WO2025164451A1 - Medicinal composition and treatment method using same - Google Patents

Abstract

The purpose of the present invention is to provide a novel drug that can be used for various medical treatments.　A medicinal composition according to the present invention is characterized by containing extracellular vesicles derived from equine mesenchymal stem cells. This medicinal composition is a tissue repair agent, an anti-inflammatory agent, an angiogenic agent, or a wound treatment agent.

Description

Medicinal compositions and treatment methods using same

The present invention relates to a medicinal composition and a treatment method using the same.

There is a need to develop new drugs for biological treatments such as tissue repair, anti-inflammation, angiogenesis, and wound healing.

The purpose of the present invention is to provide a new drug that can be used for a variety of treatments.

In order to achieve the above-mentioned objective, the medicinal composition of the present invention is characterized by containing extracellular vesicles derived from equine mesenchymal stem cells.

The biological treatment method of the present invention is characterized by including an administration step of administering the medicinal composition of the present invention to a living organism.

The medicinal composition of the present invention can, for example, heal wounds and soothe inflammation in the body, and promote angiogenesis.

FIG. 1 is a graph showing the particle size distribution of a sample of extracellular vesicles derived from equine mesenchymal stem cells in Example 1. FIG. 2 is a graph showing the results of detecting the C9 marker in a sample of extracellular vesicles derived from equine mesenchymal stem cells in Example 1. FIG. 3 is a graph showing the wound healing effect of a sample of extracellular vesicles derived from equine mesenchymal stem cells in Example 2. FIG. 4 is a graph showing cytokine concentrations in the culture supernatants in Example 3. FIG. 5 is a phase contrast micrograph of the HUVEC tube in Example 4. FIG. 6 is a graph showing the relative values of HUVEC tube length in Example 4. FIG. 7 is a graph showing the number of branching points of HUVEC tubes in Example 4. Figure 8A is a graph showing the cell viability of Dermal Fibroblast in the presence of an extracellular vesicle sample derived from equine mesenchymal stem cells in Example 5. FIG. 8B is a graph showing the cell viability of IHFPDCs (hair papilla cells) in the presence of a sample of extracellular vesicles derived from equine mesenchymal stem cells in Example 5. FIG. 8C is a graph showing the cell viability of NHEK (skin-derived keratinocytes) in the presence of a sample of extracellular vesicles derived from equine mesenchymal stem cells in Example 5.

The present invention includes, for example, the following aspects.
[1] A medicinal composition comprising extracellular vesicles derived from equine mesenchymal stem cells.
[2] The medicinal composition according to [1], which is a tissue repair agent.
[3] The medicinal composition according to [1] or [2], which is an anti-inflammatory agent.
[4] The pharmaceutical composition according to any one of [1] to [3], which is an angiogenic agent.
[5] The medicinal composition according to any one of [1] to [4], which is a wound treatment agent.
[6] A method for treating a living body, comprising administering the pharmaceutical composition according to any one of [1] to [5] to a living body.
[7] The method for treating a living body according to [6], wherein the administering step is a step of administering the medicinal composition to a living body as a tissue repair agent.
[8] The method for treating a living body according to [6], wherein the administering step is a step of administering the pharmaceutical composition to a living body as an anti-inflammatory agent.
[9] The method for treating a living body according to [6], wherein the administering step is a step of administering the pharmaceutical composition to a living body as an angiogenic agent.
[10] The method for treating a living body according to any one of [6] to [9], wherein the administering step is a step of administering the pharmaceutical composition to a living body as a wound treatment agent.
[11] The living body treatment method according to any one of [6] to [10], wherein the living body is a human or a non-human animal.
[12] The biotreatment method according to [11], wherein the non-human animal is a horse.

Unless otherwise specified, terms used in this specification may be used in the manner commonly used in the relevant technical field.

As used herein, the term "treatment" refers, for example, to treatment in the broad sense, and includes prevention in addition to treatment in the narrow sense. Treatment in the narrow sense includes, for example, curing the target disease (also known as complete recovery), alleviating or ameliorating the disease, or inhibiting the progression of the disease (preventing worsening), while prevention of disease includes, for example, preventing contraction of the disease, preventing the onset of the disease, and preventing the recurrence of the disease. Treatment or prevention of a disease can also be referred to, for example, as treatment or prevention of the symptoms of the disease.

The present invention will be explained below using specific examples, but is not limited to these. Furthermore, the examples in each invention can be used interchangeably.

(1) Medicinal Composition and Biotreatment Method As described above, the medicinal composition of the present invention is characterized by containing extracellular vesicles derived from equine mesenchymal stem cells. Mesenchymal stem cells are hereinafter also referred to as MSCs.

Extracellular vesicles are, for example, vesicles secreted from cells and are membrane vesicles surrounded by a lipid bilayer membrane. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

Mesenchymal stem cells are, for example, somatic stem cells with pluripotency and can differentiate into cells of mesenchymal tissue (mesodermal tissue). There are no particular limitations on the tissue from which mesenchymal stem cells are derived, and examples include bone marrow, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, etc.

Extracellular vesicles derived from equine mesenchymal stem cells can be prepared, for example, by culturing equine mesenchymal stem cells in a medium, recovering the supernatant containing extracellular vesicles, and then isolating a fraction of the extracellular vesicles from the supernatant.

The medicinal composition of the present invention preferably contains, for example, a fraction of extracellular vesicles isolated from equine mesenchymal stem cells, and is substantially free of equine mesenchymal stem cells. "Substantially free of equine mesenchymal stem cells" also means, for example, that no cell proliferation is observed when the medicinal composition is cultured.

Equine mesenchymal stem cells may be, for example, mesenchymal stem cells isolated from horses, or a cell line.

The medium for equine mesenchymal stem cells is not particularly limited, and any medium used for culturing stem cells can be used, preferably a medium used for culturing mesenchymal stem cells. Specific examples of medium include basal media such as DMEM and RPMI-1640, and commercially available products may also be used.

The basal medium may contain, for example, serum, plasma, or artificial serum. Serum and plasma may be derived from, for example, humans or non-human animals. Non-human animals may be derived from, for example, cows or horses. The basal medium may also be a medium that does not contain, for example, serum, plasma, or artificial serum.

Culture conditions are not particularly limited, and the culture temperature is, for example, 30 to 40°C (e.g., 37°C), and the number of days of culture is not particularly limited. It is preferable to culture by subculturing, for example, and the frequency of subculturing is not particularly limited, for example, every 4 to 5 days.

The method for separating the extracellular vesicle fraction from the supernatant is not particularly limited, and examples include ultrafiltration, ultracentrifugation, concentration gradient methods, and separation methods using a microfluidic system.

The extracellular vesicle fraction contains a plurality of extracellular vesicles. The size of the extracellular vesicles in the extracellular vesicle fraction is not particularly limited, and examples of particle sizes include 30-250 nm, 50-200 nm, and 100-150 nm. The particle size peak in the particle size distribution of the extracellular vesicle fraction is not particularly limited, and is, for example, 70-150 nm, 95-135 nm, and 100-115 nm. Furthermore, when all vesicles are taken as 100% in the particle size distribution, the proportion of vesicles at the peak (e.g., 100-115 nm) is not particularly limited, and the 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 fractionated from the supernatant to have the above particle size and particle size distribution.

The method for measuring the particle size of extracellular vesicles is not particularly limited, and can be, for example, a light scattering method, a measurement method based on Brownian motion, an electrical resistance method, etc. Measurement methods based on Brownian motion include, for example, nanoparticle tracking analysis, and a commercially available nanoparticle analyzer (trade name NanoSight, Malvern) can be used. When NanoSight is used, the following measurement conditions can be exemplified.
Measurement time: 60 seconds Number of repetitions: 3 Detection threshold: 5
Camera type:sCMOS
Laser type:Blue405
Camera level: 13
Syringe pump speed: 40

The medicinal composition of the present invention can be used, for example, as a drug for administering to a living organism. The living organism can be, for example, a human or a non-human animal. Examples of the non-human animal include mammals such as horses, mice, rats, dogs, cats, monkeys, rabbits, cows, goats, and camels.

The dosage of the medicinal composition of the present invention is not particularly limited, and it is preferable to administer a pharmaceutically effective amount. The pharmaceutically effective amount can be determined, for example, depending on the area to be treated, the severity of symptoms, etc. Furthermore, the content of the extracellular vesicles derived from equine mesenchymal stem cells in the medicinal composition of the present invention is not particularly limited, and it is preferable, for example, to administer a pharmaceutically effective amount.

The administration route of the medicinal composition of the present invention is not particularly limited and may be, for example, oral or parenteral administration. Parenteral administration includes, for example, topical, transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intraintestinal, and nasal administration. The administration route can be appropriately selected depending on, for example, the target disease, its symptoms, and the site of administration.

The medicinal composition of the present invention may contain, for example, only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or may further contain other ingredients. Furthermore, the wound treatment agent of the present invention may contain, for example, only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, and the like. Other examples of the additive components include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants. The additive components can be selected appropriately depending on, for example, the administration form of the pharmaceutical composition of the present invention.

The dosage form of the medicinal composition of the present invention is not particularly limited and can be selected appropriately depending on, for example, the mode of administration. Examples of the medicinal composition of the present invention include liquids, emulsions, gels, sols, ointments, and solid formulations. Examples of the solid formulations include tablets, tablets, and granules.

As described above, the biological treatment method of the present invention is characterized by including an administration step of administering the medicinal composition of the present invention to a living organism. The biological treatment method of the present invention is characterized by using the medicinal composition of the present invention, and other steps and conditions are not particularly limited. The description of the medicinal composition of the present invention can be used for the biological treatment method of the present invention.

The medicinal composition of the present invention has the functions of tissue repair, angiogenesis, and anti-inflammatory properties. Therefore, the medicinal composition of the present invention can be used for these purposes. Furthermore, because the medicinal composition of the present invention has these functions, it can be used, for example, in wound treatment using three approaches: tissue repair, angiogenesis, and anti-inflammatory properties. Wound healing (also known as complete healing) is generally achieved through the processes of the blood coagulation phase, the inflammation phase, the proliferation phase, and the maturation phase. As described above, the medicinal composition of the present invention has the functions of tissue repair, angiogenesis, and anti-inflammatory properties, so it can be used in wound treatment, and specifically, can function, for example, to promote wound healing.

As used herein, "tissue repair" refers to, for example, restoring lost tissue in a living organ to its original state, both in function and structure. Furthermore, as used herein, a "wound" refers to, for example, physical damage, and the site of the wound in question is not particularly limited. "Wound treatment" may refer, for example, to treatment of a wound in the surface tissue of the body (e.g., skin), treatment of a wound in muscle or an organ, or treatment of damage that reaches from the skin to muscle or an organ.

Specific examples of the medicinal composition of the present invention are listed below in (2) to (5), and each example can be used interchangeably.

(2) Tissue Repair Agent and Tissue Repair Method The pharmaceutical composition of the present invention can be used, for example, as a tissue repair agent, and can also be referred to as the tissue repair agent of the present invention.

The dosage of the tissue repair agent of the present invention is not particularly limited, and it is preferable to administer a pharmaceutically effective amount. The pharmaceutically effective amount can be determined, for example, depending on the location of the tissue to be repaired, the severity of symptoms, etc. Furthermore, the content of the extracellular vesicles derived from equine mesenchymal stem cells in the tissue repair agent of the present invention is not particularly limited, and it is preferable, for example, to administer a pharmaceutically effective amount.

The administration form of the tissue repair agent of the present invention is not particularly limited, and may be, for example, oral or parenteral administration. Parenteral administration is preferred, and a specific example is application to the tissue to be repaired.

As a specific example, when the tissue repair agent of the present invention is applied to an affected area (for example, a body surface such as the skin), the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Amount of the extracellular vesicles applied per area: 1e+8 to 1e+9 particles/cm ²
Number of applications per day: 1 to 3 times Application period: Until tissue repair is complete Application interval: Every day or every 2 to 3 days

As described above, the tissue repair agent of the present invention may contain, for example, only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or may further contain other ingredients. Furthermore, as described above, the tissue repair agent of the present invention may contain, for example, only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, and the like. Other examples of the additive components include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants. The additive components can be selected appropriately depending on, for example, the administration form of the tissue repair agent of the present invention.

The dosage form of the tissue repair agent of the present invention is not particularly limited and can be selected appropriately depending on, for example, the mode of administration. Examples of the tissue repair agent of the present invention include topical agents (external medications). From the perspective of application, examples of the tissue repair agent of the present invention include liquids, emulsions, gels, sols, ointments, etc.

The biological treatment method of the present invention can also be referred to as, for example, a tissue repair method. In this case, the tissue repair method of the present invention includes an administration step of administering the medicinal composition of the present invention (i.e., the tissue repair agent) to a living organism. The tissue repair method of the present invention is characterized by the use of the medicinal composition of the present invention, and other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or parenteral administration, and one example is application to the affected area. The description of the tissue repair agent of the present invention can be used for the tissue repair method of the present invention.

(3) Anti-inflammatory Agent and Inflammation Treatment Method The pharmaceutical composition of the present invention can be used, for example, as an anti-inflammatory agent, and can also be referred to as the anti-inflammatory agent of the present invention.

The dosage of the anti-inflammatory agent of the present invention is not particularly limited, and it is preferable to administer a pharmaceutically effective amount. The pharmaceutically effective amount can be determined, for example, depending on the site of inflammation, the degree of inflammation, etc. Furthermore, the content of the extracellular vesicles derived from equine mesenchymal stem cells in the anti-inflammatory agent of the present invention is not particularly limited, and it is preferable, for example, to administer a pharmaceutically effective amount.

The administration form of the anti-inflammatory agent of the present invention is not particularly limited, and may be, for example, oral or parenteral administration.

As a specific example, when the anti-inflammatory agent of the present invention is applied to an affected area (for example, a body surface such as the skin), the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Amount of the extracellular vesicles applied per area: 1e+8 to 1e+9 particles/cm ²
Number of applications per day: 1 to 3 times Application period: Until inflammation subsides Application interval: Every day or every 2 to 3 days

As a specific example, when the anti-inflammatory agent of the present invention is orally administered, the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Dosage frequency per day: 1-3 times Administration period: Until inflammation subsides Administration interval: Daily or every 3-7 days

As described above, the anti-inflammatory agent of the present invention may contain, for example, only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or may further contain other ingredients. Furthermore, as described above, the anti-inflammatory agent of the present invention may contain, for example, only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, and the like. Other examples of the additive components include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants. The additive components can be selected appropriately depending on, for example, the administration form of the anti-inflammatory agent of the present invention.

The dosage form of the anti-inflammatory agent of the present invention is not particularly limited and can be selected appropriately depending on, for example, the mode of administration. The dosage form is not particularly limited and includes the examples of the medicinal composition of the present invention described above, and specific examples include liquids, emulsions, gels, sols, solids, etc.

The biological treatment method of the present invention can also be referred to as, for example, a method for treating inflammation. In this case, the method for treating inflammation of the present invention includes an administration step of administering the medicinal composition of the present invention (i.e., the anti-inflammatory agent) to a living body. The wound treatment method of the present invention is characterized by using the medicinal composition of the present invention, and other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or parenteral administration, and one example is application to the affected area. The description of the anti-inflammatory agent of the present invention above can be used for the method for treating inflammation of the present invention.

(4) Angiogenic Agent and Angiogenesis Method The pharmaceutical composition of the present invention can be used, for example, as an angiogenic agent, and can also be referred to as the angiogenic agent of the present invention.

The dosage of the angiogenic agent of the present invention is not particularly limited, and it is preferable to administer a pharmaceutically effective amount. The pharmaceutically effective amount can be determined, for example, depending on the site where angiogenesis is required, the degree to which angiogenesis is required, etc. Furthermore, the content of the extracellular vesicles derived from equine mesenchymal stem cells in the angiogenic agent of the present invention is not particularly limited, and it is preferable, for example, to administer a pharmaceutically effective amount.

The administration route of the angiogenic agent of the present invention is not particularly limited, and may be, for example, oral or parenteral administration.

As a specific example, when the angiogenic agent of the present invention is applied to an affected area (for example, a body surface such as the skin), the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Amount of the extracellular vesicles applied per area: 1e+8 to 1e+9 particles/cm ²
Number of applications per day: 1 to 3 times Application period: 1 to 30 days Application interval: Every day or every 2 to 3 days

As a specific example, when the angiogenic agent of the present invention is administered by injection into the affected area, the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Dosage frequency per day: 1-3 times Dosage period: 1-30 days Dosage interval: daily or every 3-7 days

As described above, the angiogenic agent of the present invention may contain, for example, only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or may further contain other ingredients. Furthermore, as described above, the angiogenic agent of the present invention may contain, for example, only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, and the like. Other examples of the additive components include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants. The additive components can be selected appropriately depending on, for example, the administration form of the angiogenic agent of the present invention.

The dosage form of the angiogenic agent of the present invention is not particularly limited and can be selected appropriately depending on, for example, the mode of administration. The dosage form is not particularly limited, and examples thereof include those of the medicinal composition of the present invention described above, and specific examples include liquid formulations.

The biological treatment method of the present invention can also be referred to as, for example, an angiogenesis method. In this case, the angiogenesis method of the present invention includes an administration step of administering the medicinal composition of the present invention (i.e., the angiogenic agent) to a living organism. The angiogenesis method of the present invention is characterized by the use of the medicinal composition of the present invention, and other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or parenteral administration, and one example is application to the affected area. The description of the angiogenic agent of the present invention can be used for the angiogenesis method of the present invention.

(5) Wound healing agent and wound healing method The pharmaceutical composition of the present invention can be used, for example, as a wound healing agent, and can also be referred to as the wound healing agent of the present invention.

The dosage of the wound healing agent of the present invention is not particularly limited, and it is preferably administered in a pharmaceutically effective amount. The pharmaceutically effective amount can be determined, for example, depending on the site of the wound, the severity of the wound, etc. Furthermore, the content of the extracellular vesicles derived from equine mesenchymal stem cells in the wound healing agent of the present invention is not particularly limited, and it is preferably administered in a pharmaceutically effective amount, for example.

The administration form of the wound healing agent of the present invention is not particularly limited, and may be, for example, oral or parenteral administration. Parenteral administration is preferred, and a specific example is application to the wound.

As a specific example, when the wound healing agent of the present invention is applied to a wounded area (for example, a body surface such as the skin), the following conditions can be exemplified.
Total amount of extracellular vesicles per day: 1e+9 to 1e+10 particles
Amount of the extracellular vesicles applied per area: 1e+8 to 1e+9 particles/cm ²
Number of applications per day: 1 to 3 times Application period: Until the wound is completely healed Application interval: Every day or every 2 to 3 days

As described above, the wound healing agent of the present invention may contain, for example, only extracellular vesicles derived from equine mesenchymal stem cells as the active ingredient, or may further contain other ingredients. Furthermore, as described above, the wound healing agent of the present invention may contain, for example, only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; liposomes, and the like. Other examples of the additive components include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants. The additive components can be selected appropriately depending on, for example, the administration form of the wound healing agent of the present invention.

The dosage form of the wound healing agent of the present invention is not particularly limited and can be selected appropriately depending on, for example, the mode of administration. Examples of the wound healing agent of the present invention include topical preparations (external medications). From the perspective of application, examples of the wound healing agent of the present invention include liquids, emulsions, gels, sols, ointments, etc.

The biological treatment method of the present invention can also be referred to as, for example, a wound treatment method. In this case, the wound treatment method of the present invention includes an administration step of administering the medicinal composition of the present invention (i.e., the wound treatment agent) to a living body. The wound treatment method of the present invention is characterized by using the medicinal composition of the present invention, and other steps and conditions are not particularly limited. The administration step may be, for example, oral administration or parenteral administration, and one example is application to the affected area. The description of the wound treatment agent of the present invention can be used for the wound treatment method of the present invention.

As mentioned above, the medicinal composition of the present invention has the functions of tissue repair, angiogenesis, and anti-inflammatory properties, and can function, for example, to promote wound healing, and therefore can also be referred to as a wound healing agent. Furthermore, the biotreatment method of the present invention can also be referred to, for example, as a wound healing method.

(6) Uses The above description can be applied to the uses of the present invention exemplified below.

The present invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for use in tissue repair. The present invention also relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for producing a tissue repair agent.

The present invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for use in treating inflammation. The present invention also relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for producing an anti-inflammatory agent.

The present invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for use in angiogenesis. The present invention also relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for producing an angiogenic agent.

The present invention relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for use in wound treatment. The present invention also relates to the use of extracellular vesicles derived from equine mesenchymal stem cells for the manufacture of a wound treatment agent.

The present invention will be explained in detail below using examples, but the present invention is not limited to these.

[Example 1]
Extracellular vesicles derived from equine mesenchymal stem cells were prepared and characterized by the following method.

(1) Preparation Method: Equine mesenchymal stem cells (EqMSC-ad) isolated from equine adipose tissue were used. The cells were purchased from ScienCells Research Laboratories (product number H7510, lot #1396). The cell growth medium used was a basal medium for mesenchymal stem cells (product number 7501, ScienCells Research Laboratories) containing mesenchymal stem cell growth supplement (product number MSCGS, product number 7552, ScienCells Research Laboratories), antibiotics (product number P/S Solution, product number 0503, ScienCells Research Laboratories), and 10% horse serum (product number S0900, BWT).

Ten mL of the basal medium was added to each of seven 10 cm diameter culture dishes. The equine mesenchymal stem cells at passage 6 were seeded into the basal medium to a cell density of 2e+5 cells/mL and cultured at 37°C for one day. After confirming adhesion of the cells to the culture dish and removing the culture supernatant, the cells were washed with 10 mL of Ca ²⁺ - and Mg ²⁺ -free PBS(-). Ten mL of extracellular vesicle recovery medium was further added to each of the culture dishes, and the cells were cultured at 37°C for 48 hours. A phenol red-free, antibiotic-free mesenchymal stem cell basal medium (product name MSCM-prf with MSCGS, ScienCells Research Laboratories) was used as the extracellular vesicle recovery medium.

The cultures in the seven culture dishes were combined and centrifuged (2000 x g, 10 minutes, room temperature) to recover the culture supernatant. The culture supernatant was then passed through a 0.22 μm pore size filtration tool (product name Stericup®, Merck Millipore) to recover the filtrate. The filtrate was distributed into six centrifuge tubes (11 mL per tube) and subjected to ultracentrifugation in an ultracentrifuge (Beckman Coulter) at 35,000 rpm for 70 minutes at 4°C. The supernatant was removed from the tubes, and 11 mL of the PBS(-) was added to each tube to suspend the pellet. The six tubes were then again subjected to ultracentrifugation under the same conditions. The supernatant was then removed from the tubes, and the pellets were recovered from each tube (500 μL in total). This pellet was used below as an extracellular vesicle sample derived from equine mesenchymal stem cells (hereinafter referred to as equine MSC-derived EVs sample).

(2) Particle size distribution The equine MSC-derived EVs sample was subjected to a nanoparticle tracking system (trade name: Nanosight SN300, Japan Quantum Design Co., Ltd.) under the following conditions to confirm the particle size distribution of the extracellular vesicles contained in the equine MSC-derived EVs sample.
Measurement time: 60 seconds Number of repetitions: 3 Detection threshold: 5
Camera type:sCMOS
Laser type:Blue405
Camera level: 13
Syringe pump speed: 40

The particle size distribution results for the equine MSC-derived EV sample are shown in Figure 1 and the table below. Furthermore, when the equine MSC-derived EV sample was examined under an electron microscope, spheres with the particle sizes shown in Table 1 were confirmed.

(3) Identification of extracellular vesicles The culture supernatant was subjected to detection of the tetraspanin protein CD9, an exosome marker, using the ExoScreen method (Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen, Nat Commun, 2014, DOI: 10.1038/ncomms4591).

Biotin-labeled CD9 antibody and acceptor bead-labeled CD9 antibody were diluted with universal buffer (containing 1x Universal Buffer (PerkinElmer) and 1 mg/ml dextran (Sigma)) to prepare a diluted solution. 15 μL of the diluted solution and 10 μL of the culture supernatant were added to a well plate (Half Area Plate 96, PerkinElmer) and incubated at 37°C for 1 hour. 25 μL of a diluted solution of donor beads (PerkinElmer) diluted with universal buffer was then added to the plate and incubated again at 37°C for 1 hour. Fluorescence at 520-620 nm, indicating the presence of CD9, was detected from this plate using a detection system (Envision 2015 HTS, PerkinElmer). As a control, DMEM without fetal bovine serum was used instead of the culture supernatant, and fluorescence intensity was detected in the same manner.

The results of CD9 detection in the culture supernatant are shown in Figure 2. In Figure 2, the vertical axis represents the relative fluorescence intensity, which corresponds to CD9. As shown in Figure 2, CD9 was detected in the culture supernatant, confirming the presence of the extracellular vesicles.

[Example 2]
The equine MSC-derived EVs sample of Example 1 was examined for its effect on tissue repair.

Human fibroblasts were used to create a wound model. The culture medium used was DMEM (GIBCO) containing 10% fetal bovine serum and 1% antibiotic-antimycotic (trade name: Antibiotic-Antimycotic, GIBCO). The culture temperature was 37°C. Human fibroblasts were cultured in the culture medium and then seeded into a 24-well plate (trade name: NUNC® Cell-Culture Treated Multidishes, product number 142475, NUNC) at 2 x ¹⁰ cells per well. After further culture, the culture medium was removed from the plate when the plate reached 100% confluence. The surface of the cell sheet in the plate was scraped with the tip of a tip to create a scar that served as a wound model, and the cell mass was washed with 500 μL of PBS(-).

The equine MSC-derived EV sample was diluted with serum-free DMEM to 1e+9 particles/mL to prepare a diluted EV sample. The diluted EV sample was added to the wells of the plate at 500 μL/well and further cultured. 48 hours after the start of culture, the cell clusters in the wells were washed with PBS(-), fixed with 300 μL of 4% PFA at room temperature for 15 minutes, the PFA was removed, washed with PBS(-), and HE stained. The HE-stained cell clusters (cultured for 48 hours) were then photographed using a confocal microscope (MICA, Leica) (n=8). Cell clusters immediately after the addition of the diluted EV sample (cultured for 0 hours) were also similarly HE stained and photographed (n=8). As a control, PBS(-) was added instead of the extracellular vesicle sample, and the culture, HE staining, and photography were similarly performed.

Then, using free software (FIJI), the extent to which the wound in the cell cluster (cultured for 48 hours) had shrunk compared to the area of the wound in the cell cluster (cultured for 0 hours) was calculated. Specifically, the area of the wound in the cell cluster (cultured for 0 hours) was set at 100%, and the percentage of the area of the wound after culture was calculated as the degree of wound (area %). A smaller area %, which indicates the degree of wound, means that the wound has shrunk.

These results are shown in Figure 3. Figure 3 is a graph showing the extent of the wound. The vertical axis represents the extent of the wound, and indicates the ratio of the remaining wound area to the wound area before the start of culture. As shown in Figure 3, it was found that the cell clusters to which the equine MSC-derived EVs sample was added were able to reduce the wound area compared to cell clusters to which PBS (-) was added. These results confirmed that the equine MSC-derived EVs sample is able to restore the function of cells at the wound site, fill the wound with cells, regenerate, and proliferate, thereby restoring the tissue structure itself to its original state, in other words, tissue repair.

[Example 3]
The anti-inflammatory effect of equine MSC-derived EVs samples was confirmed.

The sample in Example 3 was a horse MSC-derived EVs sample, the sample in Comparative Example 3 was a human MSC-derived extracellular vesicle sample (hereinafter referred to as the human MSC-derived EVs sample), and the control sample was PBS(-).

An equine MSC-derived EV sample was prepared as follows. Specifically, the same equine-derived mesenchymal stem cells as in Example 1 were cultured, and the culture supernatant was collected from the culture at passage 7 by centrifugation (2000 x g, 10 minutes, room temperature). The culture supernatant was concentrated by tangential flow filtration (TFF, the membrane used had a molecular weight cutoff of 300 kDa), and the concentrate was further ultracentrifuged (35,000 rpm, 70 minutes, 4°C). The pellet was collected and suspended in D-PBS(-) (Nissui Pharmaceutical Co., Ltd., cat. #05913), and this was used as an equine MSC-derived EV sample. Unless otherwise specified, the medium and culture conditions were the same as in Example 1.

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

The human MSC-derived EV sample was prepared in the same manner as the equine MSC-derived EV sample, except that the culture supernatant of passage 5 human adipose-derived mesenchymal stem cells (LONZA, cat#PT-5006, lot#647217) was used.

Human peripheral blood mononuclear cells (PMBCs) were seeded into a 96-well plate at 1e+5 cells per well and cultured. The culture medium used was RPMI 1640 medium (ThermoFiosher) supplemented with 10% fetal bovine serum and 2 mM (mmol/L) L-glutamine. 24 hours after the start of culture, concanavalin A (Con-A) was added to the wells to stimulate the PMBCs and induce a cytokine storm (inflammation). Concanavalin A was added to a final concentration of 5 μg/mL per well.

24 hours after the addition of concanavalin A, inflammation-induced PMBCs were seeded onto a 96-well plate at 1e+5 cells per well, and each sample was added to the new medium in the wells and cultured. The samples from the examples and comparative examples were added so that there were 1,000 particles of extracellular vesicles per PMBC cell, and 100 μL of control saline was added per well. The supernatant was then collected after 48 hours of culture, and cytokines (IL-2, IL-5, IL-10, IL-17, IFNγ, TNFα) in the supernatant were quantified using a commercially available ELISA.

These results are shown in Figure 4, which is a graph showing the concentration of each cytokine in the supernatant. As shown in Figure 4, all cytokines were reduced in the system (n=3) to which the equine MSC-derived EVs sample (Equine MSC-EVs) of the example was added compared to the control (n=3), and were also reduced compared to the system (n=3) to which the human MSC-derived EVs sample (human MSC-EVs) of the comparative example was added.

[Example 4]
The angiogenic effect of equine MSC-derived EV samples was confirmed.

The EV sample in Example 4 was the equine MSC-derived EV sample from Example 3, and the EV sample in Comparative Example 4 was the human MSC-derived EV sample from Comparative Example 3.

EGM-2 medium was prepared by adding EGM-2 supplements (LONZA, cat# CC-4176) to EBM-2 (LONZA, cat# CC-3162). The supplements 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 supplements) at a volume ratio of 1:1. The EVs sample was then added to this mixed medium to prepare the EVs-containing medium of Example 4 and Comparative Example 4. The EVs sample was added so that the concentration of EVs per well (50 μL of medium) was 5.0e+7 particles/well. As a control, a control medium was used in which the mixed medium was supplemented with the same amount of PBS(-) as the EVs sample.

Frozen human umbilical vein endothelial cells (HUVECs) were thawed, culture medium replaced, and passaged before being suspended in each of the above media. The suspended HUVECs were seeded at 1.5e+4 cells/well onto a 96-well plate (Thermo, Nunc Microwell 96F) coated with Matrigel (Corning, cat#356231) and cultured at 37°C for 16 hours. Three wells were used for each of the above media (n=3). 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 branching points was counted using phase-contrast microscopic photographs. The average values for the systems using each medium were then compared among the control (EVs(-)), Example 4 (Equine MSC-EVs), and Comparative Example 4 (Human MSC-EVs).

These results are shown in Figures 5, 6, and 7. Figure 5 is a phase-contrast micrograph of HUVEC tubes. Figure 6 shows the relative values of HUVEC tube length, specifically, relative values with the control set at 1. Figure 7 is a graph showing the number of branch points of HUVEC tubes. As shown in these figures, when equine MSC-derived EV samples were used, the tube lengths were longer than both the control and human MSC-derived EV sample systems. Furthermore, when equine MSC-derived EV samples were used, the number of branch points was significantly higher than both the control and human MSC-derived EV sample systems. These results confirm that the equine MSC-derived EVs are capable of promoting angiogenesis.

As described above, it was confirmed that the equine MSC-derived EVs of the present invention exhibit the functions of tissue repair in Example 2, inflammation relief in Example 2, and angiogenesis promotion in Example 3. These functions enable wound treatment, demonstrating that wound treatment is possible using equine MSC-derived EVs.

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

(1) Preparation Method Equine mesenchymal stem cells (EqMSC-UC) isolated from horse umbilical cord (provided by Northern Farm) were used. The culture method and extracellular vesicle collection method were the same as those in Example 1, unless otherwise specified.

20 mL of the basal medium was added to each of 30 15 cm diameter culture dishes. The equine mesenchymal stem cells at passage 6 were seeded into the basal medium to a cell density of 3.5e+4 cells/mL and cultured at 37°C for 2 days. After confirming cell adhesion to the culture dish and removing the culture supernatant, the cells were washed with 10 mL of PBS(-) containing no Ca2 ⁺ or Mg2 ⁺ . 20 mL of extracellular vesicle recovery medium was added to each culture dish, and the culture was continued at 37°C for 48 hours. Phenol red-free, antibiotic-free DMEM (trade name: D-MEM (HIGH glucose) without L-Glutamin and Phenol Red, FujiFilm Wako) was used as the extracellular vesicle recovery medium.

The cultures in the seven culture dishes were combined and centrifuged (2000 x g, 10 minutes, room temperature) to collect the culture supernatant. The culture supernatant was then passed through a 0.22 μm pore size filtration tool (product name Stericup (registered trademark), Merck Millipore) to collect the filtrate. This filtrate was used as an equine mesenchymal stem cell-derived extracellular vesicle sample (hereinafter referred to as equine MSC-derived EVs sample (EqMSC-UC CM)). This sample was diluted 5-fold with PBS (-) and analyzed in the same manner as in Example 1 using a nanoparticle tracking system (product name Nanosight SN300, Japan Quantum Design Co., Ltd.) to confirm the particle size distribution of the extracellular vesicles (EVs).

The particle size distribution of the extracellular vesicles (EVs) in the equine MSC-derived EV sample is shown in the table below. Furthermore, when the equine MSC-derived EV sample was examined under an electron microscope, spheres with the particle sizes shown in the table below were confirmed.

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

The following three types of cells were used as target cells for evaluating proliferation.
Dermal Fibroblast (skin-derived fibroblasts): Product No. CC-2059, Lonza Inc. IHFPDC (hair papilla cells): Product No. T0501, abm Inc. NHEK (skin-derived keratinocytes): Product No. 00192907, Lonza Inc.

The target cells were individually seeded onto a 96-well plate at 1000 cells/50 μL/well and cultured overnight. The following media were used:
(Dermal Fibroblast)
DMEM (trade name: D-MEM (High Glucose) with L-Glutamin, Phenol Red, and Sodium Pyruvate, FujiFilm Wako) containing 10% fetal bovine serum and 1% antibiotic-antimycotic (trade name: Antibiotic-Antimycotic, GIBCO).
(IHFPDC)
PriGrow III (product number TM003, abm) containing 10% fetal bovine serum and 1% antibiotic-antimycotic
(NHK)
KGM Gold Keratinocyte Medium (product number 00192060, Lonza)

In the example, the diluted sample of EqMSC-UC CM was added at 50 μL/well, and the target cells were cultured. For each of the target cells, the concentrations (particles/well) of equine MSC-derived EVs in the wells were 2.5e+6, 4.10e+7, 8.20e+7, 1.6e+8, and 3.28e+8. As a control, instead of the diluted sample, phenol red-free DMEM alone was added at 50 μL/well, and the target cells were cultured. 72 hours after the addition of the diluted sample or the control sample, a reagent (product name Cell Counting Kit-8, Dojindo) was added at 10 μL/well, and the cells were incubated at 37°C for 4 hours. After the reaction, the absorbance at 450 nm was measured for each well. The absorbance of the control was set at 100%, and the relative absorbance of the example was calculated to give the cell viability (Viability (%)).

These results are shown in Figure 8. Figure 8A shows the cell viability of Dermal Fibroblasts (skin-derived fibroblasts), Figure 8B shows the cell viability of IHFPDCs (hair papilla cells), and Figure 8C shows the cell viability of NHEKs (skin-derived keratinocytes). As shown in Figures 8A, 8B, and 8C, it was confirmed that for all cells, the addition of the horse MSC-derived EVs sample increased cell viability in a concentration-dependent manner, i.e., promoted cell proliferation.

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 light, inflammation, etc., activating hair follicle cells, and activating melanocytes; and the proliferation of skin-derived keratinocytes is important, for example, from the perspective of contributing to the repair of the epidermal layer lost due to a wound. For this reason, as shown in Figure 8, the addition of the equine MSC-derived EVs sample promoted the proliferation of each cell type, demonstrating that the equine MSC-derived EVs are effective in wound treatment.

The present invention has been described above with reference to embodiments, but the present invention is not limited to the above embodiments. Various modifications that would be understandable to a person skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2024-12210, filed January 30, 2024, and Japanese Patent Application No. 2024-101298, filed June 24, 2024, the disclosures of which are incorporated herein in their entireties.

The medicinal composition of the present invention is capable of, for example, repairing tissue in the body, calming inflammation, and promoting angiogenesis, and these functions also make it possible to treat wounds.

Claims (12)

A medicinal composition characterized by containing extracellular vesicles derived from equine mesenchymal stem cells. The medicinal composition according to claim 1, which is a tissue repair agent. The medicinal composition according to claim 1 or 2, which is an anti-inflammatory agent. The medicinal composition described in any one of claims 1 to 3, which is an angiogenic agent. The medicinal composition described in any one of claims 1 to 4, which is a wound treatment agent. A biological treatment method comprising an administration step of administering the pharmaceutical composition described in any one of claims 1 to 5 to a living body. The biological treatment method according to claim 6, wherein the administration step is a step of administering the pharmaceutical composition to a living body as a tissue repair agent. The biological treatment method according to claim 6, wherein the administration step is a step of administering the pharmaceutical composition to a living body as an anti-inflammatory agent. The biological treatment method according to claim 6, wherein the administration step is a step of administering the pharmaceutical composition to a living body as an angiogenic agent. The biological treatment method according to any one of claims 6 to 9, wherein the administration step is a step of administering the pharmaceutical composition to a living body as a wound treatment agent. The biological treatment method according to any one of claims 6 to 10, wherein the living body is a human or a non-human animal. The biotreatment method according to claim 11, wherein the non-human animal is a horse.