Therapeutic agent for dilated cardiomyopathy

Abstract

To provide a therapeutic agent for dilated cardiomyopathy disease which produces excellent effects in treatment of dilated cardiomyopathy (DCM) and provides a given benefit to a large number of patients.SOLUTION: The present invention provides a therapeutic agent for dilated cardiomyopathy comprising at least one selected from a group consisting of mesenchymal stem cells and microparticles derived from the mesenchymal stem cells. It is preferable that the mesenchymal stem cells are cells having ability to contain or secrete microparticles, that the microparticles have an average particle size of 1000 nm or less, and that they are exosomes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for dilated cardiomyopathy. [Background technology]

[0002] Dilated cardiomyopathy (DCM) is a group of idiopathic cardiomyopathy characterized by myocardial contractile dysfunction and left ventricular lumen dilation, and is often progressive. DCM is also characterized by chronic heart failure symptoms and repeated acute exacerbations, resulting in a poor prognosis. Furthermore, DCM can lead to sudden death due to fatal arrhythmias and arterial thromboembolism. DCM is a mixed group of inherited and acquired diseases. By definition, DCM is classified based on morphological and functional characteristics. Clinically, there are many similar disorders that result in "left ventricular dilation" and "left ventricular systolic dysfunction." Therefore, when considering DCM, it is necessary to exclude various other similar specific cardiomyopathies.

[0003] The etiology of dilated cardiomyopathy has long been unknown, but it has become clear that genetic predisposition, viral infection, and autoimmune disorders are involved, and various cellular immune abnormalities have been observed. It has been reported that regulatory T cells are reduced and helper T cells are increased in the serum of patients with dilated cardiomyopathy, and that studies using myocardial biopsy specimens have shown the expression of MHC class II molecules. Various anti-myocardial autoantibodies are also present in the serum of patients with dilated cardiomyopathy. It has also been shown that such anti-myocardial autoantibodies are frequently detected in patients with familial dilated cardiomyopathy (Non-Patent Document 1). Furthermore, some genetic mutations have been detected in approximately 20% of patients with familial dilated cardiomyopathy, and many of these mutations are in genes encoding sarcomere proteins directly involved in cardiac contraction or genes encoding dystrophin-related proteins, which play a role in transmitting cardiac contractile force. These genetic mutations not only directly cause the onset of dilated cardiomyopathy but can also act as a genetic predisposition for the disease. It is believed that in patients with these genetic mutations, persistent autoimmune mechanisms are activated after viral infection, leading to the development of dilated cardiomyopathy. Furthermore, because patients with dilated cardiomyopathy have a poor prognosis, they are often candidates for heart transplants, but there is a shortage of donors and transplant rejection is also an issue. Therefore, the development of new treatments for dilated cardiomyopathy is desired.

[0004] The effects of mesenchymal stem cell (MSC) treatment have been investigated in animals with cardiomyopathy induced by drugs such as doxorubicin and 5-azacytidine, using these animals as models of dilated cardiomyopathy (Non-Patent Documents 2-3). However, cardiomyopathy induced by drugs such as doxorubicin and 5-azacytidine is called drug-induced cardiomyopathy. Although it is a group of cardiomyopathy diseases clinically similar to dilated cardiomyopathy, it is classified as a different category from dilated cardiomyopathy according to the Guidelines for the Treatment of Dilated Cardiomyopathy and Related Secondary Cardiomyopathy (Guidelines for the Diagnosis and Treatment of Cardiovascular Diseases 2011). Furthermore, genetic abnormalities in cytoplasmic proteins such as myocardial δ-sarcoglycan, actin, and myosin are thought to be involved in the genetic cause of dilated cardiomyopathy, which is fundamentally different from drug-induced cardiomyopathy in this respect. Therefore, even if an improvement effect is observed in a drug-induced cardiomyopathy model, it is difficult to say that it will be effective against actual dilated cardiomyopathy.

[0005] J2N hamsters lacking delta-sarcoglycan have been reported. These hamsters develop dilated cardiomyopathy at approximately 16 weeks of age, exhibiting morphological changes such as decreased cardiac function, cardiac cell hypertrophy, and fibrosis. Because these morphological changes are similar to those observed in humans, they are used as a model for human dilated cardiomyopathy. Mutations in delta-sarcoglycan have also been observed in human dilated cardiomyopathy, and it is known that impaired ANT-1 function is observed in both J2N hamsters and humans with dilated cardiomyopathy (Non-Patent Documents 4-6).

[0006] On the other hand, mesenchymal stem cells are multipotent progenitor cells that were first isolated from bone marrow by Friedenstein (1982) (Non-Patent Document 7). These mesenchymal stem cells have been shown to exist in various tissues, such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (Patent Documents 1 to 4). Recently, it has been discovered that cells with equivalent functions exist in the interstitial cells of fetal appendages, such as adipose tissue, placenta, umbilical cord, and fetal membranes. For this reason, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2002-506831 [Patent Document 2] Special Publication No. 2000-508911 [Patent Document 3] Japanese Patent Application Laid-Open No. 2012-157263 [Patent Document 4] Special Publication No. 2012-508733 [Non-patent literature]

[0008] [Non-Patent Document 1] Tomoike, H. et al., Guidelines for the diagnosis and treatment of cardiovascular disease 2011 [Non-patent document 2] Mol. Cell Biochem.,2014,387,pp.279-285 [Non-patent document 3] Med. Sci. Monit. Basic Res.,2013.14,pp.20-31 [Non-patent document 4] Circ. J.,2005,69,pp.107-113 [Non-Patent Document 5] J. Biochem.,2003,34(2),pp.269-76 [Non-patent document 6] JCI.,2000,106,pp.655-662 [Non-Patent Document 7] Pittenger FM et al.,Science,1999.284,pp.143-147 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in light of the above-mentioned circumstances, and aims to provide a therapeutic agent for dilated cardiomyopathy that is highly effective in treating dilated cardiomyopathy and that provides consistent results for many patients. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that administering mesenchymal stem cells or microparticles derived from mesenchymal stem cells to patients with dilated cardiomyopathy can significantly improve cardiac function, etc. The present invention was completed based on these findings. Specifically, the inventions made to solve the above-mentioned problems are as follows.

[0011] [1] A therapeutic agent for dilated cardiomyopathy, comprising at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells. [2] The therapeutic agent for dilated cardiomyopathy according to [1], wherein the mesenchymal stem cells have the ability to contain or secrete microparticles. [3] The therapeutic agent for dilated cardiomyopathy according to [1] or [2], wherein the microparticles have an average particle size of 1,000 nm or less. [4] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [3], wherein the microparticles are exosomes. [5] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [4], wherein the microparticles contain at least one factor selected from the group consisting of IL-10, HGF, apoprotein A-2, pigment epithelium-derived factor (PEDF), SERPINF1, and haptoglobin. [6] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [5], wherein the mesenchymal stem cells are allogeneic to the subject. [7] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [6], wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue, or bone marrow. [8] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [7], wherein the dilated cardiomyopathy is selected from the group consisting of idiopathic dilated cardiomyopathy, familial dilated cardiomyopathy, and hereditary dilated cardiomyopathy. [9] The therapeutic agent for dilated cardiomyopathy according to any one of [1] to [8], wherein the mesenchymal stem cells are cryopreserved cells.

[10] An agent for suppressing fibrosis in dilated cardiomyopathy, comprising at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells.

[11] A cardiac hypertrophy inhibitor for dilated cardiomyopathy, comprising at least one member selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells.

[12] A kit for treating dilated cardiomyopathy, comprising the therapeutic agent for dilated cardiomyopathy described in any one of [1] to [9], the fibrosis inhibitor described in

[10] , or the cardiac hypertrophy inhibitor described in

[11] , a container, and a label.

[13] A method for treating dilated cardiomyopathy, comprising administering at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells.

[14] A method for suppressing fibrosis in dilated cardiomyopathy, comprising administering at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells.

[15] A method for suppressing cardiac hypertrophy in dilated cardiomyopathy, comprising administering at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells. [Effects of the Invention]

[0012] According to the therapeutic agent for dilated cardiomyopathy of the present invention, administration of mesenchymal stem cells or mesenchymal stem cell-derived microparticles to treat dilated cardiomyopathy can significantly improve the function, structure, etc. of diseased areas such as the heart. Because the mesenchymal stem cells are less likely to cause rejection in allogeneic subjects, pre-prepared donor cells that have been expanded and cryopreserved can be used as the mesenchymal stem cells in the therapeutic agent for dilated cardiomyopathy of the present invention. Similarly, mesenchymal stem cell-derived microparticles obtained from pre-prepared donor cells that have been expanded and cryopreserved can be used as the mesenchymal stem cell-derived microparticles in the therapeutic agent for dilated cardiomyopathy of the present invention. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, commercialization is easier and a stable, consistent effect can be more easily achieved. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the therapeutic effect of the therapeutic agent for dilated cardiomyopathy of the present invention on J2N-k hamsters, an animal model for dilated cardiomyopathy (evaluation of cardiac function). [Figure 2] FIG. 2 is a graph showing the therapeutic effect of the therapeutic agent for dilated cardiomyopathy of the present invention on J2N-k hamsters, an animal model for dilated cardiomyopathy (ATP content in cardiac tissue). [Figure 3] FIG. 3 shows the therapeutic effect of the therapeutic agent for dilated cardiomyopathy of the present invention on J2N-k hamsters, an animal model for dilated cardiomyopathy (expression level of Mt.ANT-1 in mitochondria in cardiac tissue). [Figure 4] FIG. 4 is a graph showing the therapeutic effect of the therapeutic agent for dilated cardiomyopathy of the present invention on J2N-k hamsters, an animal model for dilated cardiomyopathy (heart diameter). [Figure 5]FIG. 5 shows the therapeutic effect (long-term) of the therapeutic agent for dilated cardiomyopathy of the present invention on J2N-k hamsters, an animal model of dilated cardiomyopathy (evaluation of cardiac function). [Figure 6] FIG. 6 shows the function-enhancing effect of the therapeutic agent for dilated cardiomyopathy of the present invention on human cardiomyocytes. DETAILED DESCRIPTION OF THE INVENTION

[0014] The therapeutic agent for dilated cardiomyopathy, the fibrosis suppressant, the cardiac hypertrophy suppressant, and the kit for treating dilated cardiomyopathy of the present invention will be described in detail below.

[0015] <Treatment for dilated cardiomyopathy> The therapeutic agent for dilated cardiomyopathy of the present invention contains at least one selected from the group consisting of mesenchymal stem cells and microparticles derived from mesenchymal stem cells.

[0016] <Mesenchymal stem cells> In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into cells belonging to the mesenchymal system (such as bone cells, cardiomyocytes, chondrocytes, tendon cells, and adipocytes) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as stromal cells, and does not particularly distinguish between the two. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Therefore, for example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue, and may also be referred to as adipose tissue-derived stromal cells. Of these, from the viewpoint of the effects of the dilated cardiomyopathy therapeutic agent of the present invention, such as improvement of visceral diseases, and from the viewpoint of ease of availability, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, and adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred.

[0017] The mesenchymal stem cells of the present invention are preferably allogeneic or allogeneic to the subject. Because mesenchymal stem cells are less likely to cause rejection in allogeneic subjects, donor cells prepared in advance and expanded and cryopreserved can be used as the mesenchymal stem cells in the therapeutic agent for dilated cardiomyopathy of the present invention. Therefore, compared to the case where autologous mesenchymal stem cells are prepared and used, from the viewpoints of ease of commercialization and ease of obtaining a stable and consistent effect, the mesenchymal stem cells of the present invention are more preferably allogeneic.

[0018] Because mesenchymal stem cells are less likely to cause rejection in allogeneic subjects, donor cells prepared in advance, expanded and cryopreserved, can be used as the mesenchymal stem cells in the therapeutic agent for dilated cardiomyopathy of the present invention. Similarly, mesenchymal stem cell-derived microparticles obtained from mesenchymal stem cells prepared in advance, expanded and cryopreserved donor cells, can be used as the mesenchymal stem cell-derived microparticles in the therapeutic agent for dilated cardiomyopathy of the present invention. Therefore, compared with the case of preparing and using autologous mesenchymal stem cells or microparticles, it is more preferable that the mesenchymal stem cells in the present invention are allogeneic, as this is easier to commercialize and more likely to provide a consistent effect.

[0019] In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells, in which at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% of the cell population is mesenchymal stem cells.

[0020] In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells, including microvascular cells, and is, for example, tissue obtained by surgical resection or aspiration of subcutaneous fat from a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferably obtained from the same species of animal as the recipient of the adipose tissue-derived mesenchymal stem cells described below, and considering administration to humans, human subcutaneous fat is more preferred. The individual providing the subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting from an individual, liposuction can be performed using, for example, PAL (power-assisted) liposuction, Erchoria laser liposuction, or body jet liposuction, and it is preferable not to use ultrasound in order to maintain the state of the cells.

[0021] In the present invention, the umbilical cord refers to a white tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells. The umbilical cord is preferably obtained from an animal of the same species as the subject (administration target) in whom the therapeutic agent for dilated cardiomyopathy of the present invention will be administered, and when considering administration of the therapeutic agent for dilated cardiomyopathy of the present invention to humans, a human umbilical cord is more preferred.

[0022] In the present invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

[0023] In the present invention, mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, refer to any cell population containing mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, respectively. The cell population is composed of at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0024] Mesenchymal stem cells of the present invention may be characterized by, for example, growth characteristics (e.g., population doubling ability from passage to senescence, doubling time), karyotype analysis (e.g., normal karyotype, maternal lineage or neonatal lineage), surface marker expression by flow cytometry (e.g., FACS analysis), immunohistochemistry and / or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip array; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein array, protein secretion such as cytokines (e.g., plasma clotting analysis, ELISA, cytokine array), metabolites (metabolomic analysis), or other methods known in the art.

[0025] <Method for preparing mesenchymal stem cells> The mesenchymal stem cells can be prepared by methods well known to those skilled in the art. As an example, a method for preparing adipose tissue-derived mesenchymal stem cells will be described below. Adipose tissue-derived mesenchymal stem cells may be obtained by the production method described in U.S. Patent No. 6,777,231, for example, and can be produced by a method including the following steps (i) to (iii): (i) enzymatically digesting adipose tissue to obtain a cell suspension; (ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface;

[0026] The adipose tissue used in step (i) is preferably washed. Washing can be performed by vigorous agitation and sedimentation using a physiologically compatible saline solution (e.g., phosphate-buffered saline (PBS)). This is to remove contaminants (also known as debris, such as damaged tissue, blood, and red blood cells) contained in the adipose tissue from the tissue. Therefore, washing and sedimentation are generally repeated until all debris is removed from the supernatant. Since the remaining cells exist as clumps of various sizes, it is preferable to treat the washed cell clumps with an enzyme (e.g., collagenase, dispase, or trypsin) that weakens or destroys intercellular bonds to dissociate the cells while minimizing damage to the cells themselves. The amount and duration of such enzymes vary depending on the conditions used and are known in the art. Instead of or in combination with such enzyme treatment, cell clumps can be decomposed by other treatment methods such as mechanical agitation, ultrasonic energy, or thermal energy; however, enzyme treatment alone is preferred to minimize cell damage. When an enzyme is used, it is desirable to inactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize adverse effects on cells.

[0027] The cell suspension obtained by step (i) contains a slurry or suspension of aggregated cells and various contaminating cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, these contaminating cells may be subsequently separated and removed from the aggregated cells. However, this separation and removal step may be omitted because they can be removed by adhesion and washing in step (iii) described below. Separation and removal of contaminating cells can be achieved by centrifugation, which forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing the contaminating cells is suspended in a physiologically compatible solvent. The suspended cells may contain red blood cells, but a lysis step is not necessarily required because red blood cells are excluded by selection based on adhesion to a solid surface, as described below. Methods known in the art can be used to selectively lyse red blood cells, such as ammonium chloride lysis followed by incubation in a hypertonic or hypotonic medium. After lysis, the lysate may be separated from the desired cells by, for example, filtration, centrifugal sedimentation, or density fractionation.

[0028] In step (ii), the cells in suspension may be washed once or multiple times, centrifuged, and resuspended in culture medium to increase the purity of the mesenchymal stem cells. Alternatively, the cells may be separated based on cell surface marker profiles or on cell size and granularity.

[0029] The medium used for resuspension is not particularly limited as long as it is capable of culturing mesenchymal stem cells. However, such a medium may be prepared by adding serum to a basal medium and / or one or more serum substitutes, such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. These media may further contain, as needed, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixtures thereof. Examples of serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, and rat serum. When serum is used, it may be added to the basal medium at 5 v / v% to 15 v / v%, preferably 10 v / v%. Examples of 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 lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of 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 proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, particular 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). From the perspective of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a xeno-free medium that does not contain xenogeneic components such as serum. Such media are available as pre-prepared media for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

[0030] Subsequently, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured on a solid surface without differentiation using the appropriate cell culture medium described above at an appropriate cell density and culture conditions. In the present invention, the term "solid surface" refers to any material that allows the adipose tissue-derived mesenchymal stem cells of the present invention to bind. In a specific embodiment, such a material is a plastic material that has been treated to promote the binding of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish, a flask, or the like is preferably used. After incubation, the cells are washed to remove unbound cells and cell debris.

[0031] In the present invention, cells that ultimately remain bound to the solid surface can be selected as a cell population of adipose tissue-derived mesenchymal stem cells.

[0032] To confirm that the selected cells are adipose tissue-derived mesenchymal stem cells of the present invention, surface antigens may be analyzed by conventional methods such as flow cytometry. Furthermore, the ability to differentiate into each cell lineage may be examined, and such differentiation can be carried out by conventional methods.

[0033] The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following properties: (1) Adhesion to plastic under standard culture conditions. (2) surface antigens CD44, CD73, and CD90 are positive, and CD31 and CD45 are negative; and (3) They can be differentiated into osteocytes, adipocytes, and chondrocytes under certain culture conditions.

[0034] Cryopreservation of mesenchymal stem cells The mesenchymal stem cells of the present invention may be cells that have been repeatedly cryopreserved and thawed as appropriate, as long as they have a disease therapeutic effect. In the present invention, cryopreservation can be performed by suspending the mesenchymal stem cells in a cryopreservation solution known to those skilled in the art and 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 appropriately, and then adding the cryopreservation solution.

[0035] The cryopreservation solution may contain DMSO (dimethyl sulfoxide) as a cryoprotectant. However, because DMSO is cytotoxic and has the ability to induce differentiation of mesenchymal stem cells, it is preferable to reduce the DMSO content. Examples of DMSO alternatives include glycerol, propylene glycol, and polysaccharides. When DMSO is used, it is contained in an amount of 5% to 20% v / v, preferably 5% to 10% v / v, and more preferably 10% v / v. Other additives described in WO2007 / 058308 may also be included. Examples of such cryopreservation solutions include those provided by BioVerde, Inc., Nippon Genetics Co., Ltd., ReproCell, Inc., Zenoac Corporation, Cosmo Bio, Kohjin Bio Co., Ltd., and Thermo Fisher Scientific.

[0036] When the above-described suspended cells are cryopreserved, they may be stored at a temperature between -80°C and -100°C (e.g., -80°C), and any freezer capable of achieving this temperature may be used. To avoid sudden temperature changes, the cooling rate may be appropriately controlled using a programmable freezer, although this is not particularly limited. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.

[0037] The storage period is not particularly limited as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, and may be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, the cells may be transferred to the vapor phase above liquid nitrogen (approximately −150°C or less to −180°C or less) for storage. Storage in the vapor phase above liquid nitrogen can be carried out using a storage container well known to those skilled in the art. Although not particularly limited, for example, when storing for 2 weeks or more, storage in the vapor phase above liquid nitrogen is preferred.

[0038] The thawed mesenchymal stem cells may be cultured as appropriate until the next cryopreservation. Mesenchymal stem cells are cultured using a medium capable of culturing the above-mentioned mesenchymal stem cells, and may be cultured at a temperature of, but not limited to, about 30-40°C, preferably about 37°C, in an atmosphere of CO2-containing air. The CO2 concentration is about 2-5%, preferably about 5%. After the cells reach an appropriate confluency for the culture vessel (for example, when the cells occupy 50% to 80% of the culture vessel), they may be detached with a detaching agent such as trypsin and seeded at an appropriate cell density in a separately prepared culture vessel for continued culture. When seeding the cells, a typical cell density is about 100 cells / cm. 2 ~about 100,000 cells / cm 2 , about 500 cells / cm 2 ~about 50,000 cells / cm 2 , about 1,000~10,000 cells / cm 2, about 2,000~10,000 cells / cm 2 In a specific embodiment, the cell density is 2,000 to 10,000 cells / cm. 2 The period required to reach an appropriate confluency is preferably adjusted to 3 to 7 days. During the culture, the medium may be replaced as needed.

[0039] Cryopreserved cells can be thawed by methods well known to those skilled in the art, such as by leaving the cells to stand or shaking them in a 37°C incubator or in a hot water bath.

[0040] <Microparticles derived from mesenchymal stem cells> The mesenchymal stem cell-derived microparticles of the present invention are microparticles obtained from mesenchymal stem cells and produced by mesenchymal stem cells. The mesenchymal stem cell microparticles of the present invention may be those contained in mesenchymal stem cells, those contained in mesenchymal stem cell culture supernatant, or microparticles isolated from mesenchymal stem cells or mesenchymal stem cell culture supernatant. That is, the mesenchymal stem cell-derived microparticles of the present invention may be in any form, including not only the microparticles themselves but also mesenchymal stem cells capable of containing or secreting microparticles.

[0041] Methods for isolating microparticles include ultracentrifugation, microfiltration, antibody capture, use of a microfluidic system, etc. The isolated microparticles may contain cells such as mesenchymal stem cells, or may contain a mesenchymal stem cell culture medium.

[0042] The mesenchymal stem cell-derived microparticles of the present invention can be recovered from the culture supernatant obtained by culturing mesenchymal stem cells in the above-mentioned medium. To recover the culture supernatant, for example, mesenchymal stem cells are grown to a subconfluent or confluent state in a culture vessel, the medium is replaced with new medium, and the cells are further cultured for 1 to 5 days, after which the culture supernatant can be recovered. This culture supernatant can be used as the therapeutic agent for dilated cardiomyopathy of the present invention, or the microparticles can be separated by ultracentrifugation, density gradient centrifugation, various microparticle separation kits, or the like, and then used as a material for the therapeutic agent for dilated cardiomyopathy of the present invention.

[0043] The therapeutic agent for dilated cardiomyopathy of the present invention may contain, in addition to the microparticles, mesenchymal stem cells themselves that have the ability to encapsulate or secrete the microparticles. Note that when the therapeutic agent for dilated cardiomyopathy contains mesenchymal stem cells that contain or have the ability to secrete the microparticles, the inclusion of the mesenchymal stem cells can also be interpreted as satisfying the requirement of containing the microparticles.

[0044] The mesenchymal stem cell-derived microparticles of the present invention are typically vesicles released from mesenchymal stem cells and of a size that can be confirmed by electron microscopy. The average particle diameter of the microparticles is 1 nm to 1,000 nm, preferably 10 nm to 500 nm, and more preferably 30 nm to 200 nm. Here, the average particle diameter refers to the average diameter of each particle measured by dynamic light scattering or electron microscopy. The microparticles may have a lipid bilayer surrounding biomolecules.

[0045] Examples of the microparticles include membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (average particle size: 30 to 1,000 nm), exosome-like vesicles, exosomes (average particle size: 30 to 200 nm), ectosome-like vesicles, ectosomes, and exovesicles, with exosomes being preferred. Different types of mesenchymal stem cell-derived microparticles can be distinguished based on their intracellular origin, microparticle density in sucrose, shape, sedimentation rate, lipid composition, protein markers, and secretion pattern (i.e., signal-induced or spontaneous (constitutive)). For example, by density gradient centrifugation, the microparticles are fractionated to 1.0 to 1.5 g / mL, preferably 1.1 to 1.3 g / mL. Furthermore, the microparticles contain any of phosphatidylserine, cholesterol, sphingomyelin, and ceramide as their constituent lipids.

[0046] The microparticles derived from mesenchymal stem cells in the present invention contain proteins, fatty acids, and nucleic acids such as miRNA. Examples of the above proteins and fatty acids include IL-10, HGF (Hepatocyte Growth Factor), Apoprotein A-2, Pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isoheptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, nonadecylic acid, isononadecylic acid, arachidic acid, 11Z-eicosenoic acid, dihomo-γ-linolenic acid, arachidonic acid, erucic acid, 13Z,16Z-docosadienoic acid, 13Z,16Z,19Z-docosadienoic acid, adrenic acid, and clupanodonic acid. acid, preferably IL-10, HGF, apoprotein A-2, pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isoheptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, arachidic acid, 11Z-eicosenoic acid, arachidonic acid, erucic acid, 13Z,Examples of suitable soluble ...

[0047] <Morphology of mesenchymal stem cells and mesenchymal stem cell-derived microparticles> The mesenchymal stem cells of the present invention may be in any state, including, for example, cells recovered by detaching cells during culture, or cells frozen in a cryopreservation solution. The use of cells obtained by expansion culture and then aliquoted and cryopreserved from the same lot is preferred in terms of stable effects and ease of handling. Cryopreserved mesenchymal stem cells may be thawed immediately before use and administered directly while suspended in a cryopreservation solution, or may be administered after being suspended in an infusion solution or medium. Alternatively, the cryopreservation solution may be removed by centrifugation or other methods before being mixed with an infusion solution or medium.

[0048] The mesenchymal stem cell-derived microparticles of the present invention may be in any state, including, for example, mesenchymal stem cell-derived microparticles isolated from cells in culture, or mesenchymal stem cell-derived microparticles frozen in a cryopreservation solution. The use of mesenchymal stem cell-derived microparticles isolated from the same lot of cells obtained by expansion culture, divided into small portions and cryopreserved, is preferred in terms of stable effects and ease of handling. Cryopreserved isolated mesenchymal stem cell-derived microparticles may be thawed immediately before use and administered directly while suspended in a cryopreservation solution, or may be administered after being suspended in an infusion solution or medium. Alternatively, the cryopreservation solution may be removed by centrifugation or other methods before mixing with an infusion solution or medium.

[0049] Here, the term "infusion solution" in the present invention refers to a solution used in human treatment, and includes, but is not limited to, physiological saline, Japanese Pharmacopoeia physiological saline, 5% glucose solution, Japanese Pharmacopoeia glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, No. 1 solution (initiation solution), No. 2 solution (dehydration replenishing solution), No. 3 solution (maintenance solution), No. 4 solution (postoperative recovery solution), etc. The above-mentioned infusion solutions or culture media may be prepared so as to contain other components (pharmaceutically acceptable carriers and additives) described below.

[0050] The therapeutic agent for dilated cardiomyopathy of the present invention may contain pharmaceutically acceptable carriers or additives according to the usual method depending on the intended use and form, as long as the effects of the present invention are not impaired. Examples of such carriers and additives include, but are not limited to, isotonicity agents, thickeners, sugars, sugar alcohols, preservatives, bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, acidulants, colorants, flavoring agents, fragrances, and refreshing agents.

[0051] The therapeutic agent for dilated cardiomyopathy of the present invention can be provided in various dosage forms, such as solids, semisolids, and liquids, depending on the purpose. For example, it can be used in the form of solids (tablets, powders, granules, capsules, etc.), semisolids (ointments (hard ointments, soft ointments, etc.), creams, etc.), liquids (lotions, extracts, suspensions, emulsions, syrups, injections (including infusions, implantable injections, sustained-release injections, and injections prepared immediately before use), dialysis agents, aerosols, soft capsules, drinks, etc.), patches, and poultices. The therapeutic agent for dilated cardiomyopathy of the present invention can also be used in the form of a solution or emulsion in an oily or aqueous vehicle. Furthermore, the therapeutic agent for dilated cardiomyopathy of the present invention can be applied to the affected area by spraying, or can be used in the form of a gel or sheet at the affected area after spraying. The therapeutic agent for dilated cardiomyopathy of the present invention can also be applied to the affected area after forming the mesenchymal stem cells into a sheet or three-dimensional structure.

[0052] The therapeutic agent for dilated cardiomyopathy of the present invention can be used by suspending or diluting it in an infusion solution such as physiological saline, Japanese Pharmacopoeia physiological saline, 5% glucose solution, Japanese Pharmacopoeia glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, Solution 1 (starting solution), Solution 2 (dehydration replenishment solution), Solution 3 (maintenance solution), or Solution 4 (postoperative recovery solution), or a cell culture medium such as DMEM, and can be used by suspending or diluting it preferably in physiological saline, 5% glucose solution, or Solution 1 (starting solution), more preferably in 5% glucose solution or Solution 1 (starting solution).

[0053] When the therapeutic agent for dilated cardiomyopathy of the present invention is a liquid preparation, the pH of the therapeutic agent for dilated cardiomyopathy is not particularly limited as long as it is within a medicamentarily, pharmacologically (pharmaceutical), or physiologically acceptable range, but an example is a range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0.

[0054] When the therapeutic agent for dilated cardiomyopathy of the present invention is in the form of a liquid, the osmotic pressure of the therapeutic agent for dilated cardiomyopathy is not particularly limited as long as it is within a range acceptable to the body. The osmotic pressure ratio of the composition of the present invention is preferably 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 by methods known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, saccharides, etc. The osmotic pressure ratio is defined as the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) according to the Japanese Pharmacopoeia, Fifteenth Edition, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for measuring osmolality ratios (0.9 w / v% sodium chloride aqueous solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650°C for 40-50 minutes, allowing it to cool in a desiccator (silica gel), accurately weighing 0.900 g of the solution, and dissolving it in purified water to make exactly 100 mL; alternatively, use a commercially available standard solution for measuring osmolality ratios (0.9 w / v% sodium chloride aqueous solution).

[0055] The therapeutic agent for dilated cardiomyopathy of the present invention may be in a form in which mesenchymal stem cells or mesenchymal stem cell-derived microparticles and a solution for suspending mesenchymal stem cells or mesenchymal stem cell-derived microparticles are sealed and stored in separate containers, and the two are mixed together when used. During storage, the mesenchymal stem cells or mesenchymal stem cell-derived microparticles and the solution for suspending mesenchymal stem cells or mesenchymal stem cell-derived microparticles may be frozen or refrigerated.

[0056] The therapeutic agent for dilated cardiomyopathy of the present invention can be suitably used for the treatment of dilated cardiomyopathy, and in particular, can be suitably used for the treatment of hereditary dilated cardiomyopathy, familial dilated cardiomyopathy, or idiopathic dilated cardiomyopathy.

[0057] The administration routes of the therapeutic agent for dilated cardiomyopathy of the present invention include direct administration to the cardiac surface, intracardiac administration, oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intrathecal administration, sublingual administration, rectal administration, vaginal administration, nasal administration, inhalation, and transdermal administration. From the viewpoint of the effectiveness of the therapeutic agent for dilated cardiomyopathy of the present invention, preferred are direct administration to the cardiac surface, intramuscular administration, intravenous administration, and intra-arterial administration.

[0058] In the therapeutic agent for dilated cardiomyopathy of the present invention, the dose (administration amount) of mesenchymal stem cells may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form of the therapeutic agent for dilated cardiomyopathy of the present invention, but from the viewpoint of achieving a sufficient therapeutic effect of the therapeutic agent for dilated cardiomyopathy, a larger amount of mesenchymal stem cells tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, a smaller amount of mesenchymal stem cells tends to be preferable. Usually, when administered (sprayed) to an adult, the number of cells is 1×10 3 ~1×10 12 Pieces / time, preferably 1 x 10 4 ~1×10 11 pieces / time, more preferably 1 x 10 5 ~1×10 10 pieces / time, more preferably 5 x 10 6 ~1×10 9 The amount of mesenchymal stem cells administered (sprayed) per patient's weight is 1 x 10 to 5 x 10 10 / kg, preferably 1 x 10 2 ~5×10 9 pieces / kg, more preferably 1×10 3 ~5×10 8 / kg, more preferably 1 x 10 4 ~5×10 7 This dose may be used as a single dose and the mesenchymal stem cells may be administered (sprayed) multiple times, or this dose may be divided into multiple doses and administered (sprayed) multiple times.

[0059] When the therapeutic agent for dilated cardiomyopathy of the present invention contains mesenchymal stem cells that secrete microparticles, the dose (administration amount) thereof may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form of the therapeutic agent for dilated cardiomyopathy of the present invention, but from the viewpoint of achieving a sufficient therapeutic effect of the therapeutic agent for dilated cardiomyopathy, a larger amount tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, a smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 1×10 3 ~1×10 12 Pieces / time, preferably 1 x 10 4 ~1×10 11 pieces / time, more preferably 1 x 10 5 ~1×10 10 pieces / time, particularly preferably 5 x 10 6 ~1×10 9 This dose may be administered as a single dose multiple times, or may be divided into multiple doses and administered.

[0060] When the therapeutic agent for dilated cardiomyopathy of the present invention contains mesenchymal stem cells that secrete microparticles, the dose (administration amount) may vary depending on the condition of the patient (body weight, age, symptoms, physical condition, etc.) and the dosage form of the therapeutic agent for dilated cardiomyopathy of the present invention. However, when administered to an adult, the number of cells is usually 1×10 to 5×10 10 / kg, preferably 1 x 10 2 ~5×10 9 / kg, more preferably 1 x 10 3 ~5×10 8 pieces / kg, particularly preferably 1 x 10 4 ~5×10 7 This dosage may be administered as a single dose multiple times, or may be administered in divided doses multiple times.

[0061] When the therapeutic agent for dilated cardiomyopathy of the present invention contains isolated microparticles or mesenchymal stem cell culture supernatant containing microparticles, the dose (administration amount) may vary depending on the condition of the patient (body weight, age, symptoms, physical condition, etc.) and the dosage form of the therapeutic agent for dilated cardiomyopathy of the present invention. Generally, when administered to an adult, the dose is 1×10 microparticles. 4 ~5×1013 / kg, preferably 1 x 10 5 ~5×10 12 / kg, more preferably 1 x 10 6 ~5×10 11 pieces / kg, particularly preferably 1 x 10 7 ~5×10 10 This dosage may be administered as a single dose multiple times, or may be administered in divided doses multiple times.

[0062] The therapeutic agent for dilated cardiomyopathy of the present invention may be administered together with one or more other drugs. Examples of other drugs include any drug that can be used as a therapeutic agent for dilated cardiomyopathy, a heart disease, such as ACE inhibitors, angiotensin II receptor antagonists, β-blockers, antiplatelet drugs, warfarin, calcium channel blockers, nitrates, diuretics, HMG-CoA reductase inhibitors, and ancalone. Administration of the therapeutic agent for dilated cardiomyopathy of the present invention together with one or more other drugs includes various cases, such as simultaneous use of the therapeutic agent for dilated cardiomyopathy of the present invention and other drugs, administration of one drug followed by administration of the other drug after a certain time has elapsed, and combinations of these.

[0063] The therapeutic agent for dilated cardiomyopathy of the present invention exhibits a significant therapeutic effect on subjects suffering from dilated cardiomyopathy. The therapeutic effect can be confirmed, for example, by echocardiography or histopathological examination (examination of cardiac hypertrophy and fibrosis). Specifically, the therapeutic agent for dilated cardiomyopathy of the present invention can suppress cardiac hypertrophy and cardiac tissue fibrosis in subjects suffering from dilated cardiomyopathy, thereby restoring cardiac function. Furthermore, this effect can be maintained for a long period of time. Furthermore, the therapeutic agent for dilated cardiomyopathy of the present invention can contribute to an increase in ATP content in cardiac tissue and an increase in mitochondrial expression of ANT-1 protein in subjects suffering from dilated cardiomyopathy, thereby improving survival rates.

[0064] <Method for preparing a therapeutic agent for dilated cardiomyopathy> The therapeutic agent for dilated cardiomyopathy of the present invention can be obtained by mixing mesenchymal stem cells or mesenchymal stem cell-derived microparticles with an appropriate cell or microparticle suspension liquid (including pharmaceutically acceptable carriers and additives) according to a conventional method, depending on the dosage form. One preferred example is a method in which mesenchymal stem cells or mesenchymal stem cell-derived microparticles are suspended in a fibrinogen solution, and the resulting suspension and a thrombin solution, or mesenchymal stem cells or mesenchymal stem cell-derived microparticles are suspended in a thrombin solution, and the resulting suspension and the fibrinogen solution are sprayed directly onto the diseased area substantially simultaneously, to prepare a gel-like therapeutic agent for dilated cardiomyopathy.

[0065] <Fibrosis inhibitor> The present invention also includes a fibrosis inhibitor for dilated cardiomyopathy containing mesenchymal stem cells or microparticles derived from mesenchymal stem cells. The therapeutic agent for dilated cardiomyopathy of the present invention described above has a remarkable effect of particularly inhibiting fibrosis in dilated cardiomyopathy, and is therefore also effective as a fibrosis inhibitor for dilated cardiomyopathy. Note that the description of the therapeutic agent for dilated cardiomyopathy described above can be applied to a specific description of the fibrosis inhibitor for dilated cardiomyopathy of the present invention.

[0066] <Cardiac hypertrophy inhibitor> The present invention also includes a cardiac hypertrophy inhibitor for dilated cardiomyopathy containing mesenchymal stem cells or microparticles derived from mesenchymal stem cells. The therapeutic agent for dilated cardiomyopathy of the present invention described above has the effect of particularly effectively inhibiting cardiac hypertrophy in dilated cardiomyopathy, and is therefore also effective as a cardiac hypertrophy inhibitor for dilated cardiomyopathy. Note that the description of the therapeutic agent for dilated cardiomyopathy described above can be applied to a specific description of the cardiac hypertrophy inhibitor for dilated cardiomyopathy of the present invention.

[0067] <Dilated cardiomyopathy treatment kit> The present invention also includes a kit for treating dilated cardiomyopathy, which includes the above-described dilated cardiomyopathy therapeutic agent, fibrosis inhibitor, or cardiac hypertrophy inhibitor of the present invention, a container, and a label. Suitable containers included in the kit of the present invention include, but are not limited to, cryotubes for freezing mesenchymal stem cells or mesenchymal stem cell-derived microparticles, bottles, vials, test tubes, etc. for mesenchymal stem cells or mesenchymal stem cell-derived microparticle suspension solutions. These containers may be made of a variety of materials, such as glass, metal, plastic, or a combination thereof. The labels on these containers contain information describing the contents.

[0068] <Treatment method> The present invention also includes a method for treating dilated cardiomyopathy, characterized by administering at least one selected from the group consisting of mesenchymal stem cells and mesenchymal stem cell-derived microparticles. According to the treatment method of the present invention, administration of mesenchymal stem cells or mesenchymal stem cell-derived microparticles to treat dilated cardiomyopathy can significantly improve the function, structure, etc. of diseased areas, such as the heart. Because the mesenchymal stem cells are less likely to cause rejection in allogeneic subjects, pre-prepared donor cells can be used after expansion and cryopreservation. Similarly, mesenchymal stem cell-derived microparticles obtained from pre-prepared donor cells after expansion and cryopreservation can be used. Therefore, compared to the preparation and use of autologous mesenchymal stem cells, commercialization is easier and more stable and consistent effects can be obtained. The treatment method of the present invention can also be considered a method for treating dilated cardiomyopathy by administering the above-mentioned dilated cardiomyopathy therapeutic agent of the present invention. The explanations regarding mesenchymal stem cells and mesenchymal stem cell-derived microparticles and other details apply to the above-mentioned dilated cardiomyopathy therapeutic agent.

[0069] The present invention also includes a method for suppressing fibrosis, particularly a method for suppressing fibrosis in dilated cardiomyopathy, which comprises administering at least one selected from the group consisting of mesenchymal stem cells and mesenchymal stem cell-derived microparticles. Furthermore, the present invention also includes a method for suppressing cardiac hypertrophy, particularly cardiac hypertrophy in dilated cardiomyopathy, which comprises administering at least one selected from the group consisting of mesenchymal stem cells and mesenchymal stem cell-derived microparticles. The suppression method of the present invention can also be said to be a method of causing fibrosis or cardiac hypertrophy by administering the above-mentioned therapeutic agent for dilated cardiomyopathy of the present invention, and the explanations regarding the mesenchymal stem cells and mesenchymal stem cell-derived microparticles in the suppression method of the present invention and other explanations can be applied to the explanations given in the above-mentioned section regarding the therapeutic agent for dilated cardiomyopathy.

[0070] The present kits may include other materials desirable from a commercial and user standpoint, including other additives, other agents, diluents, filters, needles, syringes, and package inserts with instructions for use. [Example]

[0071] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0072] [1] Preparation of the therapeutic agent for dilated cardiomyopathy of the present invention (Preparation of adipose tissue-derived mesenchymal stem cells) After obtaining consent from human donors, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To disrupt the extracellular matrix and isolate cells, collagenase (Roche) (solvent: physiological saline) was added and the tissue was dispersed by shaking at 37°C for 90 minutes. The cells were then harvested and centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the precipitate, and the cell suspension was centrifuged at 400g for 5 minutes. After removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto) and seeded into flasks. The cells were cultured at 37°C for several days in 5% CO2. After several days, the cultures were washed with PBS to remove blood cells and residual adipose tissue, and adipose tissue-derived mesenchymal stem cells (ADSCs) adhered to the plastic container were obtained.

[0073] (Cryopreservation of adipose tissue-derived mesenchymal stem cells) The resulting adipose tissue-derived mesenchymal stem cells were detached using trypsin, transferred to a centrifuge tube, and centrifuged at 400 g for 5 minutes to obtain a cell pellet. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER (Zenoac)) was added and the cell suspension was suspended. The cell suspension was dispensed into cryotubes and stored at -80°C in a freezer, then transferred to the vapor phase above liquid nitrogen for continued storage.

[0074] (Analysis of cell surface markers (flow cytometry)) Evaluation of various surface markers on adipose tissue-derived mesenchymal stem cells was performed by flow cytometry. Adipose tissue-derived mesenchymal stem cells were resuspended in FACS staining buffer. Antibodies used for FACS analysis were FITC (fluorescein isocyanine)- or PE (phycoerythrin)-labeled mouse anti-human antibodies CD11b, CD45, CD73, and CD90, as well as the corresponding mouse IgG1 isotype control antibodies. Cells were stained for 30 minutes at room temperature, washed, and analyzed using a BD FACSCanto II (BD Biosciences, San Jose, CA). Data were analyzed using a BD FACSDiva Software Cre (BD Biosciences). The results showed that adipose tissue-derived mesenchymal stem cells were negative for CD45 and positive for CD73 and CD90.

[0075] (Preparation of a spray-type dilated cardiomyopathy treatment agent) Fibrinogen and thrombin solutions for spraying were prepared using Beriplast RP Combi-Set Tissue Adhesion (CLS Behring, Co., Ltd.). The fibrinogen solution (Beriplast Solution A) contained 80 mg / mL fibrinogen, 60 IU Factor XIII, and 5,000 KIE bovine aprotinin. The thrombin solution (Beriplast Solution B) contained 300 units / mL thrombin. Specifically, cryopreserved adipose tissue-derived mesenchymal stem cells (ADSCs) were thawed immediately before spraying and suspended in HBSS (×1) in the amount shown in Table 1 below. Solution A was prepared by adding Beriplast Solution A. Solution B was prepared by adding Beriplast B to HBSS (×1) in the amount shown in Table 1 below. These solutions were each sealed in separate syringes and used in the transplantation test described below. [Table 1]

[0076] [2] Transplantation Study 1 (Transplantation trial protocol) After thoracotomy in J2N-k hamsters (20-week-old, male), solutions A and B, each containing the composition shown in Table 1, were directly and simultaneously dripped onto the surface of the heart (intrapericardial) where dilated cardiomyopathy had developed (ADSC group). In the vehicle-treated group, J2N-k hamsters (20-week-old, male), each containing the composition shown in Table 2, were directly and simultaneously sprayed onto the surface of the heart (intrapericardial) where dilated cardiomyopathy had developed (Control group). In the sham-operated group, J2N-k hamsters (20-week-old, male) and J2N-n hamsters (20-week-old, male), normal hamsters without cardiomyopathy, underwent thoracotomy only, without dripping any of the above drugs (Sham and Normal groups, respectively).

[0077] [Table 2]

[0078] Echocardiography was performed before transplantation (baseline) and at 1, 2, 3, and 4 weeks after transplantation. At the end of the study, animals were humanely sacrificed 4 weeks after cell transplantation for histological and biochemical analysis of cardiac tissue. The transplantation study is described in detail below.

[0079] (Adipose-derived mesenchymal stem cell transplantation experiment) J2N-k hamsters (20 weeks old, male) and J2N-n hamsters (20 weeks old, male) underwent median sternotomy under general anesthesia, and either adipose tissue-derived mesenchymal stem cells, vehicle transplantation, or sham surgery was performed. The site of myocardial infarction can be visually confirmed based on the surface scar and abnormal wall movement. Specifically, in the ADSC group, the composition (1 x 10 cells) shown in Table 1 above, sealed in separate syringes, was administered. 6 Solutions A (20 μL) and B (20 μL) containing adipose tissue-derived mesenchymal stem cells were simultaneously sprayed directly onto the surface of the myocardial infarction (the epicardial surface) to cover the affected area. J2N-k and J2N-n hamsters were allowed to recover in individual temperature-controlled cages.

[0080] (Effects of transplantation of adipose tissue-derived mesenchymal stem cells) The effects of transplanting adipose tissue-derived mesenchymal stem cells into J2N-k and J2N-n hamsters were evaluated by cardiac function and histopathological examination. LVEF was measured by echocardiography to examine cardiac function. Histopathological examination evaluated cardiac hypertrophy and fibrosis. ATP content in myocardial tissue was also measured. Furthermore, mitochondria were isolated from cardiac tissue, and adenine nucleotide translocator-1 (Mt.ANT-1, gene symbol: SLC25A4), which is expressed in mitochondria, was measured by Western blotting. Each evaluation method is explained below.

[0081] (echocardiography) J2N-k and J2N-n hamsters were anesthetized as described above. Echocardiography was performed using a commercially available echocardiography system (HITACHI: PROSOUND F75 PremierCV). An 8.0-MHz circular array transducer was used for cardiac evaluation. J2N and J2N-n hamsters were examined in the left lateral decubitus position. LV end-diastolic and end-systolic volumes (LVEDV and LVESV) were calculated using the Teichholz formula. LV ejection fraction (LVEF) was calculated using the following formula. The results are shown in Figure 1. LVEF(%)=100×(LVEDV-LVESV) / (LVEDV)

[0082] As shown in Figure 1, in the sham group, which underwent only thoracotomy, and the control group, which received the vehicle, LVEF decreased over time, and cardiac function declined. However, in the adipose tissue-derived mesenchymal stem cell transplant group, no decline in LVEF was observed, and the progression of dilated cardiomyopathy was suppressed.

[0083] (Measurement of ATP content in cardiac tissue) The ATP content in cardiac tissue two weeks after transplantation was measured using a commercially available ATP measurement kit. As shown in Figure 2, the sham group, which underwent only thoracotomy, and the control group, which received vehicle, showed a significant decrease in ATP content compared to normal hamsters. In contrast, the group transplanted with adipose tissue-derived mesenchymal stem cells showed no decrease in ATP content and showed a significant increase in ATP content compared to the sham group.

[0084] (Measurement of mitochondrial Mt.ANT-1 / COX4 in cardiac tissue) Mitochondria were isolated from cardiac tissue 2 weeks after transplantation, and the expression of adenine nucleotide translocator-1 (Mt.ANT-1, gene symbol: SLC25A4) expressed in mitochondria was measured by Western blotting. Cytochrome c oxidase 4 (COX4) was used as an endogenous control.

[0085] As shown in Figure 3, compared with normal hamsters, the sham group, which underwent only thoracotomy, and the control group, which received vehicle, had significantly lower Mt.ANT-1 expression levels. In contrast, in the group transplanted with adipose tissue-derived mesenchymal stem cells (ADSCs), Mt.ANT-1 expression levels were significantly higher than in the sham and control groups. Similarly, Mt.ANT-1 in cardiac tissue was measured 4 weeks after transplantation. As a result, as with 2 weeks after transplantation, the expression levels of Mt.ANT-1 were significantly lower in the sham and control groups, but in the group transplanted with adipose tissue-derived mesenchymal stem cells (ADSCs), the expression levels were elevated to a level that was not significantly different from the normal group.

[0086] (Heart size measurement) Four weeks after transplantation, cardiac diameter was measured under a microscope using HE-stained cardiac specimens. As shown in Figure 4, in the sham group, which underwent only thoracotomy, and the control group, which received vehicle, dilated cardiomyopathy progressed and the hearts became enlarged compared to normal hamsters. In contrast, in the adipose tissue-derived mesenchymal stem cell transplantation group, cardiac hypertrophy was significantly suppressed compared to the sham group, and dilated cardiomyopathy was improved.

[0087] (Measurement of fibrosis area) Four weeks after transplantation, the area of ​​fibrosis was measured using Sirius Red-stained cardiac specimens. Compared to the sham group, which only underwent thoracotomy, the area of ​​fibrosis in group J, which received transplantation of adipose tissue-derived mesenchymal stem cells, was 78.3%. This demonstrated that mesenchymal stem cells suppress fibrosis in dilated cardiomyopathy.

[0088] (Measurement of δ-sarcoglycan expression level) Four weeks after transplantation, the expression of δ-sarcoglycan in the heart was measured by immunohistochemistry. No expression of δ-sarcoglycan was observed in the sham, sham, or ADSC groups, demonstrating that the δ-sarcoglycan gene is not affected by adipose tissue-derived mesenchymal stem cells.

[0089] [3] Transplantation Study 2 As in Transplantation Test 1, J2N-k hamsters (20 weeks old, male) underwent a median sternotomy under general anesthesia, and adipose tissue-derived mesenchymal stem cells (ADSCs) or a medium (Control) were transplanted. Specifically, as in Transplantation Test 1, in the ADSC group, a solution of the composition shown in Table 1 above (1 x 10 6Solutions A (20 μL) and B (20 μL) containing adipose tissue-derived mesenchymal stem cells (containing 10 adipose tissue-derived mesenchymal stem cells) were simultaneously dripped directly onto the surface of the affected area (the cardiac surface) to cover the affected area. These J2N-k hamsters were allowed to recover in individual temperature-controlled cages. The effects of adipose tissue-derived mesenchymal stem cell transplantation into J2N-k hamsters were evaluated by echocardiography prior to transplantation (baseline) and at 2, 4, 8, 12, 16, and 20 weeks after transplantation. LVEF was measured by echocardiography as in Transplantation Study 1 to assess cardiac function. The animals used in this study were humanely sacrificed 20 weeks after cell transplantation.

[0090] As shown in Figure 5, in the control group administered with the vehicle, LVEF decreased over time, but in the adipose tissue-derived mesenchymal stem cell (ADSC) transplant group, no decrease in LVEF was observed and the progression of dilated cardiomyopathy symptoms was suppressed. Furthermore, this effect of suppressing the progression of dilated cardiomyopathy symptoms was maintained for as long as 20 weeks after transplantation.

[0091] [4] Transplantation Study 3 As in Transplantation Test 1, J2N-k hamsters (20 weeks old, male) underwent a median sternotomy under general anesthesia, and either adipose tissue-derived mesenchymal stem cells (ADSCs) or a vehicle (Control) were transplanted. Specifically, as in Transplantation Test 1, the ADSC group received either adipose tissue-derived mesenchymal stem cells (ADSCs) or a vehicle (Control) containing the composition shown in Table 1 (1 x 10 6 Solutions A (20 μL) and B (20 μL) containing adipose tissue-derived mesenchymal stem cells were simultaneously sprayed directly onto the surface of the myocardial infarction (the epicardial surface) to cover the affected area. J2N-k hamsters were allowed to recover in individual temperature-controlled cages.

[0092] As a result, the mortality rate after 12 weeks was 28.6% in the control group, compared to 12.5% ​​in the ADSC-administered group, demonstrating that transplantation of adipose tissue-derived mesenchymal stem cells reduced the mortality rate.

[0093] [5] Preparation of microparticles and investigation of their effects on cardiomyocytes The culture supernatant of adipose-derived mesenchymal stem cells prepared in the same manner as above was collected, filtered through a filter (0.22 μm, Merck Millipore), and then centrifuged (35,000 rpm, 70 minutes, 4°C, Beckman Optima XE-90) to collect the microparticles. The average particle size of the obtained microparticles was 155 ± 6 nm (mean ± standard error, n = 3).

[0094] Microparticles collected from 60 mL of mesenchymal stem cell culture supernatant were suspended in 0.5 mL of PBS. The resulting adipose-derived mesenchymal stem cell-derived microparticles were then transferred to human cardiomyocytes (PromoCell, 5 × 10 5 When 10μL, 30μL, and 100μL of microparticles were added to the 100μL, 30μL, and 100μL of cells / dish, respectively, and NADPH activity was measured as an indicator of energy production, the amount of energy production increased in a dose-dependent manner (Figure 6). This suggests that the microparticles secreted by mesenchymal stem cells may be involved in the cardiac function improvement effect of mesenchymal stem cells.

[0095] [6] Analysis of proteins contained in microparticles 50 mL of Total Exosome Isolation (Thermo Fisher Scientific Inc., product number 4478359) was added to 100 mL of adipose-derived mesenchymal stem cell culture supernatant (serum-free medium) and thoroughly mixed by inversion. After storing overnight in a refrigerator (2-8°C), the mixture was centrifuged (10,000 × g, 1 hour, 2-8°C) and the supernatant was removed. The resulting sediment was suspended in 500 μL of PBS, and this suspension was used as a microparticle-containing suspension. 150 μL of 20% trichloroacetic acid (TCA, Wako Pure Chemical Industries, Ltd.) was added to 150 μL of the microparticle-containing suspension to aggregate and precipitate the protein. 20 μL of Tris Hydrochloride Acid Buffer (Tris-HCl), pH 8.5, was added to the entire precipitate to dissolve the precipitate. Further, Tris buffer (Tris-HCl, pH 8.0) containing 0.01% trypsin (Aproscience) was added and allowed to react for 20 hours at 37°C. The resulting sample solution was then analyzed by LC-MS / MS (LC: Michrom BioResources, MS: ThermoFisher Scientific), and apolipoprotein A-2, pigment epithelium-derived factor (PEDF), SERPINF1, and haptoglobin were detected. [Industrial Applicability]

[0096] The therapeutic agent for dilated cardiomyopathy of the present invention can significantly improve cardiac function and other conditions associated with dilated cardiomyopathy. Because the mesenchymal stem cells are unlikely to cause rejection in allogeneic subjects, cells from a donor whose therapeutic efficacy has been confirmed can be expanded and cryopreserved and used as mesenchymal stem cells in the therapeutic agent for dilated cardiomyopathy of the present invention. Therefore, compared to the case where autologous mesenchymal stem cells are prepared and used, this method has the advantage of being easier to commercialize and more likely to produce a consistent effect.

Claims

1. A therapeutic agent for dilated cardiomyopathy, comprising at least one selected from the group consisting of adipose-derived mesenchymal stem cells and microparticles derived from adipose-derived mesenchymal stem cells.

2. A therapeutic agent for dilated cardiomyopathy as described in claim 1, wherein the adipose-derived mesenchymal stem cells have the ability to contain or secrete microparticles.

3. A therapeutic agent for dilated cardiomyopathy according to claim 1 or 2, wherein the average particle diameter of the microparticles is 1,000 nm or less.

4. A therapeutic agent for dilated cardiomyopathy described in any one of claims 1 to 3, wherein the microparticles are exosomes.

5. The therapeutic agent for dilated cardiomyopathy according to claim 1, wherein the microparticles contain at least one factor selected from the group consisting of IL-10, HGF, apolipoprotein A-2, pigment epithelium-derived factor (PEDF), SERPINF1, and haptoglobin.

6. A therapeutic agent for dilated cardiomyopathy according to any one of claims 1 to 5, wherein the adipose-derived mesenchymal stem cells are allogeneic to the subject.

7. A therapeutic agent for dilated cardiomyopathy described in any one of claims 1 to 6, wherein the dilated cardiomyopathy is selected from the group consisting of idiopathic dilated cardiomyopathy, familial dilated cardiomyopathy, and hereditary dilated cardiomyopathy.

8. A therapeutic agent for dilated cardiomyopathy according to any one of claims 1 to 7, wherein the adipose-derived mesenchymal stem cells are cryopreserved cells.

9. A fibrosis inhibitor for dilated cardiomyopathy, comprising at least one selected from the group consisting of adipose-derived mesenchymal stem cells and microparticles derived from adipose-derived mesenchymal stem cells.

10. A cardiac hypertrophy inhibitor for dilated cardiomyopathy, comprising at least one selected from the group consisting of adipose-derived mesenchymal stem cells and microparticles derived from adipose-derived mesenchymal stem cells.

11. A kit for treating dilated cardiomyopathy, comprising a therapeutic agent for dilated cardiomyopathy described in any one of claims 1 to 8, a fibrosis inhibitor described in claim 9 or a cardiac hypertrophy inhibitor described in claim 10, a container and a label.

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