WO2022103064A1 - Composition comprising corticotropin-releasing hormone as active ingredient for promoting neurosphere formation - Google Patents

Abstract

The present invention relates to a composition comprising corticotropin-releasing hormone as an active ingredient for promoting neurosphere formation. The present inventors provide a technique of forming a neurosphere, which is a cluster of neural stem cells, by using CRH and identified that not only can neurospheres be simply formed, but also the number and size of neurospheres can be increased only by adding CRH. A large number of neurospheres can be secured by using CRH and it is expected that subsequently produced neuronal stem cells can be used to effectively treat neurodegenerative disorders.

Description

Composition for promoting the formation of nerve cells comprising adrenocorticotropin-releasing hormone as an active ingredient

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

This application claims priority based on Korean Patent Application No. 10-2020-0151491 filed on November 13, 2020, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Neurodegenerative disorders are 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 the progressive loss of structure and loss of function of specific brain cell groups in the brain and spinal cord. Neuronal cell death or damage caused by degenerative nervous system disease is a problem in the formation or function of synapses that transmit information between cranial nerve cells and cranial nerve cells, which are most important for information transmission in the cranial nervous system, and ideal increase or decrease in the electrical activity of cranial nerves. known to cause Neurons in the brain and spinal cord have very diverse functions depending on their location, causing characteristic dysfunction due to damage to nerve cells in a specific area, and show a very diverse clinical picture depending on how the dysfunction progresses. These degenerative neurological diseases 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), etc.

Meanwhile, until now, for research on Alzheimer's disease, after differentiation into neurons mainly present in the forebrain, research on the etiology is being conducted. Differentiation into forebrain neurons is achieved by the neuronal differentiation method that proceeds without the addition of special external substances. Recently, the monolayer culture method through 'dual-SMAD inhibition', the method through embryoid body formation, Methods and the like are being developed, and both 'glutamatergic neurons' and 'GABAergic neurons' are widely used in research on the pathogenesis of Alzheimer's disease. According to a recently published paper, the differentiation of patient-derived pluripotent stem cells into ‘GABAergic neurons’ was confirmed to be more resistant to apoptosis by amyloid beta. That is, it is a case that shows that various phenotypes can appear according to differentiation into specific neurons.

Because of their ability to differentiate into neurons, astrocytes, and oligodendrocytes, neural stem cells have been used as a key material in stem cell therapy to treat neurodegenerative diseases such as trauma-induced nervous system damage or Alzheimer's disease. In general, neural stem cells form neural cell cells, which are thousands of neural stem cell aggregates, from one neural stem cell after about 7 days when cultured in a suspended state (see FIG. 1 ). That is, the number of neural stem cells can be increased by culturing neural cells.

Based on this fact, several approaches have been developed to enhance the growth of neuronal cells. Typically, there is a method to improve the formation of neuronal cells through genetic manipulation using retroviruses or plasmids, which is not only difficult, but also has the potential to change into cancer cells in the body. was difficult to obtain. Another method is to combine compounds or growth factors to treat cells, but it was very important and difficult to discover new effective compounds and growth factors.

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

[Prior literature]

(Non-Patent Document 1) 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 the present inventors' earnest efforts to find an efficient and economical method of forming neural cell cells with neural stem cells, it was confirmed that the adrenocorticotropic hormone-releasing hormone of the present invention can be processed to form neural cell cells from neural stem cells. , the present invention was completed.

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

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

Another object of the present invention is to provide a kit for forming nerve cell cells, comprising 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 the treatment of neurodegenerative diseases comprising the nerve cell 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 nerve cells comprising a corticotropin-releasing hormone (CRH) as an active ingredient.

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

In addition, the present invention provides the use of a composition comprising adrenocorticotropic hormone-releasing hormone as an active ingredient to form nerve cells.

In addition, the present invention provides a kit for forming nerve cell cells, comprising 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 may promote the growth of neuronal 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 relative to the total composition, but is not limited thereto.

In another embodiment of the present invention, the stem cell may be a neural stem cell, but is 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, adrenal leukoplakia, Alexander's disease, Alper's disease, vasodilatory ataxia, Batten's disease, bovine spongiform encephalopathy (BSE), Canavan disease, cortical degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal degeneration, Huntington's disease, Kennedy's disease, Crabb's disease, Lyme disease, Macado-Joseph disease, multiple sclerosis, multiple systemic atrophy , neuronal schizophrenia, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff's disease, diffuse myelinlytic sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord , spinal syphilis, Tay-Sachs disease, toxic encephalopathy, disseminated spongiform encephalopathy, and unstable Hedgehog syndrome 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 comprising adrenocorticotropic hormone-releasing hormone as an active ingredient.

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

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

The present inventors showed a technique for forming neuronal cells from neural stem cells using adrenocorticotropic hormone-releasing hormone (CRH), thereby making it possible not only to simply form neuronal cells, but also to increase the number of neuronal cells only by adding CRH. And it was confirmed that the size can be increased, it is possible to secure a large number of nerve cell cells using CRH, and it is expected that neurodegenerative diseases can be effectively treated using the neural stem cells produced later.

1 schematically shows a protocol for the formation of neural cell spheres, which are neural stem cell aggregates, through neural stem cell culture.

FIG. 2 shows that when neural stem cells were cultured in suspension in CRH-added neural stem cell culture medium, the number of neuronal cells increased nearly twice as compared to the negative control group.

3 shows that when neural stem cells are cultured in suspension in a neural stem cell culture medium to which CRH is added, the number of neural cell cells having a diameter of 50 μm or more is significantly increased.

The terms, techniques, etc. 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 of the documents mentioned in this specification are incorporated herein as documents for describing the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, "corticotropin-releasing hormone" is named as corticosteroid, CRH, CRF, etc., and is a peptide hormone involved in the 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 amino acid sequence represented by SEQ ID NO: 1 may be a variant having 80%, 90%, 95%, 97%, 98% or 99% homology thereto, and some sequences in the sequence of SEQ ID NO: 1 Deletions, substitutions or additions may also be included within the scope of the present invention.

In the present invention, the adrenocorticotropic 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 in the composition of the present invention. μM, 0.1-45 μM, 0.1-40 μM, 0.1-35 μM, 0.1-30 μM, 0.1-25 μM, 0.1-25 μM, 0.1-23 μM, 0.1-20 μM, 0.1-15 μM, 0.1-10 μM, 0.1-9 μM, 0.1-8 μM, 0.1-7 μM, 0.1-6 μM, 0.1-5 μM, 0.1-4 μM, 0.1-3 μM, 0.1-2 μM, 0.1-1.5 μM, or about 1 It may be included at a concentration of μM, but is not limited thereto.

In the present invention, the adrenocorticotropic hormone-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 cells” is not particularly limited as long as it is an undifferentiated state and a cell having indefinite proliferation and neuronal cell formation ability. Specifically, the stem cells include, but are not limited to, embryonic stem cells, adult stem cells, embryonic germ stem cells, embryonic tumor stem cells, and the like.

In the present invention, "stem cells" may be neural stem cells, but is 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, which continue to proliferate in an immature, undifferentiated state. The neural stem cells exist in various anatomical sites 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 nerve cells The term “neuron” refers to a cell belonging to the neuronal system, including neuronal cells (eg, unipolar, bipolar, and multipolar neurons) and glial cells (oligodendrocytes, Schwann cells, astrocytes, microglia).

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

In the present invention, the neuronal cells are 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 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 limited thereto not.

In the present invention, the neural cell sphere is a large cell aggregate of hundreds to thousands of neural stem cells or neurons, and has a significant difference in size from a single cell, neural stem cells or neurons.

In addition, the present invention provides a method for producing a neuronal cell comprising the step of 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. However, the present invention is not limited thereto.

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

The term "medium" of the present invention may use a basic medium known in the art without limitation. The basal medium can be artificially prepared by synthesis, and commercially prepared medium is, 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 (Nutrient Mixture). The nutrient mixture is a mixture containing various amino acids, vitamins, inorganic salts, etc. generally used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, or the like, or a commercially prepared nutrient mixture may be used. Commercially prepared nutritional mixtures include, but are not limited to, for example, Glutamax ^™ , B27, N2, F-12, M199, MCDB110, MCDB202, MCDB302, and the like.

In addition, the differentiation inducing medium of the present invention may not contain a growth factor. 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 of the medium culture medium commonly used for culturing nerve cells in the art. The culture medium used for culture generally contains a carbon source, a nitrogen source, and a trace element component.

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

The stem cells include, but are not limited to, embryonic stem cells, adult stem cells, embryonic germ stem cells, embryonic tumor stem cells, and the like.

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

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

In addition, the present invention provides a kit for the formation of nerve cells comprising 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, a culture plate for culturing a target cell or a reagent capable of evaluating the differentiation state into a target cell (eg, alizarin red, etc.) may be included, and the target cell to be cultured may be included. there is.

The provided form of the kit according to the present invention is one containing both an additive for a differentiation induction medium, or adrenocorticotropic hormone, or a nutrient mixture, a growth factor, or a basal medium and other reagents in an appropriate dose and/or form. It may be a container, or it may be provided by a different container.

The kit according to the present invention may include instructions describing the procedure for carrying out the above-described method according to the present invention.

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

The type of the 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, adrenal leukoplakia, Alexander's disease, Alper's disease, vasodilatory ataxia, Baton's disease, bovine spongiform encephalopathy (BSE), Carnaban's disease, cortical basal Degeneration, Creutzfeldt-Jakob disease, Dementia with Lewy bodies, fatal familial insomnia, frontotemporal degeneration, Huntington's disease, Kennedy's disease, Crabb's disease, Lyme disease, Macado-Joseph disease, multiple sclerosis, multiple systemic atrophy, neurostrophic cytosis , Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinlytic sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay - It may be at least one selected from the group consisting of Sachs disease, toxic encephalopathy, disseminated spongiform encephalopathy, and unstable Hedgehog syndrome, but is not limited thereto.

The present invention may also include a pharmaceutically acceptable salt of the 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. As used herein, the term “food pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable organic acids, inorganic acids, or bases. As used herein, the term "veterinary acceptable salt" includes salts derived from veterinary acceptable inorganic acids, organic acids, or bases.

Examples of suitable acids include 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 the compound in an aqueous solution of an excess of 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 an equimolar amount of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or by suction filtration of 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. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the 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. In this case, as the metal salt, it is pharmaceutically suitable 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 degree of progression 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. However, the present invention is not limited thereto. The content ratio is a value based on the dry amount from which the solvent is removed.

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

The pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, alcohols, troches, fragrances, and limonaade. , 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, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.

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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as 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 hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl 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 acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, 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, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, 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.

As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), 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, etc. can be used.

In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.

Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.

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. An agent may be used, and a surfactant, a preservative, a stabilizer, a colorant, and a fragrance may be used as needed.

Injectables according to the present invention include distilled water for injection, 0.9% sodium chloride injection, ring gel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated ring gel 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 aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethyl acetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethyl acetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone, gum; isotonic agents such as sodium chloride; 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 ), stabilizers such as ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, or aluminum monostearate.

The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro 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 IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.

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

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

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 at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.

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

The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin 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 along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

In the present invention, "individual" means a subject in need of treatment for a disease, and is not limited if it is a vertebrate, specifically, a human, a mouse, a rat, a guinea pig, a rabbit, a monkey, a pig, a horse, a cow, Applicable to sheep, antelopes, dogs, cats, fish and reptiles.

In the present invention, "administration" means providing a predetermined composition of the present invention to an individual by any suitable method.

In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, and “treatment” means that the target disease and its metabolic abnormalities are improved or It means all actions that are beneficially changed, and “improvement” means all actions that reduce the desired disease-related parameters, for example, the degree of symptoms by administration of the composition according to the present invention.

In the present invention, neural stem cells were formed into nerve cell spheres using adrenocorticotropic hormone-releasing hormone. Neural stem cells obtained from the formed neural cell cells have a very high potential for use as a cell therapy for patients.

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

As used herein, the term “cellular therapeutic agent” refers to cells and tissues isolated from humans, cultured, and manufactured through special manipulation, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention. Or through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways to restore tissue function, these cells can be used in the treatment, diagnosis and prevention of diseases. Drugs used for that purpose.

As used herein, the term "treatment" refers to inhibiting the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving the 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, may be administered intradermally, but is not limited thereto.

The composition may be formulated in a suitable form together with a pharmaceutical carrier commonly used for cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are 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 transporting a cell therapy agent to a target cell.

The cell therapy composition of the present invention may contain a therapeutically effective amount of the cell therapy agent for the treatment of a disease. "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 considered by a researcher, veterinarian, physician or other clinician. , which includes an amount that induces amelioration of the symptoms of the disease or disorder being treated.

It is apparent to those skilled in the art that the cell therapy agent included in the composition of the present invention will change depending on the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art, and 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 status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors. In consideration of all of the above factors, it is important to include an amount that can obtain the maximum effect with a minimum amount without side effects. 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 body weight, preferably 1.0×10 ⁵ to 1.0×10 ⁹ cells/kg body weight divided by one 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 several related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and, therefore, the dosage It is not intended to limit the scope of the present invention in any way.

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

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Confirmation of neurosphere formation promoting effect of adrenocorticotropic hormone-releasing hormone (CRH)

After 14 days of fertilization, a mouse fetus was isolated from the mother, and neural stem cells were extracted from the fetal brain, and then 3,000 neural stem cells were placed in a culture medium (GlutaMAX ^TM 1%, B27). 2%, heparin 3 μg/mL, fibroblast growth factor 2 (FGF2) 25 ng/mL, penicillin and streptomycin at concentrations of 100 Units/mL and 100 μg/mL, respectively, were dispensed with DMEM/F12 medium) . At this time, adrenocorticotropin-releasing hormone (CRH, SigmaAldrich) was added to the medium at a concentration of 1 μM and cultured together. As a negative control, distilled water, which is a solvent of CRH, was added in the same volume. It was cultured in an incubator at 37 °C, 5% CO ₂ for 1 week until neurospheres were formed.

As shown in FIG. 2 , as a result of measuring and quantifying the number of neuronal cells formed after 7 days, it was observed that about twice as many neuronal cells 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 was significantly increased in the presence of CRH.

From the above results, it was confirmed that only adding CRH alone to the culture medium had the effect of significantly increasing the neuronal cell formation efficiency and size of neural stem cells.

Therefore, the present invention confirms that a large number of neuronal cells can be secured 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 description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains 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, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The present inventors showed a technique for forming neuronal cells from neural stem cells using adrenocorticotropic hormone-releasing hormone (CRH), thereby making it possible not only to simply form neuronal cells, but also to increase the number of neuronal cells only by adding CRH. And it was confirmed that the size can be increased. Therefore, since a large number of nerve cell cells can be secured using CRH, and neurodegenerative diseases can be effectively treated using the neural stem cells produced later, the present invention has industrial applicability.

Claims (14)

1. A composition for forming nerve cells comprising a corticotropin-releasing hormone (CRH) as an active ingredient.
2. According to claim 1,

   The CRH is characterized in that it promotes the growth of nerve cells from neural stem cells, the composition.
3. According to claim 1,

   The composition is characterized in that the composition for adding a medium, the composition.
4. According to claim 1,

   The CRH is characterized in that contained in a concentration of 0.1 to 100 μM relative to the total composition, the composition.
5. A method for producing a neuronal cell comprising the step of culturing the composition according to claim 1 with stem cells.
6. 6. The method of claim 5,

   The stem cells are characterized in that the neural stem cells, the method.
7. 6. The method of claim 5,

   The method, characterized in that the culture is carried out for 3 to 20 days.
8. Corticotropin-releasing hormone (CRH) as an active ingredient, a kit for forming nerve cells.
9. A pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising a corticotropin-releasing hormone (CRH) as an active ingredient.
10. 10. The method of claim 9,

    The neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, adrenal leukodystrophy, Alexander's disease, Alper's disease, vasodilatory ataxia, Batten's disease, bovine spongiform encephalopathy (BSE), Canaban disease, cortical degeneration, Creutzfeldt -Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal degeneration, Huntington's disease, Kennedy's disease, Krab's disease, Lyme disease, Macado-Joseph's disease, multiple sclerosis, multiple systemic atrophy, neurostic erythrocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff's disease, diffuse myelinlytic sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay-Sachs disease, A pharmaceutical composition, characterized in that at least one selected from the group consisting of toxic encephalopathy, disseminated spongiform encephalopathy, and unstable Hedgehog syndrome.
11. A method of treating a neurodegenerative disease comprising administering to an individual a composition comprising adrenocorticotropic hormone-releasing hormone as an active ingredient.
12. Use of a composition comprising adrenocorticotropic hormone-releasing hormone as an active ingredient for treating neurodegenerative diseases.
13. Use for producing a drug used in neurodegenerative diseases of adrenocorticotropic hormone-releasing hormone.
14. Use of a composition containing adrenocorticotropic hormone-releasing hormone as an active ingredient to form nerve cells.

Images