A composition for preventing or treating occlusive vascular disease or its complications, comprising mesenchymal stem cells and vascular endothelial progenitor cells as active ingredients

Abstract

The present invention relates to a composition for preventing or treating occlusive vascular diseases or complications thereof, which contains vascular endothelial precursor cells and mesenchymal stem cells as active ingredients. It has been confirmed that when vascular endothelial precursor cells and mesenchymal stem cells are treated in combination, angiogenic ability is significantly improved, and thus it is possible to provide a new cell therapeutic agent for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, and a method for producing the same.

Description

[Technical Field]

[0001] The present invention relates to a method for treating occlusive vascular disease, comprising administering to a patient a therapeutic agent containing mesenchymal stem cells and vascular endothelial progenitor cells as active ingredients. or a composition for preventing or treating complications thereof. [Background technology]

[0002] Endothelial progenitor cells (EPCs) were first identified in peripheral blood and are cells that promote angiogenesis.

[0003] New blood vessels are generated through the processes of angiogenesis, arteriogenesis, and vasculogenesis. Angiogenesis is associated with the proliferation and migration of endothelial cells that grow from pre-existing mature endothelial cells. Arteriogenesis is the remodeling of pre-existing arteriolar connections into collateral vessels. Vasculogenesis occurs through the differentiation of endothelial progenitor cells into mature endothelial cells. Circulating endothelial progenitor cells migrate to sites of vascular injury and participate in new blood vessel formation by either directly inserting into newly formed blood vessels or by secreting a variety of angiogenic and trophic factors.

[0004] Therefore, endothelial progenitor cells (EPCs) are attracting attention as a potential target for regenerative medicine and therapeutic purposes through revascularization. Accordingly, research is underway to enhance the function of EPCs. For example, research has been conducted on the production of recombinant EPCs through ex vivo gene modification, and techniques have been studied to enhance the pro-angiogenic capacity of EPCs using vascular endothelial growth factor (VEGF) or hypoxia-inducible factor-1α.

[0005] Meanwhile, there are two main types of cells currently used in cell therapy for occlusive vascular diseases: G-CSF-mobilized MNCs and BM (Bone Marrow)-EPCs, which can directly differentiate into blood vessels in an ischemic niche, and BM-MSCs (mesenchymal stem cells) and ADSCs (adipose-derived stem cells), which use the paracrine effect to indirectly help with angiogenesis by secreting large amounts of growth factors that contribute to blood vessel formation.

[0006] However, these cell therapy methods involve the administration of a single cell, which makes it difficult to differentiate and function into blood vessels with complex structures, and it is difficult to expect therapeutic effects through paracrine effects alone without direct angiogenesis. To solve these vascular structural and functional problems, the roles of not only endothelial cells but also pericytes, especially smooth muscle cells for the regeneration of arterioles, are important, and mesenchymal stem cells are known to act as pericytes and smooth muscle cells, contributing to the stability and function of the formed blood vessels.

[0007] Therefore, the present inventors have confirmed that when vascular endothelial cells, which can directly differentiate into vascular endothelial cells in the ischemic niche, and bone marrow-derived mesenchymal stem cells, which can function as pericytes and smooth muscle cells, are combined using a combined administration method instead of the existing single administration method, stable blood vessels are formed and blood flow is restored in lower limb arterial occlusive disease, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a cell therapeutic composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises vascular endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0009] Another object of the present invention is to provide a method for producing a cell therapeutic agent for preventing or treating occlusive vascular diseases or ulcers caused by such diseases.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0011] Another object of the present invention is to provide a method for preventing or treating occlusive vascular diseases or ulcers caused by such diseases. [Means for solving the problem]

[0012] To achieve the above-mentioned objectives, the present invention provides a cell therapeutic composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises vascular endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0013] The present invention further provides a method for producing a cell therapeutic agent for preventing or treating occlusive vascular diseases or ulcers caused by the diseases, the method comprising mixing endothelial progenitor cells and mesenchymal stem cells.

[0014] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0015] Finally, the present invention provides a method for preventing or treating occlusive vascular disease or ulcers caused thereby, comprising the step of administering the pharmaceutical composition to an individual. [Effects of the Invention]

[0016] The present invention has confirmed that when vascular endothelial progenitor cells and mesenchymal stem cells are treated in combination, angiogenesis ability is significantly improved, and thus has the effect of providing a new cell therapy agent for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, and a method for producing the same. [Brief explanation of the drawings]

[0017] [Figure 1] This is a photograph showing a simplified experimental method for blood flow analysis. [Figure 2] This is a photo that shows a simplified method of testing the distribution of DiR-EL-100 using a fluorescent image analyzer. [Figure 3] 10 shows photographs showing the results of observing the ability to form a vascular network depending on the change in cell ratio. [Figure 4] 1 shows photographs showing the results of observing the ability to form a vascular network depending on the culture period. [Figure 5] Photographs showing the results of an experiment to preserve the foot by restoring blood flow using combined stem cells in an animal model of occlusive vascular disease. [Figure 6] 10 shows photographs showing the results of observing the in vivo vascular network formation ability of combined stem cells. [Figure 7] 1 shows photographs and graphs showing the results of confirming whether or not combined stem cells are present in vivo and whether or not they remain. [Figure 8] 10 shows photographs showing the results of observing the distribution of combined stem cells in organs. [Figure 9] 10 shows photographs showing the results of tissue analysis after administration of combined stem cells. [Figure 10]FIG. 1 is a schematic diagram of an experiment evaluating the therapeutic efficacy of different doses of combined stem cells. [Figure 11] 10 shows photographs and graphs showing the results of observing changes in the external shape of surgical valves according to the administration dose of compound stem cells. [Figure 12] 10 shows photographs and graphs illustrating the results of blood flow analysis according to the administration dose of combined stem cells. [Figure 13] 10 shows photographs showing the results of observing the ability of combined stem cells to form a vascular network in ischemic tissue. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below by way of examples with reference to the accompanying drawings. However, the following examples are presented as examples of the present invention, and if it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, such detailed description may be omitted and the present invention is not limited thereby. The present invention is susceptible to various modifications and applications within the scope of the claims below and the scope of equivalents analyzed therefrom.

[0019] Furthermore, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator, or the practice of the field to which the present invention pertains. Therefore, the accuracy of the terminology should be determined based on the overall content of this specification. Throughout the specification, when a part "includes" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0020] Throughout this specification, "%" used to indicate the concentration of a particular substance is (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid, unless otherwise specified.

[0021] The "thrombus" of the present invention is a blood factor aggregate, primarily composed of platelets and fibrin aggregates, which is typically formed to prevent excessive bleeding from blood vessels. When platelets come into contact with the surface of the subendothelium, a reaction to form a thrombus and block the vascular system is initiated, a process called hemostasis, which is very important for preventing excessive bleeding from wound sites. While arterial thrombosis causes serious diseases due to local occlusion, venous thrombosis primarily causes distant occlusion or embolism.

[0022] In this context, "occlusion" is a term that encompasses a state in which a blood vessel is completely or partially blocked, resulting in narrowing of the blood vessel. The degree of occlusion can be determined based on the measured blood flow. That is, the degree of occlusion is classified as partial or complete occlusion, with partial occlusion meaning that blood flow is reduced to 50-60% of baseline blood flow, and complete occlusion meaning that blood flow is reduced to 90-100%.

[0023] Currently, there are two main types of cells used in cell therapy for occlusive vascular diseases: G-CSF-mobilized MNCs and BM (Bone Marrow)-EPCs, which can directly differentiate into blood vessels in an ischemic niche, and BM-MSCs (mesenchymal stem cells) and ADSCs (adipose-derived stem cells), which use the paracrine effect to indirectly help with angiogenesis by secreting large amounts of growth factors that contribute to blood vessel formation.

[0024] However, these cell therapy methods involve the administration of a single cell, which makes it difficult to differentiate and function into blood vessels with complex structures. Therefore, it is difficult to expect therapeutic effects solely through the paracrine effect without direct angiogenesis.

[0025] Therefore, the present inventors have confirmed that when vascular endothelial cells, which can directly differentiate into vascular endothelial cells from the ischemic niche, and bone marrow-derived mesenchymal stem cells, which can function as pericytes and smooth muscle cells, are combined using a combined administration method rather than the existing single administration method, stable blood vessels are formed and blood flow is restored in lower limb arterial occlusive disease, and thus completed the present invention.

[0026] Therefore, the present invention provides a cell therapeutic composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises vascular endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0027] The term "mesenchymal stem cells (MSCs)" used herein refers to multipotent stem cells that can differentiate into various mesodermal cells, including bone, cartilage, fat, and muscle cells. The mesenchymal stem cells may be derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, chorion, decidua, or placenta, and are preferably human bone marrow-derived mesenchymal stem cells (BM-MSCs). Mesenchymal stem cells have the ability to migrate directly to the site of injury and regenerate damaged tissues and cells. They are multipotent cells that can easily proliferate outside the body and differentiate into various cell types, making them useful targets in gene therapy and cell therapy.

[0028] In one embodiment of the present invention, the endothelial precursor cells and mesenchymal stem cells may be mixed in a ratio of 2:1, but the present invention is not limited thereto.

[0029] In one embodiment of the present invention, the endothelial progenitor cells and mesenchymal stem cells may be, but are not limited to, human bone marrow-derived mesenchymal stem cells.

[0030] In one embodiment of the present invention, the occlusive vascular disease may be a disease selected from the group consisting of cerebrovascular disease (CVD), cardiovascular disease, arteriovascular disease, coronary artery disease (CAD), and peripheral artery disease (PAD), and is preferably coronary artery disease (CAD) and peripheral artery disease (PAD). Specifically, the occlusive vascular diseases include stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, lacunar infarction, acute coronary syndrome, angina pectoris, aortic stenosis, myocardial infarction, bundle branch block, cerebral ischemia, acute ischemic arteriovascular event, thrombophlebitis, venous preembolism, deep vein thrombosis, pulmonary embolism, peripheral vascular disease, atherosclerosis, vasospasm, restenosis, vascular occlusion due to vasculitis, and the like.

[0031] The term "cerebrovascular disease (CVD)" as used herein refers to arteriosclerotic vascular diseases occurring in the blood vessels that supply oxygen-rich blood to the face and brain, and generally includes comorbid diseases occurring together with CAD and / or PAD (peripheral artery disease), as well as ischemic diseases or diseases that cause insufficient blood flow. For example, CVD includes, but is not limited to, ischemic cerebrovascular disease, acute cerebral infarction, stroke, ischemic stroke, hemorrhagic stroke, varicose veins, mild cognitive impairment (MCI), and transient ischemic attacks (TIA).

[0032] The term "cardiovascular disease" or "arteriovascular disease" as used herein is a general term used to classify numerous conditions affecting the heart, heart valves, blood, and the body's vasculature, including diseases affecting the heart or blood vessels, preferably including, but not limited to, metabolic syndrome, syndrome X, atherosclerosis, atherosclerosis, coronary artery disease, stable and unstable angina, stroke, aortic disease such as aortic stenosis or aortic aneurysm, cerebrovascular disease, peripheral vascular disease, or acute ischemic atherosclerotic events.

[0033] The term "coronary artery disease (CAD)" as used herein refers to an arteriosclerotic vascular disease in which the arteries supplying blood to the heart muscle (coronary arteries) become atherosclerotic, hardened by calcium deposits, and / or narrowed. CAD leads to reduced blood flow to the heart muscle, which in turn prevents the heart muscle from receiving an adequate amount of oxygen, ultimately leading to necrosis. CAD includes, but is not limited to, acute coronary syndrome, myocardial infarction (heart attack), angina pectoris (stable and unstable), or atherosclerosis and atherosclerosis occurring in the blood vessels supplying blood to the heart.

[0034] As used herein, the term "peripheral artery disease (PAD)" refers to diseases such as atherosclerosis and atherosclerosis that occur in sites other than the heart and brain, and includes comorbid diseases that commonly occur with CAD.

[0035] The term "ischaemic ulcer" as used in the present invention refers to a condition in which tissue is lost beyond the muscular lamina mucosa, and in the present invention refers to a venous ulcer or a stasis ulcer, which is an ulcer caused by peripheral vascular disease (PVD), an ischemic ulcer, which is an arterial ulcer, or a neuropathic ulcer, which is an ulcer that often appears in diabetic patients.

[0036] Furthermore, the vascular endothelial precursor cells may be located in vascular cells when administered to an individual.

[0037] Furthermore, the mesenchymal stem cells may be located in pericytes when administered to an individual.

[0038] The term "individual" also refers to a mammal, preferably a human, who has been the object of treatment, observation or experiment.

[0039] The term "pericytes" as used herein refers to vascular wall cells located within the basement membrane of the microvasculature, which form specific local contacts with the vascular endothelium. They are connective tissue cells surrounding small blood vessels, also known as Rouget cells, adventitial cells, or mural cells, and are known to surround 10% to 50% of the outer surface of the vascular endothelium. They are elongated contractile cells that surround precapillary arterioles outside the basement membrane.

[0040] Perivascular cells are relatively undifferentiated pluripotent cells that support blood vessels and can differentiate into fibroblasts, smooth muscle cells, or macrophages as needed. Perivascular cells also play an important role in angiogenesis and blood-brain barrier stability, and regulate blood flow in the microvasculature through their adhesive force to intravascular cells.

[0041] Therefore, the cell therapy agent according to the present invention can further enhance angiogenesis, vascular regeneration, and vascular barrier stability through combined administration of endothelial progenitor cells and mesenchymal stem cells.

[0042] The term "cellular therapeutic agent" as used in the present invention refers to a pharmaceutical product (as defined by the U.S. FDA) that is used for therapeutic, diagnostic, and preventive purposes using cells and tissues isolated from an individual, cultured, and specially processed, and that is produced through a series of actions such as expanding and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological properties of the cells in other ways.

[0043] The cell therapy composition may be administered via a parenteral route as long as it can reach the target tissue, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, topical, intranasal, intrapulmonary, and rectal administration.

[0044] The cellular therapeutic compositions of the present invention can be used in an unfrozen state or frozen for future use. If frozen, a standard cryopreservative (e.g., DMSO, glycerol, Epilife Cell Freezing Medium (Cascade Biologics)) can be added to the cell population before freezing.

[0045] The cell therapy composition can be formulated and administered into a unit-dose pharmaceutical preparation suitable for administration into a patient's body using conventional methods in the pharmaceutical field. The preparation contains an effective dose for single or multiple administrations. Suitable dosage forms for this purpose include parenteral administration preparations such as injections (e.g., injection ampoules), infusion bags, and sprays (e.g., aerosol preparations). The injection ampoules can be mixed with injection fluids immediately before use, such as saline, glucose, mannitol, and Ringer's solution. Infusion bags can be made of polyvinyl chloride or polyethylene, and examples of such bags include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, and Terumo.

[0046] The pharmaceutical preparation may further contain one or more pharmaceutically acceptable conventional inert carriers in addition to the active ingredient, such as a preservative, a soothing agent, a solubilizer, or a stabilizer in the case of an injection, or a base, an excipient, a lubricant, or a preservative in the case of a preparation for topical administration.

[0047] The cell therapy composition may further include a support, preferably a biodegradable support, for containing the cells, which may be a hydrogel such as, but not limited to, fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid.

[0048] The cell therapy agent may further include a pharmaceutically acceptable carrier, which may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostats may be added as needed.

[0049] The buffer may optionally be acetate, citrate, tartrate, lactate, succinate, or phosphate. The stabilizer may be mannitol, histidine, lysine, glycine, sucrose, fructose, trehalose, lactose, or a mixture thereof. The isotonicity agent may be glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, or sorbitol. The antioxidant may be acetone, sodium bisulfite, butylated hydroxyanisole, butylated hydroxytoluene, cysteine, cysteic acid HCl, sodium dithionite, gentisic acid, ethanolamine gentisate, monosodium glutamate, sodium formaldehyde sulfoxylate, potassium metabisulfite, sodium metabisulfite, monothioglycerol, propyl gallate, sodium sulfite, sodium thioglycolate, or ascorbic acid. The bulking agent may be mannitol, glycine, lactose, sucrose, trehalose, dextrin, hydroxyethyl starch, ficoll or gelatin.

[0050] The cell therapy composition or pharmaceutical preparation of the present invention prepared in this manner can be administered together with other stem cells used for transplantation and other purposes, or in the form of a mixture with such stem cells, using administration methods commonly used in the art. Preferably, the cell therapy composition or pharmaceutical preparation can be directly engrafted or transplanted into the diseased site of a patient in need of treatment, or directly transplanted or injected into the peritoneal cavity, but is not limited to this. The administration can be non-surgical administration using a catheter or surgical administration, such as injection or transplantation after incision at the diseased site, although non-surgical administration using a catheter is more preferred. In addition to parenteral administration using conventional methods, such as direct administration into the diseased site, transplantation by intravascular injection, a common method for hematopoietic stem cell transplantation, is also possible.

[0051] The daily dose of the mixture of endothelial progenitor cells and mesenchymal stem cells was 1.0 × 10 1 ~1.0×10 10 cells / kg body weight, preferably 1.0 x 10 5 ~1.0×10 9 cells / kg body weight, more preferably 1.2 x 10 5 ~1.2×10 7 The cells / kg can be administered in one or several doses. However, it should be understood that the actual effective dose must be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the above dose does not limit the scope of the present invention in any way.

[0052] The present invention further provides a method for producing a cell therapeutic agent for preventing or treating occlusive vascular diseases or ulcers caused by the diseases, the method comprising mixing endothelial progenitor cells and mesenchymal stem cells.

[0053] Since the method for producing the cell therapeutic agent of the present invention includes the above-mentioned cell therapeutic agent, the description of the same content as the above-mentioned cell therapeutic agent of the present invention will be omitted to avoid excessive complexity of this specification due to the description of the same content.

[0054] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating occlusive vascular diseases or ulcers caused by such diseases, which comprises endothelial progenitor cells and mesenchymal stem cells as active ingredients.

[0055] Since the pharmaceutical composition of the present invention contains the above-mentioned vascular endothelial progenitor cells and mesenchymal stem cells, the description of the same as that of the above-mentioned vascular endothelial progenitor cells and mesenchymal stem cells of the present invention will be omitted to avoid excessive complexity of this specification due to the description of the same.

[0056] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0057] The composition of the present invention may comprise a pharmaceutically effective amount of a mixture of endothelial progenitor cells and mesenchymal stem cells alone or may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount refers to an amount sufficient to prevent, ameliorate, and treat symptoms of immune disorders. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause gastrointestinal disorders, dizziness, or other allergic or similar reactions when administered to humans.

[0058] The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. The carriers, excipients, and diluents may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, saline, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, dextrin, calcium carbonate, propylene glycol, and liquid paraffin, but are not limited thereto. Any commonly used carriers, excipients, or diluents may be used. The ingredients can be added independently or in combination to the active ingredient mixture of endothelial progenitor cells and mesenchymal stem cells.

[0059] Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid formulations may be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin.

[0060] Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerol, gelatin, etc.

[0061] The pharmaceutical composition of the present invention may be in any one of the dosage forms selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, analogs, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of suppository bases include witepsol, macrogol, Tween 61, cocoa butter, lauric butter, glycerol, and gelatin.

[0062] The effective dosage of the mixture of endothelial progenitor cells and mesenchymal stem cells of the present invention to the human body varies depending on the patient's age, weight, sex, dosage form, health condition, and severity of disease, but is generally about 0.01-1000 mg / kg / day, preferably 0.01-350 mg / kg / day. For an adult patient weighing 60 kg, the dosage is generally 0.6-60,000 mg / day, preferably 0.6-2100 mg / day, and can be administered once or several times a day at regular intervals as determined by a doctor or pharmacist.

[0063] Finally, the present invention provides a method for preventing or treating occlusive vascular disease or ulcers caused by the same, which comprises administering the pharmaceutical composition to an individual.

[0064] In the present invention, the term "individual" refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation to humans, dogs, monkeys, cats, rodents such as mice, transgenic mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0065] The pharmaceutical compositions of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0066] In the present invention, the term "therapeutically effective amount" refers to the amount of a pharmaceutically acceptable salt of a composition that is effective for preventing or treating a target disease. The therapeutically effective amount of the composition of the present invention varies depending on various factors, such as the administration method, the target site, and the patient's condition. Therefore, the dosage for use in humans must be determined appropriately, taking into consideration both safety and efficacy. It is also possible to extrapolate the amount used in humans from the effective amount determined through animal experiments. Such considerations when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th ed. (2001), Pergamon Press; and E.W. Martin, ed., *Remington's Pharmaceutical Sciences*, 18th ed. (1990), Mack Publishing Co.

[0067] In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, without causing adverse effects. The effective dose level can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method, time, route and excretion rate of administration, the duration of treatment, other drugs used in combination or concomitantly, and other factors well known in the medical field. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect at the minimum dose without adverse effects, which can be easily determined by one skilled in the art.

[0068] The present invention will be described in more detail with reference to the following examples. These examples are intended to explain the present invention in more detail, but the scope of the present invention is not limited to these examples. [Example]

[0069] Experimental Materials and Methods.

[0070] Human bone marrow mononuclear cells (MNCs) were prepared and seeded with the appropriate cell sorting medium to isolate bone marrow-derived endothelial progenitor cells and mesenchymal stem cells. Human bone marrow mononuclear cells (MNCs) were purchased from Stemcell Technologies Inc. (Vancouver, Canada).

[0071] For EPC culture, isolated BM-MNCs were cultured in endothelial growth medium-2 (EGM-2, Lonza, Basel, Switzerland) on 100 mm dishes coated with type I collagen (CellMatrix, VA, USA). The medium was changed daily for up to 7 days, and then every 3 days thereafter. EPCs were subcultured at 70–80% confluency.

[0072] For MSC culture, BM-MNCs were cultured in mesenchymal stem cell growth medium (Stem MACS, Miltenyi) in 100 mm dishes. The medium was replaced every 3 days, and the cells were subcultured at 70–80% confluency.

[0073] Balb / c-nu mice were anesthetized with intraperitoneal injection of ketamin (100 mg / kg) and rompun (5 mg / kg). The mice were placed in the supine position and their legs were fixed with medical tape. The skin was incised from the ankle to the thigh to expose the common femoral artery. The common femoral artery was then ligated using a needle holder and 6-0 silk suture to prevent blood flow. The proximal portion of the superficial femoral artery was ligated with 6-0 silk. During ligation, the superficial femoral artery was separated from the nerve by micro-fine forceps, and the suture needle was carefully inserted before ligation. The distal portion of the superficial femoral artery was ligated with 6-0 silk.

[0074] The distal ligation site of the superficial femoral artery was removed using micro-fine forceps, carefully separated, and lifted, and the vessel was removed using surgical scissors. The incised skin was sutured with 5-0 silk, and blood flow was measured to confirm the presence of ischemia. The suture site was then disinfected with povidone.

[0075] The administration method was to use an insulin syringe (1 mL, 29G) to administer 10 μL per spot to five separate spots (yellow spots) at 2.0-2.5 mm intervals into the ischemic area (IR) muscle, near the removed superficial femoral artery, for a total of 50 μL administered once.

[0076] After drying, the slides were deparaffinized using Xylene, then passed through high-concentration alcohol, then low-concentration alcohol, and then rinsed with water. The tissue slides were immersed in a buffer solution of 0.01M citric acid (pH 6.0), heated in a microwave oven for 10 minutes, cooled thoroughly to room temperature, and then washed with PBS. To prevent tissue autofluorescence, the slides were immersed in 0.1% sodium borohydride (in PBS) at room temperature for 1 hour, protected from light, and then thoroughly washed with PBS. To allow antibody penetration, the slides were immersed in 0.1% Triton-X for 10 minutes and then washed. A blocking solution was made with 2% NHS and incubated at room temperature for 1 hour.

[0077] Next, the diluted primary antibody was applied. The sections were stored overnight at 4°C in the dark, taking care not to let them dry out. After thorough washing with PBS, the sections were treated with the secondary antibody at room temperature for 1 hour. WGA was diluted in 2% NHS blocking solution to a concentration of 5 μg / mL and treated at 37°C for 1 hour. DAPI was diluted in DW to a concentration of 10-20 μg / mL and treated at room temperature for 5 minutes.

[0078] After installing and operating the laser Doppler device (Fig. 1A), the abdominal area of ​​the mouse model was disinfected with an alcohol swab, and anesthesia was initiated by intraperitoneal injection of ketamine and rompun (Fig. 1B).The mouse model was then placed prone, and blood flow was measured using the laser Doppler device (Fig. 1C, D).

[0079] Matrigel (Corning Inc., NY, USA) was applied to μ-slides (Ibidi, Grafelfing, Germany) at 10 μl per well. EPC and MSC mixtures were prepared at various ratios and suspended in α-MEM (GIBCO, NY, USA) medium containing 0.2% FBS. After seeding on Matrigel, images were captured using a Leica microscope.

[0080] The distribution test of DiR-EL-100 using a fluorescent image analyzer (Maestro II, Whole Body Image) was carried out as follows.

[0081] The power supply for the fluorescent image analyzer (Maestro II) and the Maestro software on the computer connected to the instrument were turned on, and the instrument was allowed to initialize for approximately 1 minute (FIG. 2A).

[0082] After disinfecting the abdominal area with alcohol swabs, anesthesia was initiated by intraperitoneal injection of ketamine and rompun. After opening the instrument gate, the anesthetized mouse was placed in the supine position and the gate was closed (Figure 2B, C). After pressing the interior illumination button on the instrument, the mouse position and imaging area were set. After selecting the appropriate filter, the imaging size and exposure sensitivity were adjusted to obtain optimal conditions (Figure 2D).

[0083] After sacrifice, the animals were photographed using an in vivo fluorescent imaging analyzer (Maestro II) and then autopsy was performed. The abdominal cavity was opened, and the saphenous vein and posterior vena cava were severed to allow the animals to die by exothermic lethality. The major organs / tissues (femur muscle at the injection site, liver, spleen, brain, kidney, heart, lung, mesenteric lymph nodes, and femur muscle on the opposite side of the injection site) were removed and thoroughly washed with cold PBS. The removed organs / tissues were photographed using a fluorescent imaging analyzer (Maestro II).

[0084] All data are presented as mean ± standard deviation, and P values ​​< 0.05 were considered significant ( * p<0.05, ** p<0.01, *** p<0.001). [Example]

[0085] Analysis of defective network formation ability according to the proportion of combined stem cells and culture period.

[0086] To confirm the change in vascular network formation ability depending on the ratio of endothelial progenitor cells and mesenchymal stem cells and the culture period, endothelial progenitor cells and mesenchymal stem cells were mixed at ratios of 0:1, 1:0, 2:1, 1:2, 5:1, 10:1, and 100:1 to prepare composite stem cells, and experimental groups with the same cell number were treated to observe whether vascular networks were formed. The results are shown in Figures 3 and 4.

[0087] When we examined the ability to form blood vessel networks by cell ratio, we found that angiogenesis was observed in the EPC-only treatment group (1:0), but superior angiogenesis was observed in the combined stem cell treatment group, which mixed endothelial progenitor cells and mesenchymal stem cells. In particular, the combined stem cells mixed with endothelial progenitor cells and mesenchymal stem cells at a ratio of 2:1 exhibited significantly superior angiogenesis ability compared to the combined stem cells mixed at ratios of 1:2, 5:1, 10:1, and 100:1 (see Figure 3).

[0088] Furthermore, when examining the ability to form vascular networks according to the culture period, sophisticated cell rearrangement was observed within a short period of time in the combined stem cell treatment group, which mixed endothelial progenitor cells and mesenchymal stem cells, but such cell rearrangement was not observed in the combined stem cell treatment group, which mixed endothelial progenitor cells and fibroblasts (LF) (see Figure 4A).Furthermore, while the ability to form vascular networks decreases due to aging of endothelial progenitor cells, when endothelial progenitor cells and mesenchymal stem cells were mixed, it was confirmed that the ability to form vascular networks that was reduced due to aging of endothelial progenitor cells was restored (see Figure 4B).

[0089] These results indicate that mesenchymal stem cells significantly improve the blood vessel formation ability of endothelial progenitor cells. In subsequent experiments, a composite stem cell mixture of endothelial progenitor cells, which have the best vascular network formation ability, and mesenchymal stem cells was used in a 2:1 ratio. [Example]

[0090] Efficacy and angiogenic potential analysis of combined stem cells in obstructive peripheral arterial disease.

[0091] To confirm the efficacy and angiogenic potential of the combined stem cells in peripheral arterial obstruction disease, we examined whether the foot could be preserved by restoring blood flow in a non-clinical animal model of lower limb arterial obstruction disease and whether the combined stem cells could form angiogenesis in vivo. The results are shown in Figures 5 and 6.

[0092] In a non-clinical animal model of lower limb arterial occlusive disease, we confirmed whether foot preservation was possible through blood flow recovery. Foot preservation was not observed in the animal model to which the compound stem cells were not administered, but was observed in the animal model to which the compound stem cells were administered (see Figure 5). Furthermore, in vivo blood vessel formation by the compound stem cells was confirmed, and it was confirmed that endothelial progenitor cells and mesenchymal stem cells were present in the foot preserved by the administration of the compound stem cells (see Figure 6). [Example]

[0093] Observation of biodistribution of combined stem cells after transplantation.

[0094] To observe the biodistribution of combined stem cells after transplantation, a total of 1.2 × 10 combined stem cells (EPC + MSC) were used. 6 After staining with DiR at a dose of 1 / mouse, the cells were injected into the thigh muscle of balb / c-nu mice. The migration and survival of the cells in vivo were monitored using the NIR wavelength of a fluorescent imaging analyzer (Maestro II) until the fluorescent signal disappeared. After the fluorescent signal disappeared, the mice were dissected to check for the survival of the administered combined stem cells in the organs and tissues. The results are shown in Figures 7 and 8.

[0095] As shown in Figures 7 and 8, the fluorescence intensity was strongest 30 minutes after cell administration into the thigh muscle, remained at a high level for up to two weeks (14 days), and then gradually decreased until three weeks (21 days). Furthermore, from four weeks (28 days), the fluorescence intensity remained at a similar level for approximately 16 weeks (116 days), indicating that the administered cells had engrafted into the body. Furthermore, after 16 weeks (116 days), autopsies of mice administered with the combined stem cells revealed that fluorescence was detected only in the thigh muscle, where the cells were administered, and not in other organs or tissues. This indicates that the combined stem cells transplanted into the body were present only at the site of administration and either migrated to other tissues or organs or did not remain.

[0096] Next, histological analysis was performed to confirm the morphology of endothelial progenitor cells and mesenchymal stem cells in the body, and the results are shown in Figure 9. As shown in Figure 9, endothelial progenitor cells (EPCs) were located in vascular cells, and mesenchymal stem cells (MSCs) were located in pericytes. These results indicate that the transplanted cells were maintained in the ischemic area and directly participated in neovascularization within the host tissue. [Example]

[0097] Evaluation of therapeutic efficacy by dose of combined stem cells.

[0098] To evaluate the therapeutic efficacy of combined stem cells (EPC+MSC) at different doses, we conducted experiments to observe changes in the external shape of surgical valves and measure blood flow rates according to the cell dose (number of transplanted cells). The experiment was designed with one control group and four experimental groups using the cell doses shown in Table 1 below. Cells were transplanted in a range of doses, from high to low, while maintaining the same overall volume, and the results were observed for two weeks (see Figure 10). The results are shown in Figures 11 and 12.

[0099] [Table 1]

[0100] Observation of changes in the external shape of the surgical valve showed that the G1 group, which was the control group, and the G5 group, which received a relatively small number of transplanted cells, showed very high severity. In contrast, the G2 to G4 groups, which received transplanted composite stem cells, all showed significant therapeutic effects (see Figure 11). Based on these results, the effective range for the therapeutic effect of composite stem cells on occlusive vascular disease was G2 (1.2 x 10 6 )~G4(1.2×10 5 ) was confirmed.

[0101] The blood flow rate measurement results for the cell dose showed that the control group G1 showed a decrease in blood flow and tissue loss over time. In contrast, the experimental groups transplanted with combined stem cells showed a gradual recovery of blood flow, indicating tissue preservation (see Figure 12A). Furthermore, the blood perfusion ratio measurement results for the G2, G3, and G4 groups showed a gradual improvement in blood flow and tissue recovery over time. Based on these results, the minimal effective dose (MED) of combined stem cells was determined to be 1.2 x 10 5 It can be seen that... [Example]

[0102] Confirmation of angiogenesis in ischemic tissues by combined stem cells.

[0103] To confirm the relationship between combined stem cells (EPCs and MSCs) and angiogenesis and improved blood flow in ischemic tissue, tissue immunohistochemistry was performed on the animal models of Groups G3 and G4 in Example 5. WGA was performed to examine muscle and vascular structures, and the vascular markers CD31 and alpha-SMA were examined as proteins. The results are shown in Figure 13.

[0104] As shown in FIG. 13, it was confirmed that both G3 and G4 were able to form angiogenesis in an ischemic environment, and no difference in angiogenesis depending on the cell number could be observed.

Claims

[Claim 1] A method for producing a cell therapeutic agent for preventing or treating obstructive peripheral arterial disease or ulcers caused by such disease, comprising a step of mixing vascular endothelial progenitor cells and mesenchymal stem cells, the vascular endothelial progenitor cells and mesenchymal stem cells are derived from human bone marrow mononuclear cells, The endothelial progenitor cells are prepared by culturing isolated human bone marrow mononuclear cells in endothelial growth medium-2 (EGM-2) on a culture dish coated with type I collagen, The mesenchymal stem cells are obtained by culturing isolated human bone marrow mononuclear cells in a culture dish using a mesenchymal stem cell growth medium; The number of the endothelial progenitor cells was 8.0 × 10 ４ and The number of mesenchymal stem cells was 4.0 × 10 ４ and The endothelial progenitor cells and mesenchymal stem cells are mixed in a ratio of 2:1, the vascular endothelial progenitor cells are located in vascular cells when administered to an individual; The method for producing a cell therapeutic agent for preventing or treating peripheral arterial obstructive disease or ulcers caused by the same, wherein the mesenchymal stem cells are located in pericytes when administered to an individual.

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