Agents for the prevention and / or treatment of organ fibrosis, and agents for inhibiting organ fibrosis.

Abstract

The present invention aims to provide a novel agent for the prevention and / or treatment of organ fibrosis. [Solution] The present invention is a preventive and / or therapeutic agent for organ fibrosis, comprising mesenchymal stem cell culture supernatant. The mesenchymal stem cell culture supernatant is preferably a culture supernatant obtained from the culture of mesenchymal stem cells derived from adipose tissue, umbilical cord tissue, or bone marrow tissue. Furthermore, the organ is preferably the kidney.

Description

Technical Field

[0001] The present invention relates to a preventive and / or therapeutic agent for organ fibrosis and an organ fibrosis inhibitor.

Background Art

[0002] Organ fibrosis is a phenomenon caused by abnormal accumulation of extracellular matrix, and it is known that chronic tissue damage occurs as this fibrosis progresses. Although tissue damage due to fibrosis has been reported in various organs, effective treatment methods have not yet been established. In addition, organ fibrosis is also a problem in animals other than humans.

[0003] Mesenchymal stem cells are pluripotent progenitor cells first isolated from bone marrow by Friedenstein (1982) (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases (see Patent Documents 1 to 2). Recently, it has been known that cells having the same functions as mesenchymal stem cells exist in stromal cells such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

[0004] It is known that mesenchymal stem cells and their culture supernatants have a therapeutic effect on kidney diseases (Patent Document 3, Non-Patent Documents 2 to 4). However, it has not been confirmed that the culture supernatant of mesenchymal stem cells is effective in suppressing fibrosis of various organs including the kidney.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] [Non-Patent Document 1] Pittenger FM et al., Science 284, pp.143-147,1999 [Non-Patent Document 2] Arianne van Koppen et al., PLos ONE vol. 7, Issue 6, e38746,2012 [Non-Patent Document 3] E. Xiang et al., Stem Cell Research & Therapy 11, 336,2020 [Non-Patent Document 4] Kanna Nagaish et al., Scientific Reports 6, 34842,DOI:10.1038 / srep34842,2016 [Overview of the project] [Problems that the invention aims to solve]

[0007] In the circumstances described above, the present invention aims to provide a novel agent for the prevention and / or treatment of organ fibrosis. [Means for solving the problem]

[0008] As a result of diligent research to solve the aforementioned problems, the inventors have discovered that the culture supernatant of mesenchymal stem (stromal) cells (MSCs) has the effect of suppressing organ fibrosis, and have completed the present invention. In other words, the gist of the present invention is as follows.

[0009] [1] A prophylactic and / or therapeutic agent for organ fibrosis, comprising mesenchymal stem cell culture supernatant. [2] The prophylactic and / or therapeutic agent according to [1], wherein the mesenchymal stem cell culture supernatant is a culture supernatant obtained from the culture of adipose tissue-derived, umbilical cord tissue-derived, or bone marrow-derived mesenchymal stem cells. [3] The prophylactic and / or therapeutic agent according to [1] or [2], wherein the organ is the kidney. [4] The therapeutic agent according to [1] or [2], characterized by being administered after the onset of fibrosis. [5] The prophylactic and / or therapeutic agent according to [1] or [2], characterized by being administered multiple times. [6] An organ fibrosis inhibitor containing mesenchymal stem cell culture supernatant. [Effects of the Invention]

[0010] According to the present invention, a novel agent for the prevention and / or treatment of organ fibrosis can be provided. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results of examining the effect of administering MSC culture supernatant to renal ischemia-reperfusion rats on blood urea nitrogen (BUN) levels (7-day rearing experiment). [Figure 2] Figure 2 shows the effect of administering MSC culture supernatant to renal ischemia-reperfused rats on the expression of inflammatory marker genes (F4 / 80) in the renal cortex (7-day rearing experiment). [Figure 3] Figure 3 shows the effect of administering MSC culture supernatant to renal ischemia-reperfused rats on the expression of fibrosis marker genes (Tgfβ1, αSMA) in the renal cortex (7-day rearing experiment). [Figure 4] Figure 4 shows the results of examining the effects of administering MSC culture supernatant to renal ischemia-reperfusion rats on body weight, food intake, and water intake (3-day rearing experiment). [Figure 5] Figure 5 shows the effect of administering MSC culture supernatant to renal ischemia-reperfused rats on the expression of inflammatory marker genes (F4 / 80, IL-1β) in the renal cortex (3-day rearing experiment). [Figure 6]Figure 6 shows the effect of administration of MSC culture supernatant on renal fibrosis marker gene expression (Tgfβ1, Fn, αSMA, Col1a1, MMP2) in the renal cortex of rats with kidney deficiency and reperfusion (3-day breeding experiment). [Figure 7] Figure 7 shows the results of gene expression analysis of Tgfβ in the renal medulla. [Figure 8-1] Figure 8-1 shows the results of Western blot showing the protein expression of TGF-β and p-SMAD2 / 3 in the renal cortex. [Figure 8-2] Figure 8-2 is a graph obtained by quantifying and graphing the results of the Western blot in Figure 8-1. [Figure 9] Figure 9 is a hematoxylin and eosin (HE) stained image of tissue sections of the kidneys of the MSC group and the Control group. [Figure 10-1] Figure 10-1 is a Sirius red stained image of tissue sections of the kidneys of the MSC group and the Control group. [Figure 10-2] Figure 10-2 is a graph of the Sirius red positive area in the Sirius red staining of Figure 10-1.

Modes for Carrying Out the Invention

[0012] Hereinafter, the novel prophylactic and / or therapeutic agent for organ fibrosis of the present invention will be described in detail.

[0013] <Prophylactic and / or Therapeutic Agent for Organ Fibrosis> The prophylactic and / or therapeutic agent for organ fibrosis of the present invention is characterized by containing a mesenchymal stem cell culture supernatant. By containing a specific mesenchymal stem cell culture supernatant, the prophylactic and / or therapeutic agent for organ fibrosis of the present invention exhibits an excellent organ fibrosis inhibitory effect.

[0014] (Mesenchymal Stem Cell Culture Supernatant) In this invention, mesenchymal stem cell culture supernatant refers to the culture supernatant obtained when mesenchymal stem cells are cultured. It is the liquid obtained by separating the culture medium, which was in direct or indirect contact with the cells via a semipermeable membrane or the like, from the cells after culturing the mesenchymal stem cells for a certain period of time. The culture supernatant is sometimes also called a culture medium conditioned or a conditioned medium.

[0015] Mesenchymal stem cell culture supernatant contains exosomes. Exosomes are cellular vesicles, approximately 50 nm to 150 nm in diameter, that exist extracellularly and are abundant in all bodily fluids such as blood, saliva, and urine. Exosomes contain cell membrane-derived lipids and proteins on their surface, and nucleic acids such as mRNA and miRNA, as well as proteins, inside, containing information originating from the released cell. Furthermore, it is known that these bioinformation molecules, consisting of nucleic acids and proteins, function in cells that take up exosomes, and it has become clear that intercellular communication occurs through the exchange of nucleic acids and proteins via exosomes. Therefore, exosomes are thought to be useful not only for diagnosis but also for the prevention and treatment of diseases. The mesenchymal stem cell culture supernatant in this invention may contain exosomes as the active component. That is, exosomes extracted from the mesenchymal stem cell culture supernatant in this invention can also exert an inhibitory effect on organ fibrosis.

[0016] In this invention, the diameter or particle size used to determine the size of extracellular vesicles and exosomes refers to the maximum dimension of the extracellular vesicle, since these are not necessarily spherical. The diameter or particle size may be measured using conventional techniques for measuring nanoparticle size, such as microscopy (e.g., transmission electron microscopy) or light scattering techniques. Furthermore, it can also be measured using nanoparticle tracking analysis (NTA), which is based on the analysis of both light scattering and Brownian motion. In addition, the diameter or particle size of extracellular vesicles and exosomes can be measured using NanoSight (LM10, Quantum Design) for extracellular vesicles with a particle size of 50 nm or more, and using a transmission electron microscope for extracellular vesicles with a particle size of less than 50 nm. Furthermore, for measuring the number of exosomes, NanoSight (LM10, Quantum Design), an exosome measurement system (ExoCounter, JVC Kenwood) that uses a sandwich detection method with antibodies on disks and nanobeads to specifically detect surface antigens, which are exosome markers, can be used.

[0017] The method for measuring the exosome content in the culture supernatant is not particularly limited, but a method for measuring the content of extracellular vesicles that are CD9 / CD63 positive is exemplified. CD9 / CD63 are tetraspanin family members present on the surface of exosomes and are exosome surface markers. Here, "CD9 / CD63" refers to CD9 and CD63.

[0018] The mesenchymal stem cell culture supernatant of the present invention can be the liquid obtained after separation from the cells, but it may also be a supernatant that has undergone some kind of treatment. The treatment of the mesenchymal stem cell culture supernatant is not particularly limited and can include purification, concentration, drying, and powdering. In the present invention, the mesenchymal stem cell culture supernatant may be a liquid, but if necessary, it may be concentrated, semi-solid, solid, or powdered by drying treatment such as vacuum drying, freeze-drying, spray drying, evaporation, high-pressure method, or ultra-high-pressure method to reduce or remove the liquid content.

[0019] In this invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to the mesenchyme (such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and that can proliferate while maintaining that ability. In this invention, the term mesenchymal stem cells refers to the same cells as stromal cells, and there is no particular distinction between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Mesenchymal stem cells in the present invention include those derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Among these, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and adipose tissue-derived mesenchymal stem cells are even more preferred.

[0020] The mesenchymal stem cells in this invention may be of the same species as the subject (test subject) treated with the organ fibrosis prevention and / or treatment agent of this invention, or they may be of a different species. Examples of mesenchymal stem cell species in this invention include human, horse, cattle, sheep, pig, dog, cat, rabbit, mouse, and rat.

[0021] Mesenchymal stem cells may be cells provided by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or cells prepared by methods well known to those skilled in the art. Furthermore, mesenchymal stem cells may be primary cells isolated from donor tissue or established cell lines.

[0022] The mesenchymal stem cells in the present invention may be cells that have been cryopreserved and thawed repeatedly as appropriate. In the present invention, cryopreservation can be performed by suspending mesenchymal stem cells in a cryopreservation solution well known to those skilled in the art and then cooling them. Suspension can be performed by detaching the cells with a detaching agent such as trypsin, transferring them to a cryopreservation container, treating them as appropriate, and then adding the cryopreservation solution.

[0023] In the present invention, the culture medium used for culturing mesenchymal stem cells is not particularly limited as long as it is a medium that can culture mesenchymal stem cells while maintaining their good condition. However, it is preferable that the medium is one that can proliferate human mesenchymal stem cells while maintaining their ability to differentiate into osteocytes, chondrocytes, and adipocytes.

[0024] The culture medium used in the present invention can be prepared by adding serum to a basal culture medium and / or by adding components other than serum.

[0025] Examples of the aforementioned basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media of these.

[0026] Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, and rat serum. When serum is used, 5 v / v% to 15 v / v%, preferably 10 v / v%, may be added to the basal medium.

[0027] In addition to the serum, one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and thioglycerol may be added to prepare the serum. Furthermore, if necessary, substances such as lipids, amino acids, proteins, sugars such as glucose, polysaccharides, vitamins such as pantothenic acid, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts such as sodium chloride and magnesium sulfate may be added.

[0028] Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of the lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of the amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of the proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharides include glycosaminoglycans, and among glycosaminoglycans, examples include, but are not limited to, hyaluronic acid and heparan sulfate. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF).

[0029] From the viewpoint of use in the treatment of humans or animals, the culture medium used for culturing mesenchymal stem cells in the present invention is preferably a xeno-free medium that does not contain heterologous components such as serum. Examples of such culture media include Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use), PromoCell, Mesenchymal Stem Cell Growth Medium XF (Ready-to-use), PromoCell, MSCGM BulletKittm, MSCGMtm Mesenchymal Stem Cell Growth Medium BulletKittm (Lonza), Xeno-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem® XF, Biological Industries), MesenCult-ACF Plus (Veritas), StemXVivotm Serum-Free Human MSC Expansion Media (R&D Systems, Corning), Serum-Free Medium for Adipose-Derived Stem Cells (KBM ADSC-4, Kojin Bio), and Serum-Free Medium for Mesenchymal Stem Cells (R:STEM Medium for hMSC High Examples include culture media provided as pre-prepared culture media for mesenchymal stem cells (stromal cells), such as Growth (manufactured by Rohto).

[0030] In particular, from the viewpoint of the therapeutic effect of the organ fibrosis prevention and / or treatment agent of the present invention, it is preferable that the culture medium for mesenchymal stem cells contains at least one, preferably two, more preferably three, even more preferably four, and especially preferably five, selected from the group consisting of EGF, bFGF, albumin, transferrin, and insulin.

[0031] (Preparation of mesenchymal stem cell culture supernatant) The supernatant of mesenchymal stem cells obtained by the following method can be used as the mesenchymal stem cell culture supernatant in this invention. Alternatively, the supernatant from which unwanted components have been removed by means of dialysis or ultrafiltration, a fraction obtained by fractionating the supernatant using a column, a fraction selected using an antibody against a specific molecule, or a fraction obtained by centrifugation (including extracted exosomes) may also be used as the mesenchymal stem cell culture supernatant in this invention.

[0032] The culture medium used to obtain the culture supernatant is the same as the medium used to culture mesenchymal stem cells. The method for obtaining the culture supernatant is not particularly limited as long as it is suitable for the culture of each type of mesenchymal stem cell, but for example, it is a method in which mesenchymal stem cells are cultured at a temperature of 20°C to 37°C, in a 2% to 7% CO2 environment, in a 5% to 21% O2 environment, preferably at room temperature to 37°C and in a 5% CO2 environment, and the culture supernatant is obtained.

[0033] The mesenchymal stem cell culture supernatant of the present invention only requires contact between the mesenchymal stem cells and the culture medium. The washing solution obtained by washing the mesenchymal stem cells with the culture medium can also be used as the culture supernatant in the present invention. The contact time between the mesenchymal stem cells and the culture medium is, for example, within 14 days, preferably within 10 days, more preferably within 7 days, and more preferably within 5 days. The culture to obtain the culture supernatant may be a planar culture performed by attaching the cells to a flask, or a suspension and agitation culture performed by attaching the cells to microbeads or the like.

[0034] The organ fibrosis prevention and / or treatment agent of the present invention may contain other components in addition to mesenchymal stem cell culture supernatant, to the extent that they do not interfere with the effects of the present invention. Examples of other components include protective agents such as dimethyl sulfoxide (DMSO) and serum albumin, antibiotics, vitamins, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, antiseptics, and physiological saline.

[0035] The dosage of the organ fibrosis prevention and / or treatment agent of the present invention can be adjusted depending on the patient's or animal's condition (weight, age, symptoms, physical condition, etc.), the dosage form of the organ fibrosis prevention and / or treatment agent of the present invention, the route of administration, etc. However, from the viewpoint of achieving sufficient therapeutic effect on the disease, a higher dose tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, a lower dose tends to be preferable. Typically, the dosage for adult humans is 0.01 mL to 3 L / dose, preferably 0.1 mL to 2 L / dose, more preferably 0.5 mL to 1 L / dose, and even more preferably 1 mL to 500 mL / dose. The dosage per body weight of the patient is 0.0001 to 100 mL / kg, preferably 0.002 to 40 mL / kg, more preferably 0.01 to 20 mL / kg, and even more preferably 0.02 to 10 mL / kg. From another perspective, the following ranges can also be presented as preferred: 0.01-100 mL / kg, 0.05-50 mL / kg, 0.1-40 mL / kg, 0.2-35 mL / kg, 0.3-30 mL / kg, and 0.4-25 mL / kg. When administered to cats or dogs, the dosage varies depending on the breed and age, but is usually 0.001-2 L / dose, preferably 0.01 mL-1 L / dose, more preferably 0.1 mL-500 mL / dose, and even more preferably 1 mL-250 mL / dose. Furthermore, the dosage per body weight of the affected animal is 0.0001-100 mL / kg, preferably 0.002-40 mL / kg, more preferably 0.01-20 mL / kg, and even more preferably 0.02-10 mL / kg. From another perspective, 0.01-100 mL / kg, 0.05-50 mL / kg, 0.1-40 mL / kg, 0.2-35 mL / kg, 0.3-30 mL / kg, and 0.4-25 mL / kg can also be presented as preferred ranges. When administered to non-mammals, the dosage varies depending on the animal species, size, age, etc., but is 0.0001-100 mL / kg, preferably 0.002-40 mL / kg, more preferably 0.01-20 mL / kg, and even more preferably 0.02-10 mL / kg. From another perspective, 0.01-100 mL / kg, 0.05-50 mL / kg, 0.1-40 mL / kg, 0.2-35 mL / kg, 0.3-30 mL / kg, and 0.4-25 mL / kg can also be presented as preferred ranges.

[0036] This dose may be administered multiple times as a single dose, or divided into multiple doses. For example, administration once a day or multiple times a day may be continued daily or at regular intervals for a certain period. The aforementioned period can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, 1 month, 2 months, 3 months, etc., with 2, 3, 4, 5, 6, 7, 8, 9, 10 days (2 to 10 days) being preferred. More preferably 3, 4, 5, 6, 7 days (3 to 7 days). In particular, it is preferable to administer the drug once a day, daily, for 3 to 7 days.

[0037] The therapeutic agent for organ fibrosis of the present invention is preferably administered after the onset of organ fibrosis.

[0038] The organ fibrosis prevention and / or treatment agent of the present invention may be administered together with one or more other agents. Examples of other agents include any agents that can be used as treatment agents for organ diseases such as kidney disease, such as corticosteroids, immunosuppressants such as mizoribine, cyclosporine, tacrolimus, and cyclophosphamide, antiplatelet agents such as dipyridamole and dilazep hydrochloride hydrate, anticoagulants such as heparin and warfarin potassium, antihypertensive agents such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), and calcium channel blockers, loop diuretics such as furosemide and azosemide, concentrated albumin preparations, dyslipidemia improving agents, phosphate binders, potassium binders, erythropoietin preparations, and beraprost sodium.

[0039] The prophylactic and / or therapeutic agent for organ fibrosis of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier, as long as it is a non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Furthermore, the pharmaceutical composition may comprise a pharmaceutically acceptable adjuvant (buffer, dispersant). Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond the range to which the target of application (prescription) can be adapted.

[0040] The organ fibrosis prevention and / or treatment agent of the present invention can be used by mixing it with an intravenous fluid. Here, "intravenous fluid" in the present invention refers to an artificial solution used in a therapeutic means of continuously injecting and supplying an artificial solution via a parenteral route (mainly intravenously), and is used for supplying water, electrolytes (salts), calories, nutrients, etc. Examples of intravenous fluids in the present invention include physiological saline, Japanese Pharmacopoeia physiological saline solution, 5% glucose solution, Japanese Pharmacopoeia glucose injection solution, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, Solution No. 1 (initial solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (postoperative recovery solution), etc.

[0041] When the prophylactic and / or therapeutic agent for organ fibrosis of the present invention is a liquid formulation, the pH of the prophylactic and / or therapeutic agent for organ fibrosis is not particularly limited as long as it is within a range that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. As an example, a range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0 is given.

[0042] When the prophylactic and / or therapeutic agent for organ fibrosis of the present invention is a liquid formulation, the osmotic pressure of the prophylactic and / or therapeutic agent for organ fibrosis is not particularly limited, as long as it is within a range acceptable to the body. An example of the osmotic pressure ratio of the composition of the present invention is preferably in the range of 0.7 to 5.0, more preferably 0.8 to 3.0, and even more preferably 0.9 to 1.4. The osmotic pressure can be adjusted using inorganic salts, polyhydric alcohols, sugar alcohols, sugars, etc., by methods known in the art. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 18th edition of the Japanese Pharmacopoeia, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. To prepare the standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution), sodium chloride (Japanese Pharmacopoeia standard reagent) is dried at 500-650°C for 40-50 minutes, then cooled in a desiccator (silica gel). 0.900g of this solution is accurately weighed and dissolved in purified water to make exactly 100mL. Alternatively, a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) can be used.

[0043] The routes of administration of the organ fibrosis prevention and / or treatment agent of the present invention include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intrathecal administration, intraperitoneal administration, sublingual administration, transrectal administration, transvaginal administration, intraocular administration, transnasal administration, inhalation, transdermal administration, implantation, direct administration by spraying onto the organ surface and applying sheets, etc. However, from the viewpoint of the effectiveness of the organ fibrosis prevention and / or treatment agent of the present invention, implantation, intra-arterial administration such as the renal artery, intravenous administration such as the renal vein, and direct administration by spraying onto the organ surface and applying sheets, etc., and from the viewpoint of reducing the burden on the subject, intravenous administration is more preferred.

[0044] The species targeted by the organ fibrosis prevention and / or treatment agent of the present invention may be mammals or non-mammals. Examples of mammals include humans, horses, cattle, sheep, pigs, dogs, cats, rabbits, mice, rats, and rare animals.

[0045] The present invention's agent for preventing and / or treating organ fibrosis can be suitably used for the prevention and / or treatment of diseases involving organ fibrosis.

[0046] Organ fibrosis can occur in one or more organs selected from the group consisting of the kidneys, liver, lungs, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles, and joints. Specifically, diseases involving organ fibrosis include fibrosis such as renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or pulmonary fibrosis, pulmonary edema, hepatic fibrosis, cystic fibrosis, endocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, myelofibrosis, articular fibrosis, lipofibrosis, cutaneous fibrosis, neurofibrosis, and muscular fibrosis. This fibrosis refers to diseases caused by the abnormal production, accumulation, and deposition of extracellular matrix by fibroblasts, resulting in fibrosis of organs or tissues. Fibrosis refers to a highly fatal disease that induces organ damage. Furthermore, the terms "fibrotic state," "fibroproliferative state," "fibrotic disease," "fibroproliferative disorder," "fibrotic disorder," and "fibroproliferative disorder" are interchangeable and may refer to a state, disease, or disorder characterized by unregulated proliferation or activity of fibroblasts, abnormal accumulation of fibronectin, and / or pathological or excessive accumulation of collagen tissue. Of these, from the viewpoint that the organ fibrosis prevention and / or treatment agent of the present invention exhibits excellent effects, diseases involving fibrosis in the kidneys, lungs, and liver are preferred, with renal fibrosis, pulmonary fibrosis, and hepatic fibrosis being more preferred.

[0047] <An inhibitor of organ fibrosis> The organ fibrosis inhibitor of the present invention is characterized by containing mesenchymal stem cell culture supernatant. Because the organ fibrosis inhibitor of the present invention contains mesenchymal stem cell culture supernatant, it suppresses organ fibrosis and is therefore effective as a preventive and / or therapeutic agent for organ fibrosis. The composition of the organ fibrosis inhibitor of the present invention is the same as that of the preventive and / or therapeutic agent for organ fibrosis described above, so for details, the description in the section on the preventive and / or therapeutic agent for organ fibrosis can be applied. [Examples]

[0048] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0049] 1. Laboratory animals Five-week-old male SD rats were purchased from Nippon SLC Co., Ltd., and after a one-week acclimatization period, renal ischemia-reperfusion surgery was performed. The rats were given ad libitum access to feed (solid lab MR stock, manufactured by Nippon Nosan Kogyo Co., Ltd.) and tap water. The rearing conditions were a 12-hour light-dark cycle (light period 8:00-20:00), temperature 22±2℃, and humidity 50±20%.

[0050] 2. Preparation of Mesenchymal Stem Cell Culture Supernatant Adipose tissue-derived mesenchymal stem cells (Lonza, hereinafter referred to as "ADSC") were cultured in RIM medium (serum-free medium for mesenchymal stem cells containing EGF, bFGF, albumin, transferrin, and insulin, Rohto) for 4 days to obtain the culture supernatant. The culture conditions at this time were 5% CO2 and 37°C.

[0051] 3. Renal ischemia reperfusion surgery Rats were anesthetized by intramuscular injection of 0.30 mL of ketamine-xylazine (6:1) into the leg of each rat. Isoflurane inhalation anesthesia was used for rats whose anesthesia was wearing off. All subsequent surgeries were performed on a hot plate set to 38°C using sterile instruments. The left flank of the rat was incised to expose the left kidney. The left renal artery and vein were clamped with clips to occlude blood flow, and the kidney was returned to the abdominal cavity. Gauze was placed over the wound to prevent drying, the entire body was wrapped in cloth, and the rat was ischemic on the hot plate for 45 minutes. After 45 minutes, the clamps were removed and reperfusion was performed, and the internal muscles were sutured with silk thread and the skin with absorbable sutures. After suturing, 0.25 mL of antibiotic (veterinary suspension procaine penicillin G) was intramuscularly injected into the rat's leg, and 0.05 mL of analgesic (Repetan injection 0.2 mg) was administered subcutaneously after the rat recovered from anesthesia.

[0052] The rats were housed in clean plastic cages, 2-3 rats per cage. On the day of surgery, a portion of the measured feed was placed on the cage floor. Weight, food intake, and water intake were measured once a day.

[0053] 4. Dosage of the sample The mesenchymal stem cell culture supernatant was stored at -80°C until the day before the first administration. After thawing, it was dispensed in a clean bench in quantities corresponding to the number of days of administration (3 vials for a 3-day experiment, 7 vials for a 7-day experiment), and stored at 4°C thereafter. On the day of surgery, the first administration was given 1-2 hours after reperfusion, and administration continued once daily until the day before dissection (MSC supernatant administration group: MSC group). Isoflurane inhalation anesthesia was used for administration, and 1 mL was administered intravenously into the tail vein using a 23-gauge needle. The control group received the same amount of sterile phosphate-buffered saline (PBS group or Control group).

[0054] 5. Collection of various samples (blood, kidney) Autopsies were performed three or seven days after surgery. During the autopsy, the animals were anesthetized with pentobarbital sodium and euthanized by total blood collection from the abdominal aorta. The left and right kidneys were collected, and the renal cortex was dissected and immersed in RNA lateral.

[0055] In experiments involving 7 days of rearing, tail vein blood sampling was performed 2 days before surgery and 1, 3, and 7 days after surgery to evaluate blood urea nitrogen levels over time. Blood sampling was performed under isoflurane anesthesia, and 0.3 mL of blood was collected before intravenous administration of the sample. Heparinized blood was centrifuged at 2,400 G for 5 minutes. Plasma components were separated into microtubes and stored at -20°C.

[0056] 6. Measurement of blood urea nitrogen (BUN) Blood urea nitrogen levels in plasma obtained by tail vein blood collection were measured by Oriental Yeast Co., Ltd., Nagahama Science Laboratory (urease-GLDH method).

[0057] 7. Methods for evaluating the expression levels of genes related to kidney damage RNA was extracted from the cortical region of kidneys immersed in RNA later and stored at -20°C, and mRNA expression levels were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Expression levels of inflammatory markers (F4 / 80, IL-1β) and fibrosis markers (Tgfβ1, αSMA, Fn, Col1a1) were analyzed. EEF1A1 (eukaryotic elongation factor 1α1) was used as an endogenous control for gene expression levels, and the relative expression level of each gene was calculated from (expression level of target gene) / (expression level of EEF1A1). The results are shown in Figures 2-7. The processing of each data point was performed as follows.

[0058] [Data Processing] The 7-day rearing experiment involved two experiments with n=5 and 2 groups under the same conditions, so the BUN graph was created by combining the results for n=10 and 2 groups. On the other hand, regarding mRNA expression levels, since only the left kidney (the one that underwent surgery) was collected in the first experiment, only the samples from the second experiment, where both kidneys were collected, were analyzed, and a graph was created for n=5 and 2 groups.

[0059] In the 3-day rearing experiment, experiments were conducted simultaneously with n=8 and 2 groups, and both kidneys were collected and analyzed. No. 17 (MSC group) in the 3-day rearing experiment was excluded from the data because it experienced a prolonged ischemic period due to complications during surgery, resulting in outliers in the expression levels of several genes. The remaining data consisted of n=8 and 2 groups (Nos. 1-16).

[0060] For BUN levels, multiple comparisons were performed using Dunnett's test for the control group (PBS group) before surgery and 1, 3, and 7 days after surgery. Additionally, t-tests were used to compare the PBS group and the MSC group at each blood sampling timing. For gene expression levels, multiple comparisons were performed using two-way ANOVA and Tukey's test.

[0061] 8. Method for evaluating the expression levels of kidney damage-related proteins SMAD2 / 3 is located downstream of the TGFβ signaling pathway and is known to act as a transcription factor upon phosphorylation, thereby promoting fibrosis. We analyzed the protein expression levels of TGF-β and p-SMAD2 / 3 in the renal cortex using Western blot (Figures 8-1, 8-2).

[0062] 9. Tissue staining The obtained kidney tissue sections were subjected to hematoxylin-eosin (HE) staining and Sirius red staining to observe the renal cortex (Figures 9 and 10-1).

[0063] The fibrotic area was quantified using ImageJ based on the staining results with Sirius Red. Specifically, 4 μm tissue sections were stained with Sirius Red, and five photographs were taken per sample for image analysis. For the analysis, the red areas were extracted with a threshold set to 30-100. After selecting areas other than blood vessels, the area within the threshold range was quantified, and the Stained Area (%) was calculated using the following [Equation 1] (Figure 10-2). Stained Area (%) = Positive Area / Selected Area [Formula 1]

[0064] In Figures 1 and 3, * "; p<0.05 vs PBS, "**"; p<0.01 vs PBS, "++"; p<0.01 vs Day 0 (Dunnett test). In Figures 6, 8-2, and 10-2, "†"; p<0.05 vs Control (R), * This shows p<0.05 vs Control(L)(Tukey's test).

[0065] 10. Evaluation Results 10-1.7 day rearing experiment BUN As shown in Figure 1, BUN levels significantly increased from the day after surgery compared to before surgery. Statistical analysis of the two experiments (n=10) revealed an improving trend in BUN levels one day after surgery (p=0.051), and significantly lower levels in the MSC group compared to the PBS group at three and seven days.

[0066] Body weight, intake No differences were observed in body weight or food intake between the MSC group and the PBS group.

[0067] Renal inflammation marker expression Gene expression analysis of F4 / 80 showed improvement in the left kidney after administration of MSC supernatant (Figure 2).

[0068] Renal fibrosis marker expression Gene expression analysis of Tgfβ1 and αSMA showed improvement in the left renal cortex following administration of MSC supernatant (Figure 3).

[0069] 10-2.3 day rearing experiment

[0070] Body weight, intake No differences were observed in body weight or food intake between the MSC group and the PBS group (Figure 4).

[0071] Expression of kidney inflammation gene markers In gene expression analysis of F4 / 80 and IL-1β, improvement was observed in the left kidney after administration of MSC supernatant (Figure 5).

[0072] Renal fibrosis gene marker expression Gene expression analysis of Tgfβ1, αSMA, Fn, Col1a1, and MMP2 in the renal cortex showed improvement in the left kidney with administration of MSC supernatant (Figure 6). Furthermore, gene expression analysis of Tgfβ in the renal medulla also showed improvement in the left kidney with administration of MSC supernatant (Figure 7).

[0073] Expression of proteins that promote renal fibrosis Protein expression levels of TGF-β and p-SMAD2 / 3 in the left kidney showed a decreasing trend in the MSC group compared to the control group (Figures 8-1, 8-2).

[0074] tissue staining HE staining revealed tubular damage and cellular infiltration in the left kidney. Furthermore, improvement in tubular damage was observed in the MSC group compared to the control group (Figure 9). Sirius red staining showed a significant decrease in the Sirius red-positive area in the MSC group compared to the control group, indicating improvement in interstitial fibrosis (Figures 10-1, 10-2).

[0075] summary BUN measurement results suggested that intravenous administration of mesenchymal stem cell culture supernatant after renal ischemia-reperfusion surgery improved renal function. mRNA expression analysis of the renal cortex three days after surgery revealed that renal fibrosis was particularly suppressed. Furthermore, improvement in the inflammatory markers F4 / 80 and IL-1β was observed, suggesting that an anti-inflammatory effect was also obtained.

[0076] Evaluations of pathological tissue using HE staining and Sirius Red staining confirmed that intravenous administration of mesenchymal stem cell culture supernatant improved tubular damage and significantly suppressed the increase in fibrotic area. Furthermore, protein expression showed that IR damage increased the expression levels of TGF-β and p-SMAD2 / 3 in the left kidney, suggesting that fibrosis progressed via the TGF-β signaling pathway. In addition, a decreasing trend in TGF-β and p-SMAD2 / 3 expression was observed in the MSC group compared to the control group. [Industrial applicability]

[0077] The present invention provides a novel agent for preventing and / or treating organ fibrosis. Furthermore, the agent for preventing and / or treating organ fibrosis of the present invention can be suitably used not only in humans but also in non-human animals for organ diseases involving fibrosis for which there are currently no effective treatments.

Claims

1. A prophylactic and / or therapeutic agent for organ fibrosis, containing mesenchymal stem cell culture supernatant.

2. The preventive and / or therapeutic agent according to claim 1, wherein the mesenchymal stem cell culture supernatant is a culture supernatant obtained from the culture of mesenchymal stem cells derived from adipose tissue, umbilical cord tissue, or bone marrow tissue.

3. The preventive and / or therapeutic agent according to claim 1 or 2, wherein the organ is the kidney.

4. The therapeutic agent according to claim 1 or 2, characterized in that it is administered after the onset of fibrosis.

5. A preventive and / or therapeutic agent according to claim 1 or 2, characterized by being administered multiple times.

6. An organ fibrosis inhibitor containing mesenchymal stem cell culture supernatant.

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