KR102536671B1 - Composition for preventing or treating Skin disease caused by hypersensitivity immune response based on pluripotent stem cells - Google Patents

Abstract

The present invention provides mesenchymal stem cells derived from human pluripotent stem cells as a cell therapy agent for preventing or treating skin diseases caused by hypersensitive immune reactions, including edema, urticaria, itching, and contact dermatitis. The present invention can effectively relieve symptoms of edema, itching, and urticaria and treat contact dermatitis with only local subcutaneous injection, and has the advantage of having no side effects compared to antihistamines that are administered systemically.

Description

Composition for preventing or treating skin disease caused by hypersensitivity immune response based on pluripotent stem cells}

The present invention relates to a pharmaceutical composition comprising human pluripotent stem cell-derived mesenchymal stem cells as an active ingredient for the prevention or treatment of skin diseases caused by hypersensitivity immune reactions, and in particular, the target disease to be prevented or treated by the present invention is edema. , urticaria, pruritus, and contact dermatitis.

Mesenchymal stem cells (MSCs) are cells with pluripotency that can be isolated from tissues such as bone marrow, cord blood, fat, and muscle, and various studies are being conducted on the functions of MSCs. For example, Nauta et al. reported the immunomodulatory properties of MSCs. According to Nauta et al., MSCs are immunomodulatory agents in maintenance of peripheral tolerance, transplantation tolerance, autoimmunity, tumor evasion, and fetal-maternal tolerance. can function as

On the other hand, hives is an intractable skin disease characterized by swelling of the skin and redness surrounding it. Although it is known, it is difficult to identify the cause, so there is no fundamental treatment method, and usually treatment methods aimed at relieving symptoms are all. For example, antihistamines are used to control the spread of histamine in the initial immune response by the hives antigen, and antihistamines are used to relieve itching symptoms, or when acute hives occur throughout the body, epinephrine is injected along with antihistamines to alleviate the symptoms. let it

As a result of intensive research on the development of cell therapy products using human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) at a clinically applicable level, the inventors of the present invention conducted human pluripotent stem cells. The present invention was completed by confirming that the derived mesenchymal stem cells could be applied to the treatment of intractable skin diseases.

Nauta AJ, et.al, Immunomodulatory properties of mesenchymal stromal cells, Blood 2007 Nov 15; 110(10): 3499-3506

While studying the various functions of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC, MMSC), the present inventors subcutaneously injected MMSC into a mouse that locally induced contact urticaria disease. As a result, It was confirmed that by suppressing the immune response of the disease-induced lesion, it was possible to reduce edema and rash and treat contact skin inflammatory diseases including pruritus, which is itching.

Accordingly, the technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating skin diseases caused by hypersensitivity immune reactions, comprising human pluripotent stem cell-derived mesenchymal stem cells (MMSC) as an active ingredient.

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

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating skin diseases caused by hypersensitivity immune reactions, comprising human pluripotent stem cell-derived mesenchymal stem cells (MMSC) as an active ingredient.

The skin disease to be prevented or treated in the present invention may be at least one disease selected from the group consisting of edema, urticaria, pruritus, and contact dermatitis.

In the present invention, human pluripotent stem cell-derived mesenchymal stem cells (MMSC) may be provided as a cell therapy agent for preventing or treating skin diseases caused by the hypersensitive immune response.

As an embodiment of the present invention, the pluripotent stem cells may be induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC), but in the specific experiments of the present invention, embryonic stem cells Mesenchymal stem cells were obtained using the cells to confirm their therapeutic effect on skin diseases.

As another embodiment of the present invention, when the pharmaceutical composition or cell therapy agent is administered to the human body, human pluripotent stem cell-derived mesenchymal stem cells (MMSC) are 2 × 10 ⁷ to 3 × 10 ⁷ Human pluripotent stem cell-derived mesenchymal stem cells may be included to be administered to dogs, preferably 2.5 × 10 ⁷ dogs.

In another embodiment of the present invention, the pharmaceutical composition or cell therapy agent is administered with 4×10 ⁵ to 5×10 ⁵ human pluripotent stem cell-derived mesenchymal stem cells (MMSC) per unit body weight (1 kg) of the subject. it could be

As another embodiment of the present invention, the pharmaceutical composition or cell therapy agent is provided in the form of an injection and may be administered subcutaneously locally to the affected area.

As another embodiment of the present invention, the pharmaceutical composition or cell therapy agent may inhibit the activity of helper T cells, and further reduce the expression level of inflammatory cytokines in a lesion. And, specifically, it may be to reduce the expression levels of IFN-γ, TNF-α, IL-4, and IL-6.

As another embodiment of the present invention, the pharmaceutical composition or cell therapy agent may inhibit the activity of mast cells in lesions.

In addition, the present invention is directed to treating at least one hypersensitive immune response selected from the group consisting of edema, urticaria, pruritus, and contact dermatitis, comprising administering human pluripotent stem cell-derived mesenchymal stem cells (MMSC) to an individual. It provides a method for treating skin diseases by

As one embodiment of the present invention, the administration may be administered by local injection under the skin of the lesion.

As another embodiment of the present invention, the subject may be a mammal, including a human, having a skin disease caused by at least one hypersensitive immune response selected from the group consisting of edema, urticaria, pruritus, and contact dermatitis.

As another embodiment of the present invention, the method is to check the degree of swelling and / or rash of the skin with the naked eye before and after the administration step, compare it with before administration, evaluate the degree of itchiness of the subject, or inflammatory reaction in the skin tissue of the subject. It may be to further include the step of measuring the degree.

In addition, the present invention is human pluripotent stem cell-derived mesenchymal stem cells for the preparation of drugs for preventing or treating skin diseases caused by at least one hypersensitive immune response selected from the group consisting of edema, urticaria, pruritus, and contact dermatitis ( MMSC).

As one embodiment of the present invention, the human pluripotent stem cell-derived mesenchymal stem cells (MMSC) may be obtained by performing the following steps:

(a) subculturing human pluripotent stem cells 70 times or less, preferably 40 to 60 times;

(b) selecting cystic embryoid bodies from among the embryoid bodies in which differentiation of the human pluripotent stem cells was induced;

(c) loading the cystic embryoid body on top of the cell-permeable 3-dimensional culture insert and separating a cluster of mesenchymal stem cells that migrated to the bottom of the culture insert; and

(d) isolating only the cluster in the form of a monolayer among the clusters of the separated mesenchymal stem cells.

The human embryonic stem cell-derived mesenchymal stem cells (MMSC) of the present invention can be locally administered to skin diseases caused by hypersensitivity immune reactions such as contact urticaria to effectively alleviate the symptoms and suppress inflammatory reactions, Compared to antihistamines that are administered and act, skin diseases can be effectively treated without side effects. In addition, the present invention provides a dosage of mesenchymal stem cells that can most effectively treat skin diseases caused by the above-mentioned hypersensitivity immune response, and can be provided as a guide for clinical application.

1 is a schematic view of the TMA/acetone-mediated contact urticaria mouse model production and human embryonic stem cell-derived mesenchymal stem cell treatment experiments.
2 and 3 are time lapses in contact urticaria disease animal models treated with human embryonic stem cell-derived mesenchymal stem cells (MMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), or cetirizine. This is a graph comparing and confirming the change in the degree of ear edema according to
Specifically, in FIG. 2, A shows the change in ear edema in all experimental groups, B shows the change in ear edema in the MMSC 10k administration group, the BM-MSC 10k administration group, and the cetirizine administration group, and C is the negative control group administered with PBS. and MMSC 10k administration group shows the change in ear edema, D shows the change in ear edema in the negative control group and the BM-MSC 10k administration group.
In addition, in FIG. 3, A shows the change in ear edema of all experimental groups, B shows the change in ear edema of the MMSC 20k administration group, the BM-MSC 20k administration group, and the cetirizine administration group, and C is the PBS-administered negative control group and It shows the change in ear edema in the MMSC 20k administration group, and D shows the change in ear edema in the negative control group and the BM-MSC 20k administration group.
Figure 4 is an image taken of the depilated buttocks, which is an initial sensitization site, over time after single administration of MMSC, BM-MSC, and cetirizine.
5 is a graph comparing and confirming the degree of hives reduction over time after single administration of MMSC, BM-MSC, and cetirizine.
6 and 7 are graphs comparing changes in the degree of itching over time in contact urticaria disease animal models treated with MMSC, BM-MSC, or cetirizine.
Specifically, in FIG. 6, A shows the change in the degree of itching in all experimental groups, B shows the degree of itching in the MMSC 10k administration group, the BM-MSC 10k administration group, and the cetirizine administration group, and C is the PBS-administered negative control group and It shows the degree of itching of the MMSC 10k-administered group, and D represents the itching degree of the negative control group and the BM-MSC 10k-administered group.
In addition, in FIG. 7, A shows the change in the degree of itching in all experimental groups, B shows the degree of itching in the MMSC 20k administration group, the BM-MSC 20k administration group, and the cetirizine administration group, and C shows the PBS-administered negative control group and the MMSC It shows the degree of itching of the 20k administration group, and D shows the itching degree of the negative control group and the BM-MSC 20k administration group.
8 is a result of comparing and confirming the degree of helper T cell activity in the spleen according to a single administration of MMSC, BM-MSC, or cetirizine in an animal model of contact urticaria disease. Specifically, in FIG. 8, A is a dot plot image of type 1/type 2 helper T cells ( _TH 1/T _H 2 ), and B is a graph showing the ratio and number of T _H 1/T _H 2 cells.
9 is a result of comparing and confirming the degree of helper T cell activity in cervical lymph nodes according to a single administration of MMSC, BM-MSC, or cetirizine in an animal model of contact urticaria disease. Specifically, in FIG. 9, A is a T _H 1 / T _H 2 dot plot image, and B is a graph showing the ratio and cell number of T _H 1 / T _H 2 cells.
10 is a result of comparing and confirming the degree of helper T cell activity in ear tissue according to a single administration of MMSC, BM-MSC, or cetirizine in an animal model of contact urticaria disease. Specifically, in FIG. 10, A is a T _H 1/T _H 2 dot plot image, and B is a graph showing the T _H 1/T _H 2 cell ratio and cell number.
11 is a graph comparing and confirming inflammatory cytokine gene expression levels in ear tissue according to a single administration of MMSC, BM-MSC, or cetirizine in an animal model of contact urticaria disease.
12 shows the results of histopathological infiltration of inflammatory cells and epidermal thickness according to single administration of MMSC, BM-MSC, or cetirizine in animal models of contact urticaria disease (A: representative histology image, B: Epidermal thickness graph ).
Figure 13 compares and confirms the degree of degranulation reaction activity of mast cells in the ear tissue according to single administration of MMSC, BM-MSC, or cetirizine in animal models of contact urticaria disease (A: representative histology imgae, B: obesity introduced into the tissue) number of cells and number of activated mast cells).

The present inventors studied various functions of human pluripotent stem cell-derived mesenchymal stem cells (MMSC), confirmed the applicability to the prevention and treatment of intractable skin diseases, and completed the present invention.

Urticaria is an intractable skin disease characterized by swelling of the skin and surrounding redness. The inventors of the present invention prepared an animal model of contact urticaria disease and as a result of topical subcutaneous administration of human embryonic stem cell-derived mesenchymal stem cells (MMSC), it was confirmed that the symptoms of the disease were significantly reduced and the hypersensitive inflammatory response caused by the urticaria antigen was reduced. did

Specifically, the inventors applied 100 μl of trimellitic anhydride (TMA) dissolved in acetone, a solvent, at a concentration of 500 mg/ml to the back and buttocks of hair-removed mice for primary sensitization, and on the 7th and 10th days after the primary sensitization Secondary sensitization was performed by applying 50 μl of TMA at a concentration of 250 mg/ml to the same area, and on the 13th day after the first sensitization, 25 μl of TMA at a concentration of 100 mg/ml was used to induce disease in the peripheral ear tissue to induce contact An animal model of urticaria disease was constructed. The prepared animal model showed hives, rash, and itching at the site where sensitization was induced, and activation of T cells in the spleen, cervical lymph node, and peripheral ear tissue, as well as activation of mast cells and inflammatory cytokines in the peripheral ear tissue, the disease-inducing site. The expression of Cain was increased.

Subsequently, the present inventors administered human embryonic stem cell-derived mesenchymal stem cells (MMSC) by subcutaneous injection to the ear of a mouse with contact urticaria disease, and over time, edema of the ear, formation of a rash on the buttocks, The degree of itch induction was observed, and the degree of activation of helper T cells and mast cells and the expression level of inflammatory cytokines were measured. A group administered with bone marrow-derived mesenchymal stem cells (BM-MSC) or an antihistamine, cetirizine, was used as a positive control group, and PBS was administered to a negative control group.

As a result, it was confirmed that ear edema was alleviated in both the positive control group and the experimental group, but the MMSC-administered group had a greater degree of relief from ear edema than the positive control group, and the MMSC-administered group had a greater degree of reduction in wheal formation than the positive control group. It was confirmed that the formation of wheal was significantly reduced, and the MMSC-administered group did not show a significant difference compared to the BM-MSC-administered group. On the other hand, as a result of measuring the incidence of pruritus, the MMSC-administered group showed an excellent anti-itching effect compared to the BM-MSC-administered group, but no significant difference was shown compared to the cetirizine-administered group.

On the other hand, the MMSC 10k-administered group showed more excellent effects in alleviating ear swelling, rash, and itching compared to the MMSC 20k-administered group.

In addition, contact urticaria disease accompanies a T cell-mediated systemic inflammatory response, and the present inventors, through specific experiments, have shown that administration of MMSCs activates helper T cells by peripheral and lymphoid tissue related to the disease in a disease-induced model and activates inflammatory cytokines. The effect on the expression level of Cain was confirmed. As a result, it was confirmed that the activity of inflammatory T cells in the cervical lymph node, which is the central immune organ, and the cervical lymph node, which is the draining lymph node, as well as in the ear tissue, which is a disease-induced peripheral tissue, was significantly reduced by MMSC administration. More specifically, it was confirmed that the MMSC-administered group suppressed T cell activity better than the BM-MSC-administered group, and furthermore, the MMSC 10k-administered group suppressed inflammatory T-cell activity better than the MMSC 20k-administered group. Furthermore, similar to the T cell activity inhibitory effect, a significant decrease in inflammatory cytokines in the ear tissue, which is a disease-causing peripheral tissue, was confirmed in the MMSC-administered group compared to other positive control groups. It was found that the width was large. On the other hand, the BM-MSC administration group showed no significant difference in IL-4, IL-6, and TNF-α expression levels compared to the negative control group.

In addition, through histopathological analysis, it was confirmed that as a result of MMSC administration, the increase in epidermal thickness of the ear tissue was significantly reduced, and the increase in the number of mast cells and the increase in the number of degranulation mast cells in the disease-induced tissue were significantly suppressed.

From the above results, the present inventors found that among mesenchymal stem cells, human embryonic stem cell-derived mesenchymal stem cells (MMSC) effectively prevent and treat skin diseases caused by hypersensitivity immune response, including edema, pruritus, hives, and contact dermatitis. It can be applied for, and thus, human embryonic stem cell-derived mesenchymal stem cells (MMSC) are intended to be provided as a cell therapy agent for the prevention or treatment of the skin disease.

In the present invention, as a cell therapy agent, MMSCs are human pluripotent stem cell-derived mesenchymal stem cells, and their preparation is the same as the preparation method of 'similar mesenchymal stem cells' described in KR 10-2280509.

More specifically, mesenchymal-like stem cells were isolated from human pluripotent stem cells and cultured. Human pluripotent stem cells were used that were subcultured 70 times or less after the cell line was established. After the establishment of the cell line, human pluripotent stem cells, subcultured less than 70 times, were maintained and cultured in a 37°C, 5% CO2 cell culture medium. The maintenance-cultured human pluripotent stem cells were separated from the culture plate by a simple enzymatic treatment. Then, only cystic embryoid bodies were selected through the formation of embryoid bodies. Before binding the cell-permeable 3D culture insert to a new culture plate (6 well plate), 2 ml of the culture medium was first put into the new culture plate only for the first time during culture. Then, the cell-permeable three-dimensional culture insert was coupled to the culture plate containing 2 ml of the culture medium. Thereafter, 2 ml of the culture medium was additionally added into the cell-permeable 3-dimensional culture insert, and the selected cystic embryoid bodies were loaded onto the combined cell-permeable 3-dimensional culture insert. EGM-2MV was used as the culture medium in the new culture plate and the culture medium in the cell-permeable 3-dimensional culture insert. The culture medium in the cell-permeable 3-dimensional culture insert loaded with cystic embryoid bodies was cultured for two days without replacement. Thereafter, the culture medium was removed, and 4 ml of the culture medium was additionally added only into the cell-permeable three-dimensional culture insert, and the culture medium was replaced every day to induce differentiation into mesenchymal-like stem cells. Cell differentiation was performed for 5-7 days. In the isolated mesenchymal-like stem cells, cells in the form of clusters that penetrated the cell-permeable 3-dimensional culture insert and migrated under the cell-permeable 3-dimensional culture insert were isolated. Among the separated clusters, multilayer clusters were removed mechanically. Among the isolated clusters, only the monolayer-type clusters were cut into a size of less than 500 μm in width and less than 500 μm in length using a micropipette tip, homogenized, and selectively cultured. More preferably, the clusters in the form of a single layer were homogenized to a size of 100 μm to 500 μm horizontally and vertically, respectively, and mesenchymal-like stem cells were cultured. After culturing for 5-7 days, only the mesenchymal stem cells protruding in the form of single cells from the population were isolated, subcultured, and proliferated. Before single cell isolation, non-single cell clusters and epithelial cells were completely removed with a micropipette tip. The isolated single cells were transferred to a new culture plate and proliferation was induced through subculture. At this time, EGM-2MV was used as the culture medium. When the cluster-like mesenchymal stem cells migrated under the cell-permeable 3-dimensional culture insert, the cell-permeable 3-dimensional culture insert was separated from the culture plate. After obtaining cluster-like mesenchymal stem cells that migrated down the separated cell-permeable 3-dimensional culture insert, the multilayer cluster was mechanically removed and the size of the monolayer cluster was uniform. and selectively cultured to obtain MMSC.

Accordingly, MMSCs as a cell therapy provided in the present invention include: (a) subculturing human pluripotent stem cells 70 times or less; (b) selecting cystic embryoid bodies from among the embryoid bodies in which differentiation of the human pluripotent stem cells was induced; (c) loading the cystic embryoid body on top of the cell-permeable 3-dimensional culture insert and separating a cluster of mesenchymal stem cells that migrated to the bottom of the culture insert; and (d) isolating only a single-layered cluster from among the clusters of the separated mesenchymal stem cells; It is preferably a mesenchymal stem cell obtained by performing.

In the present invention, the cell therapy agent may be provided as a pharmaceutical composition for preventing or treating the above-mentioned diseases.

In the present invention, "cell therapy product" refers to cells and tissues manufactured through isolation, culture, and special manipulation from a subject, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention, and is used to restore the function of cells or tissues. It refers to drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferation and selection of living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways.

In the present invention, human pluripotent stem cells derived from mesenchymal stem cells are not limited as long as they are stem cells having pluripotency, and non-limiting examples thereof include embryonic stem cells (ESCs) and/or It may be an induced pluripotent stem cell (iPSC). In the specific experiments of the present invention, embryonic stem cell-derived mesenchymal stem cells (ESC-MSC) were used, and in the present specification, embryonic stem cells are an example of pluripotent stem cells, and the present invention is not limited thereto. The term ESC-MSC can be used interchangeably with MMSC.

Diseases to be prevented and/or treated in the present invention are skin diseases including edema, pruritus (pruritus), urticaria and contact dermatitis, and the term "prevention" in the present invention refers to the treatment of the skin diseases by administration of the composition of the present invention. It means any action that inhibits or delays the occurrence, spread or recurrence, and "treatment" means any action that improves or beneficially changes the symptoms of the skin disease by administration of the composition of the present invention.

In the present invention, "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods, but preferably in the form of an injection solution for topical administration. It can be formulated and provided as

In addition, according to each formulation, pharmaceutically acceptable carriers such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc. can

On the other hand, the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is the patient's health condition, It may be determined according to severity, drug activity, drug sensitivity, administration method, administration time, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field.

Therefore, the present invention can provide a method for preventing or treating skin diseases caused by an overactive immune response by administering a composition containing human embryonic stem cell-derived mesenchymal stem cells to a subject, wherein the skin diseases caused by the overactive immune response Preferably, it may be one or more selected from the group consisting of edema, pruritus, urticaria, and contact dermatitis.

In the present invention, the term "individual" may be a mammal such as a rat, livestock, mouse, human, etc., and may specifically be a companion dog, racehorse, human, etc. in need of skin disease treatment, and preferably may be a human. When the human embryonic stem cell-derived mesenchymal stem cells according to the present invention are administered to humans as a cell therapy agent, the stem cells may be the patient's own or those derived from another person to whom the cell therapy agent is to be administered.

The pharmaceutical composition of the present invention may be formulated in various forms for administration to a subject, and a representative formulation for parenteral administration is an injectable formulation, preferably an isotonic aqueous solution or suspension. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, it may be formulated for injection by dissolving each component in saline or a buffer solution. In addition, dosage forms for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. , sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). The tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or Disintegrants such as sodium salts, absorbents, colorants, flavors and/or sweeteners may further be included. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention may further include adjuvants such as preservatives, hydrating agents, emulsification accelerators, salts or buffers for osmotic pressure control, and other therapeutically useful substances, and may be formulated according to conventional methods. .

The pharmaceutical composition according to the present invention can be administered through various routes, including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of human embryonic stem cell-derived mesenchymal stem cells depends on the route of administration, the patient's age, gender, It may be appropriately selected according to various factors such as weight and severity of the patient, but preferably, 4×10 ⁵ to 5×10 ⁵ cells per unit weight (1 kg) of the subject to be administered may be administered.

In addition, the composition of the present invention can be administered in parallel with a known compound capable of enhancing the desired effect.

The route of administration of the pharmaceutical composition according to the present invention may be administered to humans and animals orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally, but preferably administered by subcutaneous injection. .

In the pharmaceutical composition of the present invention, the total effective amount of the human embryonic stem cell-derived mesenchymal stem cells according to the present invention can be administered to the patient in a single dose, and multiple doses can be administered for a long period of time. It may be administered by a fractionated treatment protocol. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the degree of disease, but is typically administered once a day at an effective dose of 2 × 10 ⁷ to 3 × 10 ⁷ cells based on adults. may be over-dosed.

In addition, the pharmaceutical composition of the present invention may further contain, as an active ingredient, a drug that alleviates the symptoms of skin diseases caused by the above-mentioned hypersensitivity immune response known in addition to human embryonic stem cell-derived mesenchymal stem cells (MMSC), It can be used in combination with other treatments known for the treatment of these diseases.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents or substitutes to the embodiments are included within the scope of rights.

Terms used in the examples are used only for descriptive purposes and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[Experiment method and materials]

1. Selection of human embryonic stem cell-derived mesenchymal stem cells (MMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and cetirizine dosage

Human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs, MMSC) used in the experiment were provided as GMP finished products from Miraecell Bio, and human bone marrow-derived mesenchymal stem cells (BM-MSC) were purchased from Lonza. and used it. Cetirizine (sigma-aldrich) was used as a treatment drug.

Table 1 below shows the conversion of the dose in animal experiments to the clinical dose in relation to the weight of the mouse.

division treatment group clinical dose
(Human, 60kg) Animal equivalent dose (BALB/c, 20 g) preclinical dose
(Normalized) Conversion value for weight One MMSC 2.5 × 10 ⁷ 8,333 10,000 (1.0 × 10 ⁴ ) human/mouse
60,000/20
= 3,000 folds 2 5.0 × 10 ⁷ 16,667 20,000 (2.0 × 10 ⁴ ) 3 BM-MSCs 2.5 × 10 ⁷ 8,333 10,000 (1.0 × 10 ⁴ ) 4 5.0 × 10 ⁷ 16,667 20,000 (2.0 × 10 ⁴ ) 5 cetirizine 50 mg/kg po
(-1h before elicitation)

2. Production of mouse contact urticaria disease animal model and evaluation of disease severity and edema evaluation

After hair removal on the back and buttocks of Balb/c (6-8 weeks old) female mice (Orient Bio), 100 μl of 500 mg/ml TMA (trimellitic anhydride) was applied to the front of the back and buttocks to induce primary sensitization. The solvent was prepared using acetone. After primary sensitization, secondary sensitization was performed by applying 50 μl of 250 mg/ml TMA to the back and buttocks on days 7 and 10, and disease elicitation was performed on day 13.

To evaluate ear edema, 25 μl of 100 mg/ml TMA was applied to both ears, and after disease induction, the degree of edema over time was evaluated using an ear thickness gauge, and the formation of the buttocks was compared. . And the scratching behavior test was performed for 4 days. On the 4th day after disease induction, the mice were sacrificed, and the ear and cervical lymph nodes (drainage duct lymph nodes) and spleen tissues were removed to evaluate the activity, infiltration, and inflammatory response of immune cells.

3. Adoptive Immunization of MMSC in Mouse Contact Urticaria Disease Animal Model

MMSC was administered as a single dose on the 10th day (elicitation -3 days) after the first sensitization, and was administered by subcutaneous injection (s.c.) to the target tissue, the ear (Fig. 1). After the first stage experiment, 10,000 (10k) and 20,000 (20k) experimental groups were selected as the disease efficacy appropriate cell administration group, and the same amount of cells as the positive control (BM-MSC) was administered, and cetirizine was administered 1 hour before elicitation to conduct a comparative experiment performed. PBS was administered as a negative control.

4. Comparison of the area of wheal formation in the buttocks in animal models of contact urticaria

After elicitation of the TMA-induced contact urticaria disease animal model, images of the buttocks shaved for 4 days were taken to compare the degree of wheal formation (FIG. 4), and the degree of wheal formation area at 24 hours, which is the severe stage of the disease, was compared and analyzed ( Fig. 5). The area of wheal formation was calculated as the ratio of the area of wheal formation within a 3 cm ² area of the buttocks of each mouse. The calculation of the area of wheal formation was evaluated by two independent researchers and the results were derived.

5. Analysis of scratching behavior in contact urticaria disease animal models

After elicitation of the TMA-induced contact urticaria disease animal model, a 10-minute video was taken at the same time every day for 4 days, and the number of scratching behaviors (itching) was counted and evaluated. The evaluation of the number of scratching behaviors was evaluated by three independent researchers and the results were derived.

6. Assessment of inflammatory factors in target tissues in contact urticaria disease animal models

After sacrificing the TMA-induced contact urticaria disease animal model, the target tissue, the ear tissue, was freeze-fractured, mRNA was collected, and the gene expression levels of inflammatory factors were comparatively analyzed through realtime-PCR experiments. Primers for inflammatory cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α were prepared and provided by Bioneeer, and PCR results were analyzed using AriaMx from Agilent. .

7. Analysis of changes in immune cells in target and lymphoid tissues in animal models of contact urticaria disease

After sacrifice of the contact urticaria disease animal model, flow cytometry was performed using an ACEA NovoCyte Flow Cytometer (Agilent), and the influx of immune cells into the ear, the target tissue of the disease, and the activation and polarization of helper T cells were evaluated. . Antibodies for flow cytometry were Live/dead-APC/Cy7, CD3-PE/Cy7, CD4-FITC, IFN-γ-APC, and IL-4-PE, which were purchased from ebiosciences and invitrogen.

In addition, the polarization of helper T lymphocytes was also analyzed in the cervical LNs (cLN) and the spleen, a secondary lymphoid organ, of diseased animals.

8. Histopathological analysis in animal models of contact urticaria disease

After sacrificing the contact urticaria disease animal model, the target tissue, the ear, was fixed in a fixative (4% PFA in PBS) diluted with 4% paraformaldehyde in PBS for 24 hours, and paraffinization of the tissue was performed using a tissue processor (Lecia). did The paraffinized tissue was embedded, and the ear tissue was cut to a thickness of 6 μm to prepare a tissue specimen. Then, the tissue specimen was stained with hematoxylin and eosin staining reagent (sigma-aldrich), and the degree of cell invasion and epidermal thickness were compared and analyzed.

9. Analysis of mast cell activity in animal models of contact urticaria disease

After sacrificing the contact urticaria disease animal model, the target tissue, the ear, was fixed in 4% PFA in PBS for 24 hours, and paraffinization of the tissue was performed using a tissue processor (Lecia). The paraffinized tissue was embedded, cut into 6 μm thickness, and tissue specimens were prepared, and then stained with 1% toluidine blue staining reagent (sigma-aldrich) to determine the number of mast cells infiltrated into the ear tissue and activated (degranulated) mast cells. Number ratios were assessed and analyzed.

[Experiment result]

Example 1. Analysis of edema change in contact urticaria disease animal model

In order to evaluate the disease improvement effectiveness of MMSC compared to positive control BM-MSC and cetirizine drug in animal models of contact urticaria skin disease, changes in edema of the ear, the target tissue of the disease, were confirmed. The experiment was performed in a single administration (Elicitation-3d) method, and the degree of swelling of the ear was evaluated by measuring the thickness of the ear at 0, 1, 12, 24, 48, 72, and 96 hours up to the 4th day after disease induction. 10,000 (10k) and 20,000 (20k) cells of MMSC in the experimental group and BM-MSC in the positive control group were subcutaneously administered to each group, and cetirizine, a positive control treatment drug, was orally administered 1 hour before elicitation, and remained in the ear until the 4th day after disease induction. The degree of edema was observed.

The results of observation of changes in ear edema of each cell or drug (cetirizine) treatment group are shown in Table 2 below, and the results of comparing changes in ear edema of cell or drug treatment groups are shown in FIGS. 2 and 3 . 2, A shows the change in ear edema over time in the entire experimental group, B shows the change in ear edema over time in the 10k MMSC or BM-MSC administration group and the drug administration group, and C and D are A comparison of changes in ear edema in the MMSC 10k administration group, the BM-MSC 10k administration group, and the PBS administration group, respectively. 3, A shows the change in ear edema of the entire experimental group, B shows the change in ear edema over time in the MMSC or BM-MSC 20k administration group and the drug administration group, and C and D show the MMSC 20k administration group, respectively. and changes in ear edema in the BM-MSC 20k administration group and the PBS administration group were compared and shown.

time ACE+PBS TMA+PBS TMA+Cetirizine Mean sem n mean sem n mean sem n (mm) 0h 0 0 28 0 0 28 0 0 28 1h 0 0 28 0.062 0.005 28 0.082 0.012 28 12h 0 0.001 28 0.14 0.01 28 0.156 0.008 28 24h 0.003 0.001 28 0.242 0.008 28 0.22 0.008 28 48h 0.001 0.001 28 0.196 0.007 28 0.186 0.009 28 72h 0.002 0.001 28 0.183 0.007 28 0.167 0.01 28 96h 0.001 0 28 0.162 0.008 28 0.131 0.011 28 time TMA+MMSC 10K TMA+MMSC 20K TMA+BM-MSC 10K TMA+BM-MSC 20K mean sem n mean sem n mean sem n mean sem n 0h 0 0 28 0 0 28 0 0 28 0 0 28 1h 0.045 0.004 28 0.041 0.006 28 0.057 0.009 28 0.058 0.008 28 12h 0.115 0.006 28 0.113 0.008 28 0.127 0.011 28 0.133 0.01 28 24h 0.193 0.006 28 0.189 0.006 28 0.203 0.01 28 0.212 0.01 28 48h 0.161 0.003 28 0.15 0.006 28 0.177 0.008 28 0.175 0.008 28 72h 0.139 0.004 28 0.131 0.004 28 0.153 0.01 28 0.153 0.007 28 96h 0.115 0.005 28 0.102 0.005 28 0.116 0.007 28 0.124 0.007 28

TMA/aceton-induced disease in animal models showed a tendency to alleviate edema due to disease induction in all experimental groups and control groups administered with MMSC, BM-MSC, and cetirizine compared to the PBS-administered group, which is a negative control group. In addition, it was confirmed that the MMSC-administered group was more relieved of ear edema compared to the BM-MSC group, and it was confirmed that a statistically significant alleviation effect was shown when compared to the cetirizine-administered group (p<0.01).

In addition, in the MMSC 20k administration group, a statistically significant relief effect of p<0.05 and p<0.01 on ear edema was confirmed compared to the BM-MSC 20k administration group and the cetirizine administration group, respectively.

Example 2. Analysis of wheal (urticaria) formation in contact urticaria disease animal model

To evaluate wheal formation in the skin, which is a major clinical indicator in animal models of contact urticaria skin disease, the degree of wheal formation in the epilated buttocks, which is the initial sensitization site, was evaluated and compared. The disease animal model induction and the method of administering MMSC to the mouse are the same as the above-mentioned method. After the disease induction, the hip wheal formation area is checked according to the change in time, and the swelling of the ear due to the development of the disease is evaluated based on 24 hours after induction, which is the highest point of deterioration. The ratio of the area of wheal formation to the total evaluation area was converted and analyzed, and the results are shown in FIG. 5 and Table 3 below.

Group Mean
(% of area/3cm ² ) SEM N ACE+PBS 0.000 0.000 10 TMA+PBS 43.605 1.562 10 TMA+MMSC 10K 20.520 1.421 10 TMA+MMSC 20K 27.275 1.769 10 TMA+BM-MSC 10K 34.955 1.384 10 TMA+BM-MSC 20K 35.735 1.189 10 TMA+Cetirizine 28.275 1.365 10

As a result of analyzing the degree of wheal formation according to single administration of MMSC, BM-MSC, and cetirizine, both the MMSC-administered experimental group and the positive control group showed a significant reduction in the area of wheal formation compared to the negative control group. Specifically, compared to 43.605 ± 1.562% of the negative control group, the MMSC 10K administration group had 20.520 ± 1.421% and the MMSC 20K administration group had 27.275 ± 1.769%, showing an inhibitory effect on wheal formation of 53% and 38%, respectively (p <0.01).

The MMSC 10k treatment group showed a significant reduction in wheal formation compared to the BM-MSC 10k treatment group and the cetirizine treatment group (p<0.01). In addition, it was confirmed that the MMSC 20k-administered group showed a more significant reduction in wheal formation than the BM-MSC 20k-administered group (p<0.01), but no statistically significant reduction effect compared to the cetirizine-administered group.

Example 3. Analysis of itching changes in contact urticaria disease animal models

Itching caused by contact urticaria skin disease occurred in the experimental group administered with human embryonic stem cell-derived mesenchymal stem cells (MMSC), the positive control group administered with human bone marrow-derived mesenchymal stem cells (BM-MSC), and the control drug cetirizine administered group. The degree of itching was confirmed by monitoring, and the degree of prevalence of pruritus was evaluated by video recording for 10 minutes every day and the number of scratching behaviors according to itching until the 4th day after disease induction.

The results of evaluating the prevalence of itching in each cell or drug (cetirizine) treatment group are shown in Table 4 below, and the results of comparing the degree of itching in the cell or drug treatment group are shown in FIGS. 6 and 7 . 6A and 7A show the number of occurrences of itching over time in the entire experimental group, and FIG. 6B shows the frequency of occurrence of itching over time in the 10k MMSC or BM-MSC administration group and the drug administration group. In C and D, the frequency of itching in the MMSC 10k administration group, the BM-MSC 10k administration group, and the PBS administration group was compared, respectively. 7B shows the incidence of itching over time in the 20k MMSC or BM-MSC administration group and the drug administration group, and C and D in FIG. It was shown by comparing the frequency of occurrence of itching.

time ACE+PBS TMA+PBS TMA+Cetirizine Mean sem n mean sem n mean sem n (times/10min) 0h 1.429 0.542 14 1.571 0.429 14 1.071 0.399 14 1h 1.786 0.631 14 34.786 4.538 14 14.429 3.122 14 12h 0.857 0.533 14 51.429 2.963 14 22.429 2.424 14 24h 0 0 14 34.286 2.774 14 21 2.585 14 48h 0.714 0.485 14 29.429 2.824 14 16.786 1.816 14 72h 0.714 0.339 14 21.786 1.675 14 7.357 1.127 14 96h 0.786 0.447 14 17.286 2.385 14 6 1.33 14 time TMA+MMSC 10K TMA+MMSC 20K TMA+BM-MSC 10K TMA+BM-MSC 20K mean sem n mean sem n mean sem n mean sem n 0h 0.571 0.388 14 1.286 0.529 14 2.071 0.588 14 2.071 0.788 14 1h 21 3.624 14 21.214 2.798 14 20.429 3.143 14 19.357 2.324 14 12h 26.143 2.987 14 29.643 2.22 14 31.929 2.877 14 32.714 2.754 14 24h 20.429 1.991 14 21.143 2.668 14 22.643 1.295 14 22.643 2.053 14 48h 14.429 1.696 14 11.857 1.249 14 17.429 1.568 14 18.929 1.619 14 72h 10.357 1.673 14 10.357 1.117 14 11.429 1.103 14 14.857 1.64 14 96h 6.714 1.247 14 7.857 0.994 14 7.786 0.866 14 12.071 1.155 14

All cell or drug administration groups showed a lower frequency of itching compared to the negative control group (p<0.05 to p<0.01). The MMSC-administered group had a lower incidence of itching compared to the BM-MSC-administered group at all concentrations, but no significant difference was found compared to the drug-administered group.

Example 4. Analysis of T cell activity inhibitory effect in contact urticaria disease animal model

The contact urticaria disease animal model is a disease in which an immune response is recognized by pre-exposure to antigens in the mouse body through sensitization of primary and secondary antigens, and a systemic inflammatory response involving T cell-mediated cellular immune response occurs when the disease is subsequently induced. Immune cells participating in such an inflammatory response appear simultaneously with a lymphocyte-mediated cellular immune response including helper T cells and an innate immune cell-mediated innate immune response representing direct inflammation against an antigen. Therefore, we tried to evaluate whether the alleviating effect of contact urticaria disease according to the administration of the human embryonic stem cell-derived mesenchymal stem cells (MMSC) is associated with the regulation of T cell activity.

In the experiment, mice were sacrificed on the 4th day after contact urticaria disease elicitation, and the spleen, the main lymphoid organ where T cells were distributed in the body, the cervical lymph node (cLN), the draining lymph node (dLN), and the target peripheral tissue, the ear. (Ear) Single cells were extracted from tissues, stained with fluorescently labeled antibodies, and the activity of helper T cells in the body was evaluated through flow cytometry. Contact urticaria disease is a mixed inflammatory immune response in which the activities of _TH 1 and _TH 2, in which helper T cells are polarized according to the development of an inflammatory response, were evaluated and analyzed by comparing the activities of both helper T cells. After disease induction, the activity of helper T cells in the spleen, a representative secondary lymphoid organ, was evaluated in the MMSC-administered experimental group and the positive control group, BM-MSC-administered and cetirizine-administered groups, compared to the PBS-administered negative control group.

5-1. Confirmation of T cell activity in the spleen

Changes in T cells in the spleen are shown in FIG. 8 and Table 5.

Group Th1 Th2 Mean (%) SEM N Mean (%) SEM N ACE+PBS 4.181 0.397 10 0.436 0.014 10 TMA+PBS 9.377 1.173 10 1.939 0.107 10 TMA+MMSC 10K 5.153 0.416 10 0.857 0.040 10 TMA+MMSC 20K 7.289 0.342 10 1.069 0.110 10 TMA+BM-MSC 10K 5.562 0.337 10 0.874 0.072 10 TMA+BM-MSC 20K 6.390 0.181 10 0.924 0.065 10 TMA+Cetirizine 6.787 0.885 9 1.114 0.074 9

As a result of confirming the ratio and number of T _H 1 / T _H 2 cells in the spleen, compared to the animal model treated with only acetone (ACE), the T _H 1 and T _H 2 in the spleen in animals treated with TMA and administered with PBS Through the rapid increase in the number of cells, it was found that the inflammatory response through the cell-mediated immune response was active due to the disease. In addition, it was confirmed that the activity of helper T cells in the spleen was significantly suppressed in all animals administered with cells or drugs compared to the negative control group treated with PBS. Specifically, in the spleen, 5.153±0.415% in the ESC-MSC 10K administration group and 7.289±0.341% in the ESC-MSC 20K administration group compared to 9.377±1.173% of the negative control group, showing 45% and 23% of _T 1 cell activity inhibition effect, respectively. 0.857 ± 0.040% in the ESC-MSC 10K administration group and 1.069 ± 0.110% in the ESC-MSC 20K administration group, respectively, compared to 1.939 ± 0.107% of the _negative control group.

5-2. Confirmation of T cell activity in cervical lymph nodes

The results of evaluating the activity level of T cells in the cervical lymph nodes with the distribution of T _H 1 and T _H 2 and the change in cell number in the drainage duct lymph node adjacent to the ear, which is the target tissue of the contact urticaria disease animal model, are shown in FIG. 9 and Table 6.

Group Th1 Th2 Mean (%) SEM N Mean (%) SEM N ACE+PBS 0.661 0.032 10 0.085 0.010 10 TMA+PBS 1.622 0.055 10 4.365 0.201 10 TMA+MMSC 10K 1.083 0.030 10 1.637 0.302 10 TMA+MMSC 20K 1.158 0.066 10 1.598 0.278 10 TMA+BM-MSC 10K 1.232 0.036 10 2.788 0.191 10 TMA+BM-MSC 20K 1.624 0.066 10 2.730 0.112 10 TMA+Cetirizine 1.570 0.143 9 3.128 0.344 9

Similar to the result of regulating the activity of spleen-derived T cells, it was confirmed that the MMSC and BM-MSC administration groups had a significant T cell activity suppression effect compared to the negative control group. Although no significant difference could be confirmed between the MMSC-administered experimental group and the drug-administered group, it was confirmed that the distribution of _TH 1 and _TH 2 and the cell number inhibitory effect were most excellent in the MMSC 10k-administered group. Specifically, _TH 1 cell activity in the cervical lymph node was 1.083±0.030% in the MMSC 10K-administered group and 1.158±0.066% in the MMSC 20K-administered group, respectively, compared to 1.622±0.055% in the negative control group, showing 34% and 29% inhibitory effects, respectively. _TH 2 cell activity was 1.637 ± 0.302% in the MMSC 10K-administered group and 1.598 ± 0.278% in the MMSC 20K-administered group, respectively, compared to 4.365 ± 0.201% of the negative control group, showing 63% and 64% inhibitory effects, respectively.

5-3. Confirmation of T cell activity in ear tissue

Subsequently, in order to evaluate the influx or activity of T cells in the ear tissue, which is the target tissue of the urticaria disease animal model following subcutaneous administration of MMSCs in the peripheral tissue, single cells were isolated from the peripheral ear tissue and flow cytometry was used to regulate peripheral T cell activity Whether or not was evaluated, and the results are shown in FIG. 10 and Table 7.

Group Th1 Th2 Mean (%) SEM N Mean (%) SEM N ACE+PBS 1.820 0.185 10 2.803 0.270 10 TMA+PBS 8.318 0.560 10 7.552 0.458 10 TMA+MMSC 10K 5.267 0.538 10 3.841 0.232 10 TMA+MMSC 20K 4.696 0.439 10 4.393 0.442 10 TMA+BM-MSC 10K 7.314 0.943 10 3.934 0.194 10 TMA+BM-MSC 20K 8.741 0.613 10 4.494 0.234 10 TMA+Cetirizine 10.479 1.120 9 4.660 0.350 9

Similar to the results of regulating the activity of spleen and cervical lymph node-derived T cells, the MMSC and BM-MSC administration groups showed a significant _T 2 cell activity inhibition effect compared to the negative control group. Even in the ear tissues, no significant difference was found in the comparison between the MMSC-administered experimental group and the positive control cetirizine-administered group, but the MMSC 10k-administered group showed the most excellent T cell distribution and cell number suppression effect in peripheral tissues.

Specifically, _TH 1 cell activity in the ear tissue was 5.267±0.538% in the MMSC 10K administration group and 4.696±0.439% in the MMSC 20K administration group, compared to 8.318±0.560% in the negative control group, showing 37% and 64% inhibition effects, respectively. _TH 2 cell activity was 3.841 ± 0.232% in the MMSC 10K-administered group and 4.393 ± 0.442% in the MMSC 20K-administered group, respectively, compared to 7.552 ± 0.458% in the negative control group, showing 50% and 42% inhibitory effects, respectively.

Example 5. Analysis of inhibitory effect of inflammatory factors in contact urticaria disease animal models

In an animal model of urticaria disease, the secretion of TH ₁ and _TH 2 mediated inflammatory cytokines in peripheral tissues is a mixed immune response, and the effect of subcutaneous administration of MMSCs on the expression of inflammatory cytokines secreted by immune cells was investigated. To this end, after sacrifice of the disease animal model, the effect of regulating mRNA expression of representative inflammatory cytokines (IFN-γ, TNF-α, IL-4, and IL-6) in the ear tissue, which is the target tissue of the disease, was analyzed. Figure 11 is the result of confirming the expression level of inflammatory cytokines in the ear tissue of each cell or drug-administered animal, and Figure 11 and Table 8 show the cytokine expression levels of animals stimulated with acetone (ACE) and administered with PBS. As a standard, the degree of inflammatory cytokine expression in each experimental animal was compared.

Group IL-4 IL-6 IFN-γ TNF-α Mean
(fold change) SEM N Mean
(fold change) SEM N Mean
(fold change) SEM N Mean
(fold change) SEM N ACE+PBS 1.000 0.158 5 1.000 0.137 5 1.000 0.227 5 1.000 0.313 5 TMA+PBS 31.451 5.655 5 1.913 0.342 5 3.097 0.172 5 1.426 0.200 5 TMA+MMSC 10K 13.988 3.734 5 0.648 0.100 5 1.433 0.163 5 0.900 0.184 5 TMA+MMSC 20K 18.279 3.411 5 0.382 0.103 5 0.128 0.082 5 0.886 0.158 5 TMA+BM-MSC 10K 38.111 5.200 5 1.720 0.208 5 1.350 0.187 5 2.605 0.459 5 TMA+BM-MSC 20K 35.131 2.679 5 2.636 0.503 5 0.814 0.104 5 2.127 0.212 5 TMA+Cetirizine 17.040 1.735 5 1.387 0.121 5 1.174 0.081 5 2.135 0.367 5

As a result, it was confirmed that the MMSC-administered group significantly reduced the expression level of inflammatory cytokines as a whole compared to the negative control group, and in particular, the gene expression of IL-4, IL-6, and IFN-γ in the ear tissue was reduced. The degree of inhibition of IL-4, IL-6, IFN-γ, and TNF-α cytokine expression in the MMSC-administered group was similar to that of the Cetirizine-administered group, which was a positive control group. On the other hand, the BM-MSC administration group showed no significant inhibitory effect compared to the negative control group except for IFN-γ.

Specifically, in the case of IL-4, 13.988±3.734% in the MMSC 10K-administered group and 18.279 ± 3.411% in the MMSC 20K-administered group, respectively, compared to 31.451 ± 5.655% in the negative control group, showing inhibitory effects of 56% and 42%, respectively, and IL-6 In the case of , compared to 1.913 ± 0.342% of the negative control group, 0.648 ± 0.100% in the MMSC 10K administration group and 0.382 ± 0.103% in the MMSC 20K administration group, respectively, showed 67% and 81% inhibitory effects. In addition, in the case of IFN-γ, 1.433 ± 0.163% in the MMSC 10K administration group and 0.128 ± 0.082% in the MMSC 20K administration group, respectively, compared to 3.097 ± 0.172% of the negative control group, showing 54% and 96% inhibitory effects, respectively. And TNF-α expression was 0.900±0.184% in the MMSC 10K-administered group and 0.886±0.158% in the MMSC 20K-administered group, respectively, compared to 1.426±0.200% in the negative control group, showing significant inhibitory effects of 37% and 38%, respectively.

Example 6. Histopathological analysis in contact urticaria disease animal model

In animal models of contact urticaria skin disease, histological analysis of the ear, the target site of the disease, was performed to evaluate the effectiveness of MMSC compared to the positive control group BM-MSC and the cetirizine-administered group. Specifically, after disease induction, the mouse was sacrificed on the 4th day, and the target tissue, the ear, was fixed in 4% PFA in PBS for 24 hours, and the paraffinized tissue specimen was sectioned at 6 μm and stained with hematoxylin and eosin to infiltrate inflammatory cells. The degree and epidermal thickness were measured, and the results are shown in FIG. 12 and Table 9 below.

Group Mean
(μm) SEM N ACE+PBS 10.867 0.496 15 TMA+PBS 48.333 1.190 15 TMA+MMSC 10K 22.933 0.651 15 TMA+MMSC 20K 19.867 0.601 15 TMA+BM-MSC 10K 22.600 0.567 15 TMA+BM-MSC 20K 24.800 0.603 15 TMA+Cetirizine 21.733 0.686 15

After the disease was induced, a tendency for cell infiltration in the ear tissue to increase was confirmed through visual evaluation in the PBS-administered group, which was a negative control group, and the result of an increase in epidermal thickness was confirmed. In addition, it was confirmed that the increase in ear tissue epidermal thickness was generally reduced in the experimental group and the positive control group compared to the negative control group in the MMSC and BM-MSC administration group and the cetirizine administration group.

Specifically, compared to 48.333±1.190% of the negative control group, the MMSC 10K administration group was 22.933±0.651% and the MMSC 20K administration group was 19.867±0.601%, respectively, 53% and 59% of the increase in epidermal thickness of the ear tissue was significantly reduced, and the disease was improved. was able to confirm that

Example 7. Analysis of mast cell activity inhibition effect in contact urticaria disease animal model

In an animal model of contact urticaria skin disease, the increase in mast cells and degranulation activity in the ear tissue following the administration of MMSC were evaluated in comparison to the positive control group, BM-MSC and cetirizine-treated groups. Specifically, after disease induction, samples were prepared and analyzed in the same way as in histopathological analysis. Tissue samples were cut into 6 μm slices and stained with 1% toluidine blue to determine the total number of mast cells infiltrated into the target tissue, the ear tissue, and activation (degranulation). The number and ratio of mast cells were compared and analyzed, and the results are shown in FIG. 13 and Table 10 below.

Table 9 below quantifies the effect of inhibiting mast cell activity in contact urticaria disease animal models.

Group Mean
(%) SEM N ACE+PBS 4.869 1.201 15 TMA+PBS 18.061 0.815 15 TMA+MMSC 10K 8.232 0.688 15 TMA+MMSC 20K 9.553 0.791 15 TMA+BM-MSC 10K 14.507 1.109 15 TMA+BM-MSC 20K 13.448 0.915 15 TMA+Cetirizine 10.593 0.832 15

As a result of the experiment, it was confirmed that there was a significant reduction in the number of mast cells compared to the negative control group and an inhibitory effect on the activity of degranulated mast cells in all cells or drug-administered groups. In particular, it was confirmed that the number and ratio of degranulated mast cells were significantly lower in the MMSC 10k-administered group than in the BM-MSC-administered group, or a similar trend to that of the Cetirizine-administered group was confirmed.

Specifically, 8.232 ± 0.688% in the MMSC 10K group and 9.553 ± 0.791% in the MMSC 20K group compared to 18.061 ± 0.815% of the negative control group, respectively, showing 55% and 48% of the activity inhibition effect of degranulated mast cells.

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from the method described, and/or the components of the described system, structure, device, circuit, etc. may be combined or combined in a different form than the method described, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

Claims (12)

A pharmaceutical composition for preventing or treating skin diseases caused by an overactive immune response, comprising human pluripotent stem cell-derived mesenchymal stem cells as an active ingredient,
The skin disease is at least one disease selected from the group consisting of urticaria and contact dermatitis,
The composition is for topical subcutaneous administration,
The composition is a pharmaceutical composition comprising 2 × 10 ⁷ to 3 × 10 ⁷ of the mesenchymal stem cells for a single administration. According to claim 1,
The human pluripotent stem cell-derived mesenchymal stem cells are obtained by performing the following steps, a pharmaceutical composition:
(a) subculturing human pluripotent stem cells 70 times or less;
(b) selecting cystic embryoid bodies from among the embryoid bodies in which differentiation of the human pluripotent stem cells was induced;
(c) loading the cystic embryoid body on top of the cell-permeable 3-dimensional culture insert and separating a cluster of mesenchymal stem cells that migrated to the bottom of the culture insert; and
(d) isolating only the cluster in the form of a monolayer among the clusters of the separated mesenchymal stem cells. delete delete According to claim 1,
Wherein the composition inhibits the activity of helper T cells (helper T cell), the pharmaceutical composition. According to claim 1,
Wherein the composition reduces the expression level of inflammatory cytokines in the lesion (lesion), the pharmaceutical composition. According to claim 6,
The inflammatory cytokine is one or more selected from the group consisting of IFN-γ, TNF-α, IL-4, and IL-6, the pharmaceutical composition. According to claim 1,
Wherein the composition inhibits the activity of mast cells in the lesion (lesion), the pharmaceutical composition. According to claim 1,
Wherein the human pluripotent stem cells are human embryonic stem cells, the pharmaceutical composition. As a method for treating skin diseases in mammals other than humans,
The skin disease is at least one disease selected from the group consisting of urticaria and contact dermatitis,
The method comprises the step of administering human pluripotent stem cell-derived mesenchymal stem cells of an individual at least once per unit weight (1 kg) of the individual from 4 × 10 ⁵ to 5 × 10 ⁵ ,
The method of administering human pluripotent stem cell-derived mesenchymal stem cells subcutaneously to the lesion of the subject. delete delete

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