Antibacterial agent

Abstract

To develop and provide an antibacterial agent exhibiting bacterial growth inhibition or bactericidal effect, and high safety for living organisms, in bacterial infections, particularly diabetic bacterial infections.SOLUTION: Mesenchymal stem cells are cultured under hypoxic conditions, and a culture solution or a culture supernatant thereof is utilized as an antibacterial agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antimicrobial agent and a method for efficiently producing an antimicrobial peptide. [Background technology]

[0002] Diabetes is a disease that causes chronic hyperglycemia due to insufficient insulin action. It is broadly divided into type 1 diabetes, which occurs when beta cells are destroyed due to genetic factors, and type 2 diabetes, which develops when insulin secretion from beta cells is insufficient due to environmental factors such as lifestyle.

[0003] As diabetes progresses, it can cause complications such as retinopathy, nephropathy, and neuropathy, accelerating the onset and progression of cardiovascular diseases such as stroke and ischemic heart disease. Furthermore, a weakened immune system increases susceptibility to bacterial infections. Among bacterial infections, foot ulcers are particularly problematic. Diabetic foot ulcers are caused by peripheral neuropathy, peripheral arterial disease, and trauma, and are estimated to affect approximately 25% of diabetic patients (Non-Patent Document 1). When a diabetic foot ulcer becomes infected and penetrates deep into the ulcer, it can develop into necrotizing fasciitis or osteomyelitis, worsening the condition and ultimately leading to lower limb amputation. Therefore, establishing a treatment for infected diabetic foot ulcers with appropriate infection control is extremely important. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Baltzis D, et al. Adv Ther. 2014 Aug;31(8):817-36. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to develop and provide an antibacterial agent that has a bacterial growth inhibitory or bactericidal effect and is highly safe for living organisms in the treatment of bacterial infections, particularly diabetic bacterial infections. [Means for solving the problem]

[0006] To solve the above problems, the present inventors focused on mesenchymal stem cells. It is known that the culture supernatants of mesenchymal stem cells derived from various tissues have antibacterial activity. It has been suggested that part of this activity is due to cathelicidin, an antibacterial peptide produced and secreted by mesenchymal stem cells (Krasnodembskaya A et al. Stem Cells. 2010 Dec;28(12):2229-38.).

[0007] As a result of extensive research, the present inventors have found that culturing mesenchymal stem cells under low oxygen concentrations significantly enhances the antibacterial activity of the culture supernatant compared to culturing under normal conditions, and that the production of cathelicidin contained in the culture supernatant is also significantly increased. Furthermore, in vitro efficacy evaluation tests have demonstrated that the culture supernatant has significant bacterial growth inhibitory activity, and furthermore, by exerting anti-inflammatory and angiogenic effects, it contributes to the healing of various diseases, particularly wound healing, in immunocompromised patients such as diabetic foot ulcers. The present invention is based on these new findings and provides the following inventions to solve the above-mentioned problems.

[0008] (1) An antibacterial agent consisting of a culture medium of mesenchymal stem cells cultured under a low oxygen concentration of 1 to 15%. (2) The antibacterial agent according to (1), wherein the incubation time under the low oxygen concentration is 12 to 60 hours. (3) The antibacterial agent according to (1) or (2), wherein the mesenchymal stem cells are derived from amniotic membrane, bone marrow, or adipose tissue. (4) The antibacterial agent according to any one of (1) to (3), wherein the culture medium is a culture supernatant. (5) An antibacterial composition containing the antibacterial agent according to (4) as an active ingredient. (6) A wound treatment agent comprising the antibacterial agent according to (4) or the antibacterial composition according to (5). (7) A method for producing an antimicrobial peptide, the method comprising a culture step of culturing mesenchymal stem cells under a low oxygen concentration of 1 to 15%. (8) The production method according to (7), further comprising a recovery step of recovering a culture supernatant from the culture medium after the culture step. (9) The production method according to (8), further comprising a separation step of separating the antimicrobial peptide from the culture supernatant after the recovery step. (10) The production method described in any one of (7) to (9), wherein the antimicrobial peptide includes cathelicidin. (11) The method according to any one of (7) to (10), wherein the mesenchymal stem cells are amniotic mesenchymal stem cells. [Effects of the Invention]

[0009] The antibacterial agent of the present invention can provide an antibacterial agent that is safe for living organisms and has a high bacterial growth inhibitory effect.

[0010] According to the method for producing antimicrobial peptides of the present invention, antimicrobial peptides can be produced efficiently and in large quantities by culturing mesenchymal stem cells under specific culture conditions. [Brief explanation of the drawings]

[0011] [Figure 1] 10 is a graph showing the relative percentage of the number of Staphylococcus aureus colonies on amniotic membrane MSC culture supernatant plates (SCM) at a normal oxygen concentration and on culture supernatant plates (HCM) at a low oxygen concentration, when the number of Staphylococcus aureus colonies on a control plate (CtM) seeded with Staphylococcus aureus is taken as 100%. [Figure 2] This is a graph showing the amount of cathelicidin LL-37 contained per mL in a control sample of medium only (CtM), culture supernatant of amniotic membrane MSCs at normal oxygen concentration (SCM), and culture supernatant of amniotic membrane MSCs at low oxygen concentration (HCM). [Figure 3A] FIG. 10 shows the concentration of cathelicidin LL-37 in the culture supernatant when amniotic membrane MSCs were cultured under oxygen concentrations of 1%, 5%, 10%, 15%, and 21%. [Figure 3B] FIG. 1 shows the concentration of cathelicidin LL-37 in the culture supernatant when bone marrow MSCs were cultured under oxygen concentrations of 1%, 5%, 10%, 15%, and 21%. [Figure 3C] FIG. 10 shows the concentration of cathelicidin LL-37 in the culture supernatant when adipose MSCs were cultured under oxygen concentrations of 1%, 5%, 10%, 15%, and 21%. [Figure 4] Figure 1A shows the reduction rate of wound area on day 10 after application of a control gel containing medium only (CtM), a culture supernatant gel (SCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 21%, and a culture supernatant gel (HCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 1% relative to the wound area on day 1. Figure 1B shows the condition of the wound on days 1 and 10 after application of the CtM gel, SCM gel, and HCM gel. [Figure 5] Figure 1A shows the number of Staphylococcus aureus in wounds on day 10 after application of a control gel (CtM) containing medium alone, a gel containing culture supernatant (SCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 21%, and a gel containing culture supernatant (HCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 1%. Figure 1B shows the number of Staphylococcus aureus colonies grown on CtM gel, SCM gel, and HCM gel plates. [Figure 6] Figure 1 shows the Gram-stained positive area rate (a), MPO-stained positive area rate (b), CD31-stained positive area rate (c), and Ki67-stained positive area rate (d) at wound sites on day 10 after application of a control gel containing medium only (CtM), a gel containing culture supernatant (SCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 21%, and a gel containing culture supernatant (HCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 1%. [Figure 7] FIG. 1 shows the degree of wound healing on day 10 after application of a control gel containing medium only (CtM), a culture supernatant gel (SCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 21%, and a culture supernatant gel (HCM) obtained by culturing amniotic membrane MSCs at an oxygen concentration of 1%. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Antibacterial agents Overview A first aspect of the present invention is an antibacterial agent. The present invention comprises a culture medium of mesenchymal stem cells cultured under specific conditions. The antibacterial agent of the present invention can provide an antibacterial agent with high bacterial growth inhibitory activity.

[0013] 1-2.Definition of Terms As used herein, the following terms are defined. As used herein, the term "antibacterial agent" refers to a drug that has a bactericidal or growth-inhibiting effect on bacteria. Antibacterial agents are primarily used for the prevention or treatment of bacterial infections.

[0014] As used herein, the term "bacterial infection" refers to various diseases caused by bacteria invading and multiplying in multicellular organisms such as animals or plants. The organisms to be infected are preferably, but not limited to, animals, particularly mammals. Furthermore, the type of bacterial infection in this specification may be any of gram-negative bacterial infection, gram-positive bacterial infection, or spirochete infection. Non-limiting examples of Gram-negative bacterial infections include Escherichia coli infection caused by Escherichia coli, cholera caused by Vibrio cholerae, Salmonella infection or typhoid fever caused by Salmonella bacteria, bacillary dysentery caused by Shigella bacteria, Pseudomonas infection caused by Pseudomonas bacteria, gonorrhea infection caused by Neisseria gonorrhoeae, Legionella infection caused by Legionella pneumophila, plague caused by Yersinia pestis, Campylobacter infection caused by Campylobacter bacteria, and meningococcal infection caused by Neisseria meningitidis. Gram-positive bacterial infections include, for example, enterococcal infections caused by Enterococcus bacteria, Staphylococcus aureus infections caused by Staphylococcus aureus, streptococcal infections caused by Streptococcus bacteria, pneumococcal infections caused by Streptococcus pneumoniae, nocardiosis caused by Nocardia bacteria, listeriosis caused by Listeria monocytogenes, erysipelothricosis caused by Erysipelothrix rhusiopathiae, and diphtheria caused by Corynebacterium diphtheriae.Spirochete infections include, for example, Lyme disease or relapsing fever caused by Borrelia bacteria, leptospirosis caused by Leptospira bacteria, and syphilis caused by Treponema pallidum.

[0015] The cells used in the present invention are mesenchymal stem cells. Mesenchymal stem cells (MSCs) are a type of adult stem cell that are present in bone marrow, adipose tissue, dental pulp, placental tissue (including amnion and chorion), fetal membrane, umbilical cord tissue, etc., but mesenchymal stem cells derived from any tissue can be used in the present invention.

[0016] "Adult stem cells" are stem cells present in various tissues of adults, which have not yet completed terminal differentiation and have a certain degree of pluripotency. They are also called somatic stem cells or tissue stem cells. Examples of such stem cells include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, and melanocyte stem cells.

[0017] The mesenchymal stem cells used herein are, but are not limited to, cells derived from animals, preferably mammals. Mammals here include, for example, rodents such as mice, rats, hamsters, and guinea pigs, livestock or pets such as dogs, cats, rabbits, cows, horses, sheep, and goats, and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Human-derived mesenchymal stem cells are particularly preferred as mesenchymal stem cells used herein.

[0018] As used herein, the term "medium" refers to a liquid or solid substance prepared for cell culture. This term typically refers to, but is not limited to, a basal medium that contains at least the minimum necessary components for cell growth and / or maintenance and is used to culture various types of cells. Examples of basal media include, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). In addition, a dedicated medium prepared for culturing specific cells by adding various culture additives to a basal medium can also be used. For example, commercially available Mesenchymal Stem Cell Growth Medium XF (Takara Bio Inc.) and MesenPRO RS are available. TM Medium (Thermo Fisher Scientific), STK (registered trademark) 2 (Kanto Chemical Co., Ltd.), etc.

[0019] As used herein, the term "culture additive" refers to a substance other than serum that is optionally added to a culture medium for the purposes of stabilizing culture, promoting growth, activating cells, etc. Specific examples of culture additives include, but are not limited to, human platelet lysate (hPL), L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, etc. Insulin, transferrin, and cytokines may be naturally derived substances isolated from tissues or serum of animals (preferably humans, mice, rats, cows, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering. Furthermore, examples of growth factors that can be used include, but are not limited to, basic fibroblast growth factor-2 (FGF2), transforming growth factor-β1 (TGF-β1), activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7. Examples of antibiotics that can be used include, but are not limited to, penicillin, streptomycin, and amphotericin B. One or more of the culture additives may be contained in the medium.

[0020] The medium used in the present invention may be a serum-free medium or a serum medium. In the case of a serum medium, the serum may be derived from any biological species, but is preferably derived from birds or mammals. For example, fetal bovine serum (FBS) is suitable. The preferred concentration of serum is 1-20%, 5-15%, 8-12%, preferably 10% in terms of final concentration. Furthermore, unless otherwise specified, the medium used in the present invention refers to a liquid medium used for culturing animal-derived cells.

[0021] As used herein, the term "culture medium" refers to a liquid medium after cell culture. The culture medium contains, in addition to medium components, cells after culture, culture waste products, and secretions (including proteins and low-molecular-weight compounds) released from the cultured cells.

[0022] As used herein, the term "cell supernatant" refers to the liquid component remaining after removing the main solid matter, such as cells, from the culture medium. However, the supernatant may contain solid matter with a major side or major axis of 1 μm or less, 500 nm or less, 200 nm or less, or 100 nm or less. The cell supernatant can be obtained by centrifuging the culture medium to remove precipitates or by filtering the culture medium.

[0023] As used herein, "cathelicidin" refers to a type of antimicrobial peptide consisting of approximately 10 to 50 amino acids and possessing antibacterial activity. Cathelicidin contains an N-terminal cathelin domain conserved among biological species and a C-terminal region that varies widely among species. It is typically expressed as a precursor protein lacking antibacterial activity, secreted extracellularly, and then activated by the action of local proteases, which degrade and remove the C-terminal region. Cathelicidin exerts its antibacterial activity by forming pores in the cell membranes of bacteria, such as Gram-positive or Gram-negative bacteria, and lysing the bacteria. Various types of cathelicidin are known depending on the biological species, but are not particularly limited herein. Examples include human cathelicidin LL-37, which consists of 37 amino acids beginning with leucine-leucine and is shown in SEQ ID NO: 1; the mouse homolog of LL-37 shown in SEQ ID NO: 2; the rat homolog of LL-37 shown in SEQ ID NO: 3; and porcine cathelicidin PR-39, which consists of 39 amino acids and is shown in SEQ ID NO: 4.

[0024] 1-3.Configuration The antibacterial agent of the present invention is composed of a culture medium of mesenchymal stem cells cultured under specific conditions, and therefore is composed of crude components containing a mixture of multiple components.

[0025] The culture conditions for mesenchymal stem cells required for preparing the antibacterial agent of the present invention are described below. In principle, the basic culture conditions may be those known in the art. For example, mesenchymal stem cells may be collected from amniotic membrane, umbilical cord, adipose tissue, bone marrow, dental pulp, etc. of an appropriate donor after obtaining informed consent, or commercially available cells from various manufacturers. The culture medium used is not particularly limited. It may be a basal medium used for culturing animal-derived cells, or a specialized medium prepared for mesenchymal stem cells. Furthermore, the CO2 concentration and culture temperature during culture may be conditions known in the art. Typically, culture is performed at 37°C ± 1°C under a CO2 concentration of 5%.

[0026] An important point in preparing the antibacterial agent of the present invention is culturing mesenchymal stem cells under specified conditions. The "specified conditions" are specific and necessary culture conditions for producing an antibacterial agent capable of achieving the effects of the present invention, and refer to a low oxygen concentration in the medium during mesenchymal stem cell culture. Specifically, the oxygen concentration is 1-15%, 1-10%, 1-8%, 1-7%, 1-6%, or 1-5%. Culturing mesenchymal stem cells under such low oxygen concentrations results in the production of antibacterial peptides within the mesenchymal stem cells, and the amount of antibacterial peptides secreted into the culture medium increases.

[0027] The culture time under low oxygen concentration is not limited, but may be 12 to 60 hours, 18 to 54 hours, or 24 to 48 hours.

[0028] The antibacterial agent of the present invention can be made from the culture medium obtained after the above-mentioned culture as is, but it is preferable to use the culture supernatant obtained by removing solid matter such as cells present in the culture medium by centrifugation or filtration using a membrane filter, etc., i.e., the culture supernatant obtained by culturing mesenchymal stem cells under conditions in which the oxygen concentration in the medium is 1 to 15%, as the active ingredient of the antibacterial agent.

[0029] The antibacterial agent obtained as a culture supernatant is a liquid, but can be converted into a solid if necessary. The method for converting from a liquid to a solid may be any treatment method known in the art. For example, a drying treatment may be used. The drying method is not particularly limited as long as it can reduce the water content in the culture supernatant. For example, vacuum drying, air drying, freeze-drying, heat drying, or a combination thereof may be used. Alternatively, the culture supernatant may be frozen by placing it below freezing.

[0030] In addition, the antibacterial agent of the present invention is preferably stored at temperatures below 10°C, below 8°C, below 6°C, below 4°C, or below 0°C until use to prevent inactivation of the antibacterial peptide, although this is not a limitation.

[0031] One of the antibacterial components included in the antibacterial agent of the present invention is the antibacterial peptide cathelicidin. The concentration of cathelicidin included in the antibacterial agent of the present invention may be, for example, 150 pg / mL or more, 200 pg / mL or more, 250 pg / mL or more, 300 pg / mL or more, 350 pg / mL or more, 400 pg / mL or more, 450 pg / mL or more, 500 pg / mL or more, 550 pg / mL or more, 600 pg / mL or more, 650 pg / mL or more, or 700 pg / mL or more in the culture medium (or culture supernatant). The higher the upper limit, the more desirable it is, and there is no particular limitation, but it may be, for example, 100 ng / mL or less, 80 ng / mL or less, 50 ng / mL or less, 30 ng / mL or less, or 20 ng / mL or less. However, the antibacterial component in the antibacterial agent of the present invention is not limited to cathelicidin, but also includes other antibacterial substances that may be contained in the culture medium after mesenchymal stem cells have been cultured under specified conditions.

[0032] 1-4.Effects The antibacterial agent of the present invention is highly safe for living organisms and has a strong inhibitory effect on bacterial growth. Furthermore, the antibacterial agent of the present invention has an anti-inflammatory effect by inhibiting neutrophil infiltration. Furthermore, the antibacterial agent of the present invention has the effect of promoting angiogenesis and cell division. Due to these effects, the antibacterial agent of the present invention can be used not only to treat wounds on the skin and mucosa, but also as a therapeutic agent for diseases where susceptibility to infection is a problem, such as skin ulcers, particularly diabetic foot ulcers.

[0033] 2. Antimicrobial peptide production method 2-1. Overview A second aspect of the present invention is a method for producing antimicrobial peptides. In this method, the expression and secretion of antimicrobial peptides can be enhanced by culturing mesenchymal stem cells under specific conditions, resulting in efficient production of antimicrobial peptides, particularly cathelicidin.

[0034] 2-2. Method The production method of the present invention includes a culture step as an essential step, and a preculture step, a recovery step, and a separation step as selection steps. Each step will be specifically described below.

[0035] (1) Preculture step The "pre-culture step" is a step of culturing mesenchymal stem cells under general conditions as needed for the purpose of proliferation, activation, etc., of the mesenchymal stem cells to be used in the culture step described below. The culture method and conditions are not particularly limited, as long as they are methods and conditions known in the art for culturing mesenchymal stem cells. For example, as described in the first embodiment, mesenchymal stem cells, preferably amniotic mesenchymal stem cells, collected from a donor or commercially available from various manufacturers may be cultured under conditions of 5% CO2 concentration and 37°C ± 1°C using a basal medium for animal-derived cell culture or a dedicated medium prepared for mesenchymal stem cells. The culture time is not particularly limited. The mesenchymal stem cells may be cultured until they reach confluence or until they reach the logarithmic phase, or until they reach the required state.

[0036] (2)Culture process The "culture step" is a step of culturing mesenchymal stem cells under specific conditions. This step is an essential step in the production method of the present invention, and this step causes the mesenchymal stem cells to express antimicrobial peptides and secrete them extracellularly.

[0037] The predetermined conditions in this step are culturing for a certain period of time under a low oxygen concentration as described in the first embodiment. Specific conditions are the same as those described in the first embodiment, and therefore will not be described here. This step results in the production of antimicrobial peptides in the mesenchymal stem cells and their secretion into the culture medium.

[0038] (3) Recovery process The "recovery step" is a step of recovering a culture supernatant from the culture medium after the culturing step. In this step, in order to obtain antimicrobial peptides, particularly cathelicidin, secreted into the culture medium by mesenchymal stem cells from the culture medium after the culturing step, the culture supernatant is recovered from which solid matter such as mesenchymal stem cells has been removed. The method for recovering the culture supernatant is not particularly limited as long as it can remove solid matter contained in the culture medium. For example, methods include separating the precipitate and supernatant by centrifugation and then recovering the supernatant, filtering the culture medium using a filter such as a membrane filter and recovering the filtrate as the culture supernatant, or a combination thereof.

[0039] The removal of solid matter in this step only requires that the main solid matter contained in the culture medium of cells, etc. is removed, and does not necessarily require complete removal of the solid matter. For example, the recovered culture supernatant may contain solid matter with a major side or major axis of 1 μm or less, 500 nm or less, 200 nm or less, or 100 nm or less.

[0040] (4) Separation process The "separation step" is a step of separating antimicrobial peptides derived from mesenchymal stem cells from the culture supernatant after the recovery step. The antimicrobial peptides separated here are not particularly limited as long as they have antimicrobial activity, but are preferably cathelicidin. The type of cathelicidin obtained in this step depends on the biological species from which the mesenchymal stem cells used in the culture step are derived. For example, if the mesenchymal stem cells cultured in the culture step are of human origin, the cathelicidin obtained in this step will be LL-37. Furthermore, if the mesenchymal stem cells cultured in the culture step are of porcine origin, the cathelicidin obtained in this step will be PR-39. The type of cathelicidin is not important as long as it has antimicrobial activity.

[0041] The antimicrobial peptides can be separated and purified from the culture supernatant after the recovery step based on known separation methods. Examples include, but are not limited to, treatment with denaturants such as urea or surfactants, sonication, enzymatic digestion, salting out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse-phase chromatography. Specific procedures for each method can be determined according to methods known in the art.

[0042] 3. Antibacterial composition Overview A third aspect of the present invention is an antibacterial composition. The antibacterial composition of the present invention is a composition containing the antibacterial agent according to the first aspect as an active ingredient. This composition makes it possible to provide the antibacterial agent according to the first aspect stably, and also as a pharmaceutical composition, in a form that is easier to administer, reducing the burden and invasiveness when administered to a living body.

[0043] 3-2.Configuration 3-2-1. Components The components of the antibacterial composition of the present invention will be described. The antibacterial composition of the present invention contains an active ingredient as an essential component, and a solvent and / or carrier as optional components. Each component will be specifically described below.

[0044] (1) Active ingredient The antibacterial composition of the present invention contains, as an active ingredient, the antibacterial agent described in the first aspect. Since the constitution of the antibacterial agent is described in detail in the first aspect, a specific description thereof will be omitted here.

[0045] The antibacterial composition of the present invention may further contain one or more other antibacterial agents known in the art, such as amoxicillin, clindamycin, metronidazole, and vancomycin, if necessary.

[0046] The content of the active ingredient contained in the antibacterial composition of the present invention is not particularly limited, so long as it is an amount necessary for the antibacterial agent described in the first embodiment to exert its antibacterial activity and causes little or no harmful side effects to the subject to which it is applied. The content of the active ingredient generally varies depending on the type of active ingredient, the dosage form, and the type of the solvent and carrier, which are other components described below. The antibacterial agent described in the first embodiment is a crude mixture composed of multiple components, and at least the component having antibacterial activity contained in the antibacterial agent, such as cathelicidin, is the substantial active ingredient of the antibacterial agent described in the first embodiment. Therefore, when cathelicidin is used as the active ingredient in the antibacterial composition of the present invention, the antibacterial agent of the first embodiment may be contained in an amount sufficient to allow cathelicidin to exert its antibacterial activity, for example, such that the final concentration of cathelicidin in the antibacterial composition is 150 pg / mL to 1000 pg / mL, 200 pg / mL to 800 pg / mL, 230 pg / mL to 600 pg / mL, or 250 pg / mL to 400 pg / mL.

[0047] (2) Solvent The composition may contain a pharmaceutically acceptable solvent. Examples of solvents include water and liquid media. However, since the antibacterial agent described in the first embodiment, which is an essential active ingredient in the antibacterial composition of the present invention, is a mesenchymal stem cell culture medium or culture supernatant, the solvent used herein may be any other pharmaceutically acceptable aqueous solution, pharmaceutically acceptable oil, or pharmaceutically acceptable organic solvent. Examples of such solvents include pharmaceutically acceptable aqueous solutions such as saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. Examples of pharmaceutically acceptable oils include wheat germ oil, apricot oil, olive oil, camellia oil, evening primrose oil, and aloe vera oil. Examples of pharmaceutically acceptable organic solvents include ethanol.

[0048] (3) Carrier As the pharmaceutically acceptable carrier, excipients, additives, adjuvants, and / or other active ingredients can be used depending on the dosage form and application.

[0049] Examples of excipients include emulsifiers, pH adjusters, fillers, flow additive adjusters, and lubricants. Examples of emulsifiers include surfactants (e.g., polyethylene glycol, poloxamer, glyceryl acetate, isostearamide, sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters). Examples of pH adjusters include alkaline agents such as sodium hydroxide and potassium hydroxide, and acid agents such as citric acid, sodium citrate, glycolic acid, and ascorbic acid. Examples of fillers include petrolatum, the aforementioned sugars, and calcium phosphate. Examples of flow additive adjusters and lubricants include silicates, talc, stearates, and polyethylene glycol.

[0050] Additives include, for example, organic acids, fatty acids, proteins and protein hydrolysates, amino acids and their salts, vitamins, and plant extracts, and when classified according to their functions, include, for example, moisturizers, ultraviolet absorbers, antioxidants, stabilizers, preservatives, disinfectants, and fragrances.

[0051] Other active ingredients that can be blended include, for example, collagen production promoters, hyaluronic acid production promoters, whitening agents, sunscreens, astringents, and / or other cell activators, within a range that does not impair the effects of the present invention.

[0052] 3-2-2. Dosage form The dosage form of the antibacterial composition of the present invention is not particularly limited as long as it does not inactivate the antibacterial agent according to the first aspect as an active ingredient and other active ingredients, and can exert the pharmacological effects of the active ingredients in vivo after administration. Furthermore, the specific dosage form of the composition may be appropriately selected depending on the administration method and / or prescription conditions. Generally, administration methods can be broadly divided into oral administration and parenteral administration, and the composition may be in a dosage form suitable for each administration method.

[0053] For oral administration, dosage forms include solid preparations (including tablets, pills, sublingual preparations, capsules, and drops), granules, powders, dispersing agents, and liquid preparations (including oral solutions, suspensions, emulsions, and syrups). If necessary, solid preparations can be made into dosage forms coated with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets.

[0054] Parenteral administration can be divided into systemic administration and local administration, and local administration can be further subdivided into intratissue administration, transepidermal administration, transmucosal administration, and rectal administration. The composition may also be prepared in a dosage form suitable for each administration method. For example, dosage forms suitable for systemic or intratissue administration include liquid injections. Dosage forms suitable for transepidermal or transmucosal administration include solutions (including aerosols, liniments, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), powders (including nasal drops and inhalants), pastes, gels, ointments, plasters, etc. Dosage forms suitable for rectal administration include suppositories, etc.

[0055] The specific dosage form of the antibacterial composition of the present invention varies depending on the type and content of the active ingredient and solvent, as well as the application method, and can be appropriately determined taking into account each of the conditions.

[0056] 3-3.Administration method The present composition is administered for the purpose of killing or inhibiting the growth of causative bacteria in bacterial infections and the like. Bacterial infections can occur in the skin, circulatory system, digestive system, respiratory system, reproductive system, cranial nervous system, etc., depending on the type of bacteria causing the infection. Therefore, the specific administration method is preferably a method that can deliver the composition to a site where a bacterial infection has occurred or may occur. For example, administration methods are broadly divided into parenteral administration and oral administration, as described above.

[0057] Oral administration generally refers to systemic administration and is effective as a method of administration for, but not limited to, bacterial infections of the digestive, respiratory, or reproductive tracts.

[0058] Parenteral administration can be further divided into local administration and systemic administration. Local administration is effective for, but not limited to, bacterial infections on the skin or as an administration method to the site of bacterial infection by injection. Systemic administration by injection is also effective for bacterial infections at any site by passing through the circulatory system, such as by intravenous injection.

[0059] Oral administration and parenteral administration via the circulatory system are preferred administration methods because they are less invasive and easier to administer. [Example]

[0060] Example 1: Antibacterial effect of mesenchymal stem cell culture supernatant (the purpose) We will confirm whether the culture supernatant of mesenchymal stem cells cultured under hypoxic conditions for a specified culture time has a high antibacterial effect.

[0061] (method) (1) Obtaining mesenchymal stem cells Amniotic membranes were collected from pregnant women undergoing elective cesarean section who had given informed consent, and amniotic membrane-derived mesenchymal stem cells (hereinafter referred to as "amniotic membrane MSCs") were isolated using a standard method involving enzyme treatment.

[0062] (2) Preculture The isolated amniotic membrane MSCs were cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) (Thermo Fisher Scientific) containing a final concentration of 5% human platelet lysate (hPL) at 37°C in 5% CO2 until they reached confluence.

[0063] (3) Main culture Upon reaching confluence, amniotic membrane MSCs were washed with PBS, seeded in αMEM without hPL or antibiotics, and cultured at 37°C in 5% CO2 under 21% normoxia and 1% hypoxia for 48 hours.

[0064] (4) Preparation of culture supernatant After the culture, the culture supernatant was passed through a 0.2 μm membrane filter (Thermo Fisher Scientific), and the filtrate was collected as the culture supernatant.

[0065] (5) Preparation of bacterial suspension Staphylococcus aureus (S. aureus) was cultured in tryptic soy liquid medium (Becton Dickinson) at 37°C for 24 hours, and the resulting bacterial culture was centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded, and the pellet was washed with PBS and then diluted with PBS to 100 CFU / μL to prepare a bacterial suspension.

[0066] (6) Antibacterial activity of culture supernatant Each culture supernatant collected in (4) above and αMEM were mixed with tryptic soy liquid medium at a 1:1 ratio. The bacterial suspension prepared in (5) above was added to these samples to a concentration of 1,000 CFU / mL. Each sample was then cultured for 2 hours at 37°C under 5% CO2 and 21% O2, after which 50 μL of the culture was plated on tryptic soy agar medium. After 24 hours of culture at 37°C under 5% CO2 and 21% O2, the number of colonies on the plate was counted.

[0067] (result) The results are shown in Figure 1. This figure is a graph showing the relative percentage of Staphylococcus aureus colonies in the culture supernatant plate where amnion MSCs were cultured at a normal oxygen concentration of 21% (SCM: Standard Condition Medium) and the culture supernatant plate where amnion MSCs were cultured at a hypoxic concentration of 1% (HCM: Hypoxia Condition Medium), when the number of Staphylococcus aureus colonies in the control plate (CtM: Control Medium) inoculated with Staphylococcus aureus in basal medium α-MEM was taken as 100%.

[0068] Both SCM and HCM were shown to significantly inhibit the growth of Staphylococcus aureus compared to CtM, suggesting that the culture supernatant of amniotic membrane MSCs has antibacterial activity. Furthermore, the inhibitory effect of HCM on the growth of Staphylococcus aureus was significantly greater than that of SCM. This indicates that culturing amniotic membrane MSCs under low oxygen concentrations enhances the antibacterial activity of the culture supernatant.

[0069] <Example 2: Detection of cathelicidin LL-37 in the culture supernatant of mesenchymal stem cells> (the purpose) We will confirm the presence of cathelicidin LL-37 in the culture supernatant of amniotic membrane MSCs and its increase by culture at low oxygen concentrations.

[0070] (method) Each culture supernatant was prepared in accordance with Example 1. The cathelicidin LL-37 in each of the obtained culture supernatants was measured using an LL-37 ELISA kit (HycultBiotech) according to the attached protocol.

[0071] (result) The results are shown in Figure 2, which is a graph showing the amount of cathelicidin LL-37 contained per mL of each culture supernatant. The culture supernatants of both SCM and HCM were found to contain the antimicrobial peptide cathelicidin LL-37. These results indicate that amnion MSCs produce and secrete cathelicidin LL-37 into the culture medium during culture. Furthermore, the cathelicidin LL-37 concentration in HCM was more than twice as high as that in SCM, indicating that culturing amnion MSCs under low oxygen concentrations enhances the production of cathelicidin LL-37.

[0072] Example 3: Changes in oxygen concentration and LL-37 concentration in culture supernatant during culture of mesenchymal stem cells derived from various tissues (the purpose) We will confirm that LL-37 is also contained in the culture supernatant of mesenchymal stem cells derived from tissues other than amniotic membrane, and that its concentration increases when cultured under low oxygen concentrations.

[0073] (method) (1) Obtaining bone marrow-derived and adipose tissue-derived mesenchymal stem cells Human bone marrow aspirate (Veritas) was mixed with αMEM containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) at a ratio of 1:3. This mixture was cultured in a 15 cm dish (Sumitomo Bakelite) at 37°C and 5% CO2 for 13 days to obtain bone marrow-derived mesenchymal stem cells (bone marrow MSCs). Adipose tissue-derived mesenchymal stem cells (adipose MSCs) were purchased from Lonza.

[0074] (2) Preparation of culture supernatant Culture supernatants were prepared from amniotic membrane MSCs, bone marrow MSCs, and adipose MSCs. The basic procedures were similar to those described in Example 1. The only difference from Example 1 was that the oxygen concentration during culture was changed to five different levels: 1%, 5%, 10%, 15%, and 21%.

[0075] (3) Measurement of cathelicidin LL-37 in culture supernatant The cathelicidin LL-37 in each culture supernatant was measured in the same manner as in Example 2 using an LL-37 ELISA kit (HycultBiotech) according to the attached protocol.

[0076] (result) The results are shown in Figures 3A to 3C. Figure 3A shows the concentrations of cathelicidin LL-37 in the culture supernatant of amniotic MSCs, Figure 3B shows the concentrations of cathelicidin LL-37 in the culture supernatant of bone marrow MSCs, and Figure 3C shows the concentrations of cathelicidin LL-37 in the culture supernatant of adipose MSCs. For amniotic MSCs, bone marrow MSCs, and adipose MSCs, the concentrations of cathelicidin LL-37 in the culture supernatant of cultured under hypoxic conditions of 1%, 5%, 10%, and 15% were higher than those in the culture supernatant of cultured under normoxia of 21%. This indicates that, regardless of the tissue of origin, culturing mesenchymal stem cells under hypoxic conditions enhances the production of cathelicidin LL-37 and enhances their antibacterial activity.

[0077] The concentration of cathelicidin LL-37 in the culture supernatant was highest at a low oxygen concentration of 1% for amniotic membrane MSCs and 10% for bone marrow MSCs and adipose MSCs.

[0078] Example 4: In vivo efficacy of antibacterial activity in mesenchymal stem cell culture supernatant (the purpose) The results of Example 1 demonstrated that the culture supernatant of mesenchymal stem cells has an inhibitory effect on the growth of Staphylococcus aureus. Therefore, we will confirm whether the culture supernatant also has a similar bacterial growth inhibitory effect in vivo by examining its therapeutic effect on Staphylococcus aureus-infected skin ulcers in diabetic mice.

[0079] (method) (1) Preparation of culture supernatant gel The culture supernatant was prepared according to Example 1. The culture supernatant prepared in this example was obtained by culturing amniotic membrane MSCs under a hypoxic condition of 1%. The resulting culture supernatant was mixed with carboxymethylcellulose (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 7% and gelled to obtain HCM gel. Furthermore, the culture supernatant obtained by culturing amniotic membrane MSCs under a normal oxygen condition of 21% was mixed with carboxymethylcellulose at a final concentration of 7% and gelled to obtain SCM gel. As a control, basal medium αMEM was mixed with carboxymethylcellulose at a final concentration of 7% and gelled to obtain CtM gel.

[0080] (2) Creation of diabetic mice For the diabetic animal model, 9-week-old male ICR mice (Sankyo Labo Services) were used. Diabetes was induced by intraperitoneal injection of 180 mg / kg of streptozotocin (Fujifilm Wako Pure Chemical Industries, Ltd.). After 4 weeks, blood glucose levels were confirmed to be 300 mg / dL or higher, and the mice were designated as diabetic.

[0081] (3) Development of a mouse model of diabetic ulcers infected with Staphylococcus aureus Two circular wounds, each 8 mm in diameter, were excised from the dorsal skin of the diabetic mice to create full-thickness skin defects. 50 μL of a bacterial suspension of Staphylococcus aureus prepared in the same manner as in Example 2 was dropped onto each wound to infect it, creating a mouse model of diabetic ulcers infected with Staphylococcus aureus.

[0082] (4) Evaluation of the in vivo antibacterial efficacy of the culture supernatant The HCM gel, SCM gel, and control CtM gel containing the culture supernatant prepared in (1) were applied to each wound in the mouse model every other day, and each wound was photographed. Ten days after application, the wound area and bacterial count were evaluated.

[0083] The wound area was calculated by taking a photograph of the wound with a single-lens reflex camera (Canon) and analyzing it with Fiji software (National Institutes of Health).

[0084] To assess bacterial counts in the wound, the ulcer was excised over the panniculus carnosus using a 3 mm derma punch and homogenized in 100 μL of PBS. A 10 μL aliquot of the suspension was plated on tryptic soy agar. The plates were incubated at 37°C under 21% O for 16–18 hours, and the bacterial count was assessed by colony count.

[0085] In addition, samples were taken from the wound area and subjected to Gram staining to stain bacteria, MPO staining to stain neutrophils, CD31 staining to indicate angiogenesis, and Ki67 staining to indicate cell division, and the histological findings were evaluated.

[0086] Furthermore, in accordance with the SPOT skin wound scoring system (van de Vyver M, et al. Stem Cells Dev. 2021 Dec 1;30(23):1141-1152.), the HE-stained and Masson's Trichrome-stained specimens of each specimen were scored for four items: re-epithelialization rate, granulation proliferation, dermal reconstruction, and scar elevation, and the total scores were compared.

[0087] (result) The results are shown in Figures 4, 5, 6, and 7. Figure 4 shows the reduction rate of wound area on day 10 of application of culture supernatant gel relative to the wound area on day 1 of application for CtM gel, SCM gel, and HCM gel. Figure 5 shows the bacterial counts on day 10 of application for CtM gel, SCM gel, and HCM gel. Figure 6 shows a graph of the percentage of the area of ​​the wound that showed positive staining relative to the wound area on day 10 of application of each gel to the wound, as well as histological findings regarding bacterial infiltration, neutrophil infiltration, angiogenesis, and cell division in the wound. Figure 7 shows the degree of wound healing.

[0088] Figure 4 shows that amnion MSC culture supernatant gel (HCM gel) cultured under 1% hypoxia significantly reduced wound area compared with amnion MSC culture supernatant gel (SCM gel) cultured under 21% normoxia and the control gel (CtM gel). Figures 5 and 6a also demonstrate that amnion MSC culture supernatant gel (HCM gel) cultured under 1% hypoxia has bactericidal and bacterial growth-inhibitory effects on wounds. Figure 6b further demonstrates that HCM gel inhibits neutrophil infiltration. Figures 6c and 6d demonstrate that HCM gel promotes angiogenesis and cell division. Finally, Figure 7 demonstrates that HCM gel significantly promotes wound healing.

[0089] These results indicate that the conditioned medium from amniotic membrane MSCs cultured under hypoxic conditions has potent antibacterial activity against bacteria in vivo and can promote wound healing in bacterially infected tissues. This effect is not observed in conditioned medium cultured under normal oxygen concentrations of 21%, indicating that it is enhanced only by culture under hypoxic concentrations of 15% or less.

Claims

1. An antibacterial agent consisting of a culture medium of mesenchymal stem cells cultured under a low oxygen concentration of 1-15%.

2. The antibacterial agent according to claim 1, wherein the incubation time under the low oxygen concentration is 12 to 60 hours.

3. The antibacterial agent according to claim 1 or 2, wherein the mesenchymal stem cells are derived from amniotic membrane, bone marrow, or adipose tissue.

4. The antibacterial agent according to any one of claims 1 to 3, wherein the culture medium is a culture supernatant.

5. An antibacterial composition comprising the antibacterial agent according to claim 4 as an active ingredient.

6. A method for producing an antimicrobial peptide, comprising a culture step of culturing mesenchymal stem cells under a low oxygen concentration of 1 to 15%.

7. The production method according to claim 6 , further comprising a recovery step of recovering a culture supernatant from the culture medium after the culture step.

8. The production method according to claim 7, further comprising a separation step of separating the antimicrobial peptide from the culture supernatant after the recovery step.

9. The method of any one of claims 6 to 8, wherein the antimicrobial peptide comprises cathelicidin.

10. The method according to any one of claims 6 to 9, wherein the mesenchymal stem cells are amniotic mesenchymal stem cells.

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