Composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway

Abstract

The present invention relates to a composition for regulating at least one of an intracellular calcium concentration and a calcium-dependent signaling pathway, the composition containing citicoline or a salt thereof. The present invention also relates to a composition for regulating the expression of at least one of a Neuronal Calcium Sensor-1 (NCS-1) gene and a Two Pore Segment Channel 1 (TPCN1) gene, the composition containing citicoline or a salt thereof.　

Description

Composition for regulating intracellular calcium concentration and / or calcium-dependent signaling pathways

[0001] The present invention relates to a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathways.

[0002] Cells exhibit diverse physiological functions in response to various external stimuli, such as neurotransmitters, hormones, and growth factors. Calcium acts as a messenger for intracellular signal transduction, and regulation of intracellular calcium concentrations and calcium-dependent signal transduction pathways is important for the control of various physiological functions. Intracellular calcium plays an important role in the regulation of various physiological functions, such as muscle contraction, neurotransmitter release, synaptic plasticity, development, differentiation, and immunity.

[0003] The NCS-1 gene encodes Neuronal Calcium Sensor-1 (NCS-1), a calcium-binding protein that contributes to maintaining intracellular calcium homeostasis and regulating calcium-dependent signaling pathways. NCS-1 has been confirmed to be expressed in most nervous system cells in a variety of organisms, from yeast to humans, and has also been shown to be expressed in endocrine system, cardiac, smooth muscle, and gastrointestinal tissues. In humans, the highest expression level of NCS-1 is observed in the cerebral cortex. NCS-1 also regulates many different cellular functions, including exocytosis, neurite outgrowth, neuroprotection, axonal regeneration, and regulation of nuclear calcium levels, and is involved in biological development and various diseases. For example, NCS-1 is known to be upregulated in the prefrontal cortex of patients with bipolar disorder and schizophrenia. NCS-1 has also been proposed as a biomarker for aggressive breast cancer. Additionally, NCS-1 has been suggested to be associated with various diseases and symptoms such as autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED) (Non-Patent Document 1).

[0004] The TPCN1 gene encodes Two-Pore Channel 1 (TPCN1), an ion channel protein involved in regulating intracellular calcium concentration. TPCN1 has been confirmed to be expressed in many mammals, including humans, birds, and fish, and is widely expressed in many different tissues, including the brain, endocrine system, digestive system, urinary system, reproductive system, and muscle. This channel is present in the membranes of endosomes and lysosomes and is permeable to calcium and sodium, regulating intracellular signaling and organelle function. TPCN1 also regulates many different cellular functions, including intracellular vesicle transport, pancreatic B cell function, thermogenesis, nutrient sensing, endolysosomal transport, exocytosis, cytokinesis, fertilization and embryonic development, cell differentiation, angiogenesis, histamine-induced endothelial activation, smooth muscle contraction, autophagy, and skin pigmentation, and is involved in biological development and various diseases. Furthermore, since TPCN1 is required for viruses such as Ebola virus to enter cells, reducing its expression may suppress viral infection or spread. TPCN1 is also involved in the function of immune cells, and reducing its expression is expected to alter the immune response, for example, suppressing inflammatory responses and reducing the risk of autoimmune diseases. It has also been suggested that TPCN1 may be involved in the pathogenesis of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease (Non-Patent Document 2).

[0005] 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 3 and 4). However, its effects on maintaining intracellular calcium homeostasis or regulating calcium-dependent signaling pathways were unknown.

[0006] Japan Special Publication No. 2017-520513

[0007] Biochimica Biophysica Acta Molecular Cell Research, 2018, 1660-1667.Channels, 2017, 11(1), 20-33.Human Psychopharmacology, 2018, 33(4), e2662.Clinical Neuropharmacology, 2017, 40(1), p 1-5.

[0008] As described above, NCS-1 contributes to the maintenance of intracellular calcium homeostasis and the control of calcium-dependent signaling pathways and has a variety of physiological actions, and therefore, if the expression of NCS-1 can be appropriately controlled, it is believed that it will be possible to prevent, ameliorate, or treat various diseases and symptoms. Furthermore, since TPCN1 has a variety of physiological actions, if the expression of TPCN1 can be appropriately controlled, it is believed that it will be possible to prevent, ameliorate, or treat various diseases and symptoms suggested to be related to immunity, cancer, metabolism, viral infection, etc.

[0009] The present invention aims to provide a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway, a composition for regulating the expression of at least one of NCS-1 and TPCN1, a method for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway, a method for regulating the expression of at least one of NCS-1 and TPCN1, etc.

[0010] The present inventors have discovered for the first time that the expression of NCS-1 or TPCN1 can be regulated by applying citicoline to cells, i.e., that the intracellular calcium concentration and calcium-dependent signal transduction pathway can be regulated, and have thus completed the present invention.

[0011] The present disclosure includes the following: (1) A composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathways, comprising citicoline or a salt thereof. (2) A composition for regulating the expression of at least one of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene, comprising citicoline or a salt thereof. (3) The composition according to (2) above, wherein the regulating the expression is to reduce the expression of at least one of the NCS-1 gene and the TPCN1 gene. (4) A composition for use in ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED), comprising citicoline or a salt thereof. (5) The composition according to any one of (1) to (3) above, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED). (6) A composition containing citicoline or a salt thereof, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders. (7) The composition according to any one of (1) to (3) above, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders. (8) The composition according to any one of (1) to (3) above, which is used for the improvement, prevention, or treatment of an NCS-1 gene-related disease. (9) The composition according to (8) above, wherein the NCS-1 gene-related disease is at least one selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED).(10) A composition containing citicoline or a salt thereof, used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED). (11) The composition according to (8) above, wherein the NCS-1 gene-related disease is at least one selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED). (12) A composition containing citicoline or a salt thereof, used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (13) The composition according to any one of (1) to (3) above, used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (14) A composition containing citicoline or a salt thereof, which is used for ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of myocardial ischemia, fatty liver disease, type 2 diabetes, and obesity. (15) The composition according to any one of (1) to (3) above, which is used for ameliorating, preventing, or treating a disease or symptom of a TPCN1 gene-related disease. (16) The composition according to (15) above, wherein the TPCN1 gene-related disease is at least one selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (17) The composition according to (15) above, wherein the TPCN1 gene-related disease is at least one selected from the group consisting of myocardial ischemia, fatty liver disease, type 2 diabetes, and obesity. (18) The composition according to any one of (1) to (17) above, which is a food composition. (19) The composition according to any one of (1) to (17) above, which is a composition for use in a pharmaceutical or quasi-drug. (20) A method for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway, comprising administering or ingesting citicoline or a salt thereof to a subject.(21) A method for regulating expression of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene, comprising administering or ingesting citicoline or a salt thereof to a subject. (22) The composition according to (2) or (3) above, wherein the NCS-1 gene is a gene consisting of the nucleotide sequence shown by gene accession number BC004856, and the TPCN1 gene is a gene consisting of the nucleotide sequence shown by accession number BC136795. (23) The composition according to (2) or (3) above, wherein the NCS-1 gene is a gene consisting of the nucleotide sequence shown by SEQ ID NO: 1, and the TPCN1 gene is a gene consisting of the nucleotide sequence shown by SEQ ID NO: 2. (24) A method for improving, preventing or treating at least one disease or symptom selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis and erectile dysfunction (ED), comprising administering or having a subject ingest citicoline or a salt thereof. (25) The method according to (20) or (21) above, comprising improving, preventing or treating at least one disease or symptom selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis and erectile dysfunction (ED). (26) A method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED), which comprises administering or ingesting citicoline or a salt thereof to a subject. (27) The method according to (20) or (21) above, which comprises improving, preventing, or treating at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED). (28) A method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease, which comprises administering or ingesting citicoline or a salt thereof to a subject.(29) The method according to (20) or (21) above, which comprises ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (30) Use of citicoline or a salt thereof for producing a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway. (31) Use of citicoline or a salt thereof for producing a composition for regulating the expression of at least one of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene. (32) Use of citicoline or a salt thereof for producing a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED). (33) Use of citicoline or a salt thereof for producing a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED). (34) Use of citicoline or a salt thereof for producing a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (35) Use of citicoline or a salt thereof for use as a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway. (36) Use of citicoline or a salt thereof for use as a composition for regulating the expression of at least one of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene.(37) Use of citicoline or a salt thereof for use as a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED). (38) Use of citicoline or a salt thereof for use as a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED). (39) Use of citicoline or a salt thereof for use as a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease. (40) A method for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway, comprising administering to or having a subject ingest a composition containing citicoline or a salt thereof. (41) A method for regulating expression of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene, comprising administering to or having a subject ingest a composition containing citicoline or a salt thereof. (42) A method for ameliorating, preventing, or treating at least one disease or symptom selected from the group consisting of bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED), comprising administering to or having a subject ingest a composition containing citicoline or a salt thereof. (43) A method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED), comprising administering or having a subject ingest a composition containing citicoline or a salt thereof.(44) A method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease, comprising administering or having a subject ingest a composition containing citicoline or a salt thereof. (45) A method or use according to any one of (20), (21), and (24) to (44), which is a non-therapeutic method or use. (46) A method or use according to any one of (20), (21), (24), (26), (28), and (40) to (44), wherein the subject is a mammal including a human. (47) A method or use according to any one of (20), (21), (24), (26), (28), and (40) to (44), wherein the subject is a healthy individual.

[0012] The composition of the present invention containing citicoline or a salt thereof as an active ingredient has the effect of regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway. In addition, the composition containing citicoline or a salt thereof as an active ingredient has the effect of regulating the expression of at least one of Neuronal Calcium Sensor-1 (NCS-1) gene and Two Pore Segment Channel 1 (TPCN1) gene.

[0013] FIG. 1A is a portion of an abstract presented at the 2024 annual meeting of the American Society for Nutrition (ASN), "NUTRITION 2024." FIG. 1B is a portion of an abstract presented at the 2024 annual meeting of the American Society for Nutrition (ASN), "NUTRITION 2024." FIG. 2 is an overview of the procedures for the test example and the reference example. FIG. 3 is a volcano plot showing the statistical significance between each addition group in the differential gene expression analysis of the reference example. FIG. 4 is a volcano plot showing the statistical significance between each addition group in the differential gene expression analysis of the reference example. FIG. 5 is an image of the analytical roadmap for the test of the reference example. FIG. 6 is an image of the analytical roadmap for the test of the reference example. FIG. 7 is a graph showing the experimental results of the spike rate of the PBS, citicoline, or choline chloride and cytidine addition groups in the test example. FIG. 8 is a graph showing the experimental results of the firing rate of the PBS, citicoline, or choline chloride and cytidine addition groups in the test example. FIG. 9 is an image diagram showing an overview of the test example procedure. FIG. 10 is a graph of the spike rate and firing rate of the PBS or donepezil addition group in the test example. FIG. 11 is a diagram based on a slide used in a presentation at the 2024 American Society for Nutrition (ASN) annual conference, "NUTRITION 2024." FIG. 12 is a slide used in a presentation at the 2024 American Society for Nutrition (ASN) annual conference, "NUTRITION 2024."

[0014] 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.

[0015] 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.

[0016] 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 equal to or greater than 0% by mass and equal to or less than 100% by mass.

[0017] The composition of this embodiment includes a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signal transduction pathway, which contains citicoline or its salt.In this specification, regulating intracellular calcium concentration refers to reducing an increased intracellular calcium concentration, or increasing a decreased intracellular calcium concentration.Regulating intracellular calcium concentration is preferably a concentration reduction, from the viewpoint of maintaining normal calcium ion homeostasis.

[0018] As used herein, regulating a calcium-dependent signaling pathway refers to suppressing an activated calcium-dependent signaling pathway or activating a suppressed calcium-dependent signaling pathway. Regulating a calcium-dependent signaling pathway preferably involves suppressing the pathway, since excessive calcium signals can cause dysfunction of cardiomyocytes and neurons.

[0019] The composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway of this embodiment may contain citicoline or a salt thereof as an active ingredient.

[0020] In this specification, the cell is preferably a neuronal cell, and examples of the neuronal cell include glutamatergic neuronal cells, GABAergic neuronal cells, dopaminergic neuronal cells, serotonergic neuronal cells, and cholinergic neuronal cells, with glutamatergic neuronal cells being preferred.

[0021] Furthermore, the composition of the present embodiment includes a composition for regulating the expression of at least one of the Neuronal Calcium Sensor-1 (hereinafter also referred to as "NCS-1") gene and the Two Pore Segment Channel 1 (hereinafter also referred to as "TPCN1") gene, which contains citicoline or a salt thereof.

[0022] The NCS-1 gene is preferably derived from humans, and specifically may be a gene consisting of the nucleotide sequence represented by accession number BC004856. The TPCN1 gene is preferably derived from humans, and specifically may be a gene consisting of the nucleotide sequence represented by accession number BC136795. Furthermore, the NCS-1 gene may be a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1, and the TPCN1 gene may be a gene consisting of the nucleotide sequence represented by SEQ ID NO: 2. The composition for regulating the expression of at least one of the NCS-1 gene and the TPCN1 gene of this embodiment may contain citicoline or a salt thereof as an active ingredient.

[0023] Modulation of the expression of at least one of the NCS-1 gene and the TPCN1 gene includes decreasing or increasing the expression of these genes, and preferably decreasing the expression. Modulation of the expression of at least one of the NCS-1 gene and the TPCN1 gene, for example, decreasing the expression, can be confirmed by transcriptome analysis.

[0024] The reduction in expression of the NCS-1 gene and the TPCN1 gene in the composition of this embodiment is preferably such that the expression of the NCS-1 gene and the TPCN1 gene measured by the transcriptome analysis in the presence of the composition compared to the absence of the composition is at least 0.001-fold, at least 0.01-fold, or at least 0.10-fold as a lower limit, and preferably is less than 1.0-fold, at most 0.95-fold, or at most 0.90-fold as an upper limit. In other words, the expression of the NCS-1 gene and the TPCN1 gene may be at least 0.001-fold but less than 1.0-fold. These upper and lower limits can be combined in any desired manner. The reduction in expression of the NCS-1 gene and the TPCN1 gene in the composition of this embodiment means that, in the presence of the composition compared to the absence of the composition, the expression of the NCS-1 gene and the TPCN1 gene measured by the transcriptome analysis may be, for example, when the lower limit is 0.001-fold or more, the upper limit may be less than 1.0-fold, 0.95-fold or less, or 0.90-fold; when the lower limit is 0.01-fold or more, the upper limit may be less than 1.0-fold, 0.95-fold or less, or 0.90-fold; and when the lower limit is 0.10-fold or more, the upper limit may be less than 1.0-fold, 0.95-fold or less, or 0.90-fold.

[0025] As described above, the composition of this embodiment can regulate the expression of the NCS-1 gene and therefore can be used for the amelioration, prevention, or treatment of NCS-1 gene-related diseases or their symptoms. Specifically, the composition of this embodiment can be used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED). The composition of this embodiment can also be used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders. The composition of this embodiment can preferably be used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED).

[0026] Furthermore, as described above, the composition of this embodiment can regulate the expression of the TPCN1 gene, and therefore can be used for the amelioration, prevention, or treatment of a TPCN1 gene-related disease or a symptom thereof. Specifically, the composition of this embodiment can be used for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease, and preferably for the amelioration, prevention, or treatment of at least one disease or symptom selected from the group consisting of myocardial ischemia, fatty liver disease, type 2 diabetes, and obesity.

[0027] 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.

[0028] As used herein, salts of citicoline include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, etc. Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate, and organic acid salts such as acetate, maleate, fumarate, citrate, malate, lactate, α-ketoglutarate, gluconate, and caprylate.

[0029] 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.

[0030] Examples of organic amine addition salts include salts of morpholine or piperidine.

[0031] 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.

[0032] In this specification, the composition of this embodiment can contain any other component in addition to citicoline or its 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 preparations.

[0033] The amount of citicoline or its salt 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 or its salt 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 or its salt can be 1 mass% to 100 mass%.

[0034] These lower and upper limits can be combined arbitrarily. For example, when the lower limit of the mass 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 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 1 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, 97% by mass or less, or 96% by mass or less. 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; when the lower limit is 98% by mass or more, the upper limit may be less than 100% by mass, 99% by mass or less, 98% by mass or less;The dosage may be 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 the combination with other agents.

[0035] The mass of citicoline or a salt thereof relative to the total mass of the composition of the present embodiment can be calculated by liquid chromatography.

[0036] The composition of the present embodiment is not particularly limited, but examples thereof include compositions for food products, pharmaceutical products, and quasi-drug products.

[0037] The composition according to one embodiment may be a food, a medicine, or a quasi-drug. The food, the medicine, or the quasi-drug can be produced by a conventional method. The content of citicoline or its salt in the food, the medicine, or the quasi-drug is not particularly limited, and can be freely set according to the purpose. In this specification, "food" includes beverages, and can also be called "food and drink."

[0038] When the composition according to one embodiment is a food, a drug, or a quasi-drug, the food, drug, or quasi-drug may contain, in addition to citicoline or its salt, ingredients that are commonly used in food, drug, or quasi-drug.The food, drug, or quasi-drug according to one embodiment may contain bases, carriers, or additives that are commonly used in food, drug, or 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, or resins.Bases, carriers, and the various additives described above can be used in combination of two or more types as needed.

[0039] When the composition according to one embodiment is a food, the food may be a health food, a functional food, a nutritional composition, a nutritional supplement, a supplement, a health food, a food for specified health uses, a food with nutrient functions, or a food with functional claims. Such a food composition can be labeled, without being particularly limited thereto, as improving sleep quality, improving sleep quality, supporting good sleep, supporting refreshing awakening, improving mental health, improving mental health, supporting mental health, helping to improve memory, maintaining brain health, helping to reduce stress, supporting stress reduction, helping to restore mental vitality, improving mental vitality, helping mental stability, supporting mental stability, maintaining mental health, supporting social interaction, improving motor function, improving motor function, supporting heart health, maintaining heart health, helping to improve blood flow, supporting improved blood flow, maintaining cardiovascular health, supporting cardiovascular health, improving immune function, supporting the body's defenses, helping to maintain healthy cells, maintaining physical health, supporting physical health, supporting nervous function, maintaining digestive health, improving intestinal environment, supporting digestive function, supporting healthy sexual function, improving sexual health, contributing to blood sugar control, supporting maintaining a healthy weight, improving metabolic function, supporting maintaining a healthy lifestyle, helping the body's detoxification function, supporting liver health, etc. Furthermore, the composition according to one embodiment can also be used as a food additive.

[0040] 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.

[0041] When the composition according to an embodiment is a pharmaceutical or quasi-drug, the dosage form of the pharmaceutical or quasi-drug is not particularly limited, and may be, for example, a tablet, powder, granules, pill, suspension, emulsion, infusion / decoction, capsule, syrup, liquid, elixir, extract, tincture, liquid extract, eye drops, or troches, with oral preparations and eye drops being preferred. Possible indications for the pharmaceutical include NCS-1 gene-associated diseases such as bipolar disorder, schizophrenia, breast cancer, autism, depression, insomnia, Parkinson's disease, drug and nicotine addiction, neurological disorders, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED), as well as TPCN1 gene-associated diseases such as Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] For example, when the product form or formulation is a drink, the "unit package form per single ingestion or administration" may be a form in which a drink containing 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 or a salt thereof suspended or dissolved therein is contained in a bottle or the like in a single ingestion or administration. That is, the amount of citicoline or a salt thereof in the drink may be 250 mg to 1000 mg.

[0047] Regarding the amount 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; and 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.

[0048] 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 ingestion 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 or a salt thereof, the form includes a form in which 35 to 1800 tablets are packaged.

[0049] The daily dose or intake of the composition according to this embodiment is not particularly limited, and can be determined, for example, by the mass of citicoline or its salt.The daily dose or intake of the composition according to this embodiment (for example, the daily dose or intake for 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 dose or intake of the composition according to this embodiment (for example, the daily dose or intake for 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The composition of the present embodiment may be administered to or ingested by a subject in need thereof, for example, by a subject in need of the use of the composition (e.g., use in regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway). The subject in need of the use of the composition (e.g., use in regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway) may be either a healthy subject or an unhealthy subject. Subjects in need of the use of the composition (e.g., use to regulate at least one of intracellular calcium concentration and calcium-dependent signaling pathways) are not particularly limited, and examples include subjects suffering from an NCS-1 gene-related disease, such as subjects suffering from bipolar disorder, subjects suffering from schizophrenia, subjects suffering from breast cancer, subjects suffering from autism, subjects suffering from depression, subjects suffering from insomnia, subjects suffering from Parkinson's disease, subjects suffering from drug and nicotine addiction, subjects suffering from neurological disorders, subjects suffering from arrhythmia, subjects suffering from cardiac hypertrophy, subjects suffering from sepsis, subjects suffering from colitis, and subjects suffering from erectile dysfunction (ED); and subjects suffering from a TPCN1 gene-related disease, such as subjects suffering from Alzheimer's disease, subjects suffering from myocardial ischemia, subjects suffering from fatty liver disease, subjects suffering from type 2 diabetes, subjects suffering from obesity, and subjects suffering from Parkinson's disease.

[0054] In one embodiment of the present invention, the composition containing citicoline or a salt thereof may be a composition containing citicoline or a salt thereof as an active ingredient.

[0055] The method of this embodiment includes a method for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathway, which comprises administering or ingesting citicoline or a salt thereof to a subject. Also, the method of this embodiment includes a method for regulating expression of the NCS-1 gene and the TPCN1 gene, which comprises administering or ingesting citicoline or a salt thereof to a subject.

[0056] By administering or ingesting citicoline or a salt thereof to a subject, NCS-1 gene-related diseases or their symptoms can be improved, prevented, or treated. More specifically, the method of this embodiment includes a method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED), which comprises administering or ingesting citicoline or a salt thereof to a subject. Furthermore, the method of this embodiment also includes a method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders, which comprises administering or ingesting citicoline or a salt thereof to a subject. Preferably, the method of this embodiment also includes a method for improving, preventing, or treating at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED), which comprises administering or ingesting citicoline or a salt thereof to a subject.

[0057] By having a subject take citicoline or a salt thereof, it is possible to improve, prevent or treat TPCN1 gene-related diseases or their symptoms.More specifically, the method of this embodiment includes a method for improving, preventing or treating at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity and Parkinson's disease, which comprises administering or having a subject take citicoline or a salt thereof, and preferably includes a method for improving, preventing or treating at least one disease or symptom selected from the group consisting of myocardial ischemia, fatty liver disease, type 2 diabetes and obesity.

[0058] The dosage and frequency of administration or ingestion for regulating intracellular calcium concentration or calcium-dependent signaling pathways 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 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, administered or ingested 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.

[0059] 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.

[0060] The subject may be administered with or ingested by a subject in need of citicoline or a salt thereof. More specifically, the subject is the same as the subject to which the composition of this embodiment can be administered.

[0061] The use of this embodiment includes the use of citicoline or a salt thereof for producing a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathways.

[0062] The use of this embodiment includes the use of citicoline or a salt thereof for producing a composition for regulating the expression of at least one of the NCS-1 gene and the TPCN1 gene.

[0063] Uses of this embodiment include use of citicoline or a salt thereof to produce a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of NCS-1 gene-associated diseases, such as schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED). Uses of this embodiment also include use of citicoline or a salt thereof to produce a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of NCS-1 gene-associated diseases, such as bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders.

[0064] The use of this embodiment includes the use of citicoline or a salt thereof for producing a composition used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED).

[0065] The use of this embodiment includes the use of citicoline or a salt thereof for producing a composition used for improving, preventing, or treating at least one disease or symptom selected from the group consisting of TPCN1 gene-related diseases, such as Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease.

[0066] The use of this embodiment includes the use of citicoline or a salt thereof as a composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathways.

[0067] The use of this embodiment includes the use of citicoline or a salt thereof as a composition for regulating the expression of at least one of the NCS-1 gene and the TPCN1 gene.

[0068] The use of this embodiment can be comprised in the use of citicoline or its salt as a composition for improving, preventing or treating at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis and erectile dysfunction (ED).In addition, the use of this embodiment can be comprised in the use of citicoline or its salt as a composition for improving, preventing or treating at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction and neurological disorder.

[0069] The use of this embodiment includes the use of citicoline or a salt thereof as a composition for use in the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of breast cancer, autism, arrhythmia, cardiac hypertrophy, colitis, and erectile dysfunction (ED).

[0070] The use of this embodiment includes the use of citicoline or a salt thereof for use as a composition for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease.

[0071] The use of this embodiment relates to the use of citicoline or a salt thereof for use in a therapeutic method for promoting neural circuit excitation. Specific aspects of citicoline 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 the 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.

[0072] The use of this embodiment relates to the use of citicoline or a salt thereof for use in a non-therapeutic method for promoting neural circuit excitation. Specific aspects of citicoline 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 according to this disclosure may, for example, be for purposes other than medical practice and / or not involving medical practice. The non-therapeutic use according to this disclosure may not involve, for example, a medical professional administering or having a human or animal take the substance, and / or instructing a human or animal to administer or take the substance. The non-therapeutic use according to this disclosure may, for example, be for preventive or health promotion purposes, and may involve the administration or ingestion of a pharmaceutical composition or quasi-drug. The non-therapeutic use according to this disclosure may, for example, be for healthy individuals.

[0073] The relationship between citicoline and the potential for improving cognitive function and preventing brain aging was presented at the American Society for Nutrition (ASN) annual conference, "NUTRITION 2024." The conference abstract is shown in Figure 1A and Figure 1B.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] The outline of this study is also shown in Figure 2. Additionally, this study was 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.

[0080] [Test Example] 1. Research Contents 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 differentiation and maturation, baseline neural activity was recorded and the compounds described below were added. Neural activity was tracked for 24 hours and used for RNA analysis.

[0081] 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.

[0082]

[0083] 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.

[0084] 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.

[0085]

[0086] 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).

[0087]

[0088] 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.

[0089]

[0090] Cytidine was not completely soluble at 1000 mM. The 500 mM cytidine solution was clear.

[0091] 3.3 Addition of Compounds Statistical processing was performed on the following samples.

[0092]

[0093] 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.

[0094] 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.

[0095] 4. Transcriptomics 4.1 Cell Lysis Before use, 10 μL of β-mercaptoethanol (1% v / v) was added per mL of lysis buffer. Cells were lysed in the lysis buffer 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 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

[0096] 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.

[0097]

[0098] 4.3 Comparative Transcriptomics (Bulk Tissue RNA-seq) RNA-seq samples with an RNA integrity number (RIN) 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).

[0099] 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.

[0100] <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.

[0101]

[0102] 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 3 and 4. The vertical axis represents the p-value on a logarithmic scale, and the horizontal axis represents the change in expression on a logarithmic scale.

[0103] 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.

[0104]

[0105] 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.

[0106]

[0107] The analytical roadmap for the test conducted this time is shown in Figures 5 and 6.

[0108] [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 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 compounds on electrophysiological network activity were assessed using a microelectrode array. Compounds were cumulatively added to the same well 2 and 4 hours after the initial addition, and neural activity was analyzed.

[0109] The procedures from "2. Cultivation" to "3. Addition" were the same as those in the test example.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] <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.

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[0115]

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[0117]

[0118] Graphs of spike rate and firing rate in samples with PBS, citicoline, or choline chloride and cytidine added are shown in Figures 7 and 8. An image of this experiment is also shown in Figure 9. Figures 7 and 8 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.

[0119] Figure 10 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.

[0120] 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.

[0121] 11 and 12 show diagrams based on slides used in a presentation at the 2024 annual conference of the American Society for Nutrition (ASN), "NUTRITION 2024."

[0122] 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.

[0123] This application is based on a U.S. provisional application (63 / 659,740) 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.

[0124] SEQ ID NO: 1: Base sequence of human-derived NCS-1 gene SEQ ID NO: 2: Base sequence of human-derived TPCN1 gene

Claims

1. A composition for regulating at least one of intracellular calcium concentration and calcium-dependent signaling pathways, comprising citicoline or a salt thereof.

2. A composition for regulating the expression of at least one of the Neuronal Calcium Sensor-1 (NCS-1) gene and the Two Pore Segment Channel 1 (TPCN1) gene, comprising citicoline or a salt thereof.

3. The composition according to claim 2, wherein said regulating expression is reducing the expression of at least one of said NCS-1 gene and said TPCN1 gene.

4. The composition according to any one of claims 1 to 3, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of schizophrenia, breast cancer, autism, arrhythmia, cardiac hypertrophy, sepsis, colitis, and erectile dysfunction (ED).

5. The composition according to any one of claims 1 to 3, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of bipolar disorder, depression, insomnia, Parkinson's disease, drug and nicotine addiction, and neurological disorders.

6. The composition according to any one of claims 1 to 3, which is used for the improvement, prevention, or treatment of at least one disease or symptom selected from the group consisting of Alzheimer's disease, myocardial ischemia, fatty liver disease, type 2 diabetes, obesity, and Parkinson's disease.

7. The composition according to any one of claims 1 to 3, which is a food composition.

8. The composition according to any one of claims 1 to 3, which is a composition for use in pharmaceuticals or quasi-drugs.

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