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WO2023090589A1 - Composition à base de cellules souches pluripotentes pour la prévention ou le traitement d'une maladie cutanée induite par une réponse immunitaire hypersensible - Google Patents

Composition à base de cellules souches pluripotentes pour la prévention ou le traitement d'une maladie cutanée induite par une réponse immunitaire hypersensible Download PDF

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WO2023090589A1
WO2023090589A1 PCT/KR2022/011987 KR2022011987W WO2023090589A1 WO 2023090589 A1 WO2023090589 A1 WO 2023090589A1 KR 2022011987 W KR2022011987 W KR 2022011987W WO 2023090589 A1 WO2023090589 A1 WO 2023090589A1
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stem cells
cell
group
mmsc
pluripotent stem
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Korean (ko)
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김혁순
노건웅
박세필
김은영
정형민
홍기성
현승연
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Mirae Cell Bio Co Ltd
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Mirae Cell Bio Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a pharmaceutical composition
  • 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.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • Nauta et al. reported the immunomodulatory properties of MSCs.
  • MSCs are immunomodulatory agents in maintenance of peripheral tolerance, transplantation tolerance, autoimmunity, tumor evasion, and fetal-maternal tolerance. can function as
  • 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.
  • 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.
  • hESC-MSC human embryonic stem cell-derived mesenchymal stem cells
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • 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.
  • MMSC human pluripotent stem cell-derived mesenchymal stem cells
  • 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.
  • MMSC human pluripotent stem cell-derived mesenchymal stem cells
  • 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.
  • human pluripotent stem cell-derived mesenchymal stem cells may be provided as a cell therapy agent for preventing or treating skin diseases caused by the hypersensitive immune response.
  • 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.
  • iPSC induced pluripotent stem cells
  • ESC embryonic stem cells
  • human pluripotent stem cell-derived mesenchymal stem cells 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.
  • 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
  • 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.
  • 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- ⁇ , TNF- ⁇ , IL-4, and IL-6.
  • the pharmaceutical composition or cell therapy agent may inhibit the activity of mast cells in lesions.
  • 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.
  • MMSC human pluripotent stem cell-derived mesenchymal stem cells
  • the administration may be administered by local injection under the skin of the lesion.
  • 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.
  • 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.
  • 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).
  • MMSC contact dermatitis
  • the human pluripotent stem cell-derived mesenchymal stem cells may be obtained by performing the following steps:
  • 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.
  • 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.
  • FIG. 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.
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • cetirizine cetirizine
  • 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
  • C is the negative control group administered with PBS.
  • 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.
  • 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
  • C is the PBS-administered negative control group and It shows the change in ear edema in the MMSC 20k administration group
  • 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.
  • 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
  • C is the PBS-administered negative control group and It shows the degree of itching of the MMSC 10k-administered group
  • D represents the itching degree of the negative control group and the BM-MSC 10k-administered group.
  • 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
  • C shows the PBS-administered negative control group and the MMSC It shows the degree of itching of the 20k administration group
  • D shows the itching degree of the negative control group and the BM-MSC 20k administration group.
  • 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.
  • A is a T H 1 / T H 2 dot plot image
  • B is a graph showing the ratio and cell number of T H 1 / T H 2 cells.
  • A is a T H 1/T H 2 dot plot image
  • 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.
  • FIG. 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).
  • MMSC human pluripotent stem cell-derived mesenchymal stem cells
  • 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
  • TMA trimellitic anhydride
  • 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.
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • 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.
  • the MMSC 10k-administered group showed more excellent effects in alleviating ear swelling, rash, and itching compared to the MMSC 20k-administered group.
  • MMSCs activate 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.
  • 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.
  • 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- ⁇ expression levels compared to the negative control group.
  • human embryonic stem cell-derived mesenchymal stem cells 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • multilayer clusters were removed mechanically.
  • the monolayer-type clusters were cut into a size of less than 500 ⁇ m in width and less than 500 ⁇ m in length using a micropipette tip, homogenized, and selectively cultured.
  • the clusters in the form of a single layer were homogenized to a size of 100 ⁇ m to 500 ⁇ m 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.
  • 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.
  • MMSCs as a cell therapy 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.
  • the cell therapy agent may be provided as a pharmaceutical composition for preventing or treating the above-mentioned diseases.
  • 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.
  • 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).
  • ESCs embryonic stem cells
  • iPSC induced pluripotent stem cell
  • ESC-MSC embryonic stem cell-derived mesenchymal stem cells
  • 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.
  • 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
  • pharmaceutically acceptable carriers such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc.
  • the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • dosage forms for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • cetirizine dosage MMSC, bone marrow-derived mesenchymal stem cells (BM-MSC), and cetirizine dosage
  • hESC-MSCs 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.
  • TMA trimellitic anhydride
  • ear edema 25 ⁇ l 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.
  • 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).
  • 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.
  • the target tissue 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- ⁇ , and TNF- ⁇ were prepared and provided by Bioneeer, and PCR results were analyzed using AriaMx from Agilent. .
  • 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- ⁇ -APC, and IL-4-PE, which were purchased from ebiosciences and invitrogen.
  • helper T lymphocytes was also analyzed in the cervical LNs (cLN) and the spleen, a secondary lymphoid organ, of diseased animals.
  • the target tissue 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 ⁇ m 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.
  • a fixative 4% PFA in PBS
  • 4% paraformaldehyde in PBS diluted with 4% paraformaldehyde in PBS for 24 hours
  • 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 ⁇ m to prepare a
  • the target tissue 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 ⁇ m 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.
  • tissue processor Lecia
  • the paraffinized tissue was embedded, cut into 6 ⁇ m 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.
  • Example 1 Analysis of edema change in contact urticaria disease animal model
  • FIGS. 2 and 3 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 .
  • 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 administration group of MMSC or BM-MSC and the drug administration group
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • BM-MSC human bone marrow-derived mesenchymal stem cells
  • 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.
  • FIGS. 6 and 7 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
  • 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.
  • 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
  • C and D in FIG. It was shown by comparing the frequency of occurrence of itching.
  • 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.
  • the contact urticaria disease animal model is a disease in which an immune response is recognized by prior exposure to antigens in the mouse body through sensitization of primary and secondary antigens, and then a systemic inflammatory response involving T cell-mediated cellular immune response occurs when the disease is 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 alleviation 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.
  • MMSC human embryonic stem cell-derived mesenchymal stem cells
  • 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.
  • helper T cells in the spleen a representative secondary lymphoid organ
  • the activity of helper T cells in the spleen 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.
  • T H 1 / T H 2 cells in the spleen 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.
  • ACE acetone
  • 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.
  • 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.
  • T cells in the ear tissue which is the target tissue of the urticaria disease animal model according to subcutaneous administration of MMSC into the peripheral tissue
  • single cells were isolated from the peripheral ear tissue and flow cytometry was used to regulate the activity of peripheral T cells Whether or not was evaluated, and the results are shown in FIG. 10 and Table 7.
  • 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.
  • 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
  • Figure 11 is the result of confirming the expression level of inflammatory cytokines in the ear tissues 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.
  • ACE acetone
  • 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- ⁇ in the ear tissue was reduced.
  • the degree of inhibition of IL-4, IL-6, IFN- ⁇ , and TNF- ⁇ cytokine expression in the MMSC-administered group was similar to that of the Cetirizine-administered group, which was a positive control group.
  • the BM-MSC administration group showed no significant inhibitory effect compared to the negative control group except for IFN- ⁇ .
  • 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
  • Table 9 quantifies the effect of inhibiting mast cell activity in contact urticaria disease animal models.

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Abstract

La présente invention concerne la fourniture de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes humaines en guise de produit de thérapie cellulaire pour la prévention ou le traitement de maladies cutanées induites par une réponse immunitaire hypersensible, notamment l'œdème, l'urticaire, le prurit et la dermatite de contact. Capable de soulager efficacement les symptômes de l'œdème, du prurit et de l'urticaire et de traiter la dermatite de contact même avec une injection sous-cutanée topique, la présente invention présente l'avantage d'être dépourvue d'effets secondaires, par comparaison avec les antihistaminiques, qui agissent après une administration systémique.
PCT/KR2022/011987 2021-11-22 2022-08-11 Composition à base de cellules souches pluripotentes pour la prévention ou le traitement d'une maladie cutanée induite par une réponse immunitaire hypersensible Ceased WO2023090589A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101655780B1 (ko) * 2013-11-29 2016-09-09 인하대학교 산학협력단 클로날 중간엽 줄기세포를 포함하는, 아토피성 피부염 예방 또는 치료용 약학적 조성물
WO2020160457A1 (fr) * 2019-01-31 2020-08-06 Primegen Biotech, Llc Traitement de dermatite atopique au moyen de cellules souches mésenchymateuses et de la modulation immunitaire
US20210085725A1 (en) * 2012-07-11 2021-03-25 Imstem Biotechnology, Inc. Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof
KR102280509B1 (ko) * 2020-09-04 2021-07-22 건국대학교 글로컬산학협력단 전분화능 줄기세포 기반 자가면역성 및 염증성 피부질환 예방 또는 치료용 조성물 및 이의 제조방법
KR102304549B1 (ko) * 2018-05-30 2021-09-27 주식회사 강스템바이오텍 Hla 유전자가 제거된 인간 유도만능줄기세포 유래 중간엽줄기세포 및 그의 제조 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210085725A1 (en) * 2012-07-11 2021-03-25 Imstem Biotechnology, Inc. Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof
KR101655780B1 (ko) * 2013-11-29 2016-09-09 인하대학교 산학협력단 클로날 중간엽 줄기세포를 포함하는, 아토피성 피부염 예방 또는 치료용 약학적 조성물
KR102304549B1 (ko) * 2018-05-30 2021-09-27 주식회사 강스템바이오텍 Hla 유전자가 제거된 인간 유도만능줄기세포 유래 중간엽줄기세포 및 그의 제조 방법
WO2020160457A1 (fr) * 2019-01-31 2020-08-06 Primegen Biotech, Llc Traitement de dermatite atopique au moyen de cellules souches mésenchymateuses et de la modulation immunitaire
KR102280509B1 (ko) * 2020-09-04 2021-07-22 건국대학교 글로컬산학협력단 전분화능 줄기세포 기반 자가면역성 및 염증성 피부질환 예방 또는 치료용 조성물 및 이의 제조방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM EUN-YOUNG, KIM HYUK SOON, HONG KI-SUNG, CHUNG HYUNG-MIN, PARK SE-PILL, NOH GEUNWOONG: "Mesenchymal stem/stromal cell therapy in atopic dermatitis and chronic urticaria: immunological and clinical viewpoints", STEM CELL RESEARCH & THERAPY, vol. 12, no. 1, 1 December 2021 (2021-12-01), XP093067395, DOI: 10.1186/s13287-021-02583-4 *

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