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WO2018038575A1 - Composition pharmaceutique pour le traitement de la polyarthrite rhumatoïde, comprenant un protéome sécrétoire dérivé de cellules souches mésenchymateuses, et méthode thérapeutique utilisant cette dernière - Google Patents

Composition pharmaceutique pour le traitement de la polyarthrite rhumatoïde, comprenant un protéome sécrétoire dérivé de cellules souches mésenchymateuses, et méthode thérapeutique utilisant cette dernière Download PDF

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Publication number
WO2018038575A1
WO2018038575A1 PCT/KR2017/009328 KR2017009328W WO2018038575A1 WO 2018038575 A1 WO2018038575 A1 WO 2018038575A1 KR 2017009328 W KR2017009328 W KR 2017009328W WO 2018038575 A1 WO2018038575 A1 WO 2018038575A1
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Prior art keywords
mesenchymal stem
culture
pharmaceutical composition
medium
stem cells
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Korean (ko)
Inventor
박용범
김한수
문진희
최성미
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Industry Academic Cooperation Foundation of Yonsei University
Catholic Kwandong University Industry Cooperation Foundation
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Industry Academic Cooperation Foundation of Yonsei University
Catholic Kwandong University Industry Cooperation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • the present invention relates to a composition for preventing, improving or treating autoimmune diseases using mesenchymal stem cell-derived secretory proteins.
  • Rheumatoid arthritis is the most common inflammatory arthritis in adults, and its cause is not yet fully understood, but it is understood as an inflammatory response of the synovial membrane of the joint due to an autoimmune mechanism.
  • antirheumatic drugs and biological agents used to treat rheumatoid arthritis have been shown to improve arthritis through inflammation control, they are still insufficient to induce remission, the ultimate therapeutic target.
  • the therapeutic effect is incomplete, side effects associated with the therapeutic agent are impeding the treatment, and when the biological agent is used for a long time, antibodies to the drug are generated, thereby reducing the therapeutic effect.
  • Mesenchymal stem cells are undifferentiated adult stem cells that exist between differentiated cells of tissues or organs, and can be isolated from various tissues in the human body such as bone marrow, fat, and muscle, and self-renewing self-renewing. Have self-renewal In addition, since it can be easily proliferated in vitro and can be differentiated into various tissue cells such as adipocytes, bone cells, chondrocytes, and myocytes, existing stem cell researches have been concentrated on regeneration studies using differentiation functions.
  • the immunomodulatory function of mesenchymal stem cells which has been recently revealed by various studies, protects hematopoietic stem cells from damage caused by immune responses, and acts at each stage of the immune response, resulting in an immunomodulatory effect. And anti-inflammatory responses.
  • This immunomodulatory function occurs through interaction with various immune cells such as natural killer cells (NK cells), dendritic cells, macrophages (macrophage), T cells and B cells.
  • NK cells natural killer cells
  • dendritic cells dendritic cells
  • macrophages macrophages
  • the secreted protein can be manufactured and synthesized in a large amount from other (homologous) cell lines that have completed both characterization, contamination analysis, and quality control for clinical use, and thus, unlike cell therapeutics for cell replacement, It is a biologic that can easily overcome shortages or medical problems.
  • One object of the present invention is to provide a composition that can effectively prevent, ameliorate and treat autoimmune diseases by using a secretory protein derived from the culture medium of mesenchymal stem cells that do not have ethical problems and do not have immunogenicity.
  • a mesenchymal stem cell-derived secretory protein comprising a pharmaceutical composition for the prevention or treatment of autoimmune diseases.
  • the "mesenchymal stem cell” refers to an undifferentiated cell having a multipotency derived from an adult human cell of a mammal, including a human, for example, bone marrow, blood And various adult cells such as brain, skin, fat (ie, adipose tissue or adipocytes), umbilical cord blood, umbilical cord Barton's jelly and the like.
  • secretome means the sum of the protein components among the components secreted from the mesenchymal stem cells.
  • Secretory proteins refer to components that are released into the extracellular environment by a cell after transcription, translation, and post-translational modification of the gene in the cell.
  • Representative expression markers of secreted proteins correspond to growth factors such as EGF and VEGF and extracellular matrix proteins such as collagen and fibronectin.
  • the secreted protein may be isolated from the culture solution obtained by culturing the mesenchymal stem cells.
  • the method of culturing the mesenchymal stem cells in the present invention may be by a two-dimensional culture or a three-dimensional culture method.
  • the two-dimensional culture of the mesenchymal stem cells, the mesenchymal stem cells are cultured in the mesenchymal stem cell culture medium for 24 to 96 hours and then carried out in a serum-free medium for 24 to 72 hours Can be.
  • the composition of the mesenchymal stem cell culture medium is not particularly limited but may be serum medium.
  • serum medium Dulbecco's modified Eagle's medium (DMEM) or RPMI containing 5-15 wt% Fetal bovine serum (FBS) and 0.05-0.2 mM mercaptoethanol.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • -1640 medium, or the like, or serum-free medium such as StemPro medium, MSCGro medium, MesenCult medium, or NutriStem medium, but is not limited thereto, and any medium that can be used for culturing mesenchymal stem cells in the art is not limited thereto. Can be used.
  • the serum-free medium may be Dulbecco's modified Eagle's medium (DMEM) that excludes phenol red and antibiotics, but is not limited thereto. It can be used for the culture of mesenchymal stem cells, including the available medium, and any medium in which fetal bovine serum is excluded can be used without limitation.
  • DMEM Dulbecco's modified Eagle's medium
  • the secretion amount and secretion of the secreted protein may be different, the normal culture is carried out under the normal oxygen partial pressure (20% by volume level of oxygen).
  • the environment in the body is low oxygen partial pressure, by providing such an environment in vitro to cultivate stem cells when the growth and differentiation and neovascularization ability of the cells, thereby increasing the therapeutic effect of stem cells. Therefore, in the present invention, the secreted protein is not only obtained by culturing mesenchymal stem cells under normal oxygen culture conditions (20% by volume O 2 ), but also by culturing mesenchymal stem cells under hypoxic conditions (0.5-1% by volume O 2). It may be obtained by culturing under).
  • the secretory protein centrifugation of the culture solution obtained by the two-dimensional culture at 500 to 1,500 xg, the supernatant is recovered, and the concentrate of the polymer component obtained is used to suppress the expression of inflammatory cytokines.
  • Expression of anti-inflammatory cytokines is preferred because it can be activated to further suppress the immune response.
  • the concentrate of the polymer component may include filtering the supernatant from the supernatant obtained by centrifugation with a 0.1 to 0.3 ⁇ M filter, preferably a 0.2 ⁇ M filter; And filtering the molecules up to 3 kDa.
  • the method of filtering the molecules below 3 kDa may be performed by diafiltration using a tangential flow filtration (TFF) device.
  • the supernatant may be concentrated at 0 to 5 ° C. while diluting the supernatant with injection water using a peristatic tubing pump.
  • the concentrate of the polymer component may be obtained by reacting the supernatant obtained by centrifugation with an alcohol polar solvent and concentrating the active ingredient.
  • the alcohol polar solvent may be used alone or two or more of a lower alcohol having 1 to 6 carbon atoms, a dilution solution of the alcohol, for example, 95% or 90% alcohol aqueous solution, or acetone to be isopropyl alcohol by a reducing agent.
  • a dilution solution of the alcohol for example, 95% or 90% alcohol aqueous solution
  • acetone to be isopropyl alcohol by a reducing agent can be.
  • the alcohol solution in the present invention means a dilution solution of alcohol, for example, may include 95% ethanol, 90% ethanol and the like.
  • the alcohol polar solvent is preferably mixed in an amount of 2 to 5 times the weight ratio with respect to the supernatant, since only the active ingredient of the polymer concentrate in the supernatant can be effectively concentrated.
  • the reaction between the supernatant and the polar polar solvent is preferably performed at -30 to 0 ° C for 5 to 500 minutes.
  • 100% alcohol may be added to the supernatant obtained by centrifugation of the culture solution obtained by the two-dimensional culture, and left for 5 to 500 minutes at -30 to 0 ° C. Thereafter, after centrifugation, the precipitate may be centrifuged again by adding 90% alcohol to the precipitate. Alternatively, the precipitate obtained after centrifugation can be suspended by adding sterile water and frozen.
  • freeze-dried polymer concentrate of the secreted protein can be obtained in powder form.
  • the method of three-dimensional culturing the mesenchymal stem cells in the present invention after culturing the mesenchymal stem cells in the medium for mesenchymal stem cell culture for 24 to 96 hours and suspended in serum-free medium for 24 to 72 hours While incubated.
  • the composition of the mesenchymal stem cell culture medium is not particularly limited but may be serum medium.
  • it may be Dulbecco's modified Eagle's medium (DMEM) containing 5 to 15% by weight of fetal bovine serum (FBS) and 0.05 to 0.2 mM of mercaptoethanol.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • the present invention is not limited thereto, and any medium that can be used for culturing mesenchymal stem cells in the art may be used without limitation.
  • the "recombinant trypsin” may be a recombinant trypsin without an animal-derived component, for example, may be a recombinant trypsin produced in corn.
  • Recombinant trypsin without the animal-derived component is commercially available and may be, for example, TrypLETM Select (GIBCO Invitrogen), TrypLETM Express (GIBCO Invitrogen), TrypZeanTM (Sigma Aldrich) or Recombinant Trypsin SolutionTM (Biological Industries). There is no restriction in particular.
  • the serum-free medium in the present invention may be Dulbecco's modified Eagle's medium (DMEM), but is not limited thereto and may be used for culturing mesenchymal stem cells in the art. Any media that has been excluded can be used without limitation.
  • DMEM Dulbecco's modified Eagle's medium
  • the inflammatory cytokine expression is more than that in the case of stationary culture by rotating at a rotational speed of 30 to 90 rpm using a spinner flask. Is inhibited and the expression of anti-inflammatory cytokines can be activated to further suppress the immune response.
  • the secreted protein is centrifuged at 500 to 1,500 xg of the culture medium obtained by the three-dimensional culture to recover the supernatant, and then the concentration of the polymer component obtained is used to suppress the expression of inflammatory cytokines.
  • Expression of anti-inflammatory cytokines is preferred because it can be activated to further suppress the immune response.
  • the concentrate of the polymer component may include filtering the supernatant from the supernatant obtained by centrifugation with a 0.1 to 0.3 ⁇ M filter, preferably a 0.2 ⁇ M filter; And filtering the molecules up to 3 kDa.
  • the method of filtering the molecules below 3 kDa may be performed by diafiltration using a tangential flow filtration (TFF) device.
  • the supernatant may be concentrated at 0 to 5 ° C. while diluting the supernatant with injection water using a peristatic tubing pump.
  • the concentrate of the polymer component may be obtained by reacting the supernatant obtained by centrifugation with an alcohol polar solvent and concentrating the active ingredient.
  • the alcohol polar solvent may be used alone or two or more of a lower alcohol having 1 to 6 carbon atoms, a dilution solution of the alcohol, for example, 95% or 90% alcohol aqueous solution, or acetone to be isopropyl alcohol by a reducing agent.
  • a dilution solution of the alcohol for example, 95% or 90% alcohol aqueous solution
  • acetone to be isopropyl alcohol by a reducing agent can be.
  • the alcohol solution in the present invention means a dilution solution of alcohol, for example, may include 95% ethanol, 90% ethanol and the like.
  • the alcohol polar solvent is preferably mixed in an amount of 2 to 5 times the weight ratio with respect to the supernatant, since only the active ingredient of the polymer concentrate in the supernatant can be effectively concentrated.
  • the reaction between the supernatant and the polar polar solvent is preferably performed at -30 to 0 ° C for 5 to 500 minutes.
  • 100% alcohol may be added to the supernatant obtained by centrifugation of the culture solution obtained by the two-dimensional culture, and left for 5 to 500 minutes at -30 to 0 ° C. Thereafter, after centrifugation, the precipitate may be centrifuged again by adding 90% alcohol to the precipitate. Alternatively, the precipitate obtained after centrifugation can be suspended by adding sterile water and frozen.
  • freeze-dried polymer concentrate of the secreted protein can be obtained in powder form.
  • Mesenchymal stem cell-derived secretory proteins obtained as described above in the present invention can effectively prevent, improve or treat autoimmune diseases.
  • said autoimmune disease is a non-malignant disease or disorder that occurs and is directed against an individual's own tissue.
  • autoimmune disease One of the most important traits of all normal individuals is that they do not deleteriously react with the antigenic substances that make up self, while non-self antigens can be recognized and reacted to eliminate them.
  • Biological nonresponsiveness to autoantigens is called immunologic unresponsiveness or tolerance.
  • an immune response occurs to autoantigens, and as a result, an attack occurs on the tissues.
  • the disease caused by this process is called an autoimmune disease.
  • the autoimmune disease is an inflammatory disease in which antibodies are produced against its own organ tissues or components thereof, and may refer to diseases causing chronic systemic inflammation in many tissues and organs.
  • the autoimmune disease is rheumatoid arthritis, inflammatory spondyloarthritis, adult stature disease, macrophage activity syndrome, systemic sclerosis, multiple myositis, dermatitis, vasculitis, mixed connective diseases, Sjogren's syndrome, Ulcerative colitis, Crohn's disease, allergic asthma, allergic rhinitis, atopic dermatitis, gallbladder, lacrimitis, conjunctivitis, psoriasis, vulgaris ulcer, Parkinson's disease, muscular dystrophy, myasthenia gravis, multiple sclerosis, Alzheimer's disease, stroke At least one selected from the group consisting of atherosclerosis, vascular restenosis, type I diabetes, type II diabetes, diabetic retinopathy, and chronic thyroiditis, but preferably may be rheumatoid arthritis.
  • prevention means a reduction in the extent of the development of the pathological cells of the animal or damage or loss of cells. Prevention can be complete or partial. In this case, it may mean that the occurrence or abnormal immune action of pathological cells in the subject is reduced compared to the case where the composition for preventing and treating the autoimmune disease is not used.
  • the "treatment” refers to all actions that are clinically involved in altering the natural process of the subject or cell to be treated, and can be performed during or during clinical pathology.
  • the desired therapeutic effect may prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, slow the progression of the disease, or Mitigating or temporarily alleviating the condition or improving the prognosis.
  • the treatment may be interpreted as encompassing all behaviors in which the symptoms of the autoimmune disease are improved or cured by the composition.
  • the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, the pharmaceutical composition may be characterized in that it is intended for humans.
  • compositions of the present invention are not limited to these, but can be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.
  • oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc., and in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.
  • the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms.
  • solutions, suspensions, tablets, capsules, sustained release preparations and the like may be used.
  • Suitable carriers, excipients, and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
  • Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , Sublingual or rectal. Oral or parenteral release is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical composition of the present invention is dependent on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, extent of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg per day. / kg or 0.001-50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
  • a mesenchymal stem cell-derived secretory protein comprising a food composition for preventing or improving autoimmune diseases.
  • mesenchymal stem cells, secreted proteins and autoimmune diseases in the present invention is duplicated as described in the pharmaceutical composition, the following description is omitted to avoid excessive complexity of the description.
  • the food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads and the like. Since the food composition of the present invention is composed of mesenchymal stem cell-derived secretory proteins with little toxicity and side effects, it can be used with confidence even for long-term use for prophylactic purposes.
  • the amount may be added at a ratio of 0.1 to 50% of the total weight.
  • natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).
  • compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • additives can be used independently or in combination.
  • the proportion of such additives is not so critical but is usually selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.
  • composition according to the present invention increases the expression of IL-10, a representative immunomodulatory cytokine, and decreases the expression of inflammatory cytokines such as TNF- ⁇ , IL-6, IL-12 (p70), thereby reducing the expression of rheumatoid arthritis. It can effectively prevent or treat autoimmune diseases.
  • 1 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of evaluating arthritis severity after treatment with MTX.
  • 2 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the change in the foot thickness of the mouse after the MTX treatment.
  • 3 is a secreted protein isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And after treatment with MTX, the mouse's foot picture and H & E stained picture of the foot tissue are shown.
  • 4A to 4C are secreted proteins isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of quantitatively scoring the extent of inflammatory cell infiltration, synovial hyperplasia and bone erosion after MTX treatment.
  • FIG. 5 shows indocyanine green in tissues of mouse foot tissue after treatment with secreted protein and MTX isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention. Optical imaging photographs taken of fluorescence are shown.
  • 6a to 6e are secreted proteins isolated and concentrated from two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in the expression level of inflammation-related cytokines (TNF- ⁇ , IL-12 (p70), IL-6, IL-10, IL-1 ⁇ ) after treatment with MTX.
  • TNF- ⁇ , IL-12 (p70), IL-6, IL-10, IL-1 ⁇ changes in the expression level of inflammation-related cytokines after treatment with MTX.
  • Figures 7a and 7b is a collagen-induced arthritis mouse model in one embodiment of the present invention TGF after treating the secreted protein derived from human adipose-derived mesenchymal stem cells and the secreted protein isolated and concentrated from the two-dimensional culture solution TGF The change in the expression level of - ⁇ 1 and galectin-1 is shown graphically.
  • Figure 7c and 7d is a collagen-induced arthritis mouse model in the embodiment of the present invention treated with a three-dimensional rotational culture-derived secretion protein derived from human adipose-derived mesenchymal stem cells and three-dimensional stationary culture-derived secreted protein TGF- ⁇ 1 and The graph shows the change in the expression level of galectin-1.
  • 8A and 8B are secreted proteins isolated and concentrated from a two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention, respectively; Secreted protein isolated and concentrated from three-dimensional rotating culture; And the results of analyzing the number of total splenocytes and the expression of CD4 + T cells in splenocytes after treatment with MTX.
  • 9A and 9B are secreted proteins isolated and concentrated from two-dimensional cultures of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in CD4 + IFN- ⁇ + T cells and CD4 + CD25 + FoxP3 + T cells in the spleen after MTX treatment.
  • Figure 10a and Figure 10b is a secreted protein isolated and concentrated from the two-dimensional culture of human adipose-derived mesenchymal stem cells in a collagen-induced arthritis mouse model in one embodiment of the present invention, respectively; Secreted protein isolated and concentrated from three-dimensional rotating culture; And changes in dendritic cells and intraperitoneal inflammatory (M1) and anti-inflammatory (M2) macrophages in the spleen after treatment with MTX.
  • M1 and M2 intraperitoneal inflammatory
  • 11 is a biological process (biological process), the relationship between a total of 310 cytokines confirmed the expression in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional culture medium and three-dimensional rotation culture medium in one embodiment of the present invention, The results are analyzed by molecular function and cellular component.
  • 12A, 12B and 12C show biological processes, molecular functions and cytokines of cytokines expressed in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional culture medium and three-dimensional spin culture medium in one embodiment of the present invention, respectively.
  • the results show a summary of the functions that are highly relevant by cellular composition.
  • FIG. 13 shows cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotating cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures in one embodiment of the present invention; Cytokines are expressed as fold changes by the top 20.
  • 14A to 14C are cytokines whose expression is increased or decreased in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures in one embodiment of the present invention.
  • cytokines having an effective meaning in the protein network (protein network) analysis and quantitative analysis results are shown.
  • a mesenchymal stem cell-derived secretory protein comprising a pharmaceutical composition for the prevention or treatment of autoimmune diseases.
  • a mesenchymal stem cell-derived secretory protein comprising a food composition for preventing or improving autoimmune diseases.
  • DMEM Dulbecco modified Eagle's medium-low glucose
  • FBS Fetal Bovine Serum
  • penicillin / streptomycin penicillin / streptomycin
  • 2-mercaptoethanol ⁇ 1000
  • trypsin / EDTA 0.05%)
  • trypan blue 0.4%) It was purchased from Rosen.
  • Bone marrow-derived human mesenchymal stem cells were cultured at an early passage from the Yonsei University Cell Therapy Center that complies with the Korean Food and Drug Administration's standards (GMP).
  • Medium stem cell culture medium (10% FBS, DMEM low glucose with 0.1 mM mercaptoethanol added) was added to a culture dish of clinically approved human mesenchymal stem cells of KFDA, and cultured for 72 to 86 hours. Medium was exchanged every 2-3 days, cell cultures were passaged with 70-85% confluency and the fifth passage was used for the study.
  • the mesenchymal stem cells were cultured in 80% confluence, washed 4 times or more with PBS buffer to remove protein components such as fetal bovine serum, and then serum-free medium (DMEM-low glucose) excluding antibiotics and fetal bovine serum. After culturing for 48 hours, the culture was recovered.
  • PBS buffer protein components
  • DMEM-low glucose serum-free medium
  • the mesenchymal stem cells were cultured at 80% confluence and then recovered by trypsin treatment, washed twice with PBS buffer to remove serum components, and then suspended in serum-free medium, followed by 60 spinner flasks. Incubated for 48 hours while rotating at rpm to recover the culture.
  • the mesenchymal stem cells were cultured with 80% confluence, then trypsin-treated and recovered, washed twice with PBS buffer to remove serum components, and then serum-free medium. After the suspension was suspended in the culture for 48 hours, the culture solution was recovered.
  • the mesenchymal stem cell culture cultured in large quantities by the two-dimensional and three-dimensional culture method was centrifuged at 1000 x g once to remove the cell residues first. Afterwards, diafiltration was performed using a tangential flow filtration (TFF) capsule (PALL, minimate TFF capsule), which secondly filters large particles such as cell debris with a 0.2 ⁇ M filter and filters molecules of 3 kDa or less.
  • TFF tangential flow filtration
  • the culture solution was continuously concentrated at 4 ° C. with a dilution continuously with a water for injection (saline solution or Ringer injection) using a peristatic tubing pump. The concentration of the protein in the concentrated culture was confirmed by refractometer measurement and Bradford reagent, and stored at -80 ° C until the experiment.
  • the two-dimensional culture solution that did not undergo the above filtration and concentration process was also stored at ⁇ 80 ° C. until the experiment (secretory protein derived from the two-dimensional culture solution).
  • mice Six to seven week old DBA / 1 mice were used to make collagen-induced arthritis animal models. The induction of arthritis was performed by mixing quantified bovine type II collagen with an equal amount of complete Freud's adjuvant, followed by intradermal injection of the tail starting portion of the mouse by 100 ⁇ g. immunization). Two weeks later, 50 ⁇ g of bovine-derived type II collagen and an incomplete Freud's adjuvant were mixed to further inject intradermal injection into the tail of the mouse (2nd boosting). In order to confirm the therapeutic effect, when the severity of arthritis was 6-8 points, the obtained secretory protein was started to be administered and treated for 5 weeks.
  • methotrexate MTX, 35 mg / kg
  • secreted protein derived from two-dimensional culture 200 ⁇ g / mouse
  • secreted protein isolated and concentrated from two-dimensional culture 200 ⁇ g / mouse
  • secreted protein isolated and concentrated from three-dimensional rotary culture 200 ⁇ g / mouse
  • secreted protein 200 ⁇ g /
  • the severity of arthritis was measured by visual observation of foot redness, swelling, and deformity twice a week, and the severity of arthritis at each time period was quantified according to the following scores. Indicated.
  • the paw thickness was measured using a caliper twice a week, and the change of the mouse paw thickness according to each treatment is shown in FIG. 2.
  • 0 is based on the negative control and is normal
  • ICG indocyanine green
  • Inflammation-related cytokines TNF- ⁇ , IL-12 (p70), IL-6, IL-10, by collecting serum from collagen-induced mouse arthritis model treated with two-dimensional culture medium and three-dimensional culture-derived secreted protein as described above) IL-1 ⁇ ) and galectin-1 were measured by ELISA, and the results are shown in FIGS. 6A to 6E and 7A to 7D.
  • TNF- inflammatory cytokine when treated with the secreted protein (2D, 3D) separated and concentrated in the two-dimensional culture and three-dimensional rotation culture, TNF- inflammatory cytokine compared to the non-treated The expression levels of ⁇ , IL-12 (p70), IL-6 and IL-1 ⁇ were significantly decreased, and the reduction effect was superior to that of MTX.
  • IL-10 an anti-inflammatory cytokine, was treated with secreted proteins (2D and 3D) separated and concentrated in a two-dimensional culture and a three-dimensional rotary culture, and there was no treatment. It can be seen that the expression level is significantly increased compared with the case of MTX treatment.
  • the two-dimensional culture of mesenchymal stem cells after the secretion protein obtained through the separation and concentration process according to the present invention can be seen that the anti-inflammatory effect is superior to the secreted protein obtained after the two-dimensional culture, and also the mesenchymal stem cells It can be seen that the secretory protein obtained by three-dimensional rotational culture is significantly superior to the anti-inflammatory effect than the secreted protein obtained by tertiary stationary culture.
  • Splenocytes were isolated from lymphatic vessels and spleen of collagen-induced mouse arthritis model treated with 2D and 3D culture-derived secretory proteins as described above, and then, total flow was measured using a flow cytometer (FacsVerse, BD Pharmigen, USA). The number of splenocytes was measured, analyzed for the expression level of CD4, and the results were analyzed using FlowJo, and the results are shown in FIG. 8.
  • Tregs a marker of regulatory T cells (Tregs), which play an important role in immune regulation in the spleen cells
  • FlowJo The results are analyzed using and shown in FIG. 9.
  • the distribution of Th1 cells was analyzed using CD4 + IFN- ⁇ +, and the maturation and activity of dendritic cells and intraperitoneal macrophage in the spleen that regulate innate immunity were analyzed by CD86, MCH, and CD206. After analyzing the expression level, the results are analyzed using FlowJo and shown in FIG. 10.
  • CD4 + IFN- ⁇ + T cells showing Th1 cells when treated with secreted proteins (2D, 3D) isolated and concentrated in two-dimensional and three-dimensional rotary cultures were not treated. It can be seen that the fraction of was significantly reduced, as shown in Figure 9b, the fraction of CD4 + CD25 + FoxP3 + T cells showing regulatory T cells did not show a significant difference with each treatment.
  • SCK100 Fullmoon BioSystems Antibody Array
  • FIG. 11 shows the relationship between a total of 310 cytokines expressed in secreted proteins isolated and concentrated from mesenchymal stem cell-derived two-dimensional cultures and three-dimensional rotational cultures. Biological process, molecular function And analysis results with cellular components.
  • FIGS. 12A, 12B, and 12C are results of the 310 proteins identified using the DAVID program, which summarize functions related to biological processes, molecular functions, and cellular configurations.
  • Table 1 shows 55 cytokines among 85 cytokines whose expression is increased in secreted proteins isolated and concentrated from three-dimensional rotational cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. .
  • Plasminogen activator inhibitor-1 PAI-1 3.615 Ferritin 2.160 Insulin-like growth factor-binding protein 7, IGF-BP7 1.898 Osteoprotegerin, OPG 1.644 CTGFL / WISP-2 1.636 Beta-2-Microglobulin 1.517 Macrophage colony-stimulating factor, M-CSF 1.497 Tissue inhibitors of metalloproteinases-1, TIMP-1 1.441 Galectin-1 1.408 C-Kit 1.329 Insulin-like growth factor-binding protein 3, IGF-BP3 1.288 Matrix metalloproteinases 2, MMP-2 1.272 Matrix metalloproteinases 10, MMP-10 1.200 Galectin-3 1.187 Catenin-alpha 1.139 Transforming growth factor- ⁇ 2, TGF- ⁇ 2 1.108 Catenin-gamma 1.098 S 100A10 / P11 1.089 Trefoil factor 2, TFF-2
  • Table 2 shows 55 cytokines among 224 cytokines whose expression is reduced in secreted proteins isolated and concentrated from three-dimensional rotational cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. .
  • FIG. 13 is different from the secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional culture, compared to the cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotational culture, and the reduced cytokines. The result is a fold change of 20 units.
  • Cytokines with increased expression in secreted proteins isolated and concentrated from three-dimensional rotating cultures compared to secreted proteins isolated and concentrated in two-dimensional cultures include Plasminogen activator inhibitor-1 (PAI-1), Ferritin, Insulin-like growth factor-binding protein 7, IGF-BP7, Osteoprotegerin (OPG), CTGFL / WISP-2, Beta-2-micro Globulin (Beta-2-Microglobulin), Macrophage colony-stimulating factor (M-CSF), Tissue inhibitors of metalloproteinases-1 (TIMP-1), galectin -1 (Galectin-1), C-Kit, Insulin-like growth factor-binding protein 3 (IGF-BP3), Matrix metalloproteinases 2, MMP- 2), MMP-10, galectin-3, catenin-alpha, TG F- ⁇ 2, Catnin-gamma, S 100A10 / P11, TFF-2, sTNF-receptor II.
  • PAI-1 Pla
  • cytokines with reduced expression in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in two-dimensional cultures had fibroblast growth factor 5 (FGF-5) and follistatin.
  • Follistatin fibroblast growth factor
  • FGF fibroblast growth factor
  • FGF fibroblast growth factor
  • IL Interleukin
  • 4-1BB Receptor 4-1BB Receptor
  • sCD40 Ligand IGF-I, GM-CSF, EG-VEGF, G-CSF, IGF-II, sRANK Receptor
  • FGF-basic Adiponectin
  • IL-6 Eotaxin
  • IL-4 Eotaxin
  • sFas Ligand / Apo1L EGFR.
  • cytokines 14A to 14C show cytokines having an effective meaning among cytokines whose expression is increased or decreased in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures Protein network analysis and quantitative analysis results.
  • (A) is a signaling network of cytokines with an increased expression of at least 1.25-fold in secreted proteins isolated and concentrated from three-dimensional rotary cultures compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional cultures. Schematic diagram showing.
  • Figure 16 (b) is a schematic diagram showing a signal network of cytokines reduced expression less than 0.8-fold in the secreted protein isolated and concentrated from the three-dimensional rotational culture compared to the secreted protein isolated and concentrated in the mesenchymal stem cell-derived two-dimensional culture .
  • Figure 16 (c) is a cytokine that is increased by 1.25 times or more, or 0.8 or less times reduced expression in secreted proteins isolated and concentrated from the three-dimensional rotation culture medium compared to secreted proteins isolated and concentrated in mesenchymal stem cell-derived two-dimensional culture
  • the result of quantitative analysis of Cain Cytokines with a fold increase of 1.25-fold or more in three-dimensional rotational culture-derived secreted proteins compared to two-dimensional cultured-derived secreted proteins include plasminogen activator inhibitor-1 (PAI-1) and ferritin.
  • Insulin-like growth factor-binding protein 7 IGF-BP7, Osteoprotegerin (OPG), CTGFL / WISP-2, Beta-2-microglobulin (Beta- 2-Microglobulin), Macrophage colony-stimulating factor (M-CSF), Tissue inhibitors of metalloproteinases-1 (TIMP-1), and galectin-1 (Galectin) -1), C-Kit, (insulin-like growth factor-binding protein 3, IGF-BP3) and matrix metalloproteinases 2 (MMP-2) Three-dimensional rotational culture compared to secreted proteins derived from two-dimensional culture Cytokines with reduced fold values of 0.8-fold or less in derived secreted proteins include fibroblast growth factor 5 (FGF-5), follistatin, and fibroblast growth factor (FGF)- acidic), Interleukin (IL) -1RA) and Adipolean Variant.
  • FGF-5 fibroblast growth factor 5
  • FGF follistatin
  • the present invention uses mesenchymal stem cell-derived secretory proteins, rheumatoid arthritis, inflammatory spondyloarthritis, adult type S. disease, macrophage activity syndrome, systemic sclerosis, multiple myositis, dermatitis, vasculitis, mixed connective diseases, Sjogren's syndrome, Ulcerative colitis, Crohn's disease, allergic asthma, allergic rhinitis, atopic dermatitis, gallbladder, lacrimitis, conjunctivitis, psoriasis, vulgaris ulcer, Parkinson's disease, amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, Alzheimer's disease, stroke,
  • the present invention relates to a composition for preventing, ameliorating or treating various autoimmune diseases such as atherosclerosis, vascular restenosis, type I diabetes, type II diabetes, diabetic retinopathy, and chronic thyroiditis.

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Abstract

La présente invention concerne une composition visant à prévenir, améliorer ou traiter des maladies auto-immunes à l'aide d'un protéome sécrétoire dérivé de cellules souches mésenchymateuses. Une composition pharmaceutique selon la présente invention augmente l'expression de l'IL-10, une cytokine immunomodulatrice typique, et diminue l'expression de cytokines inflammatoires telles que le TNF-α, l'IL-6 et l'IL-12 (p70), et de ce fait peut efficacement prévenir ou traiter diverses maladies auto-immunes, y compris la polyarthrite rhumatoïde.
PCT/KR2017/009328 2016-08-26 2017-08-25 Composition pharmaceutique pour le traitement de la polyarthrite rhumatoïde, comprenant un protéome sécrétoire dérivé de cellules souches mésenchymateuses, et méthode thérapeutique utilisant cette dernière Ceased WO2018038575A1 (fr)

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KR102652322B1 (ko) * 2020-09-15 2024-04-01 성균관대학교 산학협력단 활성화된 대식세포를 표적하도록 표면 개질된 줄기세포 유래 엑소좀을 포함하는 염증성 대식세포 매개 자가면역질환의 예방 또는 치료용 조성물

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CN111727046A (zh) * 2018-07-17 2020-09-29 仿生技术支持有限公司 类二十烷酸产生促进剂
CN111481572A (zh) * 2019-01-29 2020-08-04 安特罗根有限公司 用于治愈肌腱或韧带损伤的自体及同种的脂肪来源间充质干细胞组合物及其的制备方法
US12213995B2 (en) 2019-07-18 2025-02-04 Direct Biologics, Llc Preparations comprising mesenchymal stem cells and cannabinoids and methods of their use
CN115896031A (zh) * 2022-07-26 2023-04-04 南京鼓楼医院 脐带间充质干细胞来源的外泌体在制备胰岛素增敏剂中的应用

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