 1 H (DMSO-d6) 5 3.41 (d, J = 8 Hz, 2H), 2.07 - 2.01 (m, 1 H), 1.79 - 1.40 (m, 10H), 1.22 - 1.13 (m, 2H).

[0114] Step 7:

[0115] 145.0 g (1.13 mol, 1.0 eq) of compound 11 are introduced into a 4 L double-walled reactor equipped with a stirrer. 2.0 L of dichloromethane are then added. 314.6 mL (1.13 mol, 1.0 eq) of triethylamine are added, and the mixture is cooled to 10°C. A solution of methanesulfonyl chloride (87.5 mL, 2.26 mol, 2.0 eq) in 400 mL of dichloromethane is added slowly over 2 hours to maintain the temperature of the mixture below 30°C. The mixture is stirred at 23°C for 18 hours. 1 L of 1 N hydrochloric acid is added. The phases are separated, and the aqueous phase is extracted with 300 mL of dichloromethane. The organic phases are combined and washed with a saturated aqueous solution of NaHCC (500ml_), water (500mL) then dried on magnesium sulfate and evaporated under reduced pressure until a pale yellow liquid (230.6g, 99%) (compound 12) is obtained.

[0116] Characterization of compound 12: (cycloheptylmethyl methylsulfonate)

[0117] 1 H NMR (DMSO-d6) 54.01 (d, J = 8 Hz, 2H), 3.01 (s, 3H), 1.93 (m, 1 H), 1.79 - 1.40 (m, 10H), 1.30 - 1.23 (m, 2H)

[0118] Step 8:

[0119] 150.0 g (0.42 mol, 1.0 eq) of compound 7 is introduced into a 4 L double-walled reactor equipped with a stirrer. 1.4 L of acetonitrile is then added. 176.0 g of K₂CO₃ (1.27 mol, 3.0 eq) is added to the suspension. A solution of compound 12 (96.3 g, 0.47 mol, 1.1 eq) in 400 mL of acetonitrile is then added. The suspension is heated to 80°C and stirred for 24 hours. 8.75 g (0.04 mol, 0.1 eq) of compound 12 is added, and heating is continued for another 24 hours. 1.4 L of acetonitrile are evaporated under reduced pressure and the reaction mixture is cooled to 40°C. 1.2 L of ethyl acetate and 1.2 L of water are added and the phases are separated. The aqueous phase is extracted with a 1:1 ethyl acetate / heptane mixture (2 x 500 mL) and the organic phases are combined and washed with a saturated aqueous solution of NH4Cl (750 mL), 500 mL of a saturated aqueous solution of NaHCO3, and 1 L of water.1.2 L of solvent is evaporated from the organic phase under reduced pressure, resulting in crystallization. The suspension is cooled to 23°C and 1 L of heptane is added. The solid is filtered, washed with 500 mL of heptane, and dried at 50°C for 18 hours. The pale yellow solid obtained is Neocoprid, a weak base.

[0120] Characterization of Neocopride (1-(4-amino-5-fluoro-2-methoxyphenyl)-3-[1-

[0121] (cycloheptylmethyl)-4-piperidinyl]propan-1 -one) NMR 1H (DMSO-d6) 5 7.32 (d, J = 12 Hz, 1 H), 6.40 (d, J = 8 Hz, 1 H), 6.08 (s, 2H), 3.79 (s, 3H), 2.83 - 2.74 (m, 4H), 1.98 (m, 2H), 1.79- 1.31 (m, 17H), 1.15 - 1.02 (m, 5H) ; NMR 13C (CDCI3) 5 199.2; 157.1, 146.5 (d, J = 232 Hz), 139.9 (d, J = 15 Hz), 117.3 (d, J = 4 Hz), 117.1 (d, J = 20 Hz), 98.6 (d, J = 15 Hz), 66.6, 55.9, 54.5, 41.1, 36.6, 35.9, 33.0, 32.4, 31.4, 28.6, 26.6 ; HRMS [M + H]+ calculated for C23H36FN2O2 391 ,2761 , obtained 391 ,2763 ; IR (□, cm) 3435, 2920, 2851, 2801, 2762, 1626, 1606, 1520, 1466, 1426.

[0122] Stage 9:

[0123] Neocopride sel de fumarate du Neocopride

[0124] 130 g of Neocopride (0.33 mol, 1.0 eq) were introduced into a 4 L double-walled reactor. 910 mL of isopropyl alcohol was added. The suspension was heated to 55°C and became a solution. 320 mL of DIE (diisopropyl ether) was added. A solution of fumaric acid (40.5 g, 0.35 mol, 1.05 eq) in isopropyl alcohol (480 mL) and N-methyl-2-pyrrolidone (40 mL) was prepared separately and heated to 80°C until clear, then cooled to 50°C. The fumaric acid solution was added to the basic Neocopride solution, and the mixture was stirred at 45°C for 1 hour, resulting in crystallization. The reaction mixture is allowed to cool to 23°C for 18 hours. The solid is separated by filtration, washed with 250 mL of diisopropyl ether, and dried under vacuum at 50°C for 18 hours. Neocopride fumarate salt is obtained as a pale yellow solid (161.0 g, 95%).The resulting product is mixed with 1.0 L of diisopropyl ether and 10 g of a reaction mixture prepared as described above. The suspension is triturated at 23°C for one hour, and the solid is filtered, washed with 250 mL of diisopropyl ether, and dried under vacuum at 50°C for 18 hours. 171 g of Neocoprid fumarate salt are obtained. The Neocoprid:fumaric acid ratio is 1.0:1.0.

[0125] Characterization of Neocopride fumarate salt (fumarate salt of 1-(4-amino-5-fluoro-2-methoxyphenyl)-3-[1-(cycloheptylmethyl)-4-piperidinyl]propan-1-one) PF = 151-153°C; NMR 1H (DMSO-d6) 5 7.33 (d, J = 12 Hz, 1 H), 6.54 (s, 2H), 6.38 (d, J = 8 Hz, 1 H), 6.09 (s, 2H), 3.80 (s, 3H), 3.05 (m, 2H), 2.82 (t, J = 8 Hz, 2H), 2.37 (m, 2H), 2.24 (m, 2H), 1.75-1.26 (m, 18H), 1.11 (m, 2H); 13C NMR (DMSO-d6, 333K) 5 197.7, 166.8, 157.7, 145.2 (d, J = 232.0 Hz), 142.8 (d, J = 13.0 Hz), 134.6, 115.1 (d, J = 19.4 Hz), 114.7, 99.0 (d, J = 4.1 Hz), 65.3, 56.4, 54.0, 40.7, 36.2, 35.3, 32.7, 31.8, 31.4, 28.5, 26.4. 19F NMR (DMSO-d6) 5 -145.43; LCMS [M + H - C4H4O4I+ 390.97; IR (KBr, cm' 1 ) 3435, 3344, 3228, 2923, 2854, 1678, 1630, 1520, 1467, 1250.

[0126] Example 2: Physicochemical characterization of Neocoprid and Neocoprid fumarate salt

[0127] If Neocopride is left for 48 hours in water at room temperature, the pH of the water is equal to 7.7.

[0128] Neocoprid fumarate salt is soluble in water. The concentration of neocoprid fumarate salt in water is 1.3 mg / mL after 24 hours at room temperature and 1.4 mg / mL after 48 hours at room temperature. The pH measured after 48 hours is 3.9.

[0129] Thermodynamic solubility tests were performed. The solubility (in mg / mL) of the compounds was measured after 48 hours at room temperature in different buffers.

[0130] The results are summarized in Table 1 below:

[0131] Table 1

[0132] It was observed that the fumarate salt of Neocoprid is more soluble than Neocoprid itself at pH 4.5. The solubility of Neocoprid and its fumarate salt depends on the pH of the medium. Neither the salt nor the base changes the pH of the buffer after 48 hours.

[0133] Example 3: Preparation of the thermo-gelling solution containing the fumarate salt of Neocoprid

[0134] 178.57 mg of P407 (Kolliphor 407® (pharmaceutical grade of P407) marketed by BASF) and 11.90 mg of P188 (Kolliphor 188® marketed by BASF) are added to 1 mL of purified water and mixed for 3 hours at 4°C with stirring at 550 rpm. The resulting composition contains 15% P407 and 1% P188 by mass. These two poloxamers correspond to the aforementioned definitions of the first and second poloxamers.

[0135] A defined mass of neocoprid fumarate is added to the solution at 4°C, and the resulting mixture is stirred for 30 minutes at 550 rpm. The resulting composition is then sonicated using an ultrasonic bath and stirred again at 550 rpm at 4°C until a homogeneous solution is obtained. The resulting composition is liquid below 31°C and gels at 31°C. Its pH is 6.2, and its osmolality is 284 mOsm / kg. The osmolality is measured using the freezing point method with a type 13 autocal micro-osmometer (marketed by Roebling).

[0136] Biological effects of the thermo-gelling solution administered intranasally: Effects in a mouse model of Alzheimer's disease

[0137] All animal tests were conducted in accordance with Directive 2010 / 63 / EU under approval number A1301337. The rodents were housed in cages containing no more than four animals. Water and food were provided ad libitum. The cage floors were covered with absorbent paper, which was changed at least three times a week.

[0138] Preparation for contamination with the AP1-42 peptide

[0139] Aged C57BI6 mice were provided by the Janvier laboratory and housed on a reversed day / night cycle. After a 5-day acclimation period, 12 animals received a 2 pL injection of peptide A|31-42 and underwent surgery. The aforementioned peptide was prepared according to the protocol described by Calizot et al. in 2013. The peptide was dissolved in a vehicle at an initial concentration of 100 pM, and the mixture was stirred for 3 days at 37°C in the dark. The mice were anesthetized with isoflurane (4%) for the induction phase and placed on a stereotaxic frame with anesthesia maintained using isoflurane (2%) and oxygen. The peptide-containing preparation is injected bilaterally into the stratum oriens, stratum pyramidale and stratum radiatum of the CA1 area of ​​the hippocampus (at three different depths, coordinates relative to Bregma: anteroposterior (AP) -2 mm; mediolateral (ML) ± 1.8 mm, Dorsoventral (DV) - 1.9, -1.7, -1.5 mm). A total of 2 pL of the peptide-containing preparation or 2 pL of the vehicle (control) were injected bilaterally using a Hamilton syringe ("Elite Nanomite syringe pump"). Anesthesia and rectal temperature were checked every 5 minutes, and the mice were able to recover before being returned to their cages.

[0140] The tested compounds were administered after peptide injection. For group 3, administration was intranasal, into a single nostril. Table 2 below summarizes the different groups and treatments. All groups consisted of 12 animals, and the treatment lasted 18 days. The exemplified thermo-gelling composition was used for intranasal administration. Table 2

[0141] Example 4: Evaluation of short-term spatial memory (Y-maze) in the mouse model of Alzheimer's disease

[0142] Before the operation, a training session was conducted using a Y-maze for acclimatization. Seven days after the operation, the mice's short-term spatial memory was tested. The test was based on two trials.

[0143] 1) The mice can freely explore two branches of the Y-shaped maze for 5 minutes. The third branch is then closed. After 5 minutes, the mice rest for 3 minutes in an empty cage. The maze is cleaned with acetic acid to eliminate odors.

[0144] 2) The mice can freely explore the two arms of the Y for 5 minutes. Both tests are filmed with a camera using the Ethovision system (Noldus). The time spent by each mouse in each arm is determined automatically.

[0145] The results are shown in Fig. 1. Fig. 1 shows that mice infected with the peptide exhibit a deficit in short-term spatial memory due to reduced exploration time in the new branch of the Y-maze (79 s ± 7 s) compared to control mice (111 s ± 9 s). Treatment with 0.3 mg / kg / day of neocopride fumarate improves short-term spatial memory regardless of whether the route of administration is intranasal or intradermal. The exploration time in the new branch of mice treated with neocopride fumarate does not differ significantly from that of the control group (112 s ± 1 s intranasally and 111 s ± 9 s intradermally).

[0146] Example 5: Evaluation of long-term memory – Morris water maze in a mouse model of Alzheimer's disease

[0147] Eleven days after surgery, the long-term spatial memory of the mice was tested using the MWM maze. The maze was filled with water at 25°C. The water was made opaque with a non-toxic dye. A platform was hidden 2 cm below the water level. Signals were placed on the walls of the maze to aid spatial orientation. The test was repeated for five consecutive days.

[0148] Days 1 to 4: Training consists of four training sessions per day, with each mouse having four trials per session. Each mouse is allowed to freely explore the maze for one minute. At the end of each trial, the mouse is left on the platform for 10 seconds. The mouse is then dried and allowed to rest for at least 10 minutes before the next trial. The test session takes place on day 5. On day 5, the platform is removed. Each mouse is placed in the maze for one minute.

[0149] The trials were filmed with a camera using the Ethovision system (Noldus). The time spent by each mouse near the platform's location (during training sessions) was recorded. The results are shown in Fig. 2. This figure shows that mice infected with the peptide exhibited a deficit in long-term spatial memory (5.4 ± 0.2 s versus 7.9 ± 0.5 s for the control group). It was also observed that intranasal administration of neocopride fumarate salt improved cognitive performance, as evidenced by a longer time spent in the platform area.

[0150] Example 6: Immunostaining - mouse model of Alzheimer's disease. Afternoon of 18 ièmeOn day 7, histological examinations were performed on seven mice. The mice were anesthetized and euthanized by perfusion through the heart with cold PBS and paraformaldehyde (4%). The brains were dissected and fixed in 4% PFA overnight at 4°C. The brains were then placed in a TBS solution containing 30% by mass of sucrose at 4°C overnight. Immunostaining was performed on five brains per group, with two brains preserved. Coronal sections 20 µm thick, including the CA1 region of the hippocampus, were cut using a cryostat and separated by at least 160 µm.

[0151] For immunostaining, the floating sections were incubated in TBS with 0.25% bovine serum albumin, 0.3% Triton X-100, and 1% goat serum at room temperature. This incubation blocks nonspecific binding sites and permeabilizes the tissues. Seven brains per group were incubated overnight at room temperature with different antibodies.

[0152] - NeuN: polyclonal anti-NeuN antibody (1 / 1000), a marker of mature neurons

[0153] - AT100: mouse monoclonal antibody, anti-AT100 (1 / 200), a marker of hyperphosphorylated Tau protein

[0154] - GFAP: a rabbit polyclonal antibody against GFAP (1 / 200), an astrocyte marker and

[0155] - Iba1: a rat polyclonal antibody against lba1 (1 / 200) a marker of microglial cells.

[0156] Antibodies were detected using Alexa fluorine 488 anti-rabbit or anti-chicken IgG dye, Alexa fluorine 568 anti-mouse or anti-rabbit IgG dye, and Alexa fluorine 647 anti-rat IgG dye at a 1 / 500 dilution in TBS with 0.3% Triton X-100 and 1% goat serum. Images were acquired using confocal laser scanning. The number of NeuN-positive pyramidal neurons in the CA1 region, the presence of hyperphosphorylated Tau protein in these pyramidal neurons, the activation of lba-1-positive microglial cells in the CA1 region, and the activation of GFAP-positive astrocytes in the CA1 region were thus determined.

[0157] The results are shown in Figures 3 to 5. Figure 3 shows that the number of neurons in the CA1 region decreased following peptide injection (59 ± 9% of the number of neurons in the control group). Intranasal or intradermal administration of Neocopride fumarate salt protects neurons (84 ± 4% of the control group). Intranasal administration with the gel also reduces the daily dose by nearly 10 times (see Figure 3B).

[0158] Neuronal loss in mice contaminated with peptide A|3I-42 is associated with hyperphosphorylation of the tau protein in pyramidal neurons. The level of hyperphosphorylated tau protein is therefore reduced by intranasal administration of Neocopride fumarate, which is not the case for its intradermal administration or for the administration of Donepezyl (see Fig. 4).

[0159] Injection of A[3i-42] triggered significant gliosis 18 days post-surgery, as shown by the increased number of astrocytes (Fig. 5) and microglia activation (Fig. 6). In mice injected with A[3i-42] and treated with the vehicle, the number of Iba1+ cells increased significantly (161 ± 6% of the control). Treatment with intranasal neocoprid fumarate (0.3 mg / kg / day) and donepezil (1 mg / kg / day) significantly reduced the number of Iba1+ cells in peptide-infected mice (117 ± 10% and 136 ± 21% of the control, respectively). However, intradermal administration of 0.3 mg / kg / day of neocopride fumarate salt did not significantly inhibit microglial activation. Intradermal treatment with neocopride fumarate salt (0.3 mg / kg / day) also did not significantly alter the number of GFAP(+) cells (Fig. 6).However, intranasal treatment with neocopride fumarate (0.3 mg / kg / day) or donepezil (1 mg / kg / day) significantly reduced astrocyte counts in peptide-infected mice (147 ± 10% and 143 ± 15% of the control, respectively) compared to mice injected with AP1-42 peptide and treated with the vehicle alone (182 ± 9% of the control). Similarly, intranasal administration with the gel resulted in a greater reduction in astrocyte counts with a daily dose ten times lower (see Fig. 5B).

[0160] Example 7: Protein Analysis - PSD95

[0161] Up to 5 mice per group underwent protein analysis. The mice were deeply anesthetized. A complete blood sample was taken via cardiac puncture and transferred to polypropylene tubes containing heparin. The tubes were centrifuged at 12,000 rpm at 4°C, and the plasma was collected. The plasma (supernatant) was transferred to tubes and frozen at -80°C. Immediately after blood collection, the mice received a transcardiac infusion of cold PBS. The brain was then removed. The right hippocampus and right cortex were microdissected, frozen with dry ice, and maintained at -80°C. A Western blot assay was performed on the total level of the PSD95 protein. The results are shown in Fig. 7. PSD95 is a key synaptic protein, classically used to quantify synapses in the hippocampus. The level of PSD95 was assessed in the hippocampus of mice in each group using WES™.

[0162] Compared to control mice, mice injected with A|3i-42 showed a significant reduction in PSD95 levels (68 ± 9% of the control). Intranasal administration of neocopride fumarate (0.3 mg / kg / day) completely prevented the reduction of PSD95 in peptide-infected mice (100 ± 6% of the control). In contrast, intradermal administration of neocopride fumarate (0.3 mg / kg / day) had no significant effect on PSD95 levels in A|3i-42-injected mice. Although hippocampal PSD95 levels increased after treatment with 1 mg / kg / day of donepezil (92 ± 5% of the control), the difference compared to the peptide-injected mice was not significant.It is also observed that intranasal administration of Neocopride fumarate salt with the gel has the same effect as oral administration but with a dose 10 times lower for the intranasal route with the intranasal gel.

[0163] Overall, the results demonstrate that intranasal administration of Neocopride fumarate salt (0.3 mg / kg / day) can prevent the loss of synapses in the hippocampus of the current mouse model of Alzheimer's disease.

[0164] Evaluation of the effect of Neocopride fumarate salt in a mouse model of Parkinson's disease

[0165] All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and current European Union regulations (Directive 2010 / 63 / EU) under agreement number A1301337. Mice were housed in a restricted-access rodent facility, with a maximum of four animals per cage. Cage trays contained absorbent paper, which was inspected and changed at least three times weekly, and drinking water and food were provided ad libitum in each cage. The aged C57BL / 6 mice were supplied by Janvier Laboratories. Experiments began after one week of acclimation in the animal facility.

[0166] Stereotaxic injections of α-syn oligomers / protofibrils.

[0167] Human α-syn peptide (stored at 69 pM in water at -20°C) was reconstituted to 50 pM in 0.9% NaCl (final concentration). Mice were briefly surgically operated on and given 2.5 pL of α-syn solution. Mice were anesthetized with isoflurane (4% for induction) in an induction chamber coupled to an oxygen vaporizer and concentrator. Mice were placed on the stereotaxic frame. Anesthesia was maintained with isoflurane (2%) via a face mask coupled to the isoflurane vaporizer and concentrator. The skull was exposed, and holes were drilled. The α-syn preparation was injected bilaterally into the SNpc at the following coordinates: AP, -0.3 cm; ML, ±0.12 cm; DV, -0.45 cm. The depth of anesthesia and rectal temperature were checked every 5 minutes. After the operation, the mice were able to recover before being returned to the cage.

[0168] CBE administration began on the day of stereotaxic injections of the a-syn preparation. Conduritol B epoxide (CBE, a GBA inhibitor) was solubilized in the vehicle (0.9% NaCl). CBE administration is daily for 28 days.

[0169] The control animals received the vehicle (NaCl 0.9%). The stock solution of neocopride fumarate salt was provided by the study sponsor (note: the concentrations of the stock solutions ranged from 1.2 mg / ml to 1.34 mg / ml for intranasal (instillation) administration). The precise concentration was specified for each new aliquot of neocopride fumarate salt. The neocopride fumarate salt was diluted in the aforementioned intranasal gel composition. The neocopride fumarate salt (group 4) was solubilized in vehicle (0.9% NaCl) from powder on a weekly basis, aliquoted, and stored at 4°C protected from light for oral administration. Table 3 below summarizes the different groups and treatments. All groups consist of 12 mice. The treatment lasts 28 days for all groups. The gelling composition from Example 3 is used for administration. intranasal.

[0170] Table 3

[0171] Example 8: Effect on motor coordination: bar test in a mouse model of Parkinson's disease

[0172] During the third post-surgical week (week 3), the mice's motor coordination was assessed using the bar test. The test involves the mice's ability to cross a horizontal bar connected to a platform (18 mm in diameter, 60 cm long). The test requires good coordination between the forelimbs and hindlimbs, as well as good balance. The mice are placed at one end of the bar and must reach a platform located on the other side. Motor coordination was assessed after two training sessions, one and two days before the test. On the day of the test, three sessions were recorded using a video camera system. The time taken to cross the bar, the number of steps taken, and the number of missed steps were recorded. The results are shown in Fig. 8.

[0173] Figure 8 shows that intranasal administration of neocopride fumarate salt reduces the time taken to reach the platform, making it the same as for mice in the control group. It also reduces the number of missteps. Example 9: Effect on motor coordination: ladder test in a mouse model of Parkinson's disease

[0174] Four (4) weeks after surgery, the mice's motor coordination was assessed using the ladder test. For this behavioral test, the mice had to walk along a horizontal ladder, and the functions of the hind and forelimbs were evaluated.

[0175] The horizontal ladder consisted of a plexiglass corridor with removable plastic rungs inserted at regular intervals to create a floor. The purpose of the test was to assess foot "faults" (incorrect placement of limbs between two rungs) made by the animal while traversing the corridor. Animals with regular / skillful motor function place their paws on each rung, while subjects with coordination or motor impairments make faults. To automatically identify and anatomically attribute the faults, infrared detectors positioned along the corridor, coinciding with the position of each rung of the ladder, identified each fault.

[0176] Before the tests, a habituation session was conducted. The mice were asked to cross the corridor once and return to their cage for approximately 30 minutes. The test then consisted of two trials, performed approximately 30 minutes apart. The number of errors made by the fore and hind paws was recorded, as well as the total number of errors and the time required to cross the corridor.

[0177] The results are visible in Fig. 9. It can be seen that intranasal administration of Neocopride fumarate salt reduces both the time and the number of false steps, which becomes the same as for the control group.

[0178] Example 10: Immunostaining in a Murine Model of Parkinson's Disease. A total of 7 mice per group were assigned to histological analysis. The mice were deeply anesthetized and subjected to a transcardiac perfusion with cold PBS and cold PFA (4%). The brains were dissected and fixed in 4% PFA overnight at 4°C. The brains were then placed in a 30% sucrose solution in trisphosphate saline (TBS) at 4°C overnight. Immunostaining was performed with n=6-7 brains / group. Coronal sections, including the SNpc, with a thickness of 20 µm were obtained using a cryostat (7 sections per mouse, each separated by at least 140 µm). For immunostaining, the floating sections were incubated in TBS with 0.25% bovine serum albumin, 0.3% Triton X-100 and 1% goat serum, for 1 hour at room temperature.This incubation allowed the blocking of non-specific binding sites and the permeabilization of tissues. Seven (n=7) brain sections per animal were treated and incubated for 24 hours at room temperature with the selected antibodies:

[0179] - TH: chicken polyclonal antibody against tyrosine hydroxylase (1 / 1000).

[0180] - a-syn: Rabbit polyclonal antibody against alpha synuclein (1 / 200).

[0181] - lbA1: Rat polyclonal antibody against IBA1 (1 / 200).

[0182] These antibodies were detected with Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 568 anti-chicken IgY, and Alexa Fluor 648 anti-rat IgG, at a 1 / 500 dilution, in TBS with 0.25% donkey serum albumin, 0.3% Triton X-100, and 1% goat serum. Images were acquired using a confocal laser scanning microscope. The following parameters were studied:

[0183] Number of positive TH cells in the SNpc (indicate what this abbreviation means).

[0184] Activation of Iba1-positive microglial cells in the SNpc.

[0185] Aggregation of a-syn in TH neurons, in the SNpc.

[0186] The results are shown in Fig. 10. This figure shows that intranasal administration of Neocopride fumarate salt increases neuronal survival compared to the control (Graph A). It also shows that it reduces the area of ​​the zone infected by the α-synuclein protein (Graph B) and that it also reduces inflammation (Graph C).

[0187] Example 11: Evaluation of plasma and hippocampal concentrations of Neocopride fumarate salt administered intranasally, orally, and intranasally using the gelling composition described in [Example 3 above]. All experiments will be conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and current European Union regulations (Directive 2010 / 63 / EU). Agreement number: B1301337.

[0188] The mice will be housed in a restricted-access rodent facility. Four mice are housed per cage. Each cage tray contains absorbent paper, which is inspected and changed at least three times a week. Fresh drinking water and food are provided ad libitum in each cage.

[0189] Aged mice will be administered a 20-day oral (PO) or intranasal gavage and a single intravenous (IV) injection of Neocopride.

[0190] The groups are all made up of 4 animals. Sample collection is carried out 2 hours after the last dosage.

[0191] Table 4 below summarizes the different groups.

[0192] Table 4

[0193] Each compound was solubilized in the vehicle (0.9% NaCl) from the powder on a weekly basis, aliquoted, and stored at 4°C protected from light. Up to 5 mice per group were assigned to optional analyses (protein, biomarker, and neurotransmitter assays). Mice were deeply anesthetized, and whole blood was collected by cardiac puncture, transferred to polypropylene tubes containing anticoagulant heparin, and centrifuged at 12,000 rpm and 4°C for 10 minutes. Plasma was collected and frozen at -80°C. Immediately after blood collection, mice were perfused transcardially with cold PBS. The brain was harvested; the right substantia nigra and striatum, as well as the left hemisphere, were microdissected, frozen on dry ice, and stored at -80°C.

[0194] Sampling: CSF, plasma, and brain. At the end of the experiment (2 hours after the last dose), 4 mice from each group will be deeply anesthetized. Cerebrospinal fluid (CSF) will be collected by puncture of the cisterna magna. CSF samples are flash-frozen and stored at -80°C.

[0195] Immediately after CSF aspiration, blood is collected via cardiac puncture and transferred to polypropylene tubes containing anticoagulant heparin, stored at 4°C and centrifuged at 12,000 rpm and 4°C for 10 minutes. Plasma is collected in a new tube and frozen on dry ice, then stored at -80°C until analysis. Immediately after plasma collection, mice are perfused with cold PBS, and the brain is dissected to separate the two hemispheres. The two hippocampi are then placed in separate tubes, instantly frozen, and stored at -80°C until the next exposure assessment.

[0196] LC-MS analysis method

[0197] An LC-MS analytical method will be developed to detect and quantify Neocopride fumarate salt in plasma and brain (hippocampus) samples, based on the following characteristics:

[0198] Neocopride: molecular molar mass 390.54 g / mol - Neocopride fumarate salt; molecular molar mass: 506.62 g / mol.

[0199] Sample preparation

[0200] Plasma. Samples were transferred from -80°C to -20°C 1 hour prior to preparation. The plasma samples were then rapidly thawed and processed sequentially. Once completely thawed, the tube was centrifuged at 12100 g for 30 seconds. 60 pL of plasma were added to an Eppendorf tube. The plasma sample was then diluted 3-fold with 180 pL of acetonitrile, vortexed for 60 seconds, incubated at 4°C for 10 min, and centrifuged at 12100 g for 5 min. 60 pL of each supernatant were collected and diluted 4-fold with 240 pL of mobile phase (water / acetonitrile solution (95 / 5; v / v)). After vortexing for 10 seconds, a 20 pL aliquot of each sample was injected and analyzed using the LC-MS method described below. Brain sample. Frozen tissue samples were homogenized in PBS (g / mL; 1:3) using a pestle.Tissue homogenates will be centrifuged for 5 minutes at 15,000 rpm at 4°C. The supernatant will be collected. Standard solutions will be prepared by diluting the stock solution of neocoprid or neocoprid fumarate in a homogenate of male mouse brain (untreated with the compound being tested). A total of 6 standard solutions will be used to generate the calibration curve. Three quality control samples will be prepared independently of those used for the calculation curves.

[0201] Chromatographic conditions

[0202] The separation will be performed using the Thermo Ultimate 3000 (UHPLC), connected to a DAD detector and a Bruker UHR-QTOF (Ultra-High Resolution Qd-Time-Of-Flight) mass spectrometer with an ESI (Electron Spray Ionization) interface. The chromatographic and detection conditions applied for the separation and detection of the compound to be tested are described below: Solvents:

[0203] Mobile phase A: water with formic acid 0.1% (v / v).

[0204] Mobile phase B: Acetonitrile (CH3CN) with formic acid at 0.1% (v / v).

[0205] Column: Acclaim RSLC 120 C18 (150x2.1 mm, 2.2pm).

[0206] The elution gradient is as described in the following Table 5:

[0207] Table 5

[0208] Flow rate: 0.4 mL / min

[0209] Injection volume: 20.0 pL

[0210] Oven temperature: 40°C

[0211] Positive ionization mode parameters: offset plate end voltage: 500V; capillary voltage: 3500V; nebulizer: 30 PSI; dry gas flow: 8.0 L / min; dry temperature: 200 °C; m / z: 100-1000; spectral acquisition rate: 2 Hz.

[0212] The results are visible in Fig. 11. We can see from this figure that the composition of example 3 makes it possible to increase the concentration of Neocopride fumarate salt in the plasma and hippocampus.

[0213] Example 12: Charcoal Test

[0214] All experiments will be conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and current European Union regulations (Directive 2010 / 63 / EU). Mice will be housed in a restricted-access rodent facility. Animals will be housed four per cage. Each cage tray will contain absorbent paper, which will be inspected and changed at least three times per week. Fresh drinking water and food will be provided ad libitum in each cage.

[0215] Preparation of compounds to be tested and administration

[0216] The control animals received the vehicle (PBS).

[0217] Neocopride fumarate stock solutions have concentrations ranging from 1.2 mg / mL to 1.34 mg / mL for intranasal administration. Neocopride fumarate is administered intranasally as described in Example 3. The fumarate was solubilized from a powder in the vehicle (0.9% NaCl) and protected from light.

[0218] All groups consisted of 12 animals. The experiment lasted one day. Table 6 below summarizes the different groups.

[0219] Table 6

[0220] The molecules to be tested and the PBS are administered at t=0 to t=20 min, the animals are gagged with a mixture of 500pL of an aqueous solution containing 20mg / ml of activated charcoal with an aqueous solution containing 0.4% by mass of methylcellulose.

[0221] All animals are fasted the day before the experiment.

[0222] On the day of the experiment, the animals received the treatment corresponding to their group. Then, 30 minutes later, they received the charcoal preparation orally via gavage. 15 minutes after this second treatment, the animals were euthanized. The distance traveled by the charcoal was measured from the pylorus of the stomach.

[0223] The results are visible in Fig. 12. This figure shows that intranasal administration via the composition of example 3 reduces the passage of Neocopride fumarate salt into the intestinal tract, thus avoiding potential side effects in the intestines.

[0224] In all the examples above, all values ​​represent the mean ± SEM (standard error of the mean) per group of animals. The statistical analysis to compare the groups was performed using a one-way ANOVA followed by the test

[0225] Fisher's LSD was analyzed using GraphPad Prism version 9 software. *p<0.05 was considered significant. Outliers were identified using Grubb's test (alpha = 0.5) and by abnormal behavior during the test (e.g., freezing behavior during the test).

Claims

Demands 1. Pharmaceutical composition formulated for intranasal administration characterized in that it comprises, as active compound(s), at least one compound selected from the compound of formula (I) following, the fumarate salt of the compound of formula (I) following: and their mixtures, at least one pharmaceutically acceptable excipient, in that it is in the form of a gel and in that it is free from hydroxypropylmethylcellulose.

2. Composition according to claim 1, characterized in that it comprises as an excipient, at least one pharmaceutically acceptable polymer and at least one pharmaceutically acceptable liquid excipient.

3. Pharmaceutical composition according to claim 1 or 2, characterized in that it is in the form of a thermosensitive gel.

4. A composition according to any one of claims 2 and 3, characterized in that said pharmaceutically acceptable polymer is selected from polysaccharides, in particular starches, cellulose, chitin, alginates, chitosans, in particular chitosans having a molecular mass less than 150 kDa, hyaluronic acid, gellan gum, galactan, polypeptides, in particular collagen, elastin, reticulin, gluten, albumin, casein, zein, glycinin, β-conglycin, modified celluloses, in particular methyl cellulose, hydroxypropylcellulose, cyclodextrins, synthetic polymers, in particular poly(N-isopropylacrylamide, poly[2-(dimethylamino)ethyl methyl methacrylate, poly(vinylcaprolactam), polyvinyl methyl ether, carboxypolymethylenes, poly(ethylene glycol) / polyester(s) copolymers, poly(ethylene glycol) / poly(D) block copolymers,L-lactide-co-glycolide), poloxamers and mixtures of these polymers.

5. Composition according to any one of claims 2 to 4, characterized in that said polymer is amphiphilic.

6. Pharmaceutical composition according to any one of claims 1 to 5, characterized in that said pharmaceutically acceptable liquid excipient is selected from water, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, methyl tert-butyl ether, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, 2-methyltertrahydrofuran, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, triethylamine and their mixtures.

7. Composition according to any one of claims 2 to 6, characterized in that said liquid excipient is water.

8. Composition according to any one of claims 1 to 7, characterized in that it comprises as pharmaceutically acceptable polymer(s), a first poloxamer of the following formula H(OCH2CH2)x(OCH(CH3)CH2)y(OCH2CH2)xOH in which x=101 and y=56 and / or a second poloxamer of the aforementioned formula in which x is equal to or greater than 75 and equal to or less than 85 and y is equal to or greater than 25 and equal to or less than 30 and preferably x=80 and y=27.

9. Composition according to claim 8, characterized in that it comprises at least 5% by mass, preferably at least 10% by mass, in particular 15%, or at least 20% by mass of said first poloxamer and from 0% to 5% by mass of said second poloxamer, in particular, 0.5%; 1%; 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3% or 4% of said second poloxamer.

10. Composition according to claim 9, characterized in that it contains 15% by mass of said first poloxamer and 1% by mass of said second poloxamer.

11. A pharmaceutical composition according to any one of the preceding claims, characterized in that it further comprises at least one agent selected from preservatives, pH buffers, in particular saline pH buffers with a pH equal to or greater than 4.5 and less than or equal to 6.5, and chloride solutions. sodium chloride, magnesium chloride and potassium chloride, humectants and lubricants.

12. Pharmaceutical composition according to any one of claims 1 to 11, characterized in that it has an osmolality greater than or equal to 200mOsm / kg and less than or equal to 600mOsm / kg and in particular equal to 284 mOsm / kg.

13. Pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is free from liposomes containing the compound of formula (I), its fumarate salt or a mixture thereof.

14. Pharmaceutical composition according to any one of the preceding claims, characterized in that it is free of liposomes.

15. Pharmaceutical composition according to any one of claims 3 to 14, characterized in that it transforms into a gel at a temperature greater than or equal to 31 °C.

16. Nasal spray device, characterized in that it contains the composition according to any one of claims 1 to 15.

17. Device according to claim 16, characterized in that it is capable of forming droplets having a size equal to or greater than 30pm and less than or equal to 800pm.

18. Product selected from an ointment, a gel, a solution, a suspension, characterized in that it contains the pharmaceutical composition according to any one of claims 1 to 17.

19. Compound chosen from the compound of the following formula (I), the fumarate salt of the compound of the following formula (I): and their mixtures characterized in that it is in a form suitable for intranasal administration, for use in the treatment of a neurodegenerative disease, chosen in particular from Alzheimer's disease and Parkinson's disease.

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