WO2018004237A1 - Composition comprenant un exosome dérivé de cellules souches traitées par la thrombine pour le traitement d'une plaie de la peau - Google Patents

Composition comprenant un exosome dérivé de cellules souches traitées par la thrombine pour le traitement d'une plaie de la peau Download PDF

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WO2018004237A1
WO2018004237A1 PCT/KR2017/006776 KR2017006776W WO2018004237A1 WO 2018004237 A1 WO2018004237 A1 WO 2018004237A1 KR 2017006776 W KR2017006776 W KR 2017006776W WO 2018004237 A1 WO2018004237 A1 WO 2018004237A1
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thrombin
stem cells
derived
exosome
exosomes
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PCT/KR2017/006776
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English (en)
Korean (ko)
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장윤실
박원순
성동경
안소윤
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사회복지법인 삼성생명공익재단
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Priority claimed from KR1020170080366A external-priority patent/KR101860266B1/ko
Application filed by 사회복지법인 삼성생명공익재단 filed Critical 사회복지법인 삼성생명공익재단
Priority to EP17820513.4A priority Critical patent/EP3479831B1/fr
Priority to CN201780047575.0A priority patent/CN109641013B/zh
Priority to US16/314,725 priority patent/US20190314469A1/en
Priority to JP2018568285A priority patent/JP6826135B2/ja
Publication of WO2018004237A1 publication Critical patent/WO2018004237A1/fr

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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
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • 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
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • 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
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug

Definitions

  • the present invention in the pharmaceutical composition for the prevention or treatment of skin wounds (Skin Wound), including a exosome derived from thrombin-treated stem cells as an active ingredient, pharmaceutical preparations containing the same, and a method for preparing the same It is about.
  • the skin is the body's primary defense against disease as a protective barrier to the body, and the epidermis provides a barrier to microbial invasion.
  • the primary objectives in the treatment of wounds, burns, abrasions and other damage to the skin are rapid closure and wound healing to prevent infection.
  • Wound healing is generally a complex process that involves three stages: inflammation, proliferation, and remodeling.
  • the first step involves clotting to achieve haemostasis, and supplementation of neutrophils to destroy bacteria and necrotic tissue, followed by supplementation of macrophages.
  • angiogenesis occurs, in which endothelial cells migrate to the wound site, and at the same time fibroblasts move to the wound site to help produce granulation tissue.
  • the formation of granulation tissue allows for re-epithelialization.
  • the level of collagen production and destruction is evened, and the wound healed is slowly changed to achieve maximum strength. Wound healing can be chronic or even delayed or damaged when one of the processes fails to function properly in a timely manner.
  • Chronic wounds are defined as open wounds that require more than 8 weeks of treatment, and the damaged healing process can often be associated with diabetes complications, with severe consequences such as high limb amputation and even death. This can be done. Approximately 15% of diabetics suffer from chronic wounds that do not heal. Therefore, wounds are an important issue not only for individuals but also for society, and early treatment should reduce the risk of secondary infection.
  • ECM extra-cellular matrix
  • stem cells are known to be involved in tissue regeneration, treatment, and immune response, as well as their multipotency, and by using such characteristics to separate and culture mesenchymal stem cells from umbilical cord blood, bone marrow, etc. Efforts have been made to develop a treatment for the disease.
  • cell therapeutics cannot rule out the possibility of tumorogenicity due to DNA transfer, and secondly, stem cells themselves can cause vascular obstruction or myocardial infarction. Third, cord blood and Rejection reaction due to surface antigen when transplanted (allograft) using the same allogeneic cells is a problem. Fourth, in general, cell therapeutics are difficult to manufacture and have high storage costs due to the limitation of storage transport. There is a limit.
  • Exosomes are small vesicles (approximately 30-100 nm in diameter) that are secreted from a variety of cells, and within the cells called multivesicular bodies (MVBs), rather than falling directly off the plasma membrane in an electron microscope study. Originated from specific compartments and released and secreted out of cells was observed. In other words, when fusion occurs between the polycystic body and the plasma membrane, the vesicles are released into the extracellular environment, which is called an exosome. It is not clear what molecular mechanism these exosomes are made of, but not only red blood cells, but also various immune cells and tumor cells, including B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, etc. Stem cells are also known to produce and secrete exosomes in living conditions.
  • exosomes derived from stem cells contain nuclear components as well as receptors and proteins are known to play an intercellular communication role.
  • the exosomes derived from the stem cells contain a relatively small amount of animal serum compared to the stem cells may also exclude the risk of symptoms (zoonosis) due to animal serum infection.
  • cell therapy using exosomes is expected to become a new paradigm that can overcome the limitations of existing stem cell therapy.
  • Korean Patent Publication No. 2012-0133709 discloses a skin regeneration effect of exosomes obtained from skin cells, but it is completely known that exosomes derived from thrombin-treated stem cells are superior to conventional skin wound treatment. There is no bar.
  • the present inventors have conducted extensive studies to improve the treatment efficacy while overcoming the limitations of the stem cell therapeutics with respect to skin wounds, and thus, exosomes derived from stem cells, especially exosomes derived from stem cells treated with thrombin.
  • the present invention was completed by confirming that this angiogenesis and skin wound healing effect was significantly increased.
  • the present invention provides a pharmaceutical composition for preventing or treating skin wounds, including an exosome derived from thrombin-treated stem cells as an active ingredient.
  • the stem cells are stem selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells and multipotent stem cells It is characterized by being a cell.
  • the mesenchymal stem cells are characterized in that derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane or placenta.
  • the treatment of the cutaneous wound is characterized by angiogenesis due to vascular endothelial cell growth promotion.
  • the skin wound is characterized by a defect in the epidermis, dermis or subcutaneous tissue.
  • the pharmaceutical composition is characterized by being administered orally or parenterally.
  • the pharmaceutical composition is characterized in that it further comprises an auxiliary component selected from the group consisting of culture medium, cytokines, growth factors and genes.
  • the exosomes are characterized by increased expression of growth factors, immunoregulatory factors, antioxidant factors or angiogenesis factors.
  • the growth factor is a brain-derived neurotropic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), or vascular endothelial growth (VEGF). factor).
  • BDNF brain-derived neurotropic factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • NGF nerve growth factor
  • VEGF vascular endothelial growth
  • the present invention also provides a pharmaceutical formulation for preventing or treating a skin wound, containing the composition.
  • the preparation is characterized in that the injection, injection, spray, liquid or patch form.
  • the preparation is characterized in that it further comprises a pharmaceutically acceptable carrier.
  • the present invention also provides a quasi-drug preparation for improving skin wounds, comprising an exosome derived from thrombin-treated stem cells as an active ingredient.
  • the improvement of the skin wound is characterized in that by angiogenesis (angiogenesis).
  • the exosomes are characterized by increased expression of growth factors, immunoregulatory factors, antioxidant factors or angiogenesis factors.
  • the quasi-drug preparation is in the form of an external preparation for skin selected from the group consisting of liquids, ointments, creams, sprays, patches, gels and aerosols.
  • the present invention (a) treating the thrombin after stem cell culture; (b) separating the exosomes from the culture medium of step (a); And (c) preparing a composition containing the exosomes separated in step (b) as an active ingredient.
  • the thrombin of step (a) is characterized in that contained in the medium at a concentration of 1-1000 unit / ml.
  • the exosome of step (c) is characterized in that using centrifugation.
  • the centrifugation is characterized in that it is performed for 10 minutes to 5 hours at 5,000-500,000g.
  • the present invention also provides a method for treating a skin wound, comprising administering to a subject an exosome derived from a thrombin-treated stem cell.
  • the present invention also provides the use of exosomes derived from thrombin treated stem cells for use in the preparation of an agent for preventing or treating a skin wound.
  • the cell-free preparation does not contain DNA, there is no risk of carcinogenesis and there is no cell surface antigen, so there is no problem of rejection reaction.
  • the size is very small than the cell there is no fear of causing blockage in the microvascular system during systemic administration, it is possible to reduce the manufacturing cost because it is possible to develop the drug to off the shelf product as a non-cell separation material.
  • the exosome derived from the stem cells treated with thrombin is superior to the untreated stem cells, the effect of treating skin wounds is excellent even with a small amount, thereby significantly reducing the amount of stem cells required to generate a therapeutic dose of exosomes. There is an advantage that can significantly lower the cost of therapeutic production.
  • the present invention can significantly improve the therapeutic efficacy while solving the problems of the conventional stem cell therapeutics, it can be usefully used for the treatment of skin wounds.
  • FIG. 2 is a Western blot result confirming that exosome markers CD63 and CD9 are normally expressed in thrombin-treated stem cell-derived exosomes.
  • BDNF growth factors
  • FGF growth factor
  • HGF growth factor
  • NGF growth factor
  • VEGF anti-inflammatory cytokines
  • IL-6 anti-inflammatory cytokines
  • FIG. 4A is a result of a tube formation assay showing that exosomes derived from thrombin-treated stem cells exert a concentration-dependent angiogenic effect
  • FIG. 4B is a graph quantifying them.
  • FIG. 5A is a result of a tube formation assay showing a difference in angiogenic effects depending on the type of cell from which the exosome is derived
  • FIG. 5B is a graph quantifying it.
  • Figure 6a is a result showing the skin wound healing effect on exosomes derived from thrombin-treated stem cells in an in vivo skin wound animal model
  • Figure 6b is a graph quantifying it.
  • FIG. 7 shows fibroblast exosomes derived from fibroblasts, naive MSC exosomes derived from untreated thrombin stem cells, thrombin MSC exosomes derived from thrombin treated stem cells and hypoxia in an in vivo skin wound animal model. This is a result of comparing the healing effect of skin wounds of stem cell-derived exosomes (hypoxia MSC exosome).
  • the present invention provides a pharmaceutical composition for preventing or treating skin wounds, including an exosome derived from thrombin-treated stem cells as an active ingredient.
  • 'stem cell' refers to a cell having an ability to differentiate into two or more different types of cells while having an autologous ability as an undifferentiated cell.
  • Stem cells of the present invention may be autologous or allogeneic stem cells, may be from any type of animal, including humans and non-human mammals, and is not limited to whether the stem cells are derived from adults or embryos Do not.
  • Stem cells of the present invention include embryonic stem cells or adult stem cells, preferably adult stem cells.
  • the adult stem cells may be mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells or multipotent stem cells, preferably mesenchymal stem cells, but is not limited thereto. It doesn't work.
  • the mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta, but is not limited thereto.
  • 'umbilical cord blood means blood collected from the umbilical vein connecting the placenta and the fetus.
  • Umbilical cord blood is a naturally occurring by-product of childbirth and is much easier to collect than normal mesenchymal tissue such as bone marrow, which requires multiple surgeries. It is also easy to get.
  • cord blood-derived cells do not express histocompatibility antigen HLA-DR (class II), which is the most important cause of rejection in tissue or organ transplantation, immunity such as rejection that has been a problem during conventional transplantation The reaction can be induced or minimized, so that not only autologous umbilical cord blood but also taga-derived cord blood can be used.
  • 'exosome' refers to a small (approximately 30-100 nm diameter) vesicle of membrane structure secreted from various cells, and the vesicle is released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane. Means.
  • the exosomes are naturally secreted or include those secreted artificially.
  • skin wound is meant to include defects of the epidermis, dermis or subcutaneous tissue.
  • prevention or treatment of skin wounds is meant to include the reduction, alleviation and improvement of symptoms of skin wounds, and to include a lowering the possibility of skin wounds.
  • vascular endothelial cells HUVEC
  • Angiogenesis is a phenomenon in which endothelial cells of existing blood vessels decompose, move, divide, and differentiate extracellular matrix to form new capillaries, which are essential in the physiological process of wound healing. By doing so, the effect of wound healing can be verified. That is, angiogenesis must be involved in the necessary wound healing process for the rejuvenation of wounded skin tissue.
  • inflammatory reactions occur due to necrosis of cells and destruction of blood vessels. After this inflammatory response, kallikrein, thrombin, and plasmin, together with devascularization of blood components, activation of platelets and coagulation, The same biological media goes through a series of processes.
  • stem cell function / efficacy is increased due to an increase in paracrine property, which is the main mechanism of action of stem cells, without altering cell stability such as cell viability and oxidative function. Can be enhanced. Furthermore, due to thrombin treatment, as well as enhancing the therapeutic efficacy of exosomes derived from stem cells, it is possible to increase the secretion amount of exosomes.
  • the paracrine may be increased growth factor, immunoregulatory factor, antioxidant factor or regenerative factor
  • growth factor refers to a proteinaceous bioactive substance that promotes cell division, growth, or differentiation, and is referred to as BDNF (brain-derived).
  • BDNF brain-derived
  • neurotropic factor fibroblast growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • NGF nerve growth factor
  • VEGF vascular endothelial growth factor
  • IL-6 Interleukin-6
  • vascular endothelial growth factor is a representative protein that regulates angiogenesis.
  • VEGF binds to VEGF receptors 1, 2, or 3 (VEGFR1, VEGFR2, VEGFR3) present in vascular endothelial cells to induce tyrosine phosphorylation of VEGF receptors (VEGFR) resulting in activation of vascular endothelial cells. This will have a significant impact on.
  • compositions of the present invention can be administered to a subject in various ways without particular limitation, for example, orally or parenterally.
  • the pharmaceutical composition of the present invention may further contain one or more known auxiliary ingredients having a therapeutic effect on the skin wound together with the stem cell-derived exosomes.
  • genes that are effective in treating skin wounds eg, anti-inflammatory cytokine genes, siRNAs for inflammatory cytokines or anti-sense primers
  • Expression vectors eg, cytokines (eg, interleukin-10), growth factors (eg, keratinocyte growth factor) that provide autocrine or paracrine effects, and these At least one auxiliary ingredient selected from the group consisting of a combination may be further included.
  • Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. Administration of the composition may be administered once a day, may be divided several times, but is not limited thereto.
  • the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the treatment of skin wounds.
  • composition of the present invention may further comprise a suitable carrier commonly used in the manufacture of pharmaceutical compositions.
  • a suitable carrier commonly used in the manufacture of pharmaceutical compositions.
  • a preservative, a painless agent, a solubilizer, or a stabilizer may be further included, and in the case of a topical administration, a base, an excipient, a lubricant, or a preservative may be further included.
  • compositions of the present invention can be administered in a dosage form, formulated in a unit dosage form suitable for administration in the body of a subject according to conventional methods in the pharmaceutical art.
  • Formulations suitable for this purpose include parenteral injectables such as injectable ampoules, injectables such as infusion bags, sprays such as aerosol preparations and the like.
  • the injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, Ringer's solution, etc. may be used as the injection solution.
  • the infusion bag may be made of polyvinyl chloride or polyethylene.
  • Administration in the present invention means providing the subject with the compositions of the present invention in any suitable manner.
  • Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. Administration of the composition may be administered once a day, may be administered several times, but is not limited thereto.
  • the present invention also provides a quasi-drug preparation for improving skin wounds, comprising an exosome derived from thrombin-treated stem cells as an active ingredient.
  • the quasi-drug preparation may be in the form of an external preparation for skin selected from the group consisting of liquids, ointments, creams, sprays, patches, gels, and aerosols, and specifically, antiseptic cleaners, shower foams, gagrins, wipes, detergent soaps, and hands. It can be used as a wash, humidifier filler, mask, ointment or filter filler composition.
  • the present invention (a) treating the thrombin after stem cell culture; (b) separating the exosomes from the culture medium of step (a); And (c) preparing a composition containing the exosomes separated in step (b) as an active ingredient.
  • the thrombin treatment concentration is sufficient to enhance the efficacy of stem cells / exosomes, and there is no particular limitation, but is preferably included in the concentration of 1-1000 unit / ml in the medium.
  • centrifugation for example, in a culture medium, centrifugation, ultrafast centrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, using a polymer Separation may be carried out using a method such as separation and a combination thereof, preferably centrifugation / supercentrifugation.
  • centrifugation / supercentrifugation is preferably performed at 4 ° C. and 3,000-100,000 g for 10 minutes to 5 hours.
  • the medium used for cell culture in the present invention is a cell such as stem cells in vitro containing elements essential for growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals, and the like. Means a mixture for growth and propagation.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Base Medium Eagle
  • RPMI1640 DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10)
  • DMEM / F-12 Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12
  • ⁇ -MEM ⁇ -Minimal Essential Medium
  • G-MEM Gasgow's Minimal Essential Medium
  • IMOC Isocove's Modified Dulbecco's Medium
  • KnockOut DMEM Commercially prepared media such as, but not limited to, artificially synthesized media, and the like.
  • the present invention also provides a method for treating a skin wound, comprising administering to a subject an exosome derived from a thrombin-treated stem cell.
  • ⁇ means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal.
  • Human umbilical cord blood-derived mesenchymal stem cells (3 ⁇ 10 5 cells) were cultured in 100 mm culture dishes (orange scientific cat # 4450200) for 1 week after dispensing. After confirming that the cells were saturated and propagated in the culture dish, the cells were replaced with MEM alpha media in which 50 unit / ml thrombin (REYON Pharmaceutical. Co., LTD) was diluted and incubated for 6 hours. .
  • the culture solution was divided into centrifuge tubes, centrifuged at 4 ° C. and 6,000 rpm for 30 minutes, and the supernatant was transferred to a new tube to remove cell debris. Again, the supernatant was ultracentrifuged at 4 ° C., 100,000 rpm for 2-4 hours, and then the supernatant was further removed to obtain an exosome (final concentration: 15 ⁇ g / ml).
  • the expression of known exosome markers CD63 and CD9 was verified through Western blot.
  • the exosomes obtained from the thrombin-treated stem cells normally expressed CD63 and CD9, confirming that the exosomes.
  • Example 1-1 Exosome obtained in Example 1-1 was confirmed whether the expression of growth factors or anti-inflammatory cytokines such as IL-6 was increased by the thrombin treatment.
  • the proteins in the exosomes are separated and the amount of BDNF, FGF, HGF, NGF, VEGF, IL-6 in the exosomes is determined using the Procarta immunoassay kit ( affymatrix, USA).
  • BDNF brain-derived neurotropic factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • NGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • IL-6 Interleukin-6 expression was increased compared to exosomes (control, normal) obtained from the thrombin untreated stem cells.
  • thrombin treatment enhances cell regeneration, vascular regeneration, and anti-inflammatory efficacy of stem cell-derived exosomes.
  • human umbilical vein endothelial cells were cultured in Medium 200PRF medium containing Low Serum Growth Supplement (LSGS) for 12 hours, then collected by trypsin treatment, and containing 5% fetal calf serum. Resuspended in M199 medium. Growth factor reduction Matrigel was aliquoted into 250 ⁇ l each in 24 cell culture wells and polymerized in a 37 ° C. CO 2 incubator.
  • LSGS Low Serum Growth Supplement
  • HUVEC cells were placed in wells with polymerized matrigel at a concentration of 4 ⁇ 10 4 cells per well, and 2.5 ug / ml, 5 ug / ml, 10 of the 'exosomes derived from thrombin-treated stem cells' obtained in Example 1 were obtained. ug / ml concentration was added. After incubation in a 37 ° C. CO 2 incubator for 24 hours, the culture was photographed ( ⁇ 40) and the length of the formed tube was measured using the Image J (National Institute of Health) open program.
  • 'exosomes derived from thrombin-treated stem cells' of the present invention induces differentiation of dose-dependent vascular endothelial cells as compared to the control group (untreated group NC) Increased tube formation.
  • exosomes derived from untreated thrombin (MSC ECV)
  • exosomes derived from thrombin treated stem cells (MSC throm ECV)
  • fibroblast ECV exosomes derived from untreated thrombin
  • exosomes derived from thrombin treated fibroblast Experiments were carried out in the same manner as in Example 2-1, except that (fibroblast throm ECV) was each treated at a concentration of 10 ug / ml.
  • exosomes obtained from the thrombin-treated stem cells of the present invention, significantly improved angiogenesis compared to exosomes (MSC ECV) obtained from thrombin untreated stem cells Showed ability.
  • the exosomes obtained from fibroblast did not exhibit any angiogenic capacity regardless of the presence or absence of thrombin treatment, and thus the specific therapeutic efficacy according to the type / capacity of the cells from which the exosomes are derived (angiogenic capacity). It can be seen that this is determined.
  • a skin model was prepared by punching the skin with a 0.8 mm biopsy punch on the back of an adult rat, and then fibroblast-derived exosomes (fibroblast exosomes), thrombin-untreated stem cells-derived exosomes (naive MSC exosomes), Thrombin-treated stem cell-derived exosomes (thrombin MSC exosomes) were each mixed with 20 ug of saline, prepared in a total of 10 ul, and then applied to the wound surface.
  • fibroblast exosomes fibroblast exosomes
  • thrombin-untreated stem cells-derived exosomes thrombin-untreated stem cells-derived exosomes
  • thrombin MSC exosomes Thrombin-treated stem cell-derived exosomes
  • the exosome (thrombin MSC exosome) obtained from the thrombin-treated stem cells of the present invention the wound significantly improved compared to the exosome (naive MSC exosome) obtained from the thrombin untreated stem cells It showed a therapeutic effect.
  • the exosomes obtained from the fibroblast was found to exhibit no wound healing effect similar to the control (saline).
  • the wound control group which was damaged by punch biopsy of the skin as compared to the normal group, showed no recovery to normal skin tissue.
  • the wound control group which was damaged by punch biopsy of the skin as compared to the normal group, showed no recovery to normal skin tissue.
  • the thrombin-treated stem cell-derived exosomes (naive MSC exosome) and hypoxia pretreated stem cell-derived exosomes (hypoxia MSC exosome) showed a tendency to improve somewhat
  • thrombin-treated stem cell-derived exosomes thrombin MSC exosome
  • the therapeutic efficacy is specifically determined according to the type / capacity of the cell from which the exosome is derived.
  • the stem cell-derived exosome-based therapeutic agent treated with the thrombin according to the present invention, it is possible to remarkably improve the treatment efficacy while solving problems such as transplant rejection reaction or manufacturing cost, which is a problem of conventional stem cell therapeutics, It can be usefully used for treatment.

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  • Neurosurgery (AREA)
  • Ophthalmology & Optometry (AREA)
  • Vascular Medicine (AREA)
  • Pregnancy & Childbirth (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une composition pharmaceutique comprenant un exosome dérivé d'une cellule souche traitée par la thrombine en tant qu'ingrédient efficace pour prévenir ou traiter une plaie cutanée, une formulation pharmaceutique le contenant, et un procédé de production de celle-ci. En tant qu'agent acellulaire, un agent thérapeutique à base d'exosome de la présente invention présente un faible risque de carcinogenèse et aucun problème de rejet de greffe ainsi que peu susceptible de se produire dans l'obstruction microvasculaire. L'agent, qui n'est pas une cellule, mais un matériau isolé à partir d'une cellule, peut être soumis à un développement de médicament en un produit hors d'étagère qui permet de réduire le coût de production. L'agent présente l'avantage de présenter des effets remarquables de traitement de l'angiogenèse et de plaie cutanée même à une faible concentration d'exosomes grâce à l'effet de traitement de la thrombine.
PCT/KR2017/006776 2016-07-01 2017-06-27 Composition comprenant un exosome dérivé de cellules souches traitées par la thrombine pour le traitement d'une plaie de la peau WO2018004237A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP17820513.4A EP3479831B1 (fr) 2016-07-01 2017-06-27 Composition comprenant un exosome dérivé de cellules souches traitées par la thrombine pour l'utilisation dans le traitement d'une plaie de la peau
CN201780047575.0A CN109641013B (zh) 2016-07-01 2017-06-27 包含凝血酶处理干细胞来源的外泌体的皮肤创伤的治疗用组合物
US16/314,725 US20190314469A1 (en) 2016-07-01 2017-06-27 Composition Comprising Thrombin-Treated Stem Cell-Derived Exosome For Treating Skin Wound
JP2018568285A JP6826135B2 (ja) 2016-07-01 2017-06-27 トロンビン処理幹細胞に由来するエクソソームを含む皮膚傷治療用組成物

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KR20160083703 2016-07-01
KR10-2016-0083703 2016-07-01
KR10-2017-0080366 2017-06-26
KR1020170080366A KR101860266B1 (ko) 2016-07-01 2017-06-26 트롬빈 처리 줄기세포에서 유래된 엑소좀을 포함하는 피부상처 치료용 조성물

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WO2020070700A3 (fr) * 2018-10-04 2020-05-14 Exogenus Therapeutics, Sa Compositions comprenant de petites vésicules extracellulaires dérivées de cellules mononucléaires du sang de cordon ombilical ayant des propriétés anti-inflammatoires et immunomodulatrices, et leur procédé d'obtention
EP4137140A4 (fr) * 2020-04-14 2024-05-22 Samsung Life Public Welfare Foundation Composition de prévention ou de traitement d'une maladie de la peau diabétique, comprenant un exosome dérivé de cellule souche traitée par de la thrombine

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020070700A3 (fr) * 2018-10-04 2020-05-14 Exogenus Therapeutics, Sa Compositions comprenant de petites vésicules extracellulaires dérivées de cellules mononucléaires du sang de cordon ombilical ayant des propriétés anti-inflammatoires et immunomodulatrices, et leur procédé d'obtention
EP4137140A4 (fr) * 2020-04-14 2024-05-22 Samsung Life Public Welfare Foundation Composition de prévention ou de traitement d'une maladie de la peau diabétique, comprenant un exosome dérivé de cellule souche traitée par de la thrombine

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