Composition for promoting excitation of neural circuit

Abstract

The present invention relates to a composition for promoting the excitation of a neural circuit, the composition containing citicoline, a decomposition product of citicoline, or a salt of citicoline or the decomposition product. The present invention also relates to a composition for promoting the excitation of a neural circuit, the composition containing citicoline or a salt thereof, the composition having such a characteristic property that the excitation promoting effect thereof on the neural circuit wears off within 24 hours.

Description

Composition for promoting neural circuit excitation

[0001] The present invention relates to a composition for promoting excitation of neural circuits.

[0002] One of the main functions of the nervous system is the transmission of information, which occurs when signals are transmitted from one end of a neuron to the other and from one neuron to another. The transmission of a signal within a neuron is called conduction, and the transmission of a signal from one neuron to another is called transmission. When a neuron becomes excited, its membrane potential changes, and an electrical signal travels from the cell body along the axon, transmitting the signal to an adjacent neuron at the end of the axon. The axon is a long projection of a neuron, and the transmission of an electrical signal from the cell body along the axon is also called conduction. The connection where signals are transmitted between neurons is called a synapse. A gap exists in the synapse, and the electrical signal transmitted along the axon is transmitted to an adjacent neuron via a chemical substance called a neurotransmitter. If conduction or transmission in a neuron does not function normally, the function of the neural circuit is affected, resulting in various diseases or symptoms. Therefore, it is important to elucidate the mechanisms that control conduction or transmission in nerve cells and to search for materials that control the mechanisms.

[0003] Glutamatergic neurons are the most common excitatory neurons in the nervous system. They primarily release the neurotransmitter glutamate and transmit information through synaptic connections with other neurons. Glutamate is known to be involved in higher brain functions such as cognition, memory, and learning. However, excessive glutamate stimulation has been suggested to be neurotoxic, and therefore glutamate concentrations in the synaptic cleft are strictly regulated (Non-Patent Document 1). Previous studies have shown that ketamine, a glutamate neuromodulator, improves scores on depressed mood, anxiety, and hopelessness, including suicidal ideation, in patients with major depression and bipolar disorder. Similarly, riluzole, another glutamate neuromodulator, is known to suppress panic symptoms (Non-Patent Document 2). A link between glutaminergic synapses and schizophrenia has also been suggested (Non-Patent Document 3). Furthermore, glutaminergic neurons have been suggested to regulate REM sleep (Non-Patent Document 4).

[0004] Cytidine 5'-diphosphocholine, also known as citicoline, is a choline-containing compound composed of choline and cytidine, and is a nucleotide essential for cellular metabolism. Previous studies have shown that citicoline improves attention and motor function in healthy individuals (Patent Document 1). It is also known to have various biological effects, such as improving depressive symptoms in patients with depression and schizophrenia (Non-Patent Documents 5 and 6).

[0005] Japan Special Table No. 2017-520513

[0006] Neurochemistry International, 2009, Vol. 55, Issues 1-3, pp. 85-97; Mental Health Research, 2013, Vol. 59, pp. 23-32; Japanese Journal of Biological Psychiatry, 2019, Vol. 30, Issue 3, pp. 94-99; Journal of Japanese Biochemical Society, 2017, 89(6), pp. 911-916; Human Psychopharmacology, 2018, 33(4), e2662. Clinical Neuropharmacology, 2017, 40(1), pp. 1-5.

[0007] The effect of citicoline in promoting the excitation of neural circuits was previously unknown.

[0008] An object of the present invention is to provide a composition for promoting excitation of neural circuits and a method for promoting excitation of neural circuits.

[0009] The present inventors have discovered for the first time that by applying citicoline, a degradation product of citicoline, or a salt thereof to glutaminergic neurons, conduction in the cells is promoted, for example, the spike rate and firing rate in the neurons are increased, and have completed the present invention.

[0010] The present disclosure includes the following: (1) A composition for promoting excitation of a neural circuit, comprising citicoline, a degradation product of citicoline, or a salt thereof. (2) The composition according to (1) above, wherein the promotion of excitation of a neural circuit is caused by promoting neuronal conduction. (3) A composition for promoting neuronal conduction, comprising citicoline, a degradation product of citicoline, or a salt thereof. (4) The composition according to (1) or (2) above, wherein the promotion of excitation of a neural circuit is caused by at least one of an increase in the firing rate and the spike rate of a neural cell. (5) A composition for increasing at least one of the firing rate and the spike rate of a neural cell, comprising citicoline, a degradation product of citicoline, or a salt thereof. (6) The composition according to any one of (1) to (5) above, wherein the degradation product of citicoline is at least one of choline and cytidine. (7) The composition according to (2) to (5) above, wherein the neural cell is a glutamatergic neuron. (8) The composition according to any one of (1) to (7) above, which is used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, lethargy, and social withdrawal, or for the control of REM sleep. (9) A composition containing citicoline, a degradation product of citicoline, or a salt thereof, which is used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, lethargy, and social withdrawal, or for the control of REM sleep. (10) A composition for promoting excitation of neural circuits, which contains citicoline or a salt thereof, wherein the effect of promoting excitation of the neural circuits by the composition disappears within 24 hours. (11) The composition according to (10) above, which is used for the amelioration, prevention, or treatment of Huntington's disease or the control of epilepsy. (12) A composition containing citicoline or a salt thereof, which is used for ameliorating, preventing, or treating Huntington's disease, or controlling epilepsy. (13) The composition according to any one of (1) to (12) above, which is a food composition.(14) The composition according to any one of (1) to (12) above, which is a pharmaceutical composition or a quasi-drug composition. (15) A method for promoting excitation of neural circuits, comprising administering or ingesting citicoline, a degradation product of citicoline, or a salt thereof to a subject. (16) A method for promoting excitation of neural circuits, comprising administering or ingesting citicoline or a salt thereof to a subject, wherein the excitation-promoting effect of the administration or ingestion on the neural circuits disappears within 24 hours. (17) The method according to (15) above, which comprises ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, emotional blunting, lethargy, apathy, and social withdrawal, or regulating REM sleep. (18) A method for ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy, and social withdrawal, or for controlling REM sleep, comprising administering or having a subject ingest citicoline, a degradation product of citicoline, or a salt thereof. (19) The method according to (16) above, comprising ameliorating, preventing, or treating Huntington's disease, or controlling epilepsy. (20) A method for ameliorating, preventing, or treating Huntington's disease, or controlling epilepsy, comprising administering or having a subject ingest citicoline or a salt thereof. (21) Use of citicoline, a degradation product of citicoline, or a salt thereof for producing a composition for promoting excitation of neural circuits. (22) Use of citicoline, a degradation product of citicoline, or a salt thereof for producing a composition used for the prevention or treatment of at least one selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy, and social withdrawal, or for the control of REM sleep. (23) Use of citicoline or a salt thereof for producing a composition for promoting the excitation of neural circuits, wherein the excitation-promoting effect of the composition on the neural circuits disappears within 24 hours.(24) Use of citicoline or a salt thereof for producing a composition used for ameliorating, preventing, or treating Huntington's disease, or controlling epilepsy. (25) Use of citicoline, the citicoline degradation product, or a salt thereof for use as a composition for promoting excitation of neural circuits. (26) Use of citicoline, the citicoline degradation product, or a salt thereof for use as a method for preventing or treating at least one condition selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy, and social withdrawal, or as a composition used for controlling REM sleep. (27) Use of citicoline or a salt thereof for use as a composition for promoting excitation of neural circuits, wherein the effect of promoting excitation of the neural circuits by the composition disappears within 24 hours. (28) Use of citicoline or a salt thereof for use as a composition for ameliorating, preventing, or treating Huntington's disease, or controlling epilepsy. (29) A method for ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy, and social withdrawal, or for controlling REM sleep, comprising administering or having a subject ingest a composition containing citicoline, a degradation product of citicoline, or a salt thereof. (30) A method for ameliorating, preventing, or treating Huntington's disease or controlling epilepsy, comprising administering or having a subject ingest a composition containing citicoline or a salt thereof. (31) A method for promoting excitation of neural circuits, comprising administering or having a subject ingest a composition containing citicoline, a degradation product of citicoline, or a salt thereof. (32) A method for promoting excitation of neural circuits, comprising administering or having a subject ingest a composition containing citicoline or a salt thereof, wherein the effect of promoting excitation of the neural circuits by the administration or ingestion disappears within 24 hours. (33) The method or use according to any one of (15) to (32), which is a non-therapeutic method or use. (34) The method or use according to any one of (15), (16), (18), (20), (29) to (32), wherein the subject is a mammal, including a human.(35) The method or use according to any one of (15), (16), (18), (20), (29) to (32), wherein the subject is a healthy individual.

[0011] The composition of the present invention containing citicoline, the degradation product of citicoline, or a salt thereof as an active ingredient has an excitatory effect on neural circuits.

[0012] FIG. 1A is a portion of the abstract of a presentation given at the 2024 American Society for Nutrition (ASN) annual conference, "NUTRITION 2024." FIG. 1B is a portion of the abstract of a presentation given at the 2024 American Society for Nutrition (ASN) annual conference, "NUTRITION 2024." FIG. 2 is an overview of the procedures for the test example and reference example. FIG. 3 is a graph showing the experimental results of the spike rate of the PBS, citicoline, or choline chloride and cytidine addition group in the test example. FIG. 4 is a graph showing the experimental results of the firing rate of the PBS, citicoline, or choline chloride and cytidine addition group in the test example. FIG. 5 is an image diagram showing an overview of the procedure for the test example. FIG. 6 is a graph showing the spike rate and firing rate of the PBS or donepezil addition group in the test example. FIG. 7 shows the statistical significance between each addition group in the differential gene expression analysis of the reference example using a volcano plot. FIG. 8 shows the statistical significance between each addition group in the differential gene expression analysis of the reference example using a volcano plot. FIG. 9 is an image of the analysis roadmap of the test of the reference example. FIG. 10 is an image of the analysis roadmap of the test of the reference example. FIG. 11 is a diagram based on slides used in a presentation at the annual conference of the American Society for Nutrition (ASN) in 2024, "NUTRITION 2024." FIG. 12 is a slide used in a presentation at the annual conference of the American Society for Nutrition (ASN) in 2024, "NUTRITION 2024."

[0013] The present invention will be described in detail below, but these are examples of preferred embodiments and the present invention is not limited to these details.

[0014] In the present disclosure, the phrase "at least one selected from the group consisting of" encompasses one element constituting the group or all combinations that can be formed by two or more elements, and may be, for example, one of the elements constituting the group, or a combination of any two, three, four, five, six, seven, eight, nine or more elements constituting the group.

[0015] The "to" in a numerical range means a range that includes the numerical values ​​before and after it. For example, "0% by mass to 100% by mass" means a range that is 0% by mass or more and 100% by mass or less.

[0016] The composition of this embodiment includes a composition for promoting excitation of neural circuits, which contains citicoline, a degradation product of citicoline, or a salt thereof.

[0017] As used herein, a neural circuit refers to a pathway through which neurons are interconnected and transmit electrical signals. Neurons have "dendrites" that receive information and "axons" that output information. Neurons function by exchanging electrical signals with other neurons through connections called synapses; this network is called a neural circuit. Neural circuits are responsible for functions such as processing sensory information, motor control, memory, and learning. As used herein, "excitation of a neural circuit" refers to a state in which a neuron generates an action potential and transmits an electrical signal to an adjacent neuron via a synapse. Furthermore, "neuronal conduction" refers to a state in which a neuron generates an action potential and transmits an electrical signal to an adjacent neuron via a synapse. In other words, neural circuit excitation causes neuronal conduction, and these processes enable information transmission within the neural circuit, supporting the function of complex neural circuits.

[0018] The promotion of excitation of neural circuits can be caused, for example, by promoting neuronal conduction or by activating microglia, and is preferably caused by promoting neuronal conduction.

[0019] The composition of this embodiment also includes a composition for promoting nerve cell conduction, which contains citicoline, a degradation product of citicoline, or a salt thereof.

[0020] Promoting excitation of neural circuits can be caused by increasing at least one of the firing rate and spike rate of neurons, promoting synaptic plasticity, or inducing long-term potentiation (LTP), but is preferably caused by increasing at least one of the firing rate and spike rate of neurons.

[0021] The composition of this embodiment also includes a composition for increasing at least one of the firing rate and the spike rate of neurons, which contains citicoline, a degradation product of citicoline, or a salt thereof.

[0022] When neurons receive strong stimulation, they generate pulses called spikes (action potentials). In this specification, spike rate means the number of spikes per second, and is the average of all spikes recorded across all neurons measured.

[0023] Neuronal firing is a phenomenon in which the intracellular potential temporarily increases. When a neuron fires in its soma upon stimulation, the firing propagates like a wave along the axon and is transmitted to the next neuron via synaptic connections. In this specification, firing rate refers to the number of firings per second, and is the average number of firings recorded in all neurons measured.

[0024] For example, if a substance or method increases the spike rate and firing rate of neurons by 1.0% or more, it can be determined that the substance or method promotes excitation of neural circuits.

[0025] The spike rate and firing rate can be measured using a multi-channel recording system, and the detailed method is described in the Examples.

[0026] The neural circuit excitation-promoting effect of the composition of this embodiment is such that the spike rate and firing rate of neurons measured by the above method in the presence of the composition, compared to those in the absence of the composition, are preferably 1.01 times or more as a lower limit, and 4.0 times or less as an upper limit. The neural circuit excitation-promoting effect of the composition of this embodiment is such that the spike rate and firing rate of neurons measured by the above method in the presence of the composition, compared to those in the absence of the composition, are, for example, 1.01 to 4.0 times.

[0027] Examples of the nerve cells include glutamatergic nerve cells, GABAergic nerve cells, dopaminergic nerve cells, serotonergic nerve cells, and cholinergic nerve cells, with glutamatergic nerve cells being preferred.

[0028] Citicoline, the degradation product of citicoline, or their salts are effective in promoting the excitation of neural circuitry, so the composition containing citicoline, the degradation product of citicoline, or their salts can be used for the improvement, prevention or treatment of emotional disorders, or psychiatric disorders or their symptoms.Specifically, the composition of this embodiment can be used for the improvement, prevention or treatment of at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, apathy, blunted affect, lethargy, lethargy and social withdrawal, or for the control of REM sleep.In this embodiment, the composition used for the improvement, prevention or treatment of at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, apathy, blunted affect, lethargy, lethargy and social withdrawal, or for the control of REM sleep, can contain citicoline, the degradation product of citicoline, or their salts as an active ingredient.

[0029] The composition of this embodiment also includes a composition for promoting the excitation of neural circuit, which contains citicoline or its salt, and the effect of promoting the excitation of neural circuit caused by said composition disappears within 24 hours.When citicoline or its salt is administered or ingested, the excitation of neural circuit is promoted as described above, but because it suppresses excessive excitation of nerve cells by post-synaptic inhibition, calcium ion regulation and receptor desensitization etc., and prevents excitotoxicity, the effect of promoting the excitation of neural circuit disappears within 24 hours.

[0030] Taking advantage of these properties of citicoline or its salt, the composition containing citicoline or its salt can be used for improving, preventing or treating neurodegenerative diseases caused by excitotoxicity or their symptoms.Specifically, the composition containing citicoline or its salt of this embodiment can be used for improving, preventing or treating Huntington's disease, or controlling epilepsy.Huntington's disease and epilepsy are caused by overproduction of glutamate, but the neuroexcitatory effect of citicoline or its salt disappears within 24 hours, so it is considered to be effective for such uses.The composition used for improving, preventing or treating Huntington's disease or controlling epilepsy in this embodiment may contain citicoline or its salt as an active ingredient.

[0031] Citicoline is a choline-containing compound composed of choline and cytidine. Citicoline may be obtained by any production method. Examples of the production method of citicoline include chemical synthesis (Japanese Patent Publication No. 39-6541, Japanese Patent Publication No. 42-1384, Japanese Patent Publication No. 63-6558, etc.), enzymatic method using microbial cells such as yeast (Japanese Patent Publication No. 48-2358, Japanese Patent Publication No. 48-40757, Japanese Patent Publication No. 48-40758, Japanese Patent Publication No. 53-109996, Japanese Patent Publication No. 54-14593, Japanese Patent Publication No. 63-313594, etc.). Commercially available citicoline can also be used, for example, Cognizin (registered trademark) manufactured by Kyowa Hakko Bio Co., Ltd. or citicoline manufactured by Sigma-Aldrich.

[0032] When citicoline is hydrolyzed or decomposed by a method simulating in vivo metabolism, it becomes choline, cytidine, phosphocholine, cytidine triphosphate, uridine, etc. That is, in this specification, the decomposition product of citicoline includes choline, cytidine, phosphocholine, cytidine triphosphate, uridine, etc., and at least one of choline and cytidine derived from hydrolysis is preferred.

[0033] In this specification, salts of citicoline or decomposition products of citicoline include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, etc. Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, etc., and organic acid salts such as acetate, maleate, fumarate, citrate, malate, lactate, α-ketoglutarate, gluconate, caprylate, etc.

[0034] Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts, etc. Examples of ammonium salts include ammonium and tetramethylammonium salts.

[0035] Examples of organic amine addition salts include salts of morpholine or piperidine.

[0036] Examples of amino acid addition salts include salts of glycine, phenylalanine, lysine, aspartic acid, glutamic acid, etc. Among the above-mentioned citrulline salts, malate is preferably used, but other salts or a suitable combination of two or more salts may also be used.

[0037] In this specification, the composition of this embodiment can contain any other component in addition to citicoline, the decomposition product of citicoline or their salt.For example, the composition can be prepared by mixing the above-mentioned active ingredient with one or more pharmacologically acceptable carriers, and by any method well known in the technical field of pharmaceutical preparation.

[0038] The amount of citicoline, the decomposition product of citicoline or their salts in the composition of the present embodiment is not particularly limited, and can be any amount as long as it can achieve the effect of the present invention.For example, with respect to the total mass of the composition of the present embodiment, the mass of citicoline, the decomposition product of citicoline or their salts can be 1 mass% or more, 10 mass% or more, 20 mass% or more, 30 mass% or more, 40 mass% or more, 50 mass% or more, 60 mass% or more, 70 mass% or more, 80 mass% or more, 90 mass% or more, 95 mass% or more, 96 mass% or more, 97 mass% or more, 98 mass% or more, 99 mass% or more, 100 mass%, or less than 100 mass%, 99 mass% or less, 98 mass% or less, 97 mass% or less, 96 mass% or less.That is, with respect to the total mass of the composition of the present embodiment, the mass of citicoline, the decomposition product of citicoline or their salts can be 1 mass% to 100 mass%.

[0039] These lower and upper limits can be combined arbitrarily. For example, when the lower limit of the mass of citicoline, the decomposition product of citicoline, or a salt thereof relative to the total mass of the composition of the present embodiment is 1% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 10% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 20% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; When the lower limit is 30% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 40% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 50% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 60% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; When the lower limit is 70% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 80% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 90% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, or 97% by mass or less. or 96% by mass or less, and when the lower limit is 95% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 96% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; when the lower limit is 97% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less; and when the lower limit is 98% by mass or more, the upper limit is less than 100% by mass;It may be 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less, and when the lower limit is 99% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less, 97% by mass or less, or 96% by mass or less. The dosage can be set depending on factors such as the health condition of the person to be administered or ingested, the method of administration or ingestion, and combination with other agents.

[0040] The mass of citicoline, the degradation product of citicoline, or a salt thereof relative to the total mass of the composition of the present embodiment can be calculated by liquid chromatography.

[0041] The composition of the present embodiment is not particularly limited, but examples thereof include compositions for food products, pharmaceutical products, and quasi-drug products.

[0042] The composition according to one embodiment may be food, medicine or quasi-drug. Food, medicine or quasi-drug can be produced by conventional methods. The content of citicoline, the degradation product of citicoline, or their salts in food, medicine or quasi-drug is not particularly limited, and can be freely set according to purpose. In this specification, "food" also includes beverages, and can also be called "food and drink."

[0043] When the composition according to one embodiment is a food, a medicine, or a quasi-drug, the food, the medicine, or the quasi-drug may contain, in addition to citicoline, the degradation product of citicoline, or their salts, ingredients that can be commonly used in food, medicine, or the quasi-drug.The food, the medicine, or the quasi-drug according to one embodiment may contain bases, carriers, additives, etc. that are commonly used in food, medicine, or the quasi-drug.Additives are not particularly limited, but can include, for example, excipients, oils, powders, buffers, solubilizers, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, antiseptics, pigments, colorants, fragrances, glossing agents, gelling agents, alcohols, water-soluble polymers, film-forming agents, resins, etc.If necessary, two or more of the bases, carriers, and the various additives described above can be used in combination.

[0044] When the composition according to one embodiment is a food product, examples of the food product include health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, and foods with functional claims. Such food compositions are not particularly limited, but may be labeled as, for example, improving sleep quality, supporting good sleep, supporting a refreshing awakening, improving mental health, improving mental health, supporting mental health, helping to restore mental vitality, improving mental vitality and improving motor function, supporting cardiac health, improving immune function, maintaining digestive health, supporting healthy sexual function, contributing to blood sugar control, supporting the maintenance of a healthy weight, and supporting liver health. Furthermore, the composition according to one embodiment can also be used as a food additive.

[0045] Such foods are not particularly limited, but examples include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria drinks, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered drinks (powdered juice, powdered soup, powdered milk, etc.), concentrated drinks, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chewable tablets, tablets, chocolate, etc.), bread, cereal, etc. Furthermore, in the case of foods for specified health uses, foods with nutrient functions, foods with functional claims, etc., they may be in the form of capsules, granules, powders, syrups, lozenges, etc.

[0046] When the composition according to one embodiment is a drug or quasi-drug, the dosage form of the drug or quasi-drug is not particularly limited, but may be, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions / decoctions, capsules, syrups, liquids, elixirs, extracts, tinctures, liquid extracts, eye drops, and lozenges, and preferably oral preparations and eye drops.When the composition according to one embodiment contains citicoline, the degradation product of citicoline, or a salt thereof, the expected indications for the drug include anxiety disorder, panic disorder, schizophrenia, loss of motivation, apathy, emotional blunting, lethargy, lethargy, social withdrawal, REM sleep, etc.When the composition according to one embodiment contains citicoline or a salt thereof, the expected indications for the drug include Huntington's disease and epilepsy, etc.

[0047] Liquid preparations suitable for oral administration or ingestion, such as syrups, can be formulated by adding water, sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol, propylene glycol, etc., oils such as sesame oil, olive oil, soybean oil, etc., preservatives such as p-hydroxybenzoic acid esters, parahydroxybenzoic acid derivatives such as methyl parahydroxybenzoate, preservatives such as sodium benzoate, flavors such as strawberry flavor or peppermint, etc.

[0048] Furthermore, tablets, powders, granules, and other preparations suitable for oral administration or ingestion can be formulated by adding sugars such as lactose, sucrose, glucose, sucrose, mannitol, sorbitol, and other sugars; starches from potato, wheat, corn, and other sources; inorganic substances such as calcium carbonate, calcium sulfate, sodium bicarbonate, and sodium chloride; excipients such as plant powders such as crystalline cellulose, licorice powder, and gentian powder; disintegrants such as starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, sodium bicarbonate, and sodium alginate; lubricants such as magnesium stearate, talc, hydrogenated vegetable oil, macrogol, and silicone oil; binders such as polyvinyl alcohol, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carmellose, gelatin, and starch paste; surfactants such as fatty acid esters; and plasticizers such as glycerin.

[0049] Furthermore, additives generally used in foods, such as sweeteners, coloring agents, preservatives, thickening and stabilizing agents, antioxidants, color formers, bleaching agents, anti-mold agents, gum bases, bittering agents, enzymes, glazing agents, acidulants, seasonings, emulsifiers, strengthening agents, manufacturing agents, fragrances, or spice extracts, may be added to formulations suitable for oral ingestion or administration.

[0050] Furthermore, product forms or formulations suitable for oral ingestion or administration can be processed and manufactured into unit package forms per ingestion or administration, such as tablets, powders, granules, pills, suspensions, emulsions, infusions / decoctions, capsules, drinks, liquids, elixirs, extracts, tinctures, liquid extracts, or lozenges, depending on the ingestion or administration period, frequency of ingestion or administration, and dosage. For example, a "unit package form per single ingestion or administration" is a form in which the amount to be ingested or administered per time is predetermined, and a "unit package form per one week to three months" is a form containing the amount to be ingested or administered over one week to three months. Examples of unit package forms include forms in which a fixed amount is specified in a pack, package, bottle, or the like.

[0051] For example, in the case where the product form or formulation is a drink, the "unit package form per one intake or administration" may be 250 mg or more, 300 mg or more, or 400 mg or more, and 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less of citicoline, the decomposition product of citicoline, or a salt thereof suspended or dissolved in a drink, which is contained in a bottle or the like in a form that can be consumed in one intake or administration. That is, the amount of citicoline, the decomposition product of citicoline, or a salt thereof in the drink may be 250 mg to 1000 mg.

[0052] Regarding the amount of citicoline, the degradation product of citicoline, or a salt thereof in the drink, when the lower limit is 250 mg or more, the upper limit may be 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less; when the lower limit is 300 mg or more, the upper limit may be 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less; when the lower limit is 400 mg or more, the upper limit may be 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less.

[0053] For example, examples of "unit packaging form per 1 week to 3 months" include a form in which 35 to 1800 tablets are packaged for once-daily intake or administration, with a daily intake or dosage of 250 mg or more, 300 mg or more, or 400 mg or more, and 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less, and in the case of tablets containing 50 mg of citicoline, a degradation product of citicoline, or a salt thereof, are packaged.

[0054] The daily dosage or intake of the composition according to this embodiment is not particularly limited, and can be determined by the mass of, for example, citicoline, the citicoline degradation product, or a salt thereof.The daily dosage or intake of the composition according to this embodiment (for example, the daily dosage or intake of an adult) is not particularly limited, and can be, for example, 250 mg or more, 300 mg or more, or 400 mg or more, and 1050 mg or less, 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less, and these upper and lower limits can be arbitrarily combined.The daily dosage or intake of the composition according to this embodiment (for example, the daily dosage or intake of an adult) can be 250 mg to 1050 mg. The daily dosage or intake of the composition according to this embodiment (e.g., daily dosage or intake for an adult) may be, for example, when the lower limit is 250 mg or more, the upper limit may be 1050 mg or less, 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less; when the lower limit is 300 mg or more, the upper limit may be 1050 mg or less, 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less; and when the lower limit is 400 mg or more, the upper limit may be 1050 mg or less, 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less.

[0055] The composition of one embodiment is preferably administered or ingested into the body. The mode of administration or ingestion may be oral administration or ingestion or parenteral administration or ingestion, with oral administration or ingestion being preferred. The composition of one embodiment may be administered or ingested only once, or multiple times, or may be administered or ingested continuously or intermittently over a certain period of time. The composition of one embodiment may be administered or ingested, for example, 1 to 5 times a day, once every 2 days, once every 3 days, once every 4 days, or once a week for one week or more, two weeks or more, one month or more, three months or more, six months or more, one year or more, three years or more, five years or more, or ten years or more.

[0056] Generally, pharmaceutical compositions are expressed as "administered to a subject," and food compositions are expressed as "ingested by a subject." These expressions both mean that the composition is taken into the body of the subject, and are therefore synonymous. Therefore, the two expressions are interchangeable and there is no essential difference between them.

[0057] The composition according to this embodiment may be intended to be administered to or ingested by a human and / or a non-human mammal, and in one aspect, may be intended to be administered to or ingested by a human. In other words, the subject to which the composition according to this embodiment is administered or ingested may be a human and / or a non-human mammal, or may be a human.

[0058] The composition of this embodiment may be administered or ingested by a subject in need thereof, for example, by a subject in need of the composition (e.g., for promoting neural circuit excitation). The subject in need of the composition (e.g., for promoting neural circuit excitation) may be a healthy individual or an unhealthy individual. The subject in need of the composition (e.g., for promoting neural circuit excitation) is not particularly limited, but examples thereof include subjects suffering from anxiety disorder, panic disorder, schizophrenia, apathy, autism, emotional blunting, lethargy, lethargy, social withdrawal, REM sleep control, or sleep regulation. Furthermore, the subject in need of citicoline or a salt thereof (e.g., for promoting neural circuit excitation and for which the effect disappears within 24 hours) is not particularly limited, but examples thereof include subjects suffering from Huntington's disease or epilepsy.

[0059] In one aspect of the present invention, the composition for promoting the excitation of neural circuit can be the composition that contains citicoline, the degradation product of citicoline, or their salt as an active ingredient.In addition, in one aspect of the present invention, the composition for promoting the excitation of neural circuit, and the composition that the excitation-promoting effect of the composition on the neural circuit disappears within 24 hours, can be the composition that contains citicoline or its salt as an active ingredient.

[0060] The method of this embodiment includes a method for promoting excitation of neural circuits, which comprises administering or ingesting citicoline, a degradation product of citicoline, or a salt thereof to a subject.

[0061] The method of this embodiment also includes a method for preventing or treating at least one selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, emotional blunting, lethargy, apathy and social withdrawal, or for controlling REM sleep, which comprises administering or making a subject ingest citicoline, the degradation product of citicoline, or a salt thereof.

[0062] The dosage and frequency of administration or ingestion for promoting the excitation of neural circuits vary depending on the administration or ingestion form, the age and body weight of the patient, and the nature or severity of the symptoms to be treated.Usually, the daily dose of citicoline, the degradation product of citicoline, or a salt thereof for an adult is usually 250 mg or more, 300 mg or more, or 400 mg or more, and 1000 mg or less, 900 mg or less, 800 mg or less, 700 mg or less, 600 mg or less, or 500 mg or less, once or several times a day.The period of ingestion or administration is not particularly limited, but is usually 1 day to 1 year, preferably 1 week to 3 months.

[0063] The method of this embodiment also includes a method for promoting excitation of neural circuits, which comprises administering or ingesting citicoline or a salt thereof to a subject, and in which the effect of promoting excitation of the neural circuits caused by the administration or ingestion disappears within 24 hours.

[0064] The method of this embodiment also includes a method for ameliorating, preventing or treating Huntington's disease or controlling epilepsy, which comprises administering or ingesting citicoline or a salt thereof to a subject.

[0065] The subject in the above method can be an animal including a human, preferably a mammal including a human, i.e., a human and a non-human mammal, with a human being being particularly preferred. Examples of non-human mammals include a mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, horse, goat, or monkey.

[0066] The subject may be administered or ingested by citicoline, the degradation product of citicoline, or a salt thereof.More specifically, the subject may be the same as the subject that can be administered the composition of the present embodiment.

[0067] The use of this embodiment includes the use of citicoline, a degradation product of citicoline, or a salt thereof for producing a composition for promoting excitation of neural circuits.

[0068] The use of this embodiment includes use of citicoline, a degradation product of citicoline, or a salt thereof for producing a composition used for the prevention or treatment of at least one selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy, and social withdrawal, or for the control of REM sleep.

[0069] The use of this embodiment includes the use of citicoline or a salt thereof to produce a composition for promoting excitation of neural circuits, wherein the effect of the composition in promoting excitation of the neural circuits disappears within 24 hours.

[0070] The use of this embodiment includes the use of citicoline or a salt thereof for producing a composition used for ameliorating, preventing or treating Huntington's disease, or controlling epilepsy.

[0071] The use of this embodiment includes the use of citicoline, a degradation product of citicoline, or a salt thereof as a composition for promoting excitation of neural circuits.

[0072] The use of this embodiment includes use of citicoline, a degradation product of citicoline, or a salt thereof for use as a method for preventing or treating at least one selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, emotional blunting, lethargy, apathy, and social withdrawal, or for use as a composition used to control REM sleep.

[0073] The use of this embodiment includes the use of citicoline or a salt thereof as a composition for promoting excitation of neural circuits, wherein the effect of the composition in promoting excitation of the neural circuits disappears within 24 hours.

[0074] The uses of this embodiment include the use of citicoline or a salt thereof for use as a composition used for ameliorating, preventing or treating Huntington's disease, or controlling epilepsy.

[0075] The use of this embodiment relates to the use of citicoline, a citicoline degradation product, or a salt thereof for use in a therapeutic method for promoting neural circuit excitation. Specific aspects of citicoline, a citicoline degradation product, or a salt thereof according to this embodiment are as described above. In this disclosure, use in a "therapeutic" method refers to use in treating a subject's body. Use in a therapeutic method according to this disclosure may be, for example, for the purpose of or accompanied by medical treatment. Use in a therapeutic method according to this disclosure may involve, for example, a medical professional administering or having a substance ingested by a human or animal, or instructing a human or animal to administer or ingest a substance. Use in a therapeutic method according to this disclosure may be, for example, for therapeutic or preventive purposes, or may be for preventive purposes involving the administration or ingestion of a food composition, or may be for therapeutic purposes. Use in a therapeutic method according to this disclosure may be, for example, for an unhealthy individual.

[0076] The use of this embodiment relates to the use of citicoline, a citicoline degradation product, or a salt thereof for use in a non-therapeutic method for promoting neural circuit excitation. Specific aspects of citicoline, a citicoline degradation product, or a salt thereof according to this embodiment are as described above. In this disclosure, "non-therapeutic" use refers to the use of a substance that does not fall under the category of therapeutic use. The non-therapeutic use of this disclosure may be, for example, a use that is not intended for medical practice and / or does not involve medical practice. The non-therapeutic use of this disclosure may not involve, for example, a medical professional administering or having a substance ingested by a human or animal, and / or instructing a human or animal to administer or ingest a substance. The non-therapeutic use of this disclosure may be, for example, a use for preventive purposes or health promotion purposes, and may involve the administration or ingestion of a pharmaceutical composition or quasi-drug. The non-therapeutic use of this disclosure may be, for example, a use in a healthy individual.

[0077] The relationship between citicoline and its potential to improve cognitive function and prevent brain aging was presented at the annual conference of the American Society for Nutrition (ASN), "NUTRITION 2024." The conference abstract is shown in Figure 1A and Figure 1B.

[0078] The present invention will be specifically explained below by way of examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the invention.

[0079] First, we outline this study. [Investigating the Specific Molecular and Functional Effects of Citicoline on Brain Health] Objective: Cytidine 5'-diphosphocholine (citicoline) is an endogenous nucleotide that serves as a key intermediate in the synthesis of phosphatidylcholine (PC), a major phospholipid component of human cell membranes. Citicoline supplements, such as Cognizin®, are widely used as dietary supplements, and citicoline supplementation has clinically demonstrated beneficial effects on cognitive function and behavior. Despite these promising results, the biological mechanisms by which citicoline exerts its neuroprotective effects on brain function and aging are not fully understood. The objective of this study was to conduct an integrated multi-omics and predictive modeling analysis to characterize the effects of citicoline administration or ingestion on brain health.

[0080] Methods: Experiments were performed using a triple culture system consisting of human iPSC-derived cortical neurons, cortical astrocytes, and microglial cells (BrainXell Inc.). Cells were cultured on MEA plates to provide a consistent substrate. Triple cultures were grown on 48-well MEA plates (Axion Biosystems) for 3-4 weeks until maturation. Mature cells were treated with citicoline and activity recordings and RNA-seq experiments were performed. The effects of compounds on cellular activity were assessed using a microelectrode array.

[0081] Results: Multiparametric characterization of cellular activity was recorded following treatment with citicoline or control. Citicoline treatment significantly altered the dose-dependent parametric responses of cellular activity compared to control. Generation and investigation of RNA-seq citicoline treatment signatures, along with investigation of comprehensive network models of neuronal cells, revealed predictive effects of citicoline.

[0082] Conclusion: This is the first study to investigate the comprehensive molecular mechanisms of citicoline in an in vitro neuronal triple culture system using iPSC-derived cells. The findings identify the potential effects and relevance of citicoline supplementation on brain health. However, further analysis is needed to elucidate the molecular basis of its mechanism of action.

[0083] The outline of this study is also shown in Figure 2. This study was also conducted in Nucleic acids research. 2016;44(D1):D574-80, Nucleic acids research. 2013;41(10):e108, Methods Mol Biol. 2018;1711:243-59, Proceedings of the National Academy of Sciences of the United States of America. 2015;112(23):7285-90, BMC bioinformatics. 2009;10:161, Journal of the Royal Statistical Society: Series B (Methodological). 1995;57(1):289-300 and Rouillard AD, Gundersen GW, Fernandez NF, Wang Z, Monteiro CD, McDermott MG, et al. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database : the journal of biological databases and This work was also carried out with reference to curation. 2016;2016 etc.

[0084] [Test Example] 1. Study Content In this test example, triple cultures of iPSC-derived cortical glutamatergic neurons, cortical astrocytes, and microglia supplied by BrainXell, Inc. (Madison, Wisconsin, USA) were cultured in 48-well MEAs (Axion Biosystems, USA). After 3-4 weeks of culture to allow differentiation and maturation, baseline neural activity was recorded and compounds were added. Neural activity was tracked for 1 hour, and the final 30 minutes of neural activity was used for analysis. All data were normalized to baseline activity. The effects of the compounds described below on electrophysiological network activity were assessed using a microelectrode array. The compounds described below were cumulatively added to the same wells 2 and 4 hours after the initial addition, and neural activity was analyzed.

[0085] 2. Culture 2.1 Cell Culture Cortical neuron: astrocyte: microglia cell lines were mixed at a ratio of 3:1:1, and 80,000 (i.e., 80K) cells of cortical neurons, 27,000 (i.e., 27K) cells of astrocytes, and 27,000 (i.e., 27K) cells of microglia were added per well and cultured. Furthermore, microglia cells were added in vitro to each well on the 7th day of culture.

[0086]

[0087] 2.2 Human iPSC-derived neuronal cell culture procedure Pre-differentiated cells and cryopreserved cells were thawed according to the standard operating procedures (SOP) of the manufacturer (i.e., BrainXell, Inc.) and suspended in seeding medium. The cell number of this suspension was counted, activity was controlled, and the required concentration was adjusted. The required amount of seeding suspension was added dropwise to the center of each well. After leaving the wells for a certain period of time in an incubator, medium was added to the wells up to the desired volume. The cells were then stored in an appropriate CO ２ The cells were cultured at 37° C. The maintenance medium was replaced at necessary intervals.

[0088] 2.3 Details of the triple culture of iPSC-derived cortical glutamatergic neurons, cortical astrocytes, and cortical microglia The details of this triple culture are shown in the table below.

[0089]

[0090] 3. Addition 3.1 Compounds Compounds used in the study were stored at the temperature specified by the manufacturer. Compounds were dissolved, aliquoted, and stored at -20°C. Citicoline was provided by Kyowa Hakko Bio Co., Ltd. (Cognizin®, Tokyo, Japan).

[0091]

[0092] 3.2 Experimental Information Each compound was added to each well at a predetermined concentration as shown in the table below. Specifically, each compound was added cumulatively to the same well 2 hours (c2), 4 hours (c3), and 24 hours (24 h) after the initial addition (c1), to reach the concentrations shown in Table 5 below.

[0093]

[0094] Cytidine was not completely soluble at 1000 mM. The 500 mM cytidine solution was clear.

[0095] 3.3 Addition of Compounds Statistical processing was performed on the following samples.

[0096]

[0097] Dilutions were prepared fresh for each day of addition. Compound stock solutions were pipetted into a 48-well working plate, and using a multichannel pipette, 100 μL of medium was aspirated from each well, mixed with the compounds listed above, and returned to the respective wells. The entire process was carried out within 3-5 minutes.

[0098] First, as shown in c1 in the table above, 1 v / v% PBS, 3 μM donepezil, 500 μM citicoline, or 500 μM each of choline chloride and cytidine were added to the cells prepared as described above. The spike and firing rates of the neurons were measured 30 minutes after the addition of each compound. Two hours after the addition, citicoline or choline chloride and cytidine were added to the cells to a concentration shown in c2 in the table above. The spike and firing rates of the neurons were measured 30 minutes after the addition of each compound. Two hours after the addition of citicoline or choline chloride and cytidine to the cells to a concentration shown in c2 in the table above (four hours after the initial addition), citicoline or choline chloride and cytidine were added to the cells to a concentration shown in c3 in the table above. The spike and firing rates of the neurons were measured 30 minutes after the addition of each compound. 24 hours after the initial addition, the spike rate and firing rate of neurons in each condition group were measured.

[0099] 4. Electrophysiological Recording and Analysis 4.1 Multichannel Recording For electrophysiological recording, a multichannel recording system, MAESTRO, from Axion Biosystems Inc. (USA) was used. For extracellular recording, 48-well MEAs were placed in a recording station and maintained at 37°C. Recordings were performed without changing the medium. pH was adjusted with appropriate CO ２ The chamber was maintained at 7.4°C under a continuous stream of filtered, humidified air containing HCl. Action potentials, or "spikes," were recorded in spike trains and grouped into so-called firing events.

[0100] 4.2 Multiparametric Data Analysis. For each stable activity phase after compound addition described above, 204 spike train parameters normalized by native activity were calculated. Bursts were quantitatively described by direct spike train analysis using the program NPWaveX (NeuroProof GmbH, Rostock, Germany). Firing was defined by the onset and end of short spike events. High-content analysis of network activity patterns yielded multiparametric descriptions characterizing activity in four categories: general activity, burst structure, synchrony, and oscillatory behavior. From the spike trains, a total of 204 spike train parameters were determined describing activity in these four categories. All compound-induced network activity was normalized to the associated spontaneous native activity, which was set to 100% in each experiment. Values ​​were obtained from 60-second bins of data acquired over a 30-minute period after activity had stabilized for 30 minutes. Data reports included the compound-induced effects of all test compounds and vehicle control on a core set of parameters representing the four activity categories. These parameters provided the most information related to the impact of the test agent on overall network activity.

[0101] 4.3 Statistics Results (parameter values) are expressed as mean ± SEM (standard error of mean). Spike rate is the number of spikes per second and is the average of all recorded spikes measured. Firing rate means the number of firings per second and is the average of the values ​​in all neurons measured. Each experiment is compared pairwise; the statistical significance of the effect of the compound and the effect of the control (vehicle-induced effect) is evaluated by Student's t-test. P<0.05 is considered statistically significant. Significance levels of p<0.05 / 0.01 / 0.001 are indicated by one, two, or three symbols (* / ** / ***), respectively.

[0102] <Results> Multiparametric Characterization The results are shown in the table below. In the table below, values ​​are shown as percentages (%). The native baseline for each experiment was set to 100%. For asterisks, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Condition numbers c1, c2, c3, and 24h in the table below correspond to condition numbers c1, c2, c3, and 24h shown in the table above.

[0103]

[0104]

[0105]

[0106]

[0107] Graphs of spike rate and firing rate in samples with PBS, citicoline, or choline chloride and cytidine added are shown in Figures 3 and 4. An image of this experiment is also shown in Figure 5. Figures 3 and 4 show multiparameter characterization of neuronal activity after the addition of PBS, citicoline, or choline chloride + cytidine. The vertical axis of the graphs shows mean ± standard error (%) (Mean ± SEM (%)). The results of Student's t-test for PBS (*) and citicoline (+) are also shown.

[0108] Figure 6 also shows graphs of spike and firing rates in samples with PBS or donepezil added. This is a multiparameter characterization of neural activity after the addition of PBS or donepezil. The vertical axis of the graph shows mean ± standard error (%) (Mean ± SEM (%)). The results of a Student's t-test against PBS are also shown.

[0109] Thus, the addition of citicoline or choline chloride plus cytidine increased spike and firing rates in conditions c1, c2, and c3 compared with the addition of PBS. This increase was comparable to that observed with donepezil, which is known to enhance neuronal activity. Furthermore, the addition of citicoline reduced spike and firing rates at 24 h compared with the addition of PBS. Citicoline, which has the effect of preventing excitotoxicity, suppressed excessive neuronal excitation by postsynaptic inhibition, calcium ion modulation, and receptor desensitization, which may be one of the reasons for the decrease in spike and firing rates at 24 h.

[0110] [Reference Example] 1. Research Contents In this reference example, triple cultures of iPSC-derived cortical glutamatergic neurons, cortical astrocytes, and microglia supplied by BrainXell, Inc. (Madison, Wisconsin, USA) were cultured in 48-well MEAs (Axion Biosystems, USA). After 3-4 weeks of differentiation and maturation, baseline neural activity was recorded and compounds were added. Neural activity was tracked for 24 hours and used for RNA analysis.

[0111] The procedures from "2. Cultivation" to "3. Addition" were the same as those in the test example.

[0112] 4. Transcriptomics 4.1 Cell Lysis Before using the lysis solution, 10 μL of β-mercaptoethanol (1 v / v%) was added per mL of lysis buffer. In the above test example, cells were lysed in the lysis solution 24 hours after the addition of compound c1 (PBS 1 v / v%, citicoline 2 mM, choline + cytidine 2 mM, donepezil 30 μM). Specifically, after washing the wells once with PBS, 200 μL of lysis buffer containing 1 v / v% β-mercaptoethanol and the above cells was added to each well of a 48-well plate and pipetted up and down five times. Samples were frozen at -80°C. Materials: Aurum Total RNA Lysis Solution, Bio-Rad Catalog No. #7326802 β-Mercaptoethanol 14.2M, Bio-Rad Catalog No. #161-0710

[0113] 4.2 RNA-seq For RNA sequencing, the DNBSEQ and DNBSEQ eukaryotic strand-specific mRNA library types were used as the platform. A total of 35 samples were sequenced with a sequencing read length of PE100 (paired-end reads). Clean fastq purged quality score encoding was set to Phred+33. Post-sequencing data filtering included removing adapter sequences, contamination, and low-quality reads from the raw reads. The following table shows the statistics of the clean RNASeq data.

[0114]

[0115] 4.3 Comparative Transcriptomics (Bulk Tissue RNA-seq) RNA-seq samples with an RNA integrity number (RIN) of less than 6 were excluded from the analysis. Single-end fastq files of samples were aligned to the Homo Sapien Ensemble Reference genome (GRCh38.97) using STAR-RNAseq read aligner (Bioinformatics. 2013;29(1):15-21.), and accepted mapping reads were aggregated into gene-level counts. Genes were automatically filtered, and expression was normalized by sequencing depth using DESeq2 package ver 1.46.0 (Nucleic acids research. 2015;43(7):e47. Smyth GK. Differences between limma voom E values ​​and edgeR cpm values? 2014 [Available from: https: / / support.bioconductor.org / p / 59846 / #59917. Genome Biology volume 15, Article number: 550 (2014)). Differential gene expression analysis was performed to identify biomolecules that were differentially expressed (FDR<0.15) across a set of comparisons and define differentially expressed genes (DEGs).

[0116] 4.4 Differential Gene Expression Analysis. A comprehensive functional molecular enrichment analysis of citicoline-related transcriptomic signatures (DEGs) was performed using the Enrichr gene set library collection (Kuleshov, 2016). Fisher's exact test was used to assess the unexpectedly high overlap between each gene set and specific DEG sets, and to identify biological pathways modulated by citicoline treatment. Raw p-values ​​were corrected using the Benjamini-Hochberg adjustment for false positive rate (FDR). Enrichments with an FDR of less than 0.05 were classified as significantly enriched. Enriched categories were also output.

[0117] <Results> The mechanism of citicoline was elucidated using a brain cell co-culture system. The results of the above-mentioned differential gene expression analysis and differential gene expression analysis obtained by comparing the in vitro citicoline-added group with the in vitro PBS-added group are shown in the following table.

[0118]

[0119] The above table shows that the addition of citicoline reduces NCS-1 expression and TPCN1 expression (underlined). The accession numbers for the NCS-1 gene and TPCN1 gene are BC004856 and BC136795, respectively. Furthermore, in the differential gene expression analysis described above, volcano plots showing the statistical significance between the groups treated with PBS, citicoline, choline and cytidine, or donepezil are shown in Figures 7 and 8. The vertical axis represents the p-value on a logarithmic scale, and the horizontal axis represents the change in expression on a logarithmic scale.

[0120] The results of differential gene expression analysis of NCS-1 expression when PBS, citicoline, or choline and cytidine were added are shown in the table below. The table below shows that the addition of citicoline or donepezil reduced NCS-1 expression compared to the addition of PBS.

[0121]

[0122] The results of the key enrichment analysis of the consensus gene signature are shown in the table below. In the table, FDR stands for false discovery rate. This indicates that the addition of citicoline regulates the expression of the NCS-1 gene and TRPC4 gene, and that calcium concentration is regulated by regulating the expression of these genes.

[0123]

[0124] The analytical roadmap for this study is shown in Figures 9 and 10. Figures 11 and 12 are based on slides used in a presentation at the annual conference of the American Society for Nutrition (ASN), "NUTRITION 2024."

[0125] It is clear that a person skilled in the art can conceive of various modifications and alterations within the scope of the claims, and it is understood that these also naturally fall within the technical scope of the present invention. Furthermore, the components in the above-described embodiments may be combined in any manner without departing from the spirit of the invention.

[0126] This application is based on a U.S. provisional application (63 / 659,725) filed on June 13, 2024, the contents of which are incorporated herein by reference. Furthermore, all publications, patent publications, and published patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A composition for promoting excitation of neural circuits, comprising citicoline, a degradation product of said citicoline, or a salt thereof.

2. The composition according to claim 1, wherein promoting excitation of the neural circuit is caused by promoting conduction of nerve cells.

3. The composition of claim 1, wherein promoting excitation of the neural circuit is caused by at least one of an increase in the firing rate and spike rate of neurons.

4. The composition according to claim 1, wherein the degradation product of citicoline is at least one of choline and cytidine.

5. The composition of claim 2, wherein the neuronal cells are glutamatergic neuronal cells.

6. The composition according to claim 1, which is used for the improvement, prevention or treatment of at least one disease or symptom selected from the group consisting of anxiety disorder, panic disorder, schizophrenia, loss of motivation, autism, blunted affect, lethargy, apathy and social withdrawal, or for the control of REM sleep.

7. A composition for promoting excitation of neural circuits, comprising citicoline or a salt thereof, wherein the effect of said composition in promoting excitation of said neural circuits disappears within 24 hours.

8. The composition according to claim 7, which is used for the amelioration, prevention or treatment of Huntington's disease or the control of epilepsy.

9. The composition according to any one of claims 1 to 8, which is a food composition.

10. The composition according to any one of claims 1 to 8, which is a pharmaceutical composition or a quasi-drug composition.

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