Pharmacodynamic compositions and treatment methods using the same

Abstract

The object of this invention is to provide novel pharmaceutical agents that can be used for various treatments. The pharmacological compositions of this invention are characterized by comprising extracellular vesicles derived from equine mesenchymal stem cells. The pharmacological compositions of this invention are tissue repair agents, anti-inflammatory agents, angiogenic agents, or wound healing agents.

Description

Technical Field

[0001] This invention relates to pharmaceutical compositions and methods of processing using such compositions. Background Technology

[0002] For the treatment of organisms involved in tissue repair, anti-inflammation, angiogenesis, and wound healing, new drugs need to be developed. Summary of the Invention The problem the invention aims to solve

[0003] The object of this invention is to provide a new medicament that can be used for various treatments. means for solving problems

[0004] To achieve the aforementioned objective, the pharmacological composition of the present invention is characterized by comprising extracellular vesicles derived from equine mesenchymal stem cells.

[0005] The biological treatment method of the present invention is characterized by including a drug delivery step of administering the pharmacological composition of the present invention to the biological organism. Invention Effects

[0006] The pharmacological composition according to the present invention can achieve, for example, relief of trauma and inflammation in organisms, and angiogenesis. Attached Figure Description

[0007] Figure 1 This is a graph showing the particle size distribution of the extracellular vesicle sample derived from equine mesenchymal stem cells in Example 1. Figure 2 This is a graph showing the detection results of CD9 markers in extracellular vesicle samples derived from equine mesenchymal stem cells in Example 1. Figure 3 This is a graph illustrating the wound healing effect of extracellular vesicle samples derived from equine mesenchymal stem cells in Example 2. Figure 4 This is a graph showing the cytokine concentrations in the culture supernatant of Example 3. Figure 5 This is a phase contrast microscope image of the HUVEC tube in Example 4. Figure 6 This is a graph showing the relative values ​​of the HUVEC tube length in Example 4. Figure 7 This is a graph showing the number of equilibrium points of the HUVEC tube in Example 4. Figure 8A This is a graph showing the cell survival rate of dermal fibroblasts coexisting with extracellular vesicle samples derived from equine mesenchymal stem cells in Example 5. Figure 8BThis is a graph showing the cell survival rate of IHFPDCs (hair papilla cells) coexisting with extracellular vesicle samples derived from equine mesenchymal stem cells in Example 5. Figure 8C This is a graph showing the cell survival rate of NHEK (skin-derived keratinocytes) coexisting with extracellular vesicle samples derived from equine mesenchymal stem cells in Example 5. Detailed Implementation

[0008] The present invention includes, for example, the following forms. [1]: The pharmacological composition is characterized by containing extracellular vesicles derived from equine mesenchymal stem cells. [2]: The pharmacological composition described in [1] is a tissue repair agent. [3]: The pharmacological composition described in [1] or [2] is an anti-inflammatory agent. [4]: The pharmacological composition described in any of [1] to [3] is an angiogenic agent. [5]: The pharmacological composition described in any of [1] to [4] is a wound treatment agent. [6]: The biological treatment method is characterized by a drug administration step of administering a pharmacologically effective composition according to any one of [1] to [5] to the biological body. [7]: According to the biological treatment method described in [6], the drug administration step is the process of administering the pharmaceutical composition as a tissue repair agent to the organism. [8]: According to the biological treatment method described in [6], the administration step is the process of administering the pharmaceutical composition as an anti-inflammatory agent to the organism. [9]: According to the biological treatment method described in [6], the administration step is the process of administering the pharmaceutical composition as an angiogenesis agent to the organism.

[10] : A biological treatment method according to any one of [6] to [9], wherein the administration step is the process of administering the pharmaceutical composition as a wound treatment agent to the organism.

[11] : A method of handling an organism according to any one of [6] to

[10] , wherein the organism is a human or a non-human animal.

[12] : According to the biological treatment method described in

[11] , the non-human animal mentioned therein is a horse.

[0009] Unless otherwise stated, the terms used in this specification may be used in the sense that are commonly used in the art.

[0010] In this specification, the term "treatment" is used in a broad sense, including prevention in addition to treatment in a narrow sense. Treatment in a narrow sense includes, for example, the cure (also known as complete cure) of a disease, the alleviation or improvement of a disease, or the suppression of the progression of a disease (prevention of deterioration). Disease prevention includes, for example, preventing the onset of a disease, preventing the onset of a disease, and preventing the recurrence of a disease. The treatment or prevention of a disease may also be referred to as the treatment or prevention of disease symptoms.

[0011] The present invention is illustrated by specific examples, but is not limited to these examples. Furthermore, the examples in each invention can be referenced interchangeably.

[0012] (1) Pharmacodynamic composition and biological treatment method As described above, the pharmacological composition of the present invention is characterized by comprising extracellular vesicles derived from equine mesenchymal stem cells. Mesenchymal stem cells are also referred to below as MSCs.

[0013] Extracellular vesicles include vesicles secreted by cells and membrane vesicles enclosed in a lipid bilayer. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

[0014] Mesenchymal stem cells are, for example, adult stem cells with multi-differentiation capacity that can differentiate into mesenchymal tissue (mesoderm). There are no particular limitations on the tissues from which mesenchymal stem cells can be derived; examples include bone marrow, adipose tissue, placental tissue, umbilical cord tissue, and dental pulp.

[0015] Extracellular vesicles derived from equine mesenchymal stem cells can be prepared, for example, by culturing equine mesenchymal stem cells in a culture medium, recovering the supernatant containing extracellular vesicles, and further separating portions of the extracellular vesicles from the supernatant.

[0016] The pharmacological composition of the present invention preferably contains, for example, a portion of the extracellular vesicles isolated from equine mesenchymal stem cells, and substantially does not contain equine mesenchymal stem cells. "Substantially does not contain equine mesenchymal stem cells" also includes, for example, the meaning that cell proliferation cannot be confirmed even if the pharmacological composition is cultured.

[0017] Horse mesenchymal stem cells can be obtained, for example, from mesenchymal stem cells isolated from horses, or from cell lines.

[0018] There are no particular restrictions on the culture medium used for equine mesenchymal stem cells; any culture medium used for culturing stem cells can be used, but it is preferred to use a culture medium used for culturing mesenchymal stem cells. Specific examples of culture media include basic media such as DMEM and RPMI-1640, and commercially available products can also be used.

[0019] The basic culture medium may, for example, contain serum, plasma, or artificial serum. Serum and plasma may be of human or non-human animal origin. Non-human animals may include, for example, cattle or horses. Alternatively, the basic culture medium may be, for example, a culture medium that does not contain serum, plasma, or artificial serum.

[0020] There are no particular restrictions on the culture conditions, such as a culture temperature of 30–40°C (37°C as a specific example), and no particular restrictions on the number of culture days. Subculture is preferred, for example, and there are no particular restrictions on the frequency of subculture, such as every 4–5 days.

[0021] There are no particular limitations on the method for separating the extracellular vesicle portion from the supernatant. Examples include ultrafiltration, ultracentrifugation, concentration gradient methods, and separation methods based on microfluidic systems.

[0022] The extracellular vesicle portion comprises a plurality of extracellular vesicles. The size of the extracellular vesicles in the extracellular vesicle portion is not particularly limited, and their particle size can be, for example, 30–250 nm, 50–200 nm, or 100–150 nm. The peak particle size distribution in the extracellular vesicle portion is not particularly limited, for example, 70–150 nm, 95–135 nm, or 100–115 nm. Furthermore, when the total vesicles in the particle size distribution are 100%, the proportion of vesicles with the peak size (e.g., 100–115 nm) is not particularly limited, but its lower limit is, for example, 30% or more, 40% or more, or 60% or more. The extracellular vesicles used as an active ingredient in the wound treatment agent of the present invention are preferably, for example, a fraction of the supernatant graded in a manner consistent with the aforementioned particle size and particle size distribution.

[0023] There are no particular limitations on the method for measuring the particle size of extracellular vesicles; it can be performed using methods such as light scattering, Brownian motion-based methods, and electrical resistance methods. Brownian motion-based methods include, for example, nanoparticle tracking analysis, which can be performed using commercially available nanoparticle analysis devices (trade name: NanoSight, Malvern). The following measurement conditions can be illustrated as an example when using the NanoSight device. Measurement time: 60 seconds Number of repetitions: 3 Detection threshold: 5 Camera type: sCMOS Laser type: Blue405 Camera rating: 13 Injection pump speed: 40

[0024] The pharmacological composition of the present invention can be used, for example, as a pharmaceutical agent for administration to organisms. The organisms include, for example, humans and non-human animals, and the non-human animals can be exemplified by mammals such as horses, rats, rats, dogs, cats, monkeys, rabbits, cattle, goats, and camels.

[0025] The dosage of the pharmacological composition of the present invention is not particularly limited, but it is preferable to administer it at a pharmaceutically effective amount. A pharmaceutically effective amount can be determined, for example, based on the site of treatment, the severity of symptoms, etc. Furthermore, the content of extracellular vesicles derived from equine mesenchymal stem cells in the pharmacological composition of the present invention is not particularly limited, but a pharmaceutically effective amount is preferred.

[0026] The administration method of the pharmacological composition of the present invention is not particularly limited; for example, it can be administered orally or non-orally. Examples of non-oral administration include local, transdermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intraintestinal, and nasal administration. The administration method can be appropriately determined based on the disease, its symptoms, and the site of administration.

[0027] The pharmacological composition of the present invention may, for example, contain only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or it may also contain other ingredients. Furthermore, the wound treatment agent of the present invention may, for example, contain only the aforementioned active ingredient, or it may contain the aforementioned active ingredient and other additives. Examples of additives include pharmaceutically permissible ingredients. Specific examples of the additives are not particularly limited; for example, excipients, carriers (base materials), etc., may be included. The excipients and carriers are, for example, aqueous solvents such as water, physiological saline, and buffer solutions; oils such as soybean oil; petrolatum; alcohols such as glycerin; sugars such as maltose, glucose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, etc. In addition, the additives may include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, vitamins, minerals, colorants, etc. The additives may be appropriately selected according to the administration method of the pharmacological composition according to the present invention.

[0028] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected according to the route of administration. Examples of the pharmaceutical compositions of the present invention include liquids, emulsions, gels, sols, ointments, and solids. Examples of solids include lozenges, tablets, and granules.

[0029] The organism processing method of the present invention, as described above, is characterized by including a drug delivery step of administering the pharmacologically effective composition of the present invention to the organism. The organism processing method of the present invention is characterized by the use of the pharmacologically effective composition of the present invention; other steps and conditions are not particularly limited. The organism processing method of the present invention may refer to the description of the pharmacologically effective composition of the present invention.

[0030] The pharmaceutical composition of the present invention has tissue repair, angiogenesis, and anti-inflammatory functions. Therefore, the pharmaceutical composition of the present invention can be used for these purposes. Furthermore, because the pharmaceutical composition of the present invention has these functions, it can be used for wound treatment, for example, through three methods: tissue repair, angiogenesis, and anti-inflammation. Wound healing (also known as complete healing) is typically achieved through a process involving a blood clotting phase, an inflammatory phase, a proliferative phase, and a maturation phase. As described above, because the pharmaceutical composition of the present invention has tissue repair, angiogenesis, and anti-inflammatory functions, it can be used for wound treatment; specifically, it can, for example, promote wound healing.

[0031] In this specification, "tissue repair" refers, for example, to restoring the function and structure of lost tissue in an organ of an organism to its original state. Additionally, in this specification, "trauma" refers, for example, to physical injury, and there are no particular limitations on the location of the injury. "Trauma treatment" refers, for example, the treatment of injuries to surface tissues (e.g., skin), the treatment of injuries to muscles or organs, and the treatment of injuries extending from the skin to muscles or organs.

[0032] The following (2) to (5) provide specific examples of the pharmacological compositions of the present invention, but each example may be cited in relation to the others.

[0033] (2) Tissue repair agents and tissue repair methods The pharmacological composition of the present invention can be used, for example, as a tissue repair agent, and may also be referred to as the tissue repair agent of the present invention.

[0034] The dosage of the tissue repair agent of the present invention is not particularly limited, but it is preferably administered at a pharmaceutically effective amount. A pharmaceutically effective amount can be determined, for example, based on the location of the tissue to be repaired, the severity of the symptoms, etc. Furthermore, the content of extracellular vesicles derived from equine mesenchymal stem cells in the tissue repair agent of the present invention is not particularly limited, but a pharmaceutically effective amount is preferred.

[0035] The administration method of the tissue repair agent of the present invention is not particularly limited. For example, it can be administered orally or non-oral, with non-oral administration being preferred. As a specific example, it is applied to the tissue to be repaired.

[0036] As a specific example, when the tissue repair agent of the present invention is applied to the affected area (e.g., the surface of the skin), the following conditions can be illustrated. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Application amount of the extracellular vesicles per unit area per application: 1e+8 to 1e+9 vesicles / cm² 2 Frequency of application: 1 to 3 times per day Application period: The period until the tissue repair is complete. Application interval: Apply daily or every 2-3 days.

[0037] As described above, the tissue repair agent of the present invention may, for example, contain only extracellular vesicles derived from equine mesenchymal stem cells as an active ingredient, or it may also contain other ingredients. Furthermore, as described above, the tissue repair agent of the present invention may, for example, contain only the aforementioned active ingredient, or it may contain the aforementioned active ingredient and other additives. Examples of additives include pharmaceutically permissible ingredients. Specific examples of the additives are not particularly limited; for example, excipients, carriers (base materials), etc., may be included. The excipients and carriers may be, for example, aqueous solvents such as water, physiological saline, buffer solutions, etc.; oils such as soybean oil; petrolatum; alcohols such as glycerin; sugars such as maltose, glucose, dextrin, etc.; sugar alcohols such as xylitol; phospholipids; liposomes, etc. In addition, examples of additives include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, vitamins, minerals, colorants, etc. The additives may be appropriately selected according to the administration method of the tissue repair agent according to the present invention.

[0038] The dosage form of the tissue repair agent of the present invention is not particularly limited, and can be appropriately selected according to the method of administration. Examples of the tissue repair agent of the present invention include topical formulations (external medications). From the viewpoint of application, examples of the tissue repair agent of the present invention include liquids, emulsions, gels, sols, ointments, etc.

[0039] The biological treatment method of the present invention may also be referred to as a tissue repair method. In this case, the tissue repair method of the present invention includes an administration step of administering the pharmaceutically effective composition of the present invention (i.e., the tissue repair agent) to the organism. The tissue repair method of the present invention is characterized by the use of the pharmaceutically effective composition of the present invention; other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or non-oral administration; as an example, application to the affected area can be cited. The tissue repair method of the present invention may refer to the description of the tissue repair agent of the present invention.

[0040] (3) Anti-inflammatory agents and treatments for inflammation The pharmacological composition of the present invention can be used, for example, as an anti-inflammatory agent, and may also be referred to as the anti-inflammatory agent of the present invention.

[0041] The dosage of the anti-inflammatory agent of the present invention is not particularly limited, but it is preferably administered at a pharmaceutically effective amount. A pharmaceutically effective amount can be determined, for example, based on the location and severity of the inflammation. Furthermore, the content of extracellular vesicles derived from equine mesenchymal stem cells in the anti-inflammatory agent of the present invention is not particularly limited, but a pharmaceutically effective amount is preferred.

[0042] The administration method of the anti-inflammatory agent of the present invention is not particularly limited; for example, it can be administered orally or non-orally.

[0043] As a specific example, when the anti-inflammatory agent of the present invention is applied to the affected area (e.g., the surface of the skin), the following conditions can be illustrated. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Application amount of the extracellular vesicles per unit area per application: 1e+8 to 1e+9 vesicles / cm² 2 Frequency of application: 1 to 3 times per day Application period: until the inflammation subsides. Application interval: Apply daily or every 2-3 days.

[0044] As a specific example, when the anti-inflammatory agent of the present invention is administered orally, the following conditions may be exemplified. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Dosage frequency: 1 to 3 times per day Duration of administration: until inflammation subsides. Dosing interval: daily or every 3 to 7 days.

[0045] As described above, the anti-inflammatory agent of the present invention may, for example, contain only extracellular vesicles derived from equine mesenchymal stem cells as its active ingredient, or it may also contain other ingredients. Furthermore, as described above, the anti-inflammatory agent of the present invention may, for example, contain only the aforementioned active ingredient, or it may contain the aforementioned active ingredient and other additives. Examples of additives include pharmaceutically permissible ingredients. Specific examples of the additives are not particularly limited; for example, excipients, carriers (base materials), etc., may be included. The excipients and carriers may be, for example, aqueous solvents such as water, physiological saline, buffer solutions, etc.; oils such as soybean oil; petrolatum; alcohols such as glycerin; sugars such as maltose, glucose, dextrin, etc.; sugar alcohols such as xylitol; phospholipids; liposomes, etc. In addition, examples of additives include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, vitamins, minerals, colorants, etc. The additives may be appropriately selected according to the administration method of the anti-inflammatory agent according to the present invention.

[0046] The dosage form of the anti-inflammatory agent of the present invention is not particularly limited, and can be appropriately selected according to the route of administration. The dosage form is not particularly limited, and examples of the pharmacologically effective compositions of the present invention can be given, such as liquids, emulsions, gels, sols, and solids.

[0047] The biological treatment method of the present invention may also be referred to as an inflammation treatment method. In this case, the inflammation treatment method of the present invention includes an administration step of administering the pharmacologically effective composition of the present invention (i.e., the anti-inflammatory agent) to the organism. The inflammation treatment method of the present invention is characterized by the use of the pharmacologically effective composition of the present invention; other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or non-oral administration; as an example, application to the affected area may be given. The description of the anti-inflammatory agent of the present invention may be cited in the inflammation treatment method of the present invention.

[0048] (4) Angiogenesis agents and methods of angiogenesis The pharmacological composition of the present invention can be used, for example, as an angiogenesis agent, and may also be referred to as the angiogenesis agent of the present invention.

[0049] The dosage of the angiogenesis agent of the present invention is not particularly limited, but it is preferably administered at a pharmaceutically effective amount. A pharmaceutically effective amount can be determined, for example, based on the site requiring angiogenesis, the degree of angiogenesis required, etc. Furthermore, the content of extracellular vesicles derived from equine mesenchymal stem cells in the angiogenesis agent of the present invention is not particularly limited, but a pharmaceutically effective amount is preferred.

[0050] The administration method of the angiogenesis agent of the present invention is not particularly limited; for example, it can be administered orally or non-orally.

[0051] As a specific example, when the angiogenesis agent of the present invention is applied to the affected area (e.g., the surface of the skin), the following conditions can be illustrated. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Application amount of the extracellular vesicles per unit area per application: 1e+8 to 1e+9 vesicles / cm² 2 Frequency of application: 1 to 3 times per day Application period: 1–30 days Application interval: Apply daily or every 2-3 days.

[0052] As a specific example, when the angiogenesis agent of the present invention is administered by injection into the affected area, the following conditions may be exemplified. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Dosage frequency: 1 to 3 times per day Duration of administration: 1–30 days Dosing interval: daily or every 3 to 7 days.

[0053] As described above, the angiogenesis agent of the present invention may, for example, contain only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or it may also contain other ingredients. Furthermore, as described above, the angiogenesis agent of the present invention may, for example, contain only the aforementioned active ingredient, or it may contain the aforementioned active ingredient and other additives. Examples of additives include pharmaceutically permissible ingredients. Specific examples of the additives are not particularly limited; examples include excipients, carriers (base materials), etc. The excipients and carriers are, for example, aqueous solvents such as water, physiological saline, and buffer solutions; oils such as soybean oil; petrolatum; alcohols such as glycerin; sugars such as maltose, glucose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, etc. In addition, other additives may include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, vitamins, minerals, colorants, etc. The additives may be appropriately selected according to the administration method of the angiogenesis agent according to the present invention.

[0054] The dosage form of the angiogenesis agent of the present invention is not particularly limited, and can be appropriately selected according to the route of administration. There are no particular limitations on the dosage form; examples of the pharmacological compositions of the present invention can be given, such as liquid formulations.

[0055] The biological treatment method of the present invention may also be referred to as an angiogenesis method. In this case, the angiogenesis method of the present invention includes an administration step of administering the pharmaceutically effective composition of the present invention (i.e., the angiogenesis agent) to the organism. The angiogenesis method of the present invention is characterized by the use of the pharmaceutically effective composition of the present invention; other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or non-oral administration; as an example, application to the affected area can be cited. The angiogenesis method of the present invention may refer to the description of the angiogenesis agent of the present invention.

[0056] (5) Trauma treatment agents and methods The pharmacological composition of the present invention can be used, for example, as a wound treatment agent, and may also be referred to as the wound treatment agent of the present invention.

[0057] The dosage of the wound treatment agent of the present invention is not particularly limited, but it is preferably administered at a pharmaceutically effective amount. A pharmaceutically effective amount can be determined, for example, based on the location and severity of the wound. Furthermore, the content of extracellular vesicles derived from equine mesenchymal stem cells in the wound treatment agent of the present invention is not particularly limited, but is preferably administered at a pharmaceutically effective amount.

[0058] The method of administration of the wound treatment agent of the present invention is not particularly limited. For example, it can be administered orally or non-orally, with non-oral administration being preferred. A specific example is the application of the agent to the wound.

[0059] As a specific example, when the wound treatment agent of the present invention is applied to the wound site (e.g., the skin or other body surface), the following conditions can be illustrated. The total number of extracellular vesicles per day: 1e+9 to 1e+10 vesicles. Application amount of the extracellular vesicles per unit area per application: 1e+8 to 1e+9 vesicles / cm² 2 Frequency of application: 1 to 3 times per day Application period: until the wound is completely healed. Application interval: Apply daily or every 2-3 days.

[0060] As described above, the wound treatment agent of the present invention may, for example, contain only extracellular vesicles derived from equine mesenchymal stem cells as an active ingredient, or it may also contain other ingredients. Furthermore, as described above, the wound treatment agent of the present invention may, for example, contain only the aforementioned active ingredient, or it may contain the aforementioned active ingredient and other additives. Examples of additives include pharmaceutically permissible ingredients. Specific examples of the additives are not particularly limited; for example, excipients, carriers (base materials), etc., may be included. The excipients and carriers may be, for example, aqueous solvents such as water, physiological saline, buffer solutions, etc.; oils such as soybean oil; petrolatum; alcohols such as glycerin; sugars such as maltose, glucose, dextrin, etc.; sugar alcohols such as xylitol; phospholipids; liposomes, etc. In addition, examples of additives include, for example, binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, wetting agents, thickeners, stabilizers, UV shielding agents, preservatives, vitamins, minerals, colorants, etc. The additives may be appropriately selected according to the administration method of the wound treatment agent according to the present invention.

[0061] The dosage form of the wound treatment agent of the present invention is not particularly limited, and can be appropriately selected according to the method of administration. Examples of the wound treatment agents of the present invention include topical agents (external medicines). From the viewpoint of application, examples of the wound treatment agents of the present invention include liquids, emulsions, gels, sols, ointments, etc.

[0062] The biological treatment method of the present invention may also be referred to as a wound treatment method. In this case, the wound treatment method of the present invention includes an administration step of administering the pharmacologically effective composition of the present invention (i.e., the wound treatment agent) to the organism. The wound treatment method of the present invention is characterized by the use of the pharmacologically effective composition of the present invention; other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or non-oral administration; as an example, application to the affected area can be cited. The wound treatment method of the present invention may refer to the description of the wound treatment agent of the present invention.

[0063] As described above, the pharmaceutical composition of the present invention has so-called tissue repair, angiogenesis, and anti-inflammatory functions; for example, it can be called a wound healing agent because it can promote wound healing. Furthermore, the biological treatment method of the present invention can also be called a wound healing method.

[0064] (6) Application The description may be referenced in relation to the following examples of the uses of the invention.

[0065] This invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for tissue repair. Additionally, this invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for the preparation of tissue repair agents.

[0066] This invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for the treatment of inflammation. Additionally, this invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for the preparation of anti-inflammatory agents.

[0067] This invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for angiogenesis. Additionally, this invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for the preparation of angiogenesis agents.

[0068] This invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for wound treatment. Additionally, this invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for preparing wound treatment agents.

[0069] The present invention will be described in detail below through examples, but the present invention is not limited thereto. Example

[0070] [Example 1] Extracellular vesicles derived from equine mesenchymal stem cells were prepared and identified using the following method.

[0071] (1) Preparation method Equine mesenchymal stem cells (EqMSC-ad) isolated from equine adipose tissue were used. These cells were purchased from ScienCells Research Laboratories (product number: H7510, lot #1396). The cell growth medium used was a mesenchymal stem cell basal medium (trade name: Mesenchymal StemCell Medium, product number: 7501, ScienCells Research Laboratories) containing mesenchymal stem cell growth supplement (trade name: MSCGS, product number: 7552, ScienCells Research Laboratories), antibiotics (trade name: P / S Solution, product number: 0503, ScienCells Research Laboratories), and 10% horse serum (trade name: Horse Serum, donor, product number: S0900, BWT).

[0072] Seven 10 cm diameter culture dishes were each supplemented with 10 mL of the aforementioned basal culture medium. Horse mesenchymal stem cells (HMSCs) of passage number 6 were seeded in the basal culture medium at a rate of 2e+5 cells / mL and cultured at 37°C for 1 day. After confirming cell adhesion to the culture dishes, the culture supernatant was removed, and the cells were then cultured with 10 mL of Ca-free medium. 2+ and Mg 2+ The cells were washed with PBS (-). 10 mL of extracellular vesicle recovery medium was added to each culture dish, and the cells were cultured at 37°C for 48 hours. The extracellular vesicle recovery medium used was phenol red-free, antibacterial-free basal medium for mesenchymal stem cells (trade name: MSCM-prf with MSCGS, ScienCells Research Laboratories).

[0073] The cultures from the seven culture dishes were combined and centrifuged (2000×g, 10 min, room temperature), and the culture supernatant was recovered. The culture supernatant was then filtered through a 0.22 μm filtration apparatus (trade name: Stericup, Merck Millipore), and the filtrate was recovered. The filtrate was aliquoted into six centrifuge tubes (11 mL / tube) and ultracentrifuged (35000 rpm, 70 min, 4 °C) using a Beckman Coulter ultracentrifuge. The supernatant was removed from the tubes, and 11 mL of the PBS(-) was added to each tube to suspend the particles. The six tubes were then ultracentrifuged again under the same conditions. The supernatant was then removed from the tubes, and the particles (total 500 μL) were recovered from each tube. These particles were used below as extracellular vesicle samples derived from equine mesenchymal stem cells (hereinafter referred to as equine MSC-derived EVs samples).

[0074] (2) Particle size distribution The equine MSC-derived EVs sample was fed into a nanoparticle tracking system (trade name: Nanosight SN300, Cantum Design, Japan), and the particle size distribution of extracellular vesicles contained in the equine MSC-derived EVs sample was confirmed under the following conditions. Measurement time: 60 seconds Number of repetitions: 3 Detection threshold: 5 Camera type: sCMOS Laser type: Blue405 Camera rating: 13 Injection pump speed: 40

[0075] Figure 1 The particle size distribution of the equine MSC-derived EVs samples is shown below. Furthermore, electron microscopy confirmed spherical particles with the particle sizes shown in Table 1.

[0076] [Table 1]

[0077] (3) Identification of extracellular vesicles The culture supernatant was used to detect CD9, a tetratransmembrane protein used as a marker for exosomes. The CD9 marker in exosomes was detected using the ExoScreen method (paper title: Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen, Nat Commun, 2014, DOI:10.1038 / ncomms4591).

[0078] Biotin-labeled CD9 antibody and receptor bead-labeled CD9 antibody were diluted using a universal buffer (containing x1 universal buffer (Perkin Elmer) and 1 mg / ml dextran (Sigma)). 15 µL of the diluted buffer and 10 µL of the culture supernatant were added to a well plate (1 / 2 Area Plate 96, Perkin Elmer) and incubated at 37°C for 1 hour. Then, 25 µL of the donor bead (Perkin Elmer) diluted with the universal buffer was added to the plate, and the plate was incubated again at 37°C for 1 hour. The fluorescence of the plate, indicating the presence of CD9, at 520–620 nm, was detected using a detection device (Envision 2015 HTS, Perkin Elmer). As a control, DMEM without fetal bovine serum was used instead of the culture supernatant, and fluorescence intensity was detected using the same method.

[0079] Figure 2 The results of CD9 detection in the culture supernatant are shown. Figure 2 In the diagram, the vertical axis represents the relative value of fluorescence intensity, equivalent to CD9. For example... Figure 2 As shown, the presence of the extracellular vesicles was confirmed by detecting CD9 in the culture supernatant.

[0080] [Example 2] The tissue repair effect was confirmed in the equine MSC-derived EVs sample of Example 1.

[0081] Human fibroblasts were used to prepare a wound model. The culture medium used was DMEM (GIBCO) containing 10% fetal bovine serum and 1% antibiotic-antifungal agent (trade name: Antibiotic-Antimycotic, GIBCO). The culture temperature was set at 37°C. Human fibroblasts were cultured in the medium and multiplied in 24-well plates (trade name: NUNC Cell-Culture Treated Multidishes, product number: 142475, NUNC) at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded in a manner similar to that used in traditional Chinese medicine. They were then cultured further, and at the point of 100% confluence, the culture medium was removed from the plate. The surface of the cell sheets within the plate was scraped off using the tip of a thin slice to form a scar, serving as a wound model. The cell masses were then washed with 500 μL of PBS (-).

[0082] The equine MSC-derived EVs sample was diluted with serum-free DMEM to a concentration of 1e+9 vesicles / mL to prepare the diluted EVs sample. This diluted EVs sample was added to the wells of the plate to a concentration of 500 μL / well and cultured further. Forty-eight hours after the start of culture, the cell blocks in the wells were washed with PBS (-), fixed with 300 μL of 4% PFA at room temperature for 15 minutes, PFA was removed, washed with PBS (-), and stained with hematoxylin and eosin (HE). The HE-stained cell blocks (48 hours of culture) were then photographed using a confocal microscope (MICA, Leica) (n=8). It should be noted that cell blocks immediately after the addition of the diluted EVs sample (0 hours of culture) were also subjected to HE staining and photographed in the same manner (n=8). Additionally, as a control, PBS (-) was added instead of the extracellular vesicle sample, and the same culture, HE staining, and photographing were performed.

[0083] Then, using free software (FIJI), the degree of reduction in the area of ​​trauma in cell blocks (cultured for 48 hours) compared to the area of ​​trauma in cell blocks (cultured for 0 hours) was calculated. Specifically, the area of ​​trauma in cell blocks (cultured for 0 hours) was set as 100%, and the proportion of the area of ​​trauma after culture was used to calculate the degree of trauma (area %). The smaller the area % representing the degree of trauma, the smaller the trauma.

[0084] These results are as follows Figure 3 As shown. Figure 3 This is a graph representing the degree of trauma. The vertical axis represents the degree of trauma, indicating the ratio of the area of ​​residual trauma to the area of ​​trauma before culture began. For example... Figure 3As shown, compared with cell blocks containing PBS(-), cell blocks containing the equine MSC-derived EVs sample exhibited a smaller wound area. These results confirm that, based on the equine MSC-derived EVs sample, by restoring the function of cells at the wound site, allowing the wound to be filled, regenerated, and proliferated by cells, the tissue structure can be restored to its original state, i.e., tissue repair can be achieved.

[0085] [Example 3] The anti-inflammatory effect was confirmed in EVs samples derived from horse MSCs.

[0086] The samples in Example 3 used horse MSC-derived EVs, the samples in Comparative Example 3 used human MSC-derived extracellular vesicles (hereinafter referred to as human MSC-derived EVs), and the control samples used PBS (-).

[0087] As shown below, equine MSC-derived EVs samples were prepared. Specifically, equine mesenchymal stem cells were cultured using the same methods as in Example 1, and the culture supernatant was recovered from a culture with a passage number of 7 by centrifugation (2000×g, 10 min, room temperature). The culture supernatant was concentrated by tangential flow filtration (TFF, using a membrane with a fractionated molecular weight of 300 kDa), and the concentrate was further subjected to ultracentrifugation (35000 rpm, 70 min, 4°C). The recovered particles were suspended in D-PBS (-) (Niksui Pharmaceutical Co., Ltd., CAT#05913) and used as the equine MSC-derived EVs sample. It should be noted that, unless otherwise specified, the culture medium and conditions are the same as in Example 1.

[0088] For the equine MSC-derived EVs samples, the particle size distribution of extracellular vesicles was confirmed in the same manner as in Example 1. The results are shown in Table 2 below.

[0089] [Table 2]

[0090] The human MSC-derived EVs samples were prepared using the same method as the horse MSC-derived EVs samples, except that they used culture supernatant from human adipose-derived mesenchymal stem cells (LONZA, cat#PT-5006, lot#647217) at passage number 5.

[0091] Human peripheral blood mononuclear cells (PMBCs) were seeded into 96-well plates at a rate of 1e+5 cells per well and cultured. The culture medium used was RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 2 mM (mmol / L) L-glutamine. Twenty-four hours after the start of culture, concanavalin A (Con-A) was added to the wells to stimulate PBMCs and induce a cytokine storm (inflammation). The final concentration of concanavalin A in each well was 5 μg / mL.

[0092] Twenty-four hours after adding concanavalin A, inflammatory PBMCs were seeded into 96-well plates at a rate of 1e+5 cells per well, and the samples were further added to fresh culture medium in the wells for culture. Samples from the examples and comparative examples were added to make 1000 extracellular vesicles per PBMC cell, and 100 μL of physiological saline was added per well for the control. The supernatant was then recovered after 48 hours of incubation, and the cytokines (IL-2, IL-5, IL-10, IL-17, IFNγ, TNFα) in the supernatant were quantified using a commercially available ELISA.

[0093] These results are as follows Figure 4 As shown. Figure 4 This is a graph representing the concentrations of various cytokines in the supernatant. (Example) Figure 4 As shown, regardless of the cytokine, the number of systems (n=3) containing the equine MSC-EVs sample of the described example was reduced compared to the control (n=3), and also reduced compared to the system containing the human MSC-EVs sample of the comparative example (n=3).

[0094] [Example 4] The angiogenesis effect was confirmed in equine MSC-derived EVs samples.

[0095] The EVs sample of Example 4 used the equine MSC-derived EVs sample of Example 3, and the EVs sample of Comparative Example 4 used the human MSC-derived EVs sample of Comparative Example 3.

[0096] EGM-2 additives (LONZA, cat# CC-4176) were added to EBM-2 (LONZA, cat# CC-3162) to prepare EGM-2 medium. The additives were hEGF, VEGF, R3-IGF-1, Ascorbic Acid, Hydrocortisone, hFGF-β, Heparin, FBS, and Gentamicin / Amphotericin-B. This EGM-2 medium was then mixed with EBM-2 medium (LONZA, cat# CC-3162, without the additives) at a 1:1 volume ratio, and the EVs sample was further added to this mixed medium to prepare the EVs-containing mediums for Example 4 and Comparative Example 4. The EVs sample was added to achieve an EVs concentration of 5.0e+7 particles / well per well (50 μL of medium). A control medium was used, in which an equal volume of PBS (-) as the EVs sample was added to the mixed medium.

[0097] Frozen human umbilical vein endothelial cells (HUVECs) were thawed, replaced with new culture medium, subcultured, and then suspended in the respective media. The suspended HUVECs were seeded onto 96-well plates (Thermo, Nunc Microwell 96F) coated with Matrigel (Corning, cat#356231) at a density of 1.5e+4 cells / well and cultured at 37°C for 16 hours. Three wells (n=3) were used for each culture medium. After culture, the total length of the tubes formed by the HUVECs was measured using an image analysis tool (ImageJ software), and the number of equilibrium points was counted based on phase-contrast microscopy photographs. The average values ​​of the systems using each culture medium were then compared among the control (EVs(-)), Example 4 (Equine MSC-EVs), and Comparative Example 4 (Human MSC-EVs).

[0098] These results are as follows Figure 5 , Figure 6 , Figure 7 As shown. Figure 5 These are phase contrast microscope images of HUVEC tubes. Figure 6 It is a relative value of the HUVEC tube length; specifically, it is a relative value with the control set to 1. Figure 7These graphs represent the number of equilibrium points for HUVEC tubes. As shown in these graphs, when using equine MSC-derived EVs samples, longer tube lengths were obtained compared to both the control and human MSC-derived EVs systems. Furthermore, regarding equilibrium points, significantly more were obtained when using equine MSC-derived EVs samples compared to both the control and human MSC-derived EVs systems. This confirms that equine MSC-derived EVs can promote angiogenesis.

[0099] As described above, Example 2 confirmed that the equine MSC-derived EVs of the present invention function in tissue repair and inflammation relief, and Example 3 confirmed that the equine MSC-derived EVs of the present invention function in promoting angiogenesis. These functions can be used to treat trauma, indicating that equine MSC-derived EVs can be used to treat trauma.

[0100] [Example 5] Extracellular vesicles derived from equine mesenchymal stem cells were prepared using the following method, and their cell proliferation capacity was confirmed.

[0101] (1) Preparation method Equine mesenchymal stem cells (EqMSC-UC) isolated from equine umbilical cords (transferred from Northern Farm) were used. Unless otherwise specified, the culture methods and methods for recovering extracellular vesicles were the same as described in Example 1.

[0102] 20 mL of the basal culture medium was added to each of 30 culture dishes with a diameter of 15 cm. Horse mesenchymal stem cells (HMSCs) of passage number 6 were seeded in the basal culture medium at a rate of 3.5e+4 cells / mL and cultured at 37°C for 2 days. After confirming cell adhesion to the culture dishes, the culture supernatant was removed, and the cells were then cultured with 10 mL of calcium-free... 2+ and Mg 2+ The cells were washed with PBS (-). 20 mL of extracellular vesicle recovery medium was added to each culture dish, and the dishes were incubated at 37°C for 48 hours. The extracellular vesicle recovery medium used was DMEM (trade name: D-MEM (HIGH glucose) without L-Glutamine and Phenol Red, FujiFilmwako).

[0103] The cultures from the seven culture dishes were combined and centrifuged (2000×g, 10 min, room temperature), and the culture supernatant was recovered. The culture supernatant was then filtered through a 0.22 μm filtration device (trade name: Stericup, registered trademark, Merck Millipore), and the filtrate was recovered. This filtrate was used as a sample of extracellular vesicles derived from equine mesenchymal stem cells (hereinafter referred to as equine MSC-derived EVs sample (EqMSC-UC CM)). This sample was diluted 5-fold with PBS(-) and analyzed using a nanoparticle tracking system (trade name: Nanosight SN300, Cantum Design, Japan) in the same manner as in Example 1, and the particle size distribution of the extracellular vesicles (EVs) was confirmed.

[0104] The following shows the results of the extracellular vesicle (EV) particle size distribution of the equine MSC-derived EVs samples. Furthermore, when examining the equine MSC-derived EVs samples using electron microscopy, spherical particles with the particle sizes shown in the table below were identified.

[0105] [Table 3]

[0106] (2) Confirmation of proliferation The horse MSC-derived EVs sample (EqMSC-UC CM) of (1) was diluted with phenol red-free DMEM, and diluted samples at multiple dilution ratios were prepared and used.

[0107] The following three cell types were used as the cells for evaluating proliferation. Dermal Fibroblast: Product number CC-2059, Lonza Corporation; IHFPDC (Hydropause Cells): Product No. T0501, abm Corporation; NHEK (Skin-derived keratinocytes): Product number 00192907, Lonza Corporation.

[0108] The target cells were individually seeded into 96-well plates at a density of 1000 cells / 50 μL / well and cultured overnight. The following culture media were used. (Skin fibroblasts) DMEM containing 10% fetal bovine serum and 1% antibiotic-antifungal agent (trade name: Antibiotic-Antimycotic, GIBCO) (trade name: D-MEM (High Glucose with L-Glutamine, Phenol Red and Sodium Pyruvate, FujiFilm Wako) (IHFPDC) PriGrow III (product number TM003, abm company) contains 10% fetal bovine serum and 1% antibiotic-antifungal agent. (NHEK) KGM Gold Keratinocyte Culture Medium (Product No. 00192060, Lonza Corporation)

[0109] Then, in the examples, a diluted sample of the EqMSC-UC CM was added to a well of 50 μL, and the target cells were cultured. For each target cell, the concentration (cells / well) of equine MSC-derived EVs in the wells was 2.5e+6, 4.10e+7, 8.20e+7, 1.6e+8, and 3.28e+8. In the control, only DMEM without phenol red was added as a control sample to a well of 50 μL, and the target cells were cultured. 72 hours after adding the diluted sample or the control sample, reagent (trade name: Cell Counting Kit-8, Dojindo) was added to a well of 10 μL, and the reaction was carried out at 37°C for 4 hours. After the reaction, the absorbance at 450 nm was measured for each well. Then, the absorbance of the control was set to 100%, and the relative value of the absorbance of the examples was calculated as the cell viability (%).

[0110] These results are shown in Figure 8. Figure 8A It is the cell survival rate of Dermal Fibroblasts (skin-derived fibroblasts). Figure 8B It refers to the cell viability of IHFPDCs (hair papilla cells). Figure 8C This refers to the cell survival rate of NHEK (skin-derived keratinocytes). For example... Figure 8A , Figure 8B , Figure 8C As shown, it can be confirmed that, regardless of the cell type, adding the equine MSC-derived EVs sample can increase cell viability in a concentration-dependent manner, i.e., promote cell proliferation.

[0111] In wound treatment, the proliferation of skin-derived fibroblasts is important, for example, from the perspective of tissue repair at the wound site, such as the epidermis. The proliferation of dermal papilla cells is important, for example, from the perspective of replenishing dermal papilla cells lost due to trauma, ultraviolet radiation, inflammation, etc., activating hair follicle cells, and activating pigment cells. The proliferation of skin-derived keratinocytes is important, for example, from the perspective of aiding in the repair of the epidermal layer lost during trauma. Therefore, as shown in Figure 8, by adding the equine MSC-derived EVs sample, the proliferation of various cells can be promoted, indicating that the equine MSC-derived EVs are effective in wound treatment.

[0112] The present invention has been described above with reference to the embodiments, but the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art will be possible within the scope of the present invention, given the structure and details of the invention.

[0113] This application claims priority based on Japanese Patent Application No. 2024-12210 filed on January 30, 2024 and Japanese Patent Application No. 2024-101298 filed on June 24, 2024, and the entire disclosure of those applications is incorporated herein. Industrial applications

[0114] The pharmacological composition according to the invention can, for example, repair tissues, relieve inflammation, promote angiogenesis in organisms, and also treat wounds based on these functions.

Claims

1. A pharmaceutical composition, characterized in that, The pharmacological composition contains extracellular vesicles derived from equine mesenchymal stem cells.

2. The pharmacological composition according to claim 1, characterized in that, The pharmaceutical composition is a tissue repair agent.

3. The pharmacological composition according to claim 1 or 2, characterized in that, The pharmaceutical composition is an anti-inflammatory agent.

4. The pharmacological composition according to any one of claims 1 to 3, characterized in that, The pharmacological composition is an angiogenesis agent.

5. The pharmacological composition according to any one of claims 1 to 4, characterized in that, The pharmaceutical composition is a wound treatment agent.

6. A method for treating organisms, characterized in that, The method includes a drug delivery process that administers the pharmaceutical composition according to any one of claims 1 to 5 to an organism.

7. The biological treatment method according to claim 6, characterized in that, The drug administration process is the process of administering the pharmaceutical composition as a tissue repair agent to an organism.

8. The biological treatment method according to claim 6, characterized in that, The drug administration process is the process of administering the pharmaceutical composition as an anti-inflammatory agent to an organism.

9. The biological treatment method according to claim 6, characterized in that, The drug delivery process is the process of administering the pharmaceutical composition as an angiogenesis agent to an organism.

10. The method for treating organisms according to any one of claims 6 to 9, characterized in that, The drug administration process is the process of administering the pharmaceutical composition as a wound treatment agent to an organism.

11. The method for treating organisms according to any one of claims 6 to 10, characterized in that, The organism in question is either a human or a non-human animal.

12. The biological treatment method according to claim 11, characterized in that, The non-human animal in question is a horse.

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