WO2021194256A1 - Pharmaceutical composition for preventing or treating retinitis pigmentosa, comprising mesenchymal stem cells derived from human embryonic stem cells - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating retinitis pigmentosa, comprising mesenchymal stem cells derived from human embryonic stem cells. The mesenchymal stem cells derived from human embryonic stem cells, according to the present invention, increase the waveform of the b-wave reflecting inner nuclear layer activity, and exhibit an effect of protecting the cell nucleus of the outer nuclear layer of the retina. Thus, the mesenchymal stem cells derived from human embryonic stem cells of the present invention are expected to be usefully employed for the development of medicine or quasi-drugs for preventing, alleviating or treating inherited retinal diseases such as retinal cell degeneration.

Description

Pharmaceutical composition for preventing or treating retinal pigmentation comprising human embryonic stem cell-derived mesenchymal stem cells

The present invention relates to a pharmaceutical composition for preventing or treating retinal pigmentation, and a quasi-drug composition for preventing or improving retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells.

The present invention claims priority based on Korean Patent Application No. 10-2020-0036338 filed on March 25, 2020, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Hereditary retinal diseases caused by mutations in genes are caused by genetic abnormalities and there is no definite treatment method yet because it is a disease in which degeneration occurs in retinal nerve cells, which are the most difficult to regenerate in our body. In order for the retina to function properly, visual cells and retinal pigment epithelial cells are required. Visual cells are sensory nerve cells that convert light into electrical nerve signals, and retinal pigment epithelial cells play an essential role in maintaining the neural retina.

Retinal pigmentation degeneration, a representative disease of hereditary retinal diseases, is a disease caused by mutations in various genes. Early symptoms of retinitis pigmentosa include deterioration of night and peripheral vision, and as the disease progresses, detailed vision, central vision and color vision may also be affected, and the age of onset of symptoms varies, but is usually 10 to I am 30 years old, and the rate of exacerbation varies from person to person. In addition, since this hereditary retinal disease usually causes severe vision loss in both eyes, the severity of the disease is serious for patients, families, and even society.

In relation to the treatment of hereditary retinal diseases, most have relied on conservative treatment because there is no treatment that can regenerate the degenerated retina and restore the lost vision so far. However, as research on cell therapy has been actively conducted in recent years, stem cell therapy has been attempted in various intractable diseases, and retinal disease is one of the fields in which this research is being actively conducted. Despite a number of different causative genes and clinical features, hereditary retinal diseases have a common etiology of retinal neuronal degeneration, and stem cell therapy can be applied regardless of the difference in mutations.

On the other hand, embryonic stem cells are formed from the inner cell mass of the blastocyte, which is the early stage of embryonic development, and have the potential to differentiate into all cells, so they can be differentiated into any tissue cell, and also die. It can be cultured in an immortal and undifferentiated state, and unlike adult stem cells, germ cells can also be produced, so it has the characteristic of being able to be inherited to the next generation.

Human embryonic stem cells are produced by isolating and culturing only the inner cell mass during the formation of human embryos. Currently, human embryonic stem cells made worldwide are obtained from frozen embryos left after sterilization. Various attempts have been made to use human embryonic stem cells with pluripotent ability to differentiate into all cells as a cell therapy, but the risk of cancer and the high barrier of immune rejection have not yet been completely controlled.

Recently, as an alternative to overcome this problem, mesenchymal stem cells without the risk of cancer with immunomodulatory function have been proposed. Mesenchymal stem cells are known to directly or indirectly provide nutritional support for neuroprotection and nerve regeneration. Compared to other organs, the retina is an ideal tissue for stem cell treatment, and it is relatively easy to access surgically. possible. In addition, the blood-retinal barrier is an organ that exhibits immune privilege in healthy eyes and is relatively less resistant to rejection. In addition, the fact that the absolute amount of cells for cell therapy is relatively small and there are two eyes is a condition for more actively trying stem cell therapy.

In previous studies, it has been reported that proliferative vitreous retinopathy appears as a complication when adult mesenchymal stem cells are transplanted. Nothing is known yet.

The present inventors confirmed that when mesenchymal stem cells derived from human embryonic stem cells were administered to a rat model inducing retinal pigmentation, the b-wave waveform was increased and the cell nucleus of the outer retinal nuclear layer was preserved. Based on the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides a method for preventing or treating retinal pigmentation, comprising administering to an individual a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides a use for preventing or treating retinitis pigmentosa of a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides the use of human embryonic stem cell-derived mesenchymal stem cells for preparing a medicament for the treatment of retinitis pigmentosa.

In one embodiment of the present invention, the human embryonic stem cell-derived mesenchymal stem cells can protect retinal neurons.

In another embodiment of the present invention, the human embryonic stem cell-derived mesenchymal stem cells may be injected into the eye subretinal or intravitreal, but is not limited thereto.

In another embodiment of the present invention, the composition may be prepared in one or more formulations selected from the group consisting of ophthalmic solutions, eye drops, eye ointments, injections, and eyewash, but is not limited thereto.

The human embryonic stem cell-derived mesenchymal stem cell according to the present invention has the effect of increasing the b-wave waveform reflecting the activity of the inner nuclear cell layer and protecting the cell nucleus of the outer retinal nuclear layer. Therefore, the human embryonic stem cell-derived mesenchymal stem cells of the present invention are expected to be usefully used in the development of pharmaceuticals or quasi-drugs for the prevention, improvement, or treatment of hereditary retinal diseases such as retinal cell degeneration.

1 is a view showing the results of confirming the electroretinal diagram by administration of human embryonic stem cell-derived mesenchymal stem cells in RCS rats according to an embodiment of the present invention.

2 is a view confirming the change in the thickness of the outer retinal nuclear layer by the administration of human embryonic stem cell-derived mesenchymal stem cells in RCS rats according to an embodiment of the present invention through H&E staining.

The present invention provides a pharmaceutical composition for preventing or treating retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

The term “stem cell” as used in the present invention is a cell that is the basis of cells or tissues constituting an individual, and its characteristics are that it can self-renew by dividing repeatedly, and has a specific function depending on the environment. It refers to a cell with multidifferentiation ability to differentiate into a cell with It occurs in all tissues during the development of the fetus and is found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissue, even in adulthood. Stem cells are totipotent stem cells, which are formed when a fertilized egg starts dividing, and pluripotent stem cells in the blastocyst, which are formed by continuing division of these cells, depending on the type of differentiated cell. And they are classified as multipotent stem cells that exist in mature tissues and organs. At this time, pluripotent stem cells are cells that can be differentiated only into cells specific to the tissues and organs that contain these cells. It is involved in maintenance and inducing regeneration in the event of tissue damage. These tissue-specific pluripotent cells are collectively referred to as adult stem cells.

Classified as adult stem cells, "mesenchymal stem cells (MSCs)" are well known as repair cells of several connective tissues, and these cells can differentiate into several types of cells of the mesenchymal system. . In addition, because of the advantages of easy acquisition and clinical application, it is difficult to obtain a sufficient amount because it is located deep in the brain and can compensate for the disadvantages of neural stem cells, which have a risk of brain damage. Cells that are in the spotlight as a material and can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord. has the ability to differentiate into In the present invention, preferably, the mesenchymal stem cells are human embryonic stem cell-derived mesenchymal stem cells.

As used herein, the term "embryonic stem cell (embryonic stem cell)" is a stem cell isolated from the inner cell mass of the blastocyst. It refers to a cell that has pluripotency, the ability to proliferate and differentiate into any type of cell or tissue necessary for our body.

As used herein, the term "retinitis pigmentosa" is one of hereditary retinal diseases, and a group of diseases characterized by hereditary and progressive retinal dysfunction, cell loss, and ultimately retinal tissue atrophy. It is a progressive disease that occurs due to problems with the function of photoreceptors.

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 according to a conventional method, such as 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. In the present invention, it may preferably have one or more formulations selected from the group consisting of ophthalmic solutions, eye drops, eye ointments, injections, and eyewash, but is not limited thereto.

When prepared as an ophthalmic solution, any dosage form used as an ophthalmic solution, for example, an aqueous ophthalmic solution such as an aqueous emulsion ophthalmic solution, a viscous ophthalmic solution, and a dissolved ophthalmic solution; or a non-aqueous ophthalmic solution such as a non-aqueous emulsion ophthalmic solution.

When prepared as an aqueous emulsion ophthalmic solution, various additives known in the art may be included as long as the purpose of the present invention is not impaired, for example, isotonic agents, buffers, stabilizers, pH adjusters, thickeners, preservatives, chelating agents, solubilizers and solvents, and the like. The buffer may be selected from, but is not limited to, the group consisting of phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer (eg sodium acetate) tromethamine and amino acids. Preferably, a phosphate buffer may be used. The isotonic agent may be selected from the group consisting of, but not limited to, sorbitol, sugars such as glucose erythritol and mannitol, polyhydric alcohols such as glycerin, polyethylene glycol and polypropylene glycol, and salts such as sodium chloride. Preservatives include benzalkonium chloride, benzethonium chloride, alkyl paraoxybenzoates such as methyl paraoxybenzoate and ethyl paraoxybenzoate, benzyl alcohol, phenethyl alcohol, sorbic acid and its salts, thimerosal, polyquaternium , may be selected from the group consisting of benzododecinium bromide, oxychlorocomplex and chlorobutanol, but is not limited thereto. The stabilizing agent may be selected from the group consisting of cyclodextrin and a water-soluble polymer such as a derivative thereof, poly (vinylpyrrolidone), and polysorbate 80 (Tween 80 ^®), Polysorbate 20, a surfactant such as tirok sapol and is not limited thereto. The pH adjusting agent may be selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, monoethanolamine, aqueous ammonia and ammonium hydroxide, but is not limited thereto. The thickener may be selected from the group consisting of hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose, polyvinyl alcohol, carbomer, povidone, poloxamer, polycarbophil and salts thereof. However, the present invention is not limited thereto. The chelating agent may be selected from, but not limited to, the group consisting of sodium edetate, sodium citrate and condensed sodium phosphate. The solubilizing agent or solvent may be selected from glycerin, DMSO, DMA, N-methylpyrrolidone, ethanol, benzyl alcohol, isopropyl alcohol, polyethylene glycol or propylene glycol of various molecular weights, but is not limited thereto. There may be some overlap between components that can be used as solvents or solubilizers, and any component may be used as either a solvent or a solubilizer. If it does not, it can be regarded as a solubilizer. Alternatively, the solubilizer may be a surfactant in some variations. Combinations of surfactants including various types of surfactants may be used. For example, nonionic, anionic (ie soap, sulfonate), cationic (ie CTAB), zwitterionic, polymeric, amphoteric surfactants can be used. For example, surfactants that may be used include, but are not limited to, those having an HLB of 10, 11, 12, 13, or 14 or higher. Examples of surfactants are polyoxyethylene products of hydrogenated vegetable oils, polyethoxylated castor oil or polyethoxylated hydrogenated castor oil, polyoxyl castor oil or derivatives thereof, polyoxyethylene-sorbitan-fatty acid esters , polyoxyethylene castor oil derivatives and the like. However, the present invention is not limited thereto. According to a specific embodiment, the ophthalmic composition of the present invention contains 0.01 to 0.1% by weight of cyclosporine, 0.5 to 7.5% by weight of trehalose, 1 to 10% by weight of a solubilizer, 0.01 to 2% by weight of a solvent, and the remaining amount based on the total weight of the composition. buffers and isotonic agents.

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 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 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), 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. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propylcellulose, 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 fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen 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, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

The liquid additives according to the present invention include water, dilute hydrochloric acid, dilute 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, and the like 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, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; 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 ₅ ), 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, and 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 such 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 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. have. 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 with 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 single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain 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 envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (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, "quasi-drug" refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, according to the Pharmaceutical Affairs Act. Quasi-drugs are products that are not used for pharmaceutical purposes. Textile and rubber products used for the treatment or prevention of diseases in humans and animals, those that have a slight or no direct action on the human body, and are not instruments or machines, and the like; This includes disinfectants and pesticides to prevent infectious diseases.

In the present invention, the quasi-drug composition may be formulated and used in the formulation of an ophthalmic composition, for example, in the formulation of one or more formulations selected from the group consisting of ophthalmic solutions, eye drops, eye ointments, injections, and eyewash. can be used, but is not limited thereto.

In addition, the present invention provides a method for preventing or treating retinal pigmentation, comprising administering to an individual a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides a use for preventing or treating retinitis pigmentosa of a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.

In addition, the present invention provides the use of human embryonic stem cell-derived mesenchymal stem cells for preparing a medicament for the treatment of retinitis pigmentosa.

As used herein, the term "individual" refers to a subject in need of prevention or treatment of retinitis pigmentosa, and more specifically, a human or non-human primate, mouse, dog, cat, horse. , and mammals such as cattle.

As used herein, the term "administration" means providing a given composition of the present invention to a subject by any suitable method.

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

In the present invention, the pharmaceutical composition or quasi-drug composition may be for topical administration to the eye, and the human embryonic stem cell-derived mesenchymal stem cells may be injected into the subretinal or intravitreal cavity of the eye. It is not limited thereto.

In the present invention, the human embryonic stem cell-derived mesenchymal stem cells may be injected into a patient's body alone or in a cultured state in an incubator.

In the present invention, the human embryonic stem cell-derived mesenchymal stem cells are 1 x 10 ⁴ cells to 7 x 10 ⁴ cells, 1 x 10 ⁴ cells to 6 x 10 ⁴ cells, 1 x 10 ⁴ cells to 5 x 10 ^{4 cells.} cells, 1 x 10 ⁴ cells to 4 x 10 ⁴ cells, 1 x 10 ⁴ cells to 3 x 10 ⁴ cells, 2 x 10 ⁴ cells to 7 x 10 ⁴ cells, 2 x 10 ⁴ cells to 6 x 10 ⁴ cells, 2 x 10 ⁴ cells to 5 x 10 ⁴ cells, 2 x 10 ⁴ cells to 4 x 10 ⁴ cells, 2 x 10 ⁴ cells to 3 x 10 ⁴ cells, 2.5 x 10 ⁴ cells to 7 x 10 ⁴ cells, 2.5 x Administer in an amount of 10 ⁴ cells to 6 x 10 ⁴ cells, 2.5 x 10 ⁴ cells to 5.5 x 10 ⁴ cells, 4 x 10 ⁴ cells to 7 x 10 ⁴ cells, or 4 x 10 ⁴ cells to 6 x 10 ^{4 cells} may be, and according to an embodiment of the present invention may be ^{administered in an amount of 2.5 x 10 4} cells to 5 x 10 ⁴ cells, but is not limited thereto.

In the present invention, when the human embryonic stem cell-derived mesenchymal stem cells were administered to a rat induced to have retinitis pigmentosa, it was confirmed that the b-wave waveform was increased through an electroretinalogram (see Example 2-2). , it was observed that the cell nucleus of the outer retinal nuclear layer was preserved (see Example 2-3), and through this, it was confirmed that the human embryonic stem cell-derived mesenchymal stem cells can protect retinal neurons.

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 1. Preparation of human embryonic stem cell-derived mesenchymal stem cells

The human embryonic stem cell-derived mesenchymal stem cells of the present invention were prepared and cultured by the following method.

1-1. embryoid body formation

Seoul National University Hospital human embryonic stem cells (Asian #1, male, STO feeder), which were maintained in an undifferentiated state, were treated with a protease (dispase, 2mg/ml), separated into fine work, and then in embryonic stem cell medium without bFGF. It was cultured for 14 days in a suspended state.

1-2. Induction of differentiation into mesenchymal stem cells

Embryos made by suspension culture for 14 days were attached to a tissue culture dish, and then the differentiation of mesenchymal stem cells was induced naturally. The induction of differentiation of mesenchymal stem cells was observed while culturing the embryo body in a medium supplemented with (10% v/v) fetal bovine serum (FBS) in DMEM (Dulbecco's Modified Eagle's Medium) for 16 days.

1-3. Maintenance and proliferation culture of differentiation-induced mesenchymal stem cells

In Example 1-2, the mesenchymal stem cells induced by culturing for 16 days after attachment to the embryonic body were treated with enzymes (Trypsin-EDTA, 0.25% Trypsin with EDTA 4Na), disassembled into single cells, and then re-organized into a tissue culture dish. attached to Then, out of a total of 500 ml of the culture medium, 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of VEGF (Vascular Endothelial Growth Factor), 2 ml of hFGF-B (human Fibroblast Growth Factor-basic), 2 ml of Insulin-like Growth Factor (IGF-1) ) 0.5 ml, hydrocortisone 0.2 ml, ascorbic acid 0.5 ml, and a medium (EGM-2MV, CC4147, Lonza) added with 470 ml of basal medium (EGM-2MV, CC4147, Lonza) were maintained and cultured at 37°C.

Example 2. Confirmation of Retinal Pigmentation Inhibitory Effects of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells

2-1. laboratory animal

The experimental animal has a de novo defect in the MertK gene, and the loss of the outer nuclear layer begins at 3 weeks of age and leads to severe degeneration in both function and structure after about 60 days of age. Royal College of Surgeons rats (RCS rats) showing similar retinal degeneration were used.

2-2. electroretinogram test

To 3-week-old RCS rats, the human embryonic stem cell-derived mesenchymal stem cells prepared in Example 1 were administered subretinal and intravitreal to 2.5 x 10 ⁴ cells and 5 x 10 ⁴ cells, respectively. After injection, visual function was evaluated 6 weeks after injection (around 60 days of age) through electroretinography.

As a result, as shown in FIG. 1 , it was confirmed that the waveform of b-wave, which decreased over time in RCS rats, appeared higher in both subretinal/intravitreal injection groups than in the control group at 6 weeks (P60) after injection. . The histogram of FIG. 1 shows the control (Untreated), subretinal 5 x 10 ⁴ cells, subretinal 2.5 x 10 ⁴ cells, intravitreal 5 x 10 ⁴ cells, and intravitreal 2.5 x 10 ⁴ cells in order from left, respectively. show the results.

2-3. Check the thickness of the outer retinal nuclear layer

Human embryonic stem cell-derived mesenchymal stem cells were injected subretinal or intravitreal into 3-week-old RCS rats, and the eyes were removed at 6 weeks after injection (around 60 days of age) and 4% paraformaldehyde After fixing for 48 hours, a paraffin block was prepared to make a tissue slide. The thickness of the outer retinal nuclear layer was confirmed by deparaffinization and tissue observation through Hematoxylin-Eosin (H&E) staining.

As a result, as shown in Fig. 2, it was found that the cell nucleus of the extraretinal nuclear layer at 6 weeks after injection was preserved in 2-4 layers in both subretinal/intravitreal injection groups, compared to the control group, which was difficult to maintain even one layer. Confirmed.

From the above results, it was found that the human embryonic stem cell-derived mesenchymal stem cells of the present invention suppressed the degeneration of retinal neurons.

The above description of the present invention 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 human embryonic stem cell-derived mesenchymal stem cell according to the present invention increases the waveform of b-wave reflecting the activity of the inner nuclear cell layer and has the effect of protecting the cell nucleus of the outer retinal nuclear layer, the retina. It is expected to be usefully used in the development of pharmaceuticals or quasi-drugs for the prevention, improvement, or treatment of hereditary retinal diseases such as cell degeneration.

Claims (11)

1. A pharmaceutical composition for preventing or treating retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.
2. According to claim 1,

   The human embryonic stem cell-derived mesenchymal stem cells are characterized in that to protect retinal neurons, a pharmaceutical composition.
3. According to claim 1,

   The human embryonic stem cell-derived mesenchymal stem cells are characterized in that the injection into the subretinal (subretinal) or vitreous cavity (intravitreal) of the eye, a pharmaceutical composition.
4. According to claim 1,

   The composition is characterized in that it is prepared in one or more formulations selected from the group consisting of ophthalmic solutions, eye drops, eye ointments, injections, and eyewash (eyewash), a pharmaceutical composition.
5. A quasi-drug composition for preventing or improving retinal pigmentation, comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.
6. 6. The method of claim 5,

   The human embryonic stem cell-derived mesenchymal stem cells are characterized in that to protect retinal neurons, quasi-drug composition.
7. 6. The method of claim 5,

   The human embryonic stem cell-derived mesenchymal stem cells is a quasi-drug composition, characterized in that the injection into the subretinal (subretinal) or vitreous cavity (intravitreal) of the eye.
8. 6. The method of claim 5,

   The composition is a quasi-drug composition, characterized in that it is prepared in one or more formulations selected from the group consisting of an ophthalmic solution, an eye ointment, an injection, and an eyewash.
9. A method for preventing or treating retinal pigmentation, comprising administering to an individual a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient.
10. Use of a composition comprising human embryonic stem cell-derived mesenchymal stem cells as an active ingredient for preventing or treating retinitis pigmentosa.
11. Use of human embryonic stem cell-derived mesenchymal stem cells for manufacturing a medicament for the treatment of retinal pigmentation.

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