Pharmaceutical composition and method for producing same

Abstract

The present invention provides a pharmaceutical composition having high tissue regeneration capacity and a method for producing the same. The present invention relates to a culture supernatant of umbilical cord blood stem cells, the culture supernatant containing exosomes at a concentration of 1.0×10 10 The present invention also relates to a method for producing a pharmaceutical composition comprising a culture supernatant, the culture supernatant being obtained by a method comprising the steps of culturing cord blood stem cells in an AOF medium that has been subjected to heavy metal removal treatment to obtain a culture solution, and obtaining a culture supernatant from the culture solution.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition comprising exosomes. [Background technology]

[0002] Exosomes are membrane vesicles consisting of lipid bilayers with diameters of about 30 to 100 nm secreted by various cells. Exosomes are produced in multivesicular endosomes and are released outside the cell by fusing the multivesicular endosomes with the cell membrane.

[0003] The lipid bilayer of exosomes has a phospholipid bilayer structure similar to that of the cell of origin. They are components of substances secreted by cells outside the cell and are known to play functional roles in cell-cell communication and cellular immune regulation.

[0004] Exosomes contain phospholipids, mRNA, miRNA, as well as various water-soluble proteins, exogenous proteins, and transmembrane protein components.

[0005] Such exosomes are excreted from all animal cells, including mast cells, lymphocytes, astrocytes, platelets, neurons, endothelial cells, and epithelial cells, and have been found in various body fluids, including blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid, and are also present in cell culture media.

[0006] In particular, exosomes secreted from stem cells are highly effective in regenerating blood vessels and skin, and suppressing various inflammations. It is believed that this is because exosomes gather in damaged cells, and the genes (mRNA, etc.) they encapsulate act to enable the targeted cells to recover on their own. mRNA is very fragile, and if released outside the cell as is, it will be broken down immediately, but exosomes encapsulate mRNA and release it outside the cell as a sack, allowing important information to be transmitted to other cells without being destroyed.

[0007] Biopharmaceuticals developed using cells, tissues, hormones, etc. derived from humans and other organisms have the advantage of being free of side effects and fundamentally solving the causes of diseases, and are being actively researched worldwide. Exosomes are also beginning to be used in the field of next-generation biopharmaceuticals.

[0008] For commercial use of exosomes, a large amount of high-quality exosomes is required, but currently, the amount of exosomes obtained from stem cells is extremely small, and there is insufficient development of substances that can increase the production of stem cell-derived exosomes.

[0009] Patent Document 1 states that 9 They disclose a culture medium containing a large amount of exosomes (number of exosomes / ml). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Special Publication No. 2023-520535 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a pharmaceutical composition having high tissue regeneration capacity and a method for producing the same. [Means for solving the problem]

[0012] The present inventors have found that the above problems can be solved by the present invention having the following aspects. Aspect 1 The culture supernatant contains exosomes at 1.0×10 10 A pharmaceutical composition comprising: Aspect 2 2. The pharmaceutical composition of embodiment 1, wherein the culture supernatant comprises an AOF medium. Aspect 3 3. The pharmaceutical composition according to aspect 2, wherein said AOF medium is substantially free of heavy metals. Aspect 4 2. The pharmaceutical composition of embodiment 1, further comprising an autologous conditioned serum. Aspect 5 The autologous adjusted serum is Incubating blood drawn from a patient using the pharmaceutical composition to enrich the blood for cytokines and / or growth factors; Separating serum from said cytokine and / or growth factor enriched blood. 5. The pharmaceutical composition according to embodiment 4, obtained by a method comprising the steps of: Aspect 6 The pharmaceutical composition according to aspect 5, wherein the step of enriching for cytokines and / or growth factors is carried out by contacting the blood with glass. Aspect 7 A method for producing a pharmaceutical composition comprising a culture supernatant, comprising: The culture supernatant is Cultivating umbilical cord blood stem cells using the AOF medium from which heavy metals have been removed to obtain a culture solution; Obtaining a culture supernatant from the culture medium; The method is obtained by a method comprising the steps of: Aspect 8 8. A method for producing a pharmaceutical composition according to embodiment 7, further comprising autologous conditioned serum, the method comprising: The autologous adjusted serum is Incubating blood drawn from a patient using the pharmaceutical composition to enrich the blood for cytokines and / or growth factors; Separating serum from said cytokine and / or growth factor enriched blood. The method is obtained by a method comprising the steps of: [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the skin condition of a patient before and after administration of the pharmaceutical composition of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The pharmaceutical composition of the present invention comprises a culture supernatant of umbilical cord blood stem cells, and the culture supernatant contains exosomes at a concentration of 1.0 × 10 10 This pharmaceutical composition contains a high concentration of exosomes and therefore has a high tissue regeneration ability.

[0015] <Culture supernatant> In this specification, the culture supernatant is a culture solution obtained by culturing cells, and usually does not substantially contain cells. In the pharmaceutical composition of the present invention, cord blood stem cells are used to obtain the culture supernatant. Cord blood stem cells can produce a high concentration of exosomes compared to adipose tissue-derived mesenchymal stromal cells, epidermis-derived epithelial cells, dental pulp-derived mesenchymal stem cells, etc.

[0016] In the pharmaceutical composition of the present invention, the culture supernatant contains exosomes at a concentration of about 1.0 × 10 10 pieces (approximately 10 billion pieces) / ml or more, approximately 3.0 × 10 10 pcs / ml or more, approximately 5.0×10 10 / ml or more, or approximately 8.0 x 10 10 / ml or more, or about 10 x 10 10 It can contain more than 30 × 10 10 pcs / ml or less, approximately 10×10 10 / ml or less, or about 7.0 x 10 10 It may contain less than 100 / ml.

[0017] The pharmaceutical composition of the present invention may contain growth factors at very high concentrations, for example, VEGF, HGF, and EGF may be contained at 0.5 ng / ml or more, 1.0 ng / ml or more, or 3.0 ng / ml or more, and 30 ng / ml or less, 10 ng / ml or less, or 8.0 ng / ml or less, respectively. These growth factors may be inherently contained in the obtained culture supernatant without being added to the pharmaceutical composition.

[0018] A known culture method can be used as a culture method for obtaining a culture supernatant, but it is preferable to use an AOF (Animal Origin Free) medium (ACF (Animal Component Free) medium) that does not contain animal-derived extracts and / or serum as a culture medium. Such media are commercially available, and an example of such a medium is StemSpan (trademark)-ACF (STEMCELL Technologies, Canada). It has been found that the use of such a medium not only allows the obtaining of an extremely high concentration of exosomes, but also reduces the risk of pathogen infection, etc.

[0019] More preferably, an AOF medium that is substantially free of heavy metals or an AOF medium that has been treated to remove heavy metals is used. Here, "heavy metals" refers to heavy metals having a density of about 4.0 g / cm 3 The above means the metal elements of groups 11 to 15 of the long periodic table. Specific examples include Se, Pb, Cr, Cd, Cu, Hg, Zn, Mn, Co, Ni, Mo, Ta, Sn, Bi, In, and As. The removal of heavy metals is not particularly limited, but examples include the use of a known heavy metal remover or adsorbent, filter treatment for removing heavy metals, and treatment by electrolytic deposition. It has been found that the use of such a medium not only allows the obtaining of an extremely high concentration of exosomes, but also eliminates toxicity.

[0020] The composition of the present invention may be a processed product obtained by removing water from the culture supernatant obtained as described above by freeze-drying, a processed product obtained by concentrating the culture supernatant under reduced pressure using an evaporator or the like, a processed product obtained by concentrating the culture supernatant using an ultrafiltration membrane or the like, a processed product obtained by separating the culture supernatant into solid and liquid using a filter, or an original solution of the culture supernatant before the above-mentioned treatment. In addition, for example, the supernatant obtained by culturing cells may be centrifuged (e.g., 1,000×g, 10 minutes), fractionated with ammonium sulfate (e.g., 65% saturated ammonium sulfate), the precipitate may be suspended in an appropriate buffer, dialyzed, and filtered with a syringe filter (e.g., 0.2 μm) to obtain a sterile culture supernatant. The collected culture supernatant may be used as it is, or may be frozen and stored and thawed when used. A pharmaceutical acceptable carrier may be added and dispensed into a sterile container in an easy-to-handle amount, such as 0.2 ml or 0.5 ml. Furthermore, to prevent the risk of infectious pathogens, the culture supernatant may be treated with a virus clearance filter or by gamma irradiation.

[0021] As described above, a freezing step of freezing the collected culture supernatant may be included after the step of obtaining the culture supernatant. To freeze the culture supernatant, for example, the culture supernatant may be cooled to a temperature of from -200°C to 0°C, or from -100°C to -5°C. The pharmaceutical composition containing the culture supernatant may be prepared by disrupting cells, centrifuging the cells, and filtering the resultant through a filter, or by freeze-drying the filtrate.

[0022] The composition of the present invention is preferably administered in a dosage unit of 0.1 mL to 3.0 mL, more preferably 0.2 mL to 2.0 mL, 0.3 mL to 1.5 mL, or 0.5 mL to 1.2 mL.

[0023] The composition of the present invention has a high regenerative effect on damaged cells and has a healing effect on various diseases. The composition of the present invention can also have a regenerative effect on periodontal tissue. Here, the periodontal tissue includes teeth and the tissues surrounding them, such as the crown part of the tooth, such as enamel, dentin, dental pulp, and gums, and the root part of the tooth, such as cementum, alveolar bone, blood vessels, and nerves. The composition of the present invention may be a composition for treating periodontal disease or alveolar pyorrhea.

[0024] <Self-adjusting serum> The present inventors have found that when the composition further contains an autologous regulating serum, a higher regenerative effect can be obtained. Here, the autologous regulating serum is a serum obtained by adjusting and separating blood drawn from the patient who uses the composition, and the cytokines and / or growth factors are much higher than the normal blood concentration. For example, the cytokines and / or growth factors contained in the autologous regulating serum are more than twice or three times, and sometimes even about five times, of the normal blood concentration. The autologous regulating serum is highly safe because it is made from blood drawn from the patient himself, and has a high concentration of cytokines and / or growth factors, so it has been found to be very useful for regenerating periodontal tissues, particularly alveolar bone.

[0025] The autologous adjusted serum can be produced by a method including, for example, a step of incubating blood drawn from a patient to enrich the blood with cytokines and / or growth factors, and a step of separating serum from the blood enriched with cytokines and / or growth factors. As such an autologous adjusted serum, the autologous adjusted serum described in the above-mentioned Patent Documents 1 and 2 can be used. Kits for producing autologous adjusted serum are also commercially available, and an example of such a product is Sanakin (trademark). Examples of cytokines include IL-1Ra, IL-4, IL-6, IL-10, TNF-α, etc. Examples of growth factors include epidermal growth factor (EGF) and fibroblast growth factor (FGF). In the autologous adjusted serum, at least one of these cytokines can be enriched 2-fold or more, 3-fold or more, or 5-fold or more compared to normal serum.

[0026] The step of warming blood drawn from a patient who uses a composition for periodontal tissues to enrich the cytokines and / or growth factors in the blood may be separated into a step of drawing blood from the patient and a step of warming the drawn blood to enrich the cytokines and / or growth factors in the blood. The step of drawing blood from a patient is a step of drawing blood from the patient who uses the composition for periodontal tissues, and for example, about 10 ml of blood can be drawn with a normal syringe.

[0027] In the step of warming the collected blood to enrich the cytokines and / or growth factors in the blood, the warming can be performed, for example, in the range of 30° C. to 45° C., 35° C. to 40° C., or 36° C. to 38° C. for 30 minutes or more, 1 hour or more, or 2 hours or more, and the warming time may be 1 day or less, half a day or less, or 5 hours or less. Furthermore, prior to this step, there may be a step of transferring the collected blood to a container.

[0028] When keeping blood warm, the blood is preferably brought into contact with a solid surface (particularly glass) such as glass or a polymer. In this way, the production of cytokines and / or growth factors can be promoted. For this reason, it is preferable to increase the surface area in contact with the blood by placing solid objects such as particles such as glass beads or fibers such as glass wool in the container in addition to the container for storing the blood.

[0029] The container for storing blood may be the syringe from which the blood was collected, or may be a container separate from the syringe. The material constituting the solid particles to be placed in the container may be a material separate from the material constituting the container body. For example, the container may be made of a polymer, and the solid objects may be, for example, glass beads. For example, the diameter of the particles may be in the range of 0.5 to 10 mm, or 1 to 5 mm, and according to a preferred embodiment, the solid objects contained in the syringe may be less than 70% or less than 50% by volume of the internal volume of the syringe. The container may have a volume of, for example, 10 to 100 ml.

[0030] In a particularly advantageous configuration of the invention, the container and the solid article may be made of glass, quartz glass, corundum, quartz, plastics such as polystyrene, polyethylene, polyvinyl chloride, polypropylene, etc., or may essentially contain these materials or mixtures thereof.

[0031] In particular, when the solid surface in contact with blood is glass, it has been found that the effect of producing cytokines and / or growth factors is very strong, and IL-6 receptor antibodies, which have a very strong effect on periodontal tissue regeneration, are particularly produced in large amounts. Therefore, it has been found that the autologous adjusted serum thus produced is very effective when used as a pharmaceutical composition for periodontal tissue.

[0032] The step of separating serum from blood enriched in cytokines and / or growth factors can be carried out by centrifugation. Centrifugation may be carried out at a normal speed used for obtaining serum, for example, at 500 to 2000 g for several minutes. For example, when 10 ml of blood is collected, about 3 to 5 ml of serum enriched in cytokines and / or growth factors can be collected after centrifugation. It is preferable to remove platelets and blood cell components from the collected serum using a cell filter so that they are not included.

[0033] <Hyaluronic Acid> The composition of the present invention can be combined with a composition containing hyaluronic acid to form a treatment kit, particularly when used for wound treatment or periodontal tissue regeneration.In particular, it is preferable that the composition of the present invention contains the above-mentioned culture supernatant, autologous adjusted serum, and hyaluronic acid.In this specification, hyaluronic acid includes all modifications of hyaluronic acid, hyaluronate, or hyaluronan, including various chain lengths and charge states, and various chemical modifications including crosslinking, and combinations of these modifications.

[0034] The above-mentioned pharmaceutical composition is very costly to produce and is expensive, but it may take a long time for the effect to appear, so it is expected that some patients will stop treatment before the effect appears, but by combining the composition of the present invention with hyaluronic acid, the effect can be felt early.The point that the effect can be felt early is very important for continuing treatment, and the kit containing the composition of the present invention has become a composition that is very satisfactory for patients.

[0035] Without being bound by theory, the use of hyaluronic acid can eliminate the black triangle caused by the thinning of the gums by swelling the hyaluronic acid injected part. Hyaluronic acid is absorbed over time, but it does not return to its original position after absorption. This is thought to be because administration of hyaluronic acid swells the gums, giving stretching stimulation to the gum cells, and enhancing the activity of fibroblasts, such as collagen synthesis ability and proliferation ability, to promote gum regeneration, but continuous administration is required to achieve this effect. On the other hand, the above-mentioned culture supernatant and optional self-adjusted serum do not have an immediate effect, but they can act on cells near the wound and promote tissue regeneration, and it is thought that these tissue regeneration abilities are effectively exerted around the tissue stretched by hyaluronic acid, resulting in a synergistic effect.

[0036] The term "hyaluronic acid" is a type of glycosaminoglycan that is mainly composed of glucuronic acid and N-acetylglucosamine.As mentioned above, "hyaluronic acid" includes all the variants of hyaluronic acid, hyaluronate or hyaluronan, including various chain lengths and charge states, and various chemical modifications, including cross-linking, and combinations of these variants.That is, this term also includes various hyaluronates of hyaluronic acid with various counterions, such as sodium hyaluronate. Also, various modifications of hyaluronic acid are encompassed by terms such as oxidation (e.g., oxidation of -CHOH to -CHO and / or -COOH); periodate oxidation of adjacent hydroxyl groups, optionally followed by reduction (e.g., reduction of -CHO to -CHOH), or coupling with amine to form imine and reduction to secondary amine; sulfation; deamidation, optionally followed by deamination or amide formation with new acid; esterification; crosslinking; substitution with various compounds, e.g., using crosslinking agents or carbodiimide-assisted coupling; coupling with different molecules, such as proteins, peptides and active pharmaceutical ingredients, to hyaluronic acid. Other examples of modifications are isourea, hydrazide, bromoane, monoepoxide and monosulfone coupling.

[0037] Hyaluronic acid can be obtained from a variety of sources, both animal and non-animal. Non-animal sources include yeast, and preferably bacteria. The molecular weight of a single hyaluronic acid molecule typically ranges from 0.1 to 10 MDa, although other molecular weights are possible.

[0038] In certain embodiments, the concentration of the hyaluronic acid is in the range of 1-100 mg / ml. In some embodiments, the concentration of the hyaluronic acid is in the range of 2-50 mg / ml. In certain embodiments, the concentration of the hyaluronic acid is in the range of 5-30 mg / ml, or in the range of 10-30 mg / ml. In certain embodiments, the hyaluronic acid is crosslinked. Crosslinked hyaluronic acid includes covalent crosslinks, physical enmeshing of hyaluronic acid chains, and crosslinks between hyaluronic acid chains that create a continuous network of hyaluronic acid molecules held together by various interactions such as electrostatic interactions, hydrogen bonds, and van der Waals forces.

[0039] Crosslinking of hyaluronic acid may be achieved by modification with a chemical crosslinking agent. The chemical crosslinking agent may be selected from the group consisting of, for example, divinylsulfone, multiepoxide and diepoxide. According to an embodiment, the chemical crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane. According to a preferred embodiment, the chemical crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0040] The crosslinked hyaluronic acid product is preferably biocompatible, which implies that no or only mild immune response is generated in the treated individual, i.e., no or only mild undesirable local or systemic effects occur in the treated individual.

[0041] The crosslinked hyaluronic acid product of the present invention may be a gel or hydrogel, i.e., it may be considered water insoluble, but when subjected to a liquid, typically an aqueous liquid, it is capable of substantially diluting the crosslinked system of hyaluronic acid molecules.

[0042] Gels are mostly liquid by weight, e.g., they can contain 90-99.9% water, and behave like solids due to a three-dimensional cross-linked hyaluronic acid network in the liquid. Due to their significant liquid content, gels are structurally flexible, similar to natural tissues, making them very useful as scaffolds in tissue engineering and for tissue augmentation.

[0043] As mentioned above, cross-linking of hyaluronic acid to form cross-linked hyaluronic acid gel can be achieved by modification with chemical cross-linkers such as BDDE (1,4-butanediol diglycidyl ether). Hyaluronic acid concentration and the degree of cross-linking affect the mechanical properties, such as the elastic modulus G' and stability of the gel.

[0044] Crosslinked hyaluronic acid gels are often characterized in terms of their "degree of modification". The degree of modification of hyaluronic acid gels generally ranges between 0.1 and 15 mol%. The degree of modification of hyaluronic acid gels is advantageously 2 mol% or less, such as 1.5 mol% or less, such as 1.25 mol% or less, such as in the range of 0.1-2 mol%, such as in the range of 0.2-1.5 mol%, such as in the range of 0.3 mol-1.25 mol% compared to more crosslinked hyaluronic acid gels. The degree of modification (mol%) indicates the amount of crosslinker(s) bound to HA relative to the total molar amount of repeating disaccharide units of HA, i.e. the molar amount of bound crosslinker(s). The degree of modification reflects the extent to which HA has been chemically modified by chemical crosslinkers. The reaction conditions for the crosslinking technique and suitable analytical techniques for determining the degree of modification are all well known to those skilled in the art who can easily adjust these and other related factors to provide suitable conditions to obtain a degree of modification in the range of 0.1-2% and to modify the obtained product characteristics regarding the degree of modification. BDDE (1,4-butanediol diglycidyl ether) crosslinked hyaluronic acid gel may be prepared, for example, according to the method described in Examples 1 and 2 of International Publication WO97 / 04012A.

[0045] In a preferred embodiment, the hyaluronic acid is present in the form of a crosslinked hyaluronic acid gel crosslinked by a chemical crosslinking agent, wherein the concentration of the hyaluronic acid is in the range of 10 to 30 mg / ml, and the degree of modification by the chemical crosslinking agent is in the range of 0.1 to 2 mol %.

[0046] The hyaluronic acid gel may also contain portions of hyaluronic acid that are not crosslinked, i.e., not bound to a three-dimensional crosslinked hyaluronic acid network, but preferably at least 50% by weight, preferably at least 60% by weight, more preferably at least 70% by weight, and most preferably at least 80% by weight of the hyaluronic acid forms part of a crosslinked hyaluronic acid network.

[0047] The type of hyaluronic acid used can vary depending on the area of ​​the periodontal tissue to which the composition of the present invention is applied. For example, when injecting into the gums, it is preferable to use hyaluronic acid gel with relatively small and soft particles, such as Restylane Vital Skin Boosters (registered trademark).

[0048] <others> The composition of the present invention and the kit containing the same may be prepared together with a pharma- ceutically acceptable carrier or vehicle. Examples of pharma- ceutically acceptable carriers or vehicles include pharma- ceutically acceptable substances such as stabilizers, solubilizers, suspending agents, buffers, isotonicity agents, antioxidants, or preservatives. Examples of isotonicity agents are sodium chloride and glucose. Examples of buffers are citrate, acetate, boric acid, and phosphate. As an aqueous medium for suspending cells, for example, an aqueous solution for injection or infusion in which the osmotic pressure and pH are adjusted to be close to those of blood and the salt concentration is adjusted may be appropriately used, and examples of the aqueous medium for suspending cells include, but are not limited to, Ringer's solution such as acetated Ringer's solution and glucose-added acetated Ringer's solution, other infusion solutions, physiological saline, or glucose solution. For example, when Ringer's solution for infusion is used, an acceptable amount of dimethyl sulfoxide (DMSO) or human serum albumin (HSA) may be added thereto. Examples of antioxidants are ascorbic acid, sodium bisulfite, and sodium pyrosulfite.

[0049] The present specification also discloses a method for treating a subject (a human or a non-human mammal) using a composition containing the above-mentioned culture supernatant and a kit containing the same. When treating a subject using the composition, the composition can be administered to the affected area by injection or the like, or can be administered by drip infusion.

[0050] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. EXAMPLES

[0051] Preparation of culture supernatant Cord blood stem cells were cultured in StemSpan™-ACF (STEMCELL Technologies, Canada) from which heavy metals had been removed using a heavy metal removal filter in a 5% CO2 incubator at 37°C for 1-4 days. After that, the culture solution was centrifuged for 5 minutes, and the supernatant was separated and filtered through a filter to remove cells, yielding a culture supernatant.

[0052] The exosome concentration in the culture supernatant was measured using a measuring device (NanoSight NS300), and was found to be approximately 5 × 10 10 The growth factors VEGF, HGF, and EGF were found to be contained at approximately 3.5 ng / ml, approximately 6.1 ng / ml, and approximately 5.9 ng / ml, respectively.

[0053] Preparation of autologous serum To prepare autologous adjusted serum for use in the composition of the present invention, 10 ml of blood was drawn from each patient using a syringe.

[0054] The collected blood was transferred from the syringe to a Sanakin (trademark) tube, the tube was inverted 3 to 4 times to mix, and then stored in a storage cabinet maintained at 37°C for 3 hours.

[0055] The Sanakin™ tube was then transferred to a centrifuge and centrifuged at 3000 rpm (1200 G) for 5 minutes, thereby sedimenting platelets and blood cell components suspended in the serum.

[0056] The serum in the supernatant separated by centrifugation was collected in an amount of approximately 3 to 5 ml using a Luer-lock syringe equipped with a Cathelan needle, and then any small amounts of platelets and blood cell components that may have been present were removed using a cell filtration filter to obtain autologous adjusted serum.

[0057] Preparation of the Composition The above culture supernatant was used as the composition of Example 1. The above autologous adjusted serum was used as the composition of Reference Example 1. Furthermore, a mixture of the above culture supernatant and autologous adjusted serum in a volume ratio of 1:1 was used as the composition of Example 2.

[0058] Pharmacological Effects of the Compositions of the Examples <Diabetes> 1 cc of the composition of Example 1 was diluted with an infusion solution and administered by infusion once a week for six weeks to two diabetic patients with HbA1c levels of 8.2 and 8.7, respectively. As a result, the HbA1c levels were reduced to 6.7 and 7.3, respectively.

[0059] <Liver disease> 1 cc of the composition of Example 1 was diluted with an infusion solution and administered by infusion once a week for 6 weeks to a patient whose γ-GTP level was 242. As a result, the γ-GTP level eventually decreased to 55.

[0060] <Lung disease> 1 cc of the composition of Example 1 was diluted with an infusion solution and administered once a week for six weeks to a patient who had a persistent cough due to the aftereffects of COVID-19 and who quickly became short of breath even after exercise. As a result, the patient's symptoms improved to the same level as before infection with COVID-19.

[0061] <Aftereffects of cerebral infarction> 1 cc of the composition of Example 1 was diluted with an infusion solution and administered once a week for 8 weeks to a patient who had become paralyzed on the right side of his body due to cerebral infarction and had difficulty walking due to uncontrollable hand tremors. As a result, his hand tremors improved to the extent that he was able to use a smartphone, and his walking speed recovered to the same level as a healthy individual.

[0062] <Rough skin> 1 cc of the composition of Example 1 was diluted with an infusion solution for drip infusion, and a patient with rough skin due to acne or the like was given a drip infusion once a week for six weeks. The results before and after are shown in Figure 1. After administration of the composition of Example 1, the skin became very clean, and it was clear that a skin-beautifying effect was achieved.

[0063] Furthermore, when 1 cc of the composition of Example 2 was diluted with an infusion solution and administered to a patient with rough skin, it was found to have a higher skin-beautifying effect than the composition of Example 1. The composition of Example 2 also had a higher skin-beautifying effect than the composition of Reference Example 1.

Claims

1. Exosomes at 1.0 x 10 １０ A method for producing a pharmaceutical composition comprising a culture supernatant containing 50 μg / ml or more of the above-mentioned The culture supernatant is Cultivating umbilical cord blood stem cells in an AOF medium that has been treated to remove heavy metals and does not contain an animal-derived extract or serum to obtain a culture solution; Obtaining a culture supernatant from the culture medium; (provided that in the step of obtaining the culture medium, lipopolysaccharide or lipoic acid is not added to the medium when the umbilical cord blood stem cells are cultured).

2. 10. A method for producing the pharmaceutical composition of claim 1, further comprising: The autologous adjusted serum is a step of incubating blood drawn from a patient using the pharmaceutical composition to enrich the blood for cytokines and / or growth factors; Separating serum from said cytokine and / or growth factor enriched blood. The method is obtained by a method comprising the steps of:

3. The method of claim 2 , wherein the step of enriching for cytokines and / or growth factors is carried out by contacting the blood with glass.

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