Pharmaceutical composition comprising inferior turbinate stem cells as active ingredient for preventing or treating secondary atrophic rhinitis

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating secondary atrophic rhinitis, the composition comprising inferior turbinate stem cells as an active ingredient. It was found that excellent therapeutic effects are exhibited when inferior turbinate stem cells are treated in order to treat secondary atrophic rhinitis. The inferior turbinate stem cells according to the present invention not only cure submucosal fibrosis and regenerated cilia, but also cure squamous metaplasia. In addition, the inferior turbinate stem cells according to the present invention were shown to remain at an injection site for a long period of time, thereby significantly enhancing such effects, and are thus expected to be effectively used as a substance for preventing or treating secondary atrophic rhinitis.

Description

Pharmaceutical composition for preventing or treating secondary atrophic rhinitis containing inferior turbinate stem cells as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating secondary atrophic rhinitis, comprising inferior turbinate stem cells as an active ingredient.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0122882, filed February 16, 2024, and Korean Patent Application No. 10-2025-0018975, filed February 13, 2025, the entire contents of which are incorporated herein by reference.

[0003] Advances in biotechnology have opened up possibilities in cell therapy and regenerative medicine, and clinical trials and research in the field of stem cell therapy for human disease are currently underway at an astonishing pace. Stem cells, which possess the ability to self-replicate and differentiate into cells of various tissues, possess the ability to regenerate damaged body tissues and cells. They are recognized as a next-generation technology with significant potential for medical applications in the treatment of difficult-to-treat injuries, such as degenerative diseases for which there are currently no definitive treatments, and severe trauma.

[0004] Meanwhile, atrophic rhinitis (AR) is a condition that affects the inside of the nose. It occurs when the tissue known as the nasal mucosa and the underlying bone shrink, a process known as atrophy. This shrinkage alters the function of the nasal passages, resulting in a variety of symptoms.

[0005] Atrophic rhinitis is classified into primary and secondary atrophic rhinitis. Primary atrophic rhinitis is known to be caused by a virus. Patients suffering from secondary atrophic rhinitis experience a paradoxical sensation of nasal obstruction despite the anatomically intact nasal passages, including difficulty breathing, a perception of excessive nasal airflow, dyspnea, impaired lung expansion, and a subjective feeling of suffocation, all of which significantly impact their quality of life.

[0006] Despite the urgent need for development of a treatment for secondary atrophic rhinitis, no treatment for secondary atrophic rhinitis using inferior turbinate stem cells has been reported to date.

[0007] One object of the present invention is to provide a pharmaceutical composition for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate as an active ingredient.

[0008] Another object of the present invention is to provide a stem cell treatment for preventing or treating atrophic rhinitis, which comprises stem cells derived from inferior turbinate as an active ingredient.

[0009] Another object of the present invention is to provide a kit for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate and an instruction manual.

[0010]

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0012] The present invention provides a pharmaceutical composition for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate as an active ingredient.

[0013] In one embodiment of the present invention, the inferior turbinate-derived stem cell may be in the form of a single cell or a spheroid, but is not limited thereto.

[0014] In one embodiment of the present invention, the inferior turbinate-derived stem cells may be mixed with type 1 collagen, but is not limited thereto.

[0015] In one embodiment of the present invention, the inferior turbinate-derived stem cells may have differentiation potential into at least one cell selected from the group consisting of adipocytes, chondrocytes, and osteocytes, but are not limited thereto.

[0016] In one embodiment of the present invention, the composition may include, but is not limited to, 1X10^5 to 1X10^10 inferior turbinate-derived stem cells per 1 mL of type 1 collagen.

[0017] In one embodiment of the present invention, the composition may be, but is not limited to, an injectable composition.

[0018] In one embodiment of the present invention, the composition may be administered by intranasal injection, but is not limited thereto.

[0019] In one embodiment of the present invention, the inferior turbinate-derived stem cells may be human nasal turbinate-derived stem cells (hNTSCs), but are not limited thereto.

[0020] In one embodiment of the present invention, the inferior turbinate-derived stem cells may be characterized by negative expression of at least one marker selected from the group consisting of IgG-PE, CD14, and CD34, or positive expression of at least one marker selected from the group consisting of CD73, CD90, and CD105, but are not limited thereto.

[0021] In one embodiment of the present invention, the atrophic rhinitis may be secondary atrophic rhinitis, but is not limited thereto.

[0022] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0023] a) Regenerate ciliated cells and increase the number of ciliated cells;

[0024] b) Long-term presence in the treatment area;

[0025] c) Restoration of squamous metaplasia, submucosal fibrosis, and stratified ciliated columnar epithelium;

[0026] d) Increases nasal mucosal thickness;

[0027] e) increasing the gene expression level of FOXJ1; and

[0028] f) Regenerates nasal mucosal tissue.

[0029] The present invention provides a stem cell therapeutic agent for preventing or treating atrophic rhinitis, which comprises stem cells derived from inferior turbinate as an active ingredient.

[0030] A kit for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate and an instruction manual, is provided.

[0031]

[0032] In addition, the present invention provides a method for preventing or treating secondary atrophic rhinitis, comprising a step of administering a pharmaceutically effective amount of inferior turbinate-derived stem cells or a composition containing the same as an active ingredient to a subject in need thereof.

[0033] In addition, the present invention provides a use of a stem cell derived from the inferior turbinate or a composition containing the same as an active ingredient for preventing or treating secondary atrophic rhinitis.

[0034] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating secondary atrophic rhinitis using stem cells derived from inferior turbinate or a composition containing the same as an active ingredient.

[0035] According to a pharmaceutical composition for preventing or treating secondary atrophic rhinitis containing inferior turbinate stem cells as an active ingredient, it was confirmed that excellent therapeutic effects were exhibited when inferior turbinate stem cells were administered to treat secondary atrophic rhinitis. The inferior turbinate stem cells of the present invention not only restored submucosal fibrosis and regenerated cilia, but also restored squamous metaplasia. In addition, since the inferior turbinate stem cells of the present invention were found to exist at the injection site for a long period of time, and thus the above effects were significantly enhanced, it is expected that the composition will be usefully utilized as a substance for preventing or treating secondary atrophic rhinitis.

[0036] Figure 1 is a schematic diagram of an experiment to confirm the therapeutic effect of hNTSC on secondary atrophic rhinitis in the present invention.

[0037] Figure 2a shows the tissue including the surgical site, and the picture on the right is a photograph of the entire tissue shape at 10x magnification (turbinate; NT, septum; S).

[0038] Figure 2b shows the H&E staining results of the ciliated pseudostratified columnar epithelium of a normal control group and an animal model of secondary atrophic rhinitis.

[0039] Figure 2c shows squamous cell metaplasia (arrow), submucosal fibrosis (arrowhead), linear atrophy (chevron), and goblet cell metaplasia (thin arrow) taken at 200X magnification (upper image) and 600X magnification (lower image).

[0040] Figure 2d shows the results of MT staining of a normal control group and a secondary atrophic rhinitis animal model, showing that submucosal fibrosis occurred in the secondary atrophic rhinitis animal model.

[0041] The upper row images in Figure 2e show that ciliated cells were significantly reduced in the secondary atrophic rhinitis model as a result of IHC analysis to identify respiratory epithelial cell markers (Acetyl-α-Tubulin).

[0042] The lower row images in Fig. 2f show the results of IHC analysis of MUC5AC in a normal control group and a secondary atrophic rhinitis animal model, photographed at 400X, respectively.

[0043] Figure 2g shows a graph quantifying the mRNA expression levels of ZEB-1, FOXJ1, KRT14, and MUC5AC in a normal control group and a secondary atrophic rhinitis animal model.

[0044] Figure 3a shows endoscopic and microscopic photographs of hNTSC sampled in the present invention.

[0045] Figure 3b shows the experimental results confirming the hNTSC surface markers (IgG-PE, CD14, CD34, CD73, CD90, CD105) selected in the present invention.

[0046] Figure 3c shows the results of an experiment verifying the differentiation ability of hNTSCs selected in the present invention into adipocytes, chondrocytes, and osteocytes.

[0047] Figure 4a shows the results of manufacturing spheroids using hNTSCs selected in the present invention.

[0048] Figure 4b is an experimental result confirming the cell viability of the above spheroid, and Figure 4c is a graph quantifying this.

[0049] Figure 4d shows an injectable formulation manufactured using the hNTSC selected in the present invention.

[0050] Figure 4e is an experimental result evaluating the stability of the injection-type formulation over time as a cell viability rate, and Figure 4f is a graph quantifying this.

[0051] Figure 5a shows a time series of the surgical procedure for producing a secondary atrophic rhinitis animal model and the production process of the vehicle group and the treatment group.

[0052] Figure 5b shows the therapeutic effect of an injectable preparation using a single cell of stem cells derived from the inferior turbinate. When the preparation was injected into each of the Control group, Sham group, Vehicle group, and Treatment group, the presence of type 1 collagen at the injection site in the tissue was shown by MT staining.

[0053] Figures 5c and 5d show the therapeutic effect of an injectable preparation using single cells of inferior turbinate-derived stem cells. While loss of cilia was observed in the vehicle group, regeneration of cilia was confirmed in the treatment group.

[0054] Figure 5e shows the therapeutic effect of an injectable preparation using single cells of stem cells derived from the inferior turbinate, and shows the results of confirming ciliary markers through IHC analysis for the Sham group, Vehicle group, and Treatment group.

[0055] Figure 5f shows the therapeutic effect of an injectable preparation using single cells of inferior turbinate-derived stem cells, and analyzes the average nasal mucosal thickness of the Sham group, Vehicle group, and Treatment group.

[0056] Figures 5g and 5h show the therapeutic effect of an injectable preparation using single cells of inferior turbinate-derived stem cells, showing the labeling of hNTSCs through HuNu staining in the treatment group and the therapeutic effect of the corresponding area.

[0057] Figure 5i shows the therapeutic effect of an injectable formulation using spheroids of inferior turbinate-derived stem cells, and shows the mRNA expression levels of FOXJ1 in the nasal mucosal tissues of the Sham group, Vehicle group, and Treatment group (low, middle, high).

[0058] Figure 5j shows the therapeutic effect of an injectable preparation using spheroids of stem cells derived from the inferior turbinate, and the results of H&E and MT analysis show the mucosal regeneration effect in the treatment group.

[0059] The present invention provides a pharmaceutical composition for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate as an active ingredient.

[0060] The term "stem cell" used in the present invention refers to a cell that serves as the foundation for the cells or tissues that constitute an organism, capable of self-renewal through repeated division, and possessing the ability to differentiate into cells with specific functions depending on the environment. Stem cells are produced in all tissues during fetal development, and are also found in certain tissues where cells are actively replaced, such as bone marrow and epithelial tissues, even in adults.

[0061] Stem cells can be categorized into embryonic stem cells, derived from embryos in their early developmental stages; adult stem cells, derived from cells that form tissues after development; and induced pluripotent stem cells, derived using gene editing technology. Adult stem cells exist in various tissues of the body and play a crucial role in maintaining bodily homeostasis. Depending on the tissue's origin, they are classified as endoderm-, mesoderm-, or ectoderm-derived tissue stem cells.

[0062] Alternatively, stem cells can be classified into totipotent stem cells, which are formed when the fertilized egg begins its first division, pluripotent stem cells, which are formed by these cells continuously dividing and are present in the blastocyst lining, and multipotent stem cells, which exist in mature tissues and organs. Pluripotent stem cells are cells that can differentiate only into cells specific to the tissues and organs they contain, and are involved in the growth and development of each tissue and organ in the fetal, neonatal, and adult stages, as well as in maintaining homeostasis in adult tissues and inducing regeneration when tissues are damaged. These tissue-specific pluripotent cells are collectively called adult stem cells.

[0063] Among adult stem cells, surgeries to obtain bone marrow-derived stem cells and adipose tissue-derived stem cells are extremely painful and time-consuming, and the amount of stem cells obtained is very small. It takes a lot of time and money to culture clinically sufficient amounts, and there is a high risk of infection and cell loss. In the case of human inferior turbinate-derived stem cells, the surgery to obtain them is very short, with very little bleeding and pain. In addition, stem cells can be continuously secured through recycling of the discarded inferior turbinate tissue from the inferior turbinate surgery (rhinitis surgery), which is the most frequently performed procedure in the field of otolaryngology. In addition, the proliferative capacity of the stem cells is higher than that of the bone marrow-derived and adipose tissue-derived stem cells.

[0064] In one embodiment of the present invention, in order to confirm the use of inferior turbinate-derived stem cells for treating secondary atrophic rhinitis, inferior turbinate-derived stem cells were treated in the form of single cells or spheroids, and it was confirmed that in each case, they exhibited a therapeutic effect on secondary atrophic rhinitis. Therefore, in one embodiment of the present invention, the inferior turbinate-derived stem cells may be in either the form of single cells or spheroids, but are not limited thereto.

[0065] In the present invention, "spheroid" refers to a three-dimensional cell aggregate capable of self-organization. A spheroid is a miniature, simplified, in vitro three-dimensional organ that mimics the anatomical structure of actual tissues and can be used for various drug screening and disease models. A spheroid generally refers to a three-dimensional structure in which cells are aggregated to the extent that their cross-section appears circular or oval. However, it should be noted that this shape should be determined by considering the characteristics of the cell or cell aggregate, and does not necessarily imply a perfect spheroid or sphere.

[0066] In the present invention, all inferior turbinate-derived stem cells in the form of single cells or spheroids are mixed with type 1 collagen. Therefore, in one embodiment of the present invention, the inferior turbinate-derived stem cells may be mixed with type 1 collagen, but is not limited thereto. In this case, “mixing” may mean that type 1 collagen in the composition can be used as a solvent to achieve an appropriate concentration of inferior turbinate-derived stem cells in the form of single cells or spheroids. In this case, the inferior turbinate-derived stem cells and type 1 collagen do not necessarily need to be in a form that requires chemical, physical bonding, etc. In addition, in the present invention, if the inferior turbinate-derived stem cells are not delivered together with a carrier / carrier comprising type 1 collagen of the present invention, the therapeutic effect of secondary atrophic rhinitis may not occur.

[0067] In the present invention, "collagen" is a major protein in the extracellular matrix (ECM), providing structural support to skin, bones, cartilage, tendons, and other tissues. It is naturally produced in the body and plays a role in maintaining tissue elasticity and strength, making it suitable for use in various industries, including cosmetics, medical care, and food, to improve skin elasticity and promote wound healing.

[0068] At this time, type 1 collagen may be atelocollagen, but is not limited thereto, and any type 1 collagen with the same function may be applied. In the present invention, “atelocollagen” refers to a form of collagen in which the telopeptide, which induces an immune response, is removed, and the atelocollagen of the present invention may include any substance generally referred to as atelocollagen in the art.

[0069] In one embodiment of the present invention, type 1 collagen was used as collagen, and the atelocollagen form of type 1 collagen was used to control the concentration of inferior turbinate-derived stem cells. At this time, the concentration of type 1 collagen used may be 1% to 10%, for example, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 2% to 6%, 2% to 5%, 2% to 4%, 2% to 3%, 3% to 10%, 3% to 9%, 3% to 8%, 3% to 7%, 3% to 6%, 3% to 5%, 3% to 4%, or 3%, but is not limited thereto. Additionally, the above concentration of type 1 collagen can be manufactured using hyaluronic acid as a solvent, but is not limited thereto.

[0070] In the present invention, tri-lineage differentiation potential refers to the ability of stem cells to differentiate into adipocytes, chondrocytes, and osteocytes. Adipocytes are responsible for energy storage and metabolic regulation, chondrocytes play a role in movement and forming cartilage tissue and absorbing physical shock, osteocytes contribute to the formation and function of bone tissue, and the multipotent differentiation potential of stem cells is known to play an important role in tissue damage treatment and migration. In one embodiment of the present invention, tri-lineage differentiation potential was analyzed to verify the stem cell nature of inferior turbinate-derived stem cells. As a result, it was demonstrated that the inferior turbinate-derived stem cells of the present invention have the potential to differentiate into all of adipocytes, chondrocytes, and osteocytes. Therefore, in one embodiment of the present invention, the inferior turbinate-derived stem cells may have differentiation potential into any one or more selected from the group consisting of adipocytes, chondrocytes, and osteocytes, but is not limited thereto.

[0071] In one embodiment of the present invention, the composition may include, but is not limited to, 1X10^5 to 1X10^10 inferior turbinate-derived stem cells per 1 mL of type 1 collagen.

[0072] For example, 5X10^5 to 1X10^10, 6X10^5 to 1X10^10, 7X10^5 to 1X10^10, 8X10^5 to 1X10^10, 9X10^5 to 1X10^10, 1X10^6 to 1X10^10, 1.5X10^6 to 1X10^10, 2X10^6 to 1X10^10, 1X10^5 to 1X10^9, 5X10^5 to 1X10^9, 6X10^5 to 1X10^9, 7X10^5 to 1X10^9, 8X10^5 to 1X10^9, 9X10^5 to 1X10^9, It may be, but is not limited to, 1X10^6 to 1X10^9, 1.5X10^6 to 1X10^9, 2X10^6 to 1X10^9, 1X10^5 to 1X10^8, 5X10^5 to 1X10^8, 6X10^5 to 1X10^8, 7X10^5 to 1X10^8, 8X10^5 to 1X10^8, 9X10^5 to 1X10^8, 1X10^6 to 1X10^8, 1.5X10^6 to 1X10^8, or 2X10^6 to 1X10^8.

[0073] At this time, in the present invention, the inferior turbinate-derived stem cells were used in either the form of a single cell or a spheroid. At this time, the number of inferior turbinate-derived stem cells contained in the spheroid is the same as when the inferior turbinate-derived stem cells were used in the form of a single cell, and only the form of the cells may differ.

[0074] In one embodiment of the present invention, the composition may be, but is not limited to, an injectable composition. In one embodiment of the present invention, the composition may be administered by intranasal injection, but is not limited thereto.

[0075] In the present invention, “injection” may refer to a process of reaching the disease site of secondary atrophic rhinitis, which is a disease of the present invention, through the nasal cavity when injected into the nasal cavity by a specific method. Therefore, the “injection composition” may include any composition characterized by any formulation, concentration, and density that can be injected through the nasal cavity, and is not limited to specific examples. In one embodiment of the present invention, a method of injecting into the nasal cavity using a syringe was used, so the injection composition may include an injectable composition, but is not limited thereto. In addition, in the present invention, “intranasal injection” may include all administration routes that inject into the nasal cavity. In this case, as an example of intranasal injection, in one embodiment of the present invention, intranasal injection was successfully performed through injection into the nasal mucosa, but is not limited thereto.

[0076] In one embodiment of the present invention, the inferior turbinate-derived stem cells may be human nasal turbinate-derived stem cells (hNTSCs), but are not limited thereto.

[0077] In one embodiment of the present invention, the inferior turbinate-derived stem cells may be characterized by negative expression of at least one marker selected from the group consisting of IgG-PE, CD14, and CD34, or positive expression of at least one marker selected from the group consisting of CD73, CD90, and CD105, but are not limited thereto.

[0078] At this time, in the present invention, CD14 and CD34 may be hematopoietic cell markers, and CD73, CD90, and CD105 may be mesenchymal stem cell markers, but are not limited thereto.

[0079] In one embodiment of the present invention, the atrophic rhinitis may be secondary atrophic rhinitis, but is not limited thereto.

[0080] In one embodiment of the present invention, the secondary atrophic rhinitis may be treated by turbinate surgery, but is not limited thereto. Secondary atrophic rhinitis is known to commonly result from turbinate surgery, and up to 10% of patients undergoing turbinate surgery are known to develop secondary atrophic rhinitis due to excessive destruction of the inferior turbinate.

[0081] In the present invention, secondary atrophic rhinitis may be characterized by, but is not limited to, submucosal fibrosis localized by spindle-shaped cells.

[0082] In one embodiment of the present invention, the composition is a pharmaceutical composition characterized by at least one selected from the group consisting of:

[0083] a) Regenerate ciliated cells and increase the number of ciliated cells;

[0084] b) Long-term presence in the treatment area;

[0085] c) Restoration of squamous metaplasia, submucosal fibrosis, and stratified ciliated columnar epithelium;

[0086] d) Increases nasal mucosal thickness;

[0087] e) increasing the gene expression level of FOXJ1; and

[0088] f) Regenerates nasal mucosal tissue.

[0089] The present invention provides a stem cell therapeutic agent for preventing or treating atrophic rhinitis, which comprises stem cells derived from inferior turbinate as an active ingredient.

[0090] In one embodiment of the present invention, the atrophic rhinitis may be secondary atrophic rhinitis, but is not limited thereto.

[0091] In the present invention, “squamous metaplasia” is a transformation of the shape of squamous epithelial cells due to chronic inflammation or continuous stimulation. When squamous metaplasia occurs, it may partially take on the shape of squamous cell carcinoma, but it is known that when the cause is removed, it can be restored to the original cell, squamous epithelial cell.

[0092] In the present invention, "MUC5AC (MUC-5AC)" refers to a protein encoded by the MUC5AC gene in humans, a large, gel-forming glycoprotein. In the respiratory tract, it binds to inhaled pathogens that are removed by mucociliary clearance, thereby protecting against infection. Overproduction of MUC-5AC can cause diseases such as asthma and chronic obstructive pulmonary disease, and is also known to be associated with greater protection against influenza infection.

[0093] In the present invention, “ZEB-1 (Zinc finger E-box-binding homeobox 1, ZEB1) (formerly known as TCF8)” is a protein encoded by the ZEB1 gene in humans. ZEB-1 is known to be an important transcription factor in epithelial-mesenchymal transition (EMT) that participates in various life processes such as embryonic development, fibrosis, and tumor progression, and in particular, it has been shown to contribute to the development of fibrosis, cancer, and chemoresistance, and the fibrosis ultimately develops into cancer, and is therefore considered in the art as a biomarker of fibrosis and cancer.

[0094] In the present invention, ciliated pseudostratified columnar epithelium refers to the shape of a tissue in which various types of cells such as ciliated cells, goblet cells, and basal cells gather to form a single tissue. At this time, ciliated cells can be identified with acetyl-a-tubulin as a marker, goblet cells with FOXJ1, and basal cells with KRT14 as a marker. Ciliated pseudostratified columnar epithelium may refer to a type of respiratory epithelium found in the inner walls of the trachea and other airways that allows filtration and humidification of incoming air. In one embodiment of the present invention, it was confirmed that the expression of KRT14 or FOXJ1, which are markers of cells forming the ciliated pseudostratified columnar epithelium structure, was decreased, which may prove dryness, which is a clinical characteristic of secondary atrophic rhinitis.

[0095] Since the presence can be confirmed by positive staining by KRT14, which is encoded by a gene included in the bronchial signature of epithelial cells, “KRT14” can be used as a biomarker for epithelial cells in the present invention.

[0096] In the present invention, “FOXJ1 (Forkhead Box Protein J1)” and “Acetyl-α-Tubulin” are transcription factors related to ciliogenesis and are known as markers of epithelial cells, which are airway cells. FOXJ1 is located in the nucleus, and acetyl-α-tubulin can be identified by staining cilia, etc., rather than the nucleus of ciliated cells.

[0097] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0098] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, 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.

[0099] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0100] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (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, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0101] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0102] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0103] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0104] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0105] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0106] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as 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), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0107] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0108] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0109] 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. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0110] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0111] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0112] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0113] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0114] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0115] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0116] The present invention provides a stem cell therapeutic agent for preventing or treating atrophic rhinitis, which comprises stem cells derived from the inferior turbinate as an active ingredient.

[0117] In the present invention, "cell therapy" refers to a therapeutic agent that utilizes autologous, allogenic, or xenogenic cells to restore tissue function. In the present invention, it refers to a therapeutic agent that utilizes animal cells to regenerate injured tissue. In the present invention, "stem cell therapy" may refer to a cell therapy agent in which the cells are stem cells.

[0118] The present invention provides a kit for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate and an instruction manual.

[0119] In one embodiment of the present invention, the atrophic rhinitis may be secondary atrophic rhinitis, but is not limited thereto.

[0120] In the present invention, the "kit" refers to a tool that can prevent or treat secondary atrophic rhinitis using the inferior turbinate-derived stem cells of the present invention. In addition to the above-mentioned substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for the storage and processing methods thereof. As a specific example, each component may be applied at least once without limitation in the number of times, there is no restriction on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0121] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0122] In addition, the present invention provides a method for preventing or treating secondary atrophic rhinitis, comprising a step of administering a pharmaceutically effective amount of inferior turbinate-derived stem cells or a composition containing the same as an active ingredient to a subject in need thereof.

[0123] In addition, the present invention provides a use of a stem cell derived from the inferior turbinate or a composition containing the same as an active ingredient for preventing or treating secondary atrophic rhinitis.

[0124] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating secondary atrophic rhinitis using stem cells derived from inferior turbinate or a composition containing the same as an active ingredient.

[0125] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0126]

[0127] [Example]

[0128]

[0129] Preparation of white rabbits for animal model use

[0130] New Zealand white rabbits (SPF NZW; Kangda, Qingdao, China) were divided into four groups: a model group that underwent turbinate surgery, a sham group that received surgery and intraoperative type I collagen injection, a treatment group that received surgery and hNTSC, and a normal control (NC) group that received no intervention. All procedures were conducted in compliance with relevant ethical guidelines, including approval from the Institutional Animal Care and Use Committee (IACUC) of The Catholic University of Korea (CUMS-2022-0289-03).

[0131]

[0132] Isolation and culture of hNTSCs from human turbinate tissue

[0133] Fresh hNTSCs were sampled from the inferior turbinate tissue of a 22-year-old male. This procedure was performed at Seoul St. Mary's Hospital and received ethical approval from the Institutional Review Board of The Catholic University of Korea (KC18TESI0167). Initially, the tissue was rinsed with gentamicin solution in the operating room and washed with antibiotic-antimycotic solution (Gibco, Gaithersburg, MD, USA) and Dulbecco's phosphate-buffered saline (D-PBS; Welgene, Namcheon, South Korea).

[0134] Then cut the tissue into 0.5mm 3 The slices were cut into thick fragments and cultured at 37°C in 10% fetal bovine serum and α-MEM (Minimum Essential Medium Eagle - alpha modification). The culture environment was maintained at 5% CO2 and sealed with sterilized glass slides.

[0135] After a 2-week culture period, cells were harvested from the plates and subsequently dissociated using a 1 mL solution of 0.25% trypsin / 1 mM ethylenediaminetetraacetic acid (EDTA), and cell morphology and concentration were examined at 40x magnification.

[0136]

[0137] Flow cytometry analysis

[0138] Single-cell suspensions of hNTSCs were prepared and incubated with phycoerythrin-conjugated antibodies specifically targeting mesenchymal stem cell surface markers, including CD14, CD34, CD73, CD90, and CD105 (BD Pharmingen, San Diego, CA, USA). After 1 hour of incubation, the cells were washed and resuspended in D-PBS.

[0139]

[0140] Preparation of the remedy

[0141] To prepare the therapeutic agent to be administered to the treatment group, hNTSCs were harvested from the culture dish and pelleted. These cells were then cultured in cold type I collagen (COLTRIX ® (TendoRegen), Ubiosis, Sungnam, South Korea) were fused at a concentration of 2 × 10^6 cells / mL. 100 μL aliquots were accurately administered using a 1 mL insulin syringe with a 29-gauge needle (BD Pharmingen). Accordingly, 100 μL of type I collagen was dispensed to prepare the sham group to ensure uniformity of the administration process.

[0142]

[0143] Surgical procedure

[0144] To create an animal model of empty nose syndrome (ENS) using rabbits, a bone flap from the nasal dorsum was first removed. A 3-cm incision was made along the midline from the tip of the nose, and the skin flap and periosteum were carefully separated. A disk burr was then attached to a micro handpiece to incise a 0.5 x 2.5 cm square shape on both sides of the nasal septum. The removed bone flap was stored in saline solution, and electrocautery was performed using Bovie bipolar forceps to create a defect in the nasal turbinate. After creating the defect, collagen type I hydrogel (COLTRIX) was applied to the wound site at a density of 2 x 10^6 cells per mL. ® After mixing TendoRegen and injecting 100 μL into each turbinate, the bone flap stored in saline was placed in place, and the capsule and periosteum were sutured to finish the procedure. Observation was conducted for two months.

[0145]

[0146] Organizational processing

[0147] Rabbits were euthanized with potassium chloride. Samples were fixed overnight in 4% formaldehyde and rinsed with PBS. After decalcification in 1% EDTA (pH 8) for 2 months, they were embedded in paraffin.

[0148]

[0149] Histological analysis

[0150] Sections 4.5 μm thick were deparaffinized in xylene and rehydrated through graded ethanol. Hematoxylin (Abcam, Cambridge, UK) and Eosin-Y (Siheung, South Korea) were used along with Masson's trichrome stain (Abcam, Cambridge, UK) for collagen visualization.

[0151]

[0152] Immunohistochemistry (IHC) and immunofluorescence (IF) analysis

[0153] Sections were subjected to epitope retrieval in 0.01 M sodium citrate buffer (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Endogenous peroxidase activity was neutralized with 3% H2O2. Sections were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h.

[0154] For IHC, primary antibodies, anti-Mucin5AC (1:100; Abcam) and anti-acetylated-α-tubulin (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), were applied for a 2-h incubation period. Sections were washed with phosphate-buffered saline (PBS) and treated with a mixture of anti-rabbit and anti-mouse horseradish peroxidase polymer (GBI Labs, Bothell, WA, USA) for 30 min. Diaminobenzidine (DAB; GBI Labs) staining and hematoxylin counterstaining were then performed.

[0155] For IF analysis, sections were incubated primarily with anti-HuNu (1:200; Sigma-Aldrich) for 2 h, washed, and treated with secondary antibody, goat anti-mouse IgG Alexa Fluor™ 555 (1:1000; Invitrogen, Carlsbad, CA, USA), for 1 h. DAPI (1 μg / mL; Invitrogen) counterstaining was performed. Slides were examined using a confocal LASER microscope (LSM800; Carl Zeiss, Oberkochen, Germany).

[0156]

[0157] RNA extraction and real-time quantitative polymerase chain reaction

[0158] Total RNA was extracted from unfixed tissues using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using iScript™ (Bio-Rad, Hercules, CA, USA) and amplified on a CFX96™ (Bio-Rad). Relative mRNA levels were calculated using the 2-ΔΔCt method, with HPRT1 as a control. Primers are listed in Table 1.

[0159]

[0160] NO.NAMESEQUENCE1HPRT1_FGAC CAG TCA ACA GGG GAC AT2HPRT1_RCTT GCG ACC TTG ACC ATC TT3FOXJ1_FTCG ACT GGG AAG CCA TCT4FOXJ1_RGTC GAA GTC CAG GCT GTT G5Keratin 14_FGAA GGA GGA ACT GGC CTA CC6Keratin 14_RTCT CGT ACT GGT CAC GCA TC7MUC5AC_FTCT GCT GTC CCG AGA GAA CAC8MUC5AC_RCTG CCA TCA CAA ATG ACC AC9ZEB-1_FGCC TAC AGA ACC CAA CTG GA10ZEB-1_RTCT CTC CGC TGT GAA TCC TT

[0161]

[0162] Statistical analysis

[0163] All statistical evaluations were performed using Prism 5.0 software (GraphPad Software, San Diego, CA, USA) using two-way analysis of variance.

[0164]

[0165] Example 1. Confirmation of histological changes in a secondary atrophic rhinitis model

[0166] Example 1-1. Validation of an animal model of secondary atrophic rhinitis based on H&E analysis.

[0167] To verify the animal model of secondary atrophic rhinitis developed in the present invention, complete nasal extraction was performed via conical cross-section (Fig. 2a). Subsequently, H&E staining was used to analyze whether the histological characteristics of secondary atrophic rhinitis of the present invention were reflected. A normal control (NC) was used as a control.

[0168]

[0169] As a result, ciliated pseudostratified columnar epithelium was confirmed in the normal control group and the secondary atrophic rhinitis animal model, as shown in Figure 2b. In addition, squamous cell metaplasia (arrow), submucosal fibrosis (arrowhead), linear atrophy (chevron), and goblet cell metaplasia (thin arrow) were confirmed in the normal control group and the secondary atrophic rhinitis animal model, as shown in Figure 2c. In addition, a comparative evaluation of the histological parameters for secondary atrophic rhinitis in both groups is shown in Table 2.

[0170]

[0171] VariablesNormal Controls, n / N (%)Models, n / N (%)Squamous cell metaplasia0 / 3 (0%)2 / 3 (66.7%)Submucosal fibrosis0 / 3 (0%)3 / 3 (100%)Glandular atrophy0 / 3 (0%)3 / 3 (100%)Goblet cell metaplasia0 / 3 (0%)2 / 3 (66.7%)

[0172]

[0173] According to these results, the normal control group exhibited well-preserved pseudostratified epithelium with cilia. In contrast, the secondary atrophic rhinitis model group exhibited significant histological changes characteristic of secondary atrophic rhinitis, including squamous metaplasia, submucosal fibrosis, glandular atrophy, and goblet cell metaplasia. Notably, as shown in Figure 2b, the glandular structure was completely reduced in areas with prominent submucosal fibrosis.

[0174]

[0175] Example 1-2. Validation of an animal model of secondary atrophic rhinitis based on marker analysis.

[0176] To elucidate histological alterations associated with secondary atrophic rhinitis, specific markers were analyzed. Marker analysis was performed using Masson's trichrome (MT) staining and IHC analysis.

[0177]

[0178] Visualization of collagen fibrosis, indicated by blue staining using Masson's trichrome (MT) staining, revealed that high concentrations of collagen were confined to fibrotic cells. Comparative analysis between the normal control group and the secondary atrophic rhinitis model revealed fibrosis, indicated by blue, within the submucosal layer of the secondary atrophic rhinitis model (Fig. 2d).

[0179]

[0180] IHC analysis was used to identify respiratory epithelial cell markers. Ciliated cells were targeted using an antibody against acetylated-α-tubulin. Results showed that the presence of ciliated cells was sporadic, but significantly reduced in a model of secondary atrophic rhinitis (Figure 2e).

[0181] MUC5AC staining revealed two distinct expression patterns in goblet cells. In areas where the pseudostratified columnar structure was deteriorated due to squamous metaplasia, MUC5AC expression was not significantly evident. However, in areas exhibiting goblet cell metaplasia, MUC5AC expression was concentrated (Fig. 2f).

[0182] Additionally, upregulation of the fibrosis marker zinc-finger E-box-binding homeobox 1 (ZEB-1) was observed. Markers associated with ciliated pseudostratified columnar epithelium, with the exception of MUC5AC, were found to exhibit reduced expression, as evidenced by KRT14 and FOXJ1. Nevertheless, these differences between the two groups did not reach statistical significance (Fig. 2g).

[0183]

[0184] Example 2. Selection of stem cells derived from the inferior turbinate and verification of differentiation potential.

[0185] Example 2-1. Selection of stem cells derived from the inferior turbinate

[0186] The hNTSCs sampled according to the experimental method of the present invention are as shown in Fig. 3a. The collected hNTSCs were subjected to flow cytometry analysis (BD FACS Canto II, Becton Dickinson, Franklin Lakes, NJ, USA) to evaluate the constituent cell populations.

[0187]

[0188] As a result, hNTSCs used in the present invention were selected based on confirmed negative expression for hematopoietic cell markers CD14 and CD34 and positive expression for mesenchymal stem cell markers CD73, CD90, and CD105, as described in FIG. 3b.

[0189]

[0190] Example 2-2. Verification of differentiation potential of stem cells derived from the inferior turbinate

[0191] To verify the inferior turbinate-derived stem cells selected in Example 2-1, the trilineage differentiation potential into adipocytes, chondrocytes, and osteocytes was confirmed in passage 6 hNTSCs. First, the method for adipogenesis analysis is as follows. hNTSCs were seeded at 1.5 × 10^4 cells per well in a 4-well plate, and the medium was replaced with the StemPro™ Adipocyte Differentiation Kit (Gibco). After 2 weeks, the cells were fixed with 2% paraformaldehyde (PFA) and stained with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) to evaluate differentiation into adipocytes. For chondrogenesis analysis, hNTSCs were first grown to form spheroids using the hanging-drop technique for 2 days to differentiate into chondrocytes. Afterwards, the generated hNTSC spheroids were transferred to StemPro™ chondrogenic differentiation medium (Gibco), and after 2 weeks, the spheroids were fixed with 2% PFA, embedded in paraffin, and sectioned at 4.5 μm thickness. The sections were deparaffinized with xylene and rehydrated through a graded ethanol series. Afterwards, they were stained with Alcian Blue (Abcam) to evaluate chondrogenic differentiation according to the manufacturer's instructions. Finally, for osteogenic differentiation analysis, hNTSCs were induced to differentiate into the osteocyte lineage using the StemPro™ osteogenic differentiation kit (Gibco). The cells were cultured in osteogenic differentiation medium for 3 weeks, fixed with 2% PFA, and stained with 2% Alizarin Red (Sigma-Aldrich) to evaluate cellular differentiation and to determine whether calcium deposition occurred.

[0192]

[0193] As a result, it was proven that the inferior turbinate-derived stem cells of the present invention have multipotent differentiation potential that can differentiate into adipocytes, chondrocytes, and osteocytes (Fig. 3c).

[0194]

[0195] Example 3. Preparation of spheroids and injectable preparations using inferior turbinate-derived stem cells

[0196] Example 3-1. Production of spheroids using inferior turbinate-derived stem cells and confirmation of their survival rate.

[0197] Spheroids (cell aggregates) were prepared using the inferior turbinate-derived stem cells identified in Example 2. The spheroids were prepared using StemFIT 3D ® It was manufactured using plate (H853400, Microfit, Gyeonggi, Korea).

[0198] As a result, the size of the spheroids was 251.6 ± 10.4 μm, and the average size was 250 μm, confirming that spheroids of uniform size were formed by the method of the present invention (Fig. 4a).

[0199]

[0200] Next, the cell viability was checked to confirm the safety of the above spheroids. Specifically, 1.5X10^6 cells were seeded in Stemfit (gridded wells), and spheroid formation was confirmed after overnight (14-16 hours), and then harvested and used for viability analysis. The spheroids were stained with Calcein-AM (green - live) / EthD-1 (red - dead) to check the cell viability.

[0201] As a result, 98% of the cells in the entire spheroids survived, confirming a remarkably high cell survival rate (Figs. 4b and 4c). Since a high cell survival rate indicates that the cells used for treatment can efficiently maintain their function in the body and minimize side effects, these experimental results suggest that the inferior turbinate-derived stem cells of the present invention have excellent safety as a cell therapy agent.

[0202]

[0203] Example 3-2. Preparation of an injectable formulation using inferior turbinate-derived stem cells and confirmation of survival rate.

[0204] An injectable formulation was prepared using spheroids derived from the inferior turbinate stem cells prepared in Example 3-1. At this time, the inferior turbinate stem cells alone and type 1 collagen (collagen hydrogel, COLTRIX) serving as a support for each spheroid were prepared. ® By mixing stemfit and TendoRegen, we developed an injectable formulation by manufacturing an advanced bio-convergence formulation. Specifically, after seeding cells in stemfit and forming spheroids, the spheroids were collected by pipetting or scraping with a tip. The spheroids were collected at the bottom of the tube by gently centrifuging (200 g, 3 minutes), the medium was removed, and type 1 collagen was added according to the concentration. At this time, the following method was applied to add according to the concentration. For example, since 1.5X10^6 inferior turbinate-derived stem cells are obtained from one stemfit product, in order to adjust the concentration of 2X10^6 cells / mL in the low-concentration group among the treatment groups, 750uL of type 1 collagen was added to the tube where the spheroids were collected and pipetted to adjust the concentration. For the injection-type preparation, an insulin syringe (30G) was used to develop an injection form to minimize damage to the nasal mucosa when injecting the preparation into the nose (Fig. 4c). Only the amount of the well-mixed preparation to be injected once was put into the injection form and stored in the refrigerator.

[0205]

[0206] Next, to confirm the stability of the manufactured injectable formulation, cell viability was analyzed over time. Specifically, the injectable formulation, manufactured by mixing type I collagen and spheroids, was stained with Calcein-AM (green—live) / EthD-1 (red—dead), and cell viability was analyzed for 5 and 10 hours.

[0207]

[0208] As a result, the injectable formulation using spheroids demonstrated excellent cell viability for up to 10 hours, demonstrating its stability as a stable injectable formulation (Figs. 4d and 4e). Considering the time gap required between the preparation of the injectable formulation for the treatment of secondary atrophic rhinitis and its injection into the nasal mucosa due to transport time and surgical time, this suggests that the excellent stability will lead to therapeutic activity in secondary atrophic rhinitis.

[0209]

[0210] Example 4. Confirmation of the therapeutic effect of inferior turbinate-derived stem cells on secondary atrophic rhinitis.

[0211] According to the experimental method of the present invention and Fig. 5a, the secondary atrophic rhinitis model of Example 1 was used to observe the model group, the sham group, and the treatment group, respectively. At this time, in the case of the treatment group, 50 μL of the injection-type preparation was each administered at the following concentrations to the area where electrocautery was performed: low concentration: 2X10^6 cells / mL, medium concentration: 1X10^7 cells / mL, and high concentration: 1X10^8 cells / mL. The injection-type preparation using single cells (hTNSC) was injected at a low concentration, and in the vehicle group, collagen type I (collagen type I, COLTRIX ® The same amount of TendoRegen was administered to the area where electrocautery was performed.

[0212]

[0213] Example 4-1. Confirmation of the therapeutic effect of secondary atrophic rhinitis following single-cell administration of inferior turbinate-derived stem cells.

[0214] First, MT staining was performed after administering the preparation to the control group, sham group, vehicle group, and treatment group.

[0215] As a result, the presence of type I collagen was confirmed at the injection site within the tissue, and accordingly, it was investigated that all preparations using type I collagen were well injected within each experimental group (Fig. 5b) (blue: collagen, red: cytoplasm, and black: cell nuclei). In addition, it was confirmed that submucosal fibrosis localized to spindle-shaped cells was well-appeared in the Sham group, which is a secondary atrophic rhinitis model group of the present invention.

[0216]

[0217] Additionally, the treatment effects in the vehicle group and the treatment group were compared and analyzed.

[0218] As a result (Figs. 5c and 5d), unlike the Sham group and the Vehicle group, which showed no therapeutic effect with type I collagen, the treatment group showed ciliary regeneration promoted by the injected hNTSCs. Furthermore, these different areas were distinguished based on the presence or absence of nuclei detected by hematoxylin staining. In particular, while exogenous type I collagen lacked nuclei, the submucosal fibrosis area contained nuclei.

[0219]

[0220] To further investigate ciliary regeneration, the ciliary regeneration effect of hNTSCs was analyzed by confirming cilia in tissues using immunohistochemistry (IHC) staining for acetylated-α-tubulin, a protein marker of cilia, in each experimental group.

[0221] As a result of the analysis (Fig. 5e), cilia regrowth in the squamous metaplasia area was confirmed by acetylated-α-tubulin immunohistochemical staining (brown: acetylated-α-tubulin, purple: cell nuclei).

[0222]

[0223] Meanwhile, after administering the formulation to the sham, vehicle, and treatment groups, H&E analysis was performed, and the data were used to analyze nasal mucosal thickness. Specifically, nasal mucosal thickness was measured within tissue photographs, the average was calculated, and this was quantified.

[0224] As a result (Fig. 5f), when the nasal mucosal thickness of the Sham group was set as 100 as the reference point, the nasal mucosal thicknesses of the Vehicle group and the treatment group were 117.9 (%) and 140.4 (%), respectively, indicating that the average nasal mucosal thickness increased, and accordingly, it was confirmed that the nasal mucosal tissue was restored in secondary atrophic rhinitis when the inferior turbinate-derived stem cells of the present invention were treated.

[0225]

[0226] Finally, in the treatment group, the labeling in hNTSCs and the therapeutic effect in the corresponding area were confirmed through staining with HuNu (human nuclei), a human nuclear marker.

[0227] As a result (Figs. 5g and 5h), staining for human nuclei (HuNu) demonstrated migration of hNTSCs within the epithelial layer of the squamous metaplasia area, demonstrating that the injected cells in the treatment group remained at the injection site even after 2 months, demonstrating long-term presence of hNTSCs (Fig. 5g). In addition, localized regeneration of cilia was observed adjacent to the HuNu-confined area (Fig. 5h).

[0228]

[0229] Example 4-2. Confirmation of the therapeutic effect of secondary atrophic rhinitis following administration of a spheroid injection-type preparation of inferior turbinate-derived stem cells.

[0230] First, we analyzed the transcript expression levels of FOXJ1, a ciliated cell marker, and MUC5AC, a mucus cell marker, in the nasal mucosal tissues of the Sham group, the Vehicular group, and the treatment groups (low, middle, and high concentrations). Specifically, RT-qPCR was performed to analyze the expression levels (Table 1), and after injecting the corresponding preparations for each experimental group, nasal mucosa was obtained, RNA was isolated with trozol, and cDNA was synthesized to confirm the gene expression levels of the above markers in the nasal mucosal tissues.

[0231]

[0232] As a result, it was confirmed that the expression level of FOXJ1 mRNA was high in the order of high group, middle group, low group, Vehicle group, and sham group in a dose-dependent manner of hNSTCs. In the experimental group injected with the inferior turbinate-derived stem cell spheroid injection preparation of the present invention, the FOXJ1 mRNA expression level was found to be higher than that of the sham group and Vehicle group regardless of the concentration, and in particular, the mRNA expression level of FOXJ1 in the high group was confirmed to be statistically significant. These experimental results correspond to the occurrence of a ciliary regeneration effect when a single stem cell derived from inferior turbinate is injected, suggesting that ciliary regeneration occurs as ciliated cells are regenerated and their number increases (Fig. 5i).

[0233]

[0234] Finally, after the treatment group was injected into rabbits, the histology of the nasal mucosal tissue was analyzed using H&E and MT staining to confirm the regenerative effect of the nasal mucosal tissue.

[0235] As a result, it was confirmed that the mucosa was not regenerated in the vehicle group, whereas in the treatment group, the mucosa was regenerated to an excellent level through cilia formation (low concentration) / cilia formation and restoration of the stratified ciliated columnar epithelium morphology (medium concentration, high concentration) (Fig. 5j).

[0236]

[0237] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0238] The present invention relates to a pharmaceutical composition for preventing or treating secondary atrophic rhinitis, comprising inferior turbinate stem cells as an active ingredient. It was confirmed that when inferior turbinate stem cells were treated to treat secondary atrophic rhinitis induced through turbinate surgery, an excellent therapeutic effect was exhibited. The inferior turbinate stem cells of the present invention not only restored submucosal fibrosis and regenerated cilia, but also restored squamous metaplasia. In addition, since the inferior turbinate stem cells of the present invention were shown to exist for a long period of time at the injection site, and such effect was significantly enhanced, it is expected that the composition will be usefully utilized as a substance for preventing or treating secondary atrophic rhinitis, and thus its industrial applicability is recognized.

Claims

1. A pharmaceutical composition for preventing or treating atrophic rhinitis, comprising stem cells derived from the inferior turbinate as an active ingredient.

2. In paragraph 1, A pharmaceutical composition wherein the stem cells derived from the inferior turbinate are in either a single cell or spheroid form.

3. In paragraph 2, A pharmaceutical composition wherein the above-mentioned inferior turbinate-derived stem cells are mixed with type 1 collagen.

4. In paragraph 1, A pharmaceutical composition wherein the stem cells derived from the inferior turbinate have the ability to differentiate into at least one cell selected from the group consisting of adipocytes, chondrocytes, and osteocytes.

5. In paragraph 3, A pharmaceutical composition comprising 1X10^5 to 1X10^10 inferior turbinate-derived stem cells per 1 mL of type 1 collagen.

6. In paragraph 1, A pharmaceutical composition, wherein the composition is a composition for injection.

7. In paragraph 6, A pharmaceutical composition, wherein the composition is administered by intranasal injection.

8. In paragraph 1, A pharmaceutical composition, wherein the above inferior turbinate-derived stem cells are human nasal turbinate-derived stem cells (hNTSCs).

9. In paragraph 1, A pharmaceutical composition, wherein the above-mentioned inferior turbinate-derived stem cells are characterized in that they negatively express at least one marker selected from the group consisting of IgG-PE, CD14, and CD34, or positively express at least one marker selected from the group consisting of CD73, CD90, and CD105.

10. In paragraph 1, A pharmaceutical composition wherein the above atrophic rhinitis is secondary atrophic rhinitis.

11. In paragraph 1, A pharmaceutical composition characterized in that the composition comprises at least one selected from the group consisting of: a) Regenerate ciliated cells and increase the number of ciliated cells; b) Long-term presence in the treatment area; c) Restoration of squamous metaplasia, submucosal fibrosis, and stratified ciliated columnar epithelium; d) Increases nasal mucosal thickness; e) increasing the gene expression level of FOXJ1; and f) Regenerates nasal mucosal tissue.

12. A stem cell treatment for preventing or treating atrophic rhinitis, comprising stem cells derived from the inferior turbinate as an active ingredient.

13. A kit for preventing or treating atrophic rhinitis, comprising stem cells derived from inferior turbinate and an instruction manual.

14. A method for preventing or treating secondary atrophic rhinitis, comprising administering a pharmaceutically effective amount of stem cells derived from inferior turbinate or a composition containing the same as an active ingredient to a subject in need thereof.

15. Use of stem cells derived from inferior turbinate or a composition containing them as an active ingredient for preventing or treating secondary atrophic rhinitis.

16. Use for manufacturing a preparation for preventing or treating secondary atrophic rhinitis using stem cells derived from inferior turbinate or a composition containing the same as an active ingredient.

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