KR102551880B1 - Composition for promoting the formation of neurosphere comprising Corticotropin-releasing hormone as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for stimulating the formation of nerve cell spheres containing corticotropin-releasing hormone (CRH) as an active ingredient. By showing the technique for forming spheres, it was confirmed that not only can neurospheres be formed simply, but also the number and size of neurospheres can be increased only by the addition of CRH. It can be secured in large numbers, and it is expected that neurodegenerative diseases can be effectively treated using the neural stem cells produced later.

Description

Composition for promoting the formation of neurosphere comprising Corticotropin-releasing hormone as an active ingredient}

The present invention relates to a composition for promoting the formation of neurospheres containing corticotropin-releasing hormone (CRH) as an active ingredient.

Neurodegenerative disorders refer to diseases that occur in the brain or spinal cord among degenerative diseases that occur with aging, and are caused by unknown causes or genetic defects or environmental factors. It refers to a disease caused by progressive structural loss and loss of function of specific brain cell populations in the brain and spinal cord. Nerve cell death or damage caused by degenerative nervous system disease causes problems in the formation or function of synapses that transmit information between brain nerve cells, which are most important for information transmission in the brain nervous system, and abnormal increase or decrease in electrical activity of brain nerves. known to cause Nerve cells in the brain and spinal cord have very diverse functions depending on their location, so damage to nerve cells in a specific area causes characteristic dysfunction, and shows very diverse clinical aspects depending on the form of these dysfunctions. These degenerative diseases of the nervous system include Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD), multiple sclerosis ( Multiple sclerosis: MS).

Meanwhile, for research on Alzheimer's disease, research on pathogenesis has been conducted after differentiation into neurons, which are mainly present in the forebrain. Differentiation into forebrain neurons is achieved by a neuronal differentiation method that proceeds without the addition of special external substances. methods are being developed, and both 'glutamatergic neurons' and 'GABAergic neurons' are widely used in research on the discovery of Alzheimer's pathogenesis. According to a recently published paper, it was confirmed that when patient-derived induced pluripotent stem cells were differentiated into 'GABAergic neurons', they were more resistant to cell death by amyloid beta. In other words, it was shown that various phenotypes can appear depending on the differentiation into specific neurons.

Because of their ability to differentiate into neurons, astrocytes, and oligodendrocytes, neural stem cells have been used as key materials in stem cell therapy to treat neurodegenerative diseases such as traumatic nervous system damage or Alzheimer's disease. In general, when neural stem cells are cultured in a floating state, a neural cell sphere, which is a cluster of thousands of neural stem cells, is formed from one neural stem cell after about 7 days (see FIG. 1). That is, the number of neural stem cells can be increased through the culture of neural cell spheres.

Based on this fact, several approaches have been developed to increase the growth of neuronal cells. Representatively, there is a method to improve the formation of neurospheres through genetic manipulation using retroviruses or plasmids. This method is not only difficult, but also genetically engineered neural stem cells have the possibility of changing into cancer cells in the body, so they are safe in clinical practice. had difficulty obtaining Another method is to treat cells with a combination of compounds or growth factors, but it was very important and difficult to discover new efficient compounds and growth factors.

On the other hand, it has not been known whether corticotropin-releasing hormone (CRH) is effective in forming neural cell spheres from neural stem cells.

Yin L, Hu Q (2014). "CYP17 inhibitors-abiraterone, C17,20-lyase inhibitors and multi-targeting agents". Nat Rev Urol. 11 (1): 32-42.

As a result of diligent efforts to find an efficient and economical method for forming neural cell spheres from neural stem cells, the inventors of the present invention confirmed that the corticotropin-releasing hormone of the present invention can be processed to form nerve cell spheres from neural stem cells. , completed the present invention.

Accordingly, an object of the present invention is to provide a composition for forming neurospheres containing corticotropin-releasing hormone (CRH) as an active ingredient.

Another object of the present invention is to provide a method for producing neuronal cells, comprising the step of culturing the composition with stem cells.

Another object of the present invention is to provide a kit for forming neurospheres containing corticotropin-releasing hormone (CRH) as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising corticotropin-releasing hormone (CRH) as an active ingredient.

Another object of the present invention is to provide a cell therapy agent for treating neurodegenerative diseases comprising the neuronal cells prepared by the above method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a composition for forming neurospheres containing corticotropin-releasing hormone (CRH) as an active ingredient.

In addition, the present invention provides a method for producing neuronal cells, comprising culturing the composition with stem cells.

In addition, the present invention provides a kit for forming neurospheres containing corticotropin-releasing hormone (CRH) as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising corticotropin-releasing hormone (CRH) as an active ingredient.

In one embodiment of the present invention, the CRH can promote the growth of neural cell cells from neural stem cells, but is not limited thereto.

In another embodiment of the present invention, the composition may be a composition for adding a medium, but is not limited thereto.

In another embodiment of the present invention, the CRH may be included in a concentration of 0.1 to 100 μM compared to the total composition, but is not limited thereto.

In another embodiment of the present invention, the stem cells may be neural stem cells, but are not limited thereto.

In another embodiment of the present invention, the culture may be performed for 3 to 20 days, but is not limited thereto.

In another embodiment of the present invention, the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander's disease, Alper's disease, vasodilatory ataxia, Batten's disease, bovine spongiform encephalopathy (BSE), Canavan disease, cortical basal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal degeneration, Huntington's disease, Kennedy's disease, Crab's disease, Lyme disease, Machado-Josef disease, multiple sclerosis, multiple systemic atrophy , neuroelectrocytosis, Niemann-Pick disease, Parkinson disease, Pick disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinolytic sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord , spinal cord syphilis, Tay-Sachs disease, toxic encephalopathy, dissemable spongiform encephalopathy, and unstable hedgehog syndrome, but may be one or more selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a method for treating a neurodegenerative disease comprising administering to a subject a composition containing adrenocorticotropic hormone-releasing hormone as an active ingredient.

In addition, the present invention provides a use of a composition containing adrenocorticotropic hormone-releasing hormone as an active ingredient for treating neurodegenerative diseases.

In addition, the present invention provides the use of adrenocorticotropic hormone-releasing hormone for producing a drug used for neurodegenerative diseases.

The present inventors show a technique for forming neural cell cells from neural stem cells using corticotropin-releasing hormone (CRH), so that not only can the nerve cell cells be formed simply, but also the number of nerve cell cells can be increased only by the addition of CRH. and size can be increased, it is expected that a large number of neuronal cells can be obtained using CRH, and that neurodegenerative diseases can be effectively treated using the neural stem cells produced later.

1 schematically illustrates a protocol for forming neural cell spheres, which are neural stem cell clusters, through neural stem cell culture.
FIG. 2 shows that when the neural stem cells were cultured in a suspension culture in a neural stem cell culture medium to which CRH was added, the number of neural cell cells nearly doubled compared to the negative control group.
3 shows that the number of neural cell spheres having a diameter of 50 μm or more increased significantly when neural stem cells were cultured in a suspension culture in a neural stem cell culture medium to which CRH was added.

Terms, technologies, and the like used in this specification are used in the meaning generally used in the technical field to which the present invention belongs unless there is a special limitation. In addition, all documents mentioned in this specification are included in this specification as documents for explaining the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, "corticotropin-releasing hormone" is named corticoliberin, CRH, CRF, etc., and is a peptide hormone involved in stress response. A releasing hormone belonging to the corticotropin releasing factor family, which in humans is encoded by the CRH gene. Its main function is to stimulate the pituitary synthesis of ACTH as part of the HPA axis. CRH is a 41 amino acid peptide derived from 196-amino acid preprohormone, and is known to be secreted from the periventricular nucleus (PVN) of the hypothalamus in response to stress.

The CRH of the present invention may include or consist of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, the corticotropin-releasing hormone is 0.1 to 100 μM, 0.1 to 90 μM, 0.1 to 80 μM, 0.1 to 70 μM, 0.1 to 60 μM, 0.1 to 55 μM, 0.1 to 50 μM in the composition of the present invention. μM, 0.1 to 45 μM, 0.1 to 40 μM, 0.1 to 35 μM, 0.1 to 30 μM, 0.1 to 25 μM, 0.1 to 25 μM, 0.1 to 23 μM, 0.1 to 20 μM, 0.1 to 15 μM, 0.1 to 10 0.1 to 9 μM, 0.1 to 8 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1.5 μM, or about 1 It may be included at a concentration of μM, but is not limited thereto.

In the present invention, the corticotropin-releasing hormone may be included in the composition of the present invention at a concentration of 0.1 to 10 μM, but is not limited thereto.

In the present invention, the term "stem cell" is not particularly limited as long as it is a cell in an undifferentiated state and has the ability to proliferate indefinitely and form neurospheres. Specifically, stem cells include, but are not limited to, embryonic stem cells, adult stem cells, embryonic reproductive stem cells, embryonic tumor stem cells, and the like.

In the present invention, “stem cells” may be neural stem cells, but are not limited thereto. The neural stem cells are primitive cells mainly present in the nervous system, and have the ability to differentiate into various neuronal phenotypes and self-renewal that continues to proliferate in an immature, undifferentiated state. The neural stem cells exist in various anatomical regions throughout the fetal nervous system of mammals, including humans, and neural stem cells exist in the nervous system of adults as well as fetuses. create new neurons The term "nerve cell" refers to a cell belonging to the neuronal cell lineage, including neuronal cells (eg, unipolar, bipolar, and multipolar neurons) and glial cells (oligodendrocytes, Schwann cells, astrocytes, and microglia).

In the present invention, “neuronocyte” may have a diameter of 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, but is not limited thereto.

In the present invention, the neurosphere is 20 to 200 μm, 20 to 150 μm, 20 to 100 μm, 20 to 90 μm, 20 to 80 μm, 20 to 70 μm, 30 to 100 μm, 30 to 90 μm, 30 to 90 μm It may have a diameter size of 80 μm, 40 to 100 μm, 40 to 90 μm, 40 to 80 μm, 50 to 200 μm, 50 to 150 μm, 50 to 100 μm, or 50 to 90 μm, but is not limited thereto no.

In the present invention, neural cell spheres are large cell clusters in which hundreds to thousands of neural stem cells or nerve cells are aggregated, and are significantly different in size from single cells such as neural stem cells or nerve cells.

In addition, the present invention provides a method for producing neuronal cells, comprising culturing the composition with stem cells.

In the present invention, the culture period is 3 to 20 days, 3 to 15 days, 4 to 13 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 5 to 8 days, 6 to 8 days or about 7 days It may be, but is not limited thereto.

The term 'medium' used in the present invention refers to a medium containing essential elements such as sugar, amino acids, various nutrients, serum, growth factors, and minerals for the formation of neuronal cells in vitro. It refers to a mixture, and in particular, the medium of the present invention is a medium for increasing the number and/or size of neuronal cells. The 'cultivation' is meant to include the formation of neuronal cells, that is, an increase in the number or size of neuronal cells.

The term "medium" of the present invention can be used without limitation a basal medium known in the art. The basal medium may be artificially synthesized, and commercially produced mediums include, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10 , F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium, but are not limited thereto.

In addition, the culture medium of the present invention may further include a nutrient mixture. The nutrient mixture is a mixture containing various amino acids, vitamins, inorganic salts, etc. commonly used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, etc., or commercially prepared nutrient mixtures may be used. Commercially prepared nutrient mixtures include, for example, Glutamax (Glutamax ^TM ), B27, N2, F-12, M199, MCDB110, MCDB202, MCDB302, etc., but are not limited thereto.

In addition, the differentiation induction medium of the present invention may not contain growth factors. The growth factor may be, for example, EGF or FGF2, but is not limited thereto.

The culture medium for culturing the stem cells includes all medium culture medium commonly used for culturing nerve cells in the art. A culture medium used for culture generally includes a carbon source, a nitrogen source, and trace element components.

More specifically, it may be a method for producing neuronal cells comprising culturing stem cells in a differentiation medium containing a composition containing adrenocorticotropic hormone-releasing hormone as an active ingredient to form neuronal cells. .

Examples of the stem cells include embryonic stem cells, adult stem cells, embryonic reproductive stem cells, and embryonic tumor stem cells, but are not limited thereto.

The medium may contain one or more selected from the group consisting of antibiotics, Glutamax ^™ , B27, heparin, and FGF2 (Fibroblast growth factor 2), but is not limited thereto.

The medium is glutamax (Glutamax ^TM ) 0.1 to 5% (v / v); Antibiotic 0.1 to 5% (v/v); B27 0.5 to 5% (v/v); It may include one or more selected from the group consisting of 1 to 5 μg/mL of heparin and 20 to 30 ng/mL of FGF2, but is not limited thereto.

In addition, the present invention provides a kit for forming neurospheres containing adrenocorticotropic hormone-releasing hormone as an active ingredient.

The kit is not limited to the above, and may include other reagents or instruments. For example, it may contain a culture plate for culturing target cells or a reagent (eg, alizarin red, etc.) capable of evaluating the state of differentiation into chalk blast cells, and may contain stem cells to be cultured. there is.

The provision form of the kit according to the present invention is one containing all of the additives for differentiation induction medium, corticotropin-releasing hormone, or nutrient mixture, growth factor, or basal medium and other reagents in an appropriate amount and / or form. It can be a container, or it can be provided by different containers.

The kit according to the present invention may include instructions describing procedures and the like for carrying out the method according to the present invention described above.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising corticotropin-releasing hormone or a pharmacologically acceptable salt thereof as an active ingredient.

The type of term "neurodegenerative disease" used in the present invention is not particularly limited. Non-limiting examples of the neurodegenerative disease include Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodysplasia, Alexander's disease, Alper's disease, vasodilatory ataxia, Batten's disease, bovine spongiform encephalopathy (BSE), Canavan's disease, and cortical basal Degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal degeneration, Huntington's disease, Kennedy's disease, Krab's disease, Lyme disease, Machado-Josef disease, multiple sclerosis, multiple systemic atrophy, neurostimuli , Niemann-Pick disease, Parkinson's disease, Pick disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinolytic sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal cord syphilis, Tay - It may be one or more selected from the group consisting of Sax's disease, toxic encephalopathy, transmissible spongiform encephalopathy, and unstable hedgehog syndrome, but is not limited thereto.

The present invention may also include a pharmaceutically acceptable salt of an active ingredient as an active ingredient. As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. In the present invention, the term "acceptable salt in food science" includes salts derived from organic acids, inorganic acids, or bases acceptable in food science. As used herein, the term "veterinarily acceptable salt" includes a salt derived from a veterinarily acceptable inorganic acid, organic acid, or base.

Examples of suitable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Acid addition salts can be prepared by conventional methods, for example, by dissolving a compound in an aqueous solution of excess acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering the precipitated salt.

Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. An alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable for pharmaceutical purposes to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

The content of the CRH in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition. It may, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents. , lotion, liniment, pasta, or cataplasma may have formulations such as the like.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl excipients such as cellulose, HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, A lubricant such as sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Agents may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₃ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.

In the present invention, "subject" means a subject in need of treatment of a disease, and is not limited as long as it is a vertebrate, but specifically, humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cows, It can be applied to sheep, antelope, dogs, cats, fish and reptiles.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of a desired disease, and “treatment” means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and "improvement" means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.

In the present invention, neural stem cells were formed into neural cell spheres using adrenocorticotropic hormone-releasing hormone. Neural stem cells obtained from the formed neural cell sphere are highly likely to be used as cell therapy agents for patients.

Accordingly, the present invention relates to a cell therapy agent for treating neurodegenerative diseases comprising the neuronal cells prepared by the above method.

The term "cellular therapeutic agent" in the present invention refers to cells and tissues prepared by isolation, culture, and special manipulation from humans, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention. Or, through a series of actions such as proliferating autologous, allogeneic, or xenogeneic living cells in vitro to restore tissue function, or changing the biological characteristics of cells in other ways, these cells can be used for treatment, diagnosis, and prevention of diseases. Means medicines used for their intended purpose.

As used herein, the term 'treatment' refers to inhibition of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction of the rate of disease progression, improvement of a disease state, amelioration, alleviation, or improved prognosis. means

The administration route of the cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration may be administered, but is not limited thereto.

The composition may be formulated in a suitable form together with a pharmaceutical carrier generally used for cell therapy. 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or similar reaction when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, the composition may be administered by any device capable of moving a cell therapy agent to a target cell.

The cell therapy composition of the present invention may contain a therapeutically effective amount of a cell therapy agent for the treatment of diseases. 'Therapeutically effective amount' means the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, physician or other clinician. , which includes an amount that induces relief of the symptoms of the disease or disorder being treated.

It is obvious to those skilled in the art that the cell therapy agent included in the composition of the present invention will be changed according to the desired effect. Therefore, the optimal amount of cell therapy agent can be easily determined by a person skilled in the art, and depends on the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health condition, sex and diet of the patient. , It can be adjusted according to various factors, including administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. It is important to include the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors. For example, the daily dose of the stem cells of the present invention is 1.0×10 ⁴ to 1.0×10 ¹¹ cells/kg of body weight, preferably 1.0×10 ⁵ to 1.0×10 ⁹ cells/kg of body weight divided once or several times. can be administered. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age and sex of the patient, and therefore, the dosage is It does not limit the scope of the present invention in any way.

The present invention provides a treatment method comprising administering the cell therapy composition of the present invention in a therapeutically effective amount to a mammal. As used herein, the term mammal refers to a mammal that is a subject of treatment, observation, or experimentation, and preferably refers to a human.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Confirmation of the effect of promoting corticotropin-releasing hormone (CRH) on neurosphere formation

A mouse fetus 14 days after ^{fertilization} was separated from the mother, and neural stem cells were extracted from the brain of the fetus. 2%, heparin 3 μg/mL, fibroblast growth factor 2 (FGF2) 25 ng/mL, penicillin and streptomycin were added at concentrations of 100 Units/mL and 100 μg/mL, respectively (DMEM/F12 medium)). . At this time, corticotropin-releasing hormone (CRH, SigmaAldrich) was added to the medium at a concentration of 1 μM and cultured together. As a negative control, distilled water, a solvent of CRH, was added in the same volume. The cells were cultured in an incubator at 37°C and 5% CO ₂ for one week until neurospheres were formed.

As shown in FIG. 2, as a result of measuring and quantifying the number of neurospheres formed after 7 days, it was observed that about twice as many neurospheres were formed in the CRH-added medium than in the negative control group.

In addition, as shown in FIG. 3 , it was confirmed that not only the number of neuronal cells having a diameter of 50 μm or more increased significantly, but also the overall number of neuronal cells significantly increased in the presence of CRH.

Through the above results, it was confirmed that the addition of CRH alone to the culture medium had the effect of significantly increasing the efficiency and size of neural cell sphere formation of neural stem cells.

Therefore, the present invention has confirmed that a large number of neuronal cell cells can be obtained only by adding CRH, and is expected to contribute to stem cell treatment of various brain diseases using neural stem cells in the future.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Composition for promoting the formation of neurosphere comprising Corticotropin-releasing hormone as an active ingredient <130> MP20-170 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 196 <212> PRT <213> artificial sequence <220> <223> CRH <400> 1 Met Arg Leu Pro Leu Leu Val Ser Ala Gly Val Leu Leu Val Ala Leu 1 5 10 15 Leu Pro Cys Pro Pro Cys Arg Ala Leu Leu Ser Arg Gly Pro Val Pro 20 25 30 Gly Ala Arg Gln Ala Pro Gln His Pro Gln Pro Leu Asp Phe Phe Gln 35 40 45 Pro Pro Pro Gln Ser Glu Gln Pro Gln Gln Pro Gln Ala Arg Pro Val 50 55 60 Leu Leu Arg Met Gly Glu Glu Tyr Phe Leu Arg Leu Gly Asn Leu Asn 65 70 75 80 Lys Ser Pro Ala Ala Pro Leu Ser Pro Ala Ser Ser Leu Leu Ala Gly 85 90 95 Gly Ser Gly Ser Arg Pro Ser Pro Glu Gln Ala Thr Ala Asn Phe Phe 100 105 110 Arg Val Leu Leu Gln Gln Leu Leu Leu Pro Arg Arg Ser Leu Asp Ser 115 120 125 Pro Ala Ala Leu Ala Glu Arg Gly Ala Arg Asn Ala Leu Gly Gly His 130 135 140 Gln Glu Ala Pro Glu Arg Glu Arg Arg Ser Glu Glu Pro Pro Ile Ser 145 150 155 160 Leu Asp Leu Thr Phe His Leu Leu Arg Glu Val Leu Glu Met Ala Arg 165 170 175 Ala Glu Gln Leu Ala Gln Gln Ala His Ser Asn Arg Lys Leu Met Glu 180 185 190 Ile Ile Gly Lys 195

Claims (11)

In the composition for forming neurospheres,
The composition contains corticotropin-releasing hormone (CRH) as an active ingredient,
The composition is characterized in that it does not contain EGF (Epidermal growth factor), a composition for forming neurospheres.
According to claim 1,
The CRH is a composition characterized in that it promotes the growth of neural cell cells from neural stem cells.
According to claim 1,
The composition is a composition for adding a medium, characterized in that the composition.
According to claim 1,
The CRH is characterized in that contained in a concentration of 0.1 to 100 μM compared to the total composition, the composition.
Culturing the composition according to claim 1 with stem cells,
The composition is characterized in that it does not contain EGF (Epidermal growth factor), a method for producing nerve cells.
According to claim 5,
The stem cells are characterized in that the neural stem cells, the method.
According to claim 5,
Characterized in that the culture is carried out for 3 to 20 days, the method.
In the kit for forming neurospheres,
The kit contains corticotropin-releasing hormone (CRH) as an active ingredient,
Characterized in that the kit does not contain EGF (Epidermal growth factor), a kit for forming neurospheres.
delete delete According to claim 1,
The composition is characterized in that it comprises FGF (fibroblast growth factor), the composition.

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