WO2017110425A1 - Therapeutic agent for liver disease and method for treating liver disease - Google Patents

Abstract

The purpose of the present invention is to provide a novel therapeutic agent for a liver disease. Provided is a therapeutic agent for liver disease that comprises microparticles derived from interstitial cells. The microparticles preferably have an average particle size of 1,000 nm or smaller. The therapeutic agent for a liver disease according to the present invention preferably comprises interstitial cells that contain the aforesaid microparticles or are capable of secreting the microparticles.

Description

Liver disease therapeutic agent and method for treating liver disease

The present invention relates to a therapeutic agent for liver disease and a method for treating liver disease.

Acute hepatitis is an acute diffuse disease with symptoms such as jaundice, loss of appetite, nausea and vomiting, general malaise, and fever. Prognosis is generally good, but about 1-2% of patients become fulminant, and once fulminant, die at a high rate. As the pathogenesis of hepatitis, hepatitis virus is mainly known. It is said that hepatitis caused by this hepatitis virus is caused not by the virus itself destroying hepatocytes but by the destruction of hepatocytes infected with the virus by an immunological mechanism (Non-Patent Document). 1).

On the other hand, autoimmune hepatitis is known as a hepatitis other than viruses. This autoimmune hepatitis has both acute and chronic clinical symptoms, but is a disease caused by an autoimmune reaction due to failure of immunological tolerance to the hepatocytes by some mechanism. The involvement of preceding infections and drugs has also been suggested as triggers for the onset. The number of autoimmune hepatitis patients in Japan is about 20,000, accounting for about 1.8% of chronic hepatitis. About 30% of deaths due to autoimmune hepatitis are deaths within half a year from diagnosis, and treatment for acute liver failure at the time of diagnosis is important for improving prognosis (see Non-Patent Document 2). . As a result of repeated liver regeneration and connective tissue renewal due to chronic inflammation, an increase in the extracellular matrix mainly composed of collagen (liver fibrosis) is observed, leading to cirrhosis. When the compensation period for cirrhosis progresses to the non-compensation period with a higher degree of fibrosis, existing drugs and therapies cannot adequately cope with it. Therefore, development of a novel therapeutic agent having a different mechanism of action from the conventional one is desired.

Mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (1982) (see Non-Patent Document 3). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases ( (See Patent Documents 1 and 2). Recently, it is known that cells having equivalent functions exist in stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, the mesenchymal stem cell may be referred to as a stromal cell (Mesenchymal Stroma Cell).

JP 2012-157263 A Special table 2012-508733 gazette

Liver specialist text, pp. 186-190, Nankodo Co., Ltd., issued on March 30, 2013 Liver specialist text, pp. Issued 205-208, Nanedo Co., Ltd., March 30, 2013 Pittenger F. M.M. et al. , Science 284, pp. 143-147 (1999)

An object of the present invention is to provide a novel therapeutic agent and a novel treatment method for liver diseases in the above situation.

In order to solve the above problems, the present inventors examined the effect of stromal cells, focusing on hepatitis that causes cirrhosis, particularly autoimmune hepatitis. In addition, we focused on microparticles such as extracellular vesicles that play an important role in cell-to-cell information transmission, and worked to elucidate the mechanism of action. As a result, it has been found that a composition containing microparticles derived from stromal cells is effective in treating liver diseases. That is, the gist of the present invention is as follows.

(1) A therapeutic agent for liver disease, comprising stromal cell-derived microparticles.
(2) The therapeutic agent for liver disease according to (1), wherein the average particle size of the fine particles is 1,000 nm or less.
(3) The therapeutic agent for liver disease according to (1) or (2), comprising stromal cells having the ability to contain or secrete the microparticles.
(4) The therapeutic agent for liver disease according to any one of (1) to (3), wherein the microparticle is an exosome.
(5) The microparticles are composed of IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1), and haptoglobin (Haptoglobin). The therapeutic agent for liver disease according to any one of (1) to (4), which comprises at least one factor selected from the group.
(6) The therapeutic agent for liver disease according to any one of (1) to (5), wherein the stromal cells are derived from adipose tissue.
(7) The therapeutic agent for liver disease according to any one of (1) to (6), wherein the liver disease is an autoimmune liver disease.
(8) The therapeutic agent for liver disease according to (7), wherein the autoimmune liver disease is autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), or primary biliary cirrhosis (PSC).
(9) A method for treating liver disease by administering stromal cell-derived microparticles to a patient.
(10) The method according to (9), wherein the average particle size of the fine particles is 1,000 nm or less.
(11) The method according to (9) or (10), wherein stromal cells having the ability to contain or secrete the microparticles are administered to a patient.
(12) The method according to any one of (9) to (11), wherein the microparticle is an exosome.
(13) The microparticles include IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1), and haptoglobin (Haptoglobin). The method according to any one of (9) to (12), comprising at least one factor selected from the group.
(14) The method according to any one of (9) to (13), wherein the stromal cells are derived from adipose tissue.
(15) The method according to any one of (9) to (14), wherein the liver disease is an autoimmune liver disease.
(16) Any of (9) to (15), wherein the autoimmune liver disease is autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), or primary biliary cirrhosis (PSC). the method of.

According to the present invention, a novel therapeutic agent for liver disease and a novel treatment method for liver disease can be provided.

Fig. 1 shows that in the adipose-derived stromal cells (KD-hASC) in which nSMase2 was knocked down, the production of microparticles was suppressed and the secretion amount of the microparticles was significantly reduced compared to the control cells (Mock-hASC). It is a figure which shows doing. FIG. 2 shows the amount of cytokine (IL-10) in microparticles in the culture supernatant of fat-derived stromal cells (KD-hASC) and control cells (Mock-hASC) knocked down by nSMase2 by ELISA. It is a figure which shows a result. FIG. 3 shows the results of measuring the amount of cytokine (HGF) in microparticles in the culture supernatant of fat-derived stromal cells (KD-hASC) and control cells (Mock-hASC) knocked down by nSMase2 by ELISA. FIG. FIG. 4 is a graph showing the therapeutic effect of adipose-derived stromal cells on autoimmune liver disease by measuring the blood AST value of model mice with autoimmune liver disease. The adipose-derived stromal cells knocked down nSMase2 (KD-hASC) have a reduced therapeutic effect compared to control cells (Mock-hASC). FIG. 5 is a graph showing the therapeutic effect of adipose-derived stromal cells on autoimmune liver disease by measuring the blood ALT value of model mice with autoimmune liver disease. The adipose-derived stromal cells knocked down nSMase2 (KD-hASC) have a reduced therapeutic effect compared to control cells (Mock-hASC). FIG. 6 is a graph showing the therapeutic effect of adipose-derived stromal cells on the liver tissue of a model mouse with autoimmune liver disease. It has been shown that adipose-derived stromal cells (KD-hASC) knocked down nSMase2 have a reduced therapeutic effect on lymphocyte infiltration compared to control cells (Mock-hASC).

Hereinafter, the therapeutic agent for liver disease of the present invention will be described in detail.

The therapeutic agent for liver disease of the present invention contains stromal cell-derived microparticles. The therapeutic agent for liver disease according to the present invention preferably contains stromal cells having the ability to contain or secrete the microparticles. In the therapeutic agent for liver disease of the present invention, the microparticles may be contained in any form, including including in the form of stromal cells having the ability to contain or secrete the microparticles. That is, the therapeutic agent for liver disease of the present invention may contain microparticles alone, or may contain stromal cells having the ability to contain or secrete the microparticles together with the microparticles. Stromal cells having the ability to contain or secrete may be contained alone. Furthermore, other components may be included as long as the effects of the present invention are not impaired. The disease therapeutic agent of the present invention will be described in detail below.

<Stromal cells>
The stromal cell in the present invention is a cell that has the ability to differentiate into cells belonging to the mesenchymal system (bone cells, cardiomyocytes, chondrocytes, tendon cells, fat cells, etc.) and can proliferate while maintaining this differentiation ability. Means. For example, bone marrow, fat, blood, periosteum, dermis, umbilical cord, umbilical cord blood, placenta, amniotic membrane, chorion, decidua, muscle, synovial membrane, synovial fluid, dental pulp, nerve, menstrual blood, peripheral blood, etc. Preferably, it is a stromal cell derived from fat, more preferably an adult fat. Here, “derived” means stromal cells contained in the tissue. For example, adipose-derived stromal cells are differentiated into cells belonging to the mesenchymal system existing in adipose tissue. It means a cell having the ability.

The stromal cells in the present invention may be derived from the same species as the subject (subject) to be treated, or may be derived from different species. Examples of the stromal cell species in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, and preferably cells derived from the same species as the subject to be treated (subject). The stromal cells in the present invention may be derived from a subject (subject) to be treated, that is, autologous cells, or may be derived from another subject of the same type, that is, autologous cells. An allogeneic cell is preferable.

In addition to having the ability to contain or secrete the microparticles described above, the stromal cells in the present invention include, for example, growth characteristics (eg, population doubling ability from passage to aging, doubling time), karyotype analysis (eg, Normal karyotype, maternal or neonatal strain), surface marker expression by flow cytometry (eg FACS analysis), immunohistochemistry and / or immunocytochemistry (eg epitope detection), gene expression profiling (eg 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 cytokine (eg, plasma coagulation analysis, ELISA, cytokine array), metabolite (metabolome analysis) ), Other methods known in the field By, it may be characterized.

The stromal cell acquisition method and culture method in the present invention are not particularly limited, and for example, the following methods can be used. That is, (a) a step of treating a tissue containing stromal cells with an enzyme or the like, (b) a step of suspending the cell suspension obtained by the enzyme treatment in an appropriate culture medium and performing an adhesion culture, The stromal cells can be obtained and cultured by a method including a step of c) removing floating cells and a step of (d) culturing stromal cells. Each step will be described in detail below.

In step (a), the tissue containing stromal cells such as adipose tissue is washed with physiological saline (for example, phosphate buffered saline (PBS)) and the like by stirring and sedimenting. Is preferred. By this operation, contaminants (also referred to as debris, such as damaged tissue, blood, and red blood cells) contained in the tissue are removed from the tissue. Accordingly, washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. The remaining cells exist as clumps of various sizes, so that the cell clumps after washing are weakened or disrupted in order to dissociate while minimizing damage to the cells themselves. It is preferable to treat with an enzyme (for example, collagenase, dispase, trypsin, etc.). The amount of enzyme used and the treatment period vary depending on the conditions used, but can be performed within the scope of common general technical knowledge in the art. Instead of or in combination with such enzyme treatment, the cell mass can be decomposed by other treatment methods such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal cell damage In order to suppress it, it is preferable to carry out only by enzyme treatment. When an enzyme is used, in order to minimize harmful effects on cells, it is desirable to inactivate the enzyme using a medium or the like after treatment with the enzyme.

The cell suspension obtained by the step (a) includes a slurry or suspension of aggregated cells and various contaminated cells such as erythrocytes, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the cells in the aggregated state and these contaminating cells may be subsequently separated and removed, but since they can be removed by removing floating cells or the like in step (c) described later, the separation and removal are You may omit it. When separating and removing contaminating cells, it can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically compatible solvent. Suspended cells may contain erythrocytes, but erythrocytes are excluded by selection by adhesion to the individual surface described later, and thus a lysis step is not always necessary. As a method for selectively lysing red blood cells, for example, a method well known in the art such as incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride can be used. After lysis, the lysate may be separated from the desired cells, for example, by filtration, centrifugation, or density fractionation.

In step (b), in order to increase the purity of the stromal cells in the suspended cells, the cells may be washed once or continuously several times, centrifuged, and resuspended in the medium. Alternatively, cells may be separated based on cell surface marker profile or based on cell size and granularity.

The medium used for resuspension is not particularly limited as long as it can culture stromal cells. For example, it can be prepared by adding serum and / or a serum substitute to a basic medium for animal cells. . Moreover, you may use what is marketed as a culture medium suitable for culture | cultivation of a stromal cell. In the present invention, since the stromal cells and the culture supernatant thereof are used for the treatment of diseases of animals (including humans), it should be a medium (eg, serum-free medium) that contains as little biological material as possible. preferable. In particular, a medium containing no xenogeneic component (xeno-free) is preferred.

The composition of the basal medium can be appropriately selected according to the type of cells to be cultured. For example, Minimum Essential Medium (MEM) such as Eagle Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium α (MEM-α), Mesenchymal Cell Basal Medium (MSCBM), Ham's F-12 and F -10 medium, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified Dulbecco medium (IMDM), McCoy modified medium, and the like.

Examples of 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, rat serum and the like. When using serum, 0.5% to 15%, preferably 5% to 10%, may be added to the basal medium.

Examples of the serum substitute added to the basal medium include albumin, transferrin, fatty acid, insulin, sodium selenite, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiolglycerol, and the like.

In the basal medium, if necessary, amino acids, inorganic salts, vitamins, growth factors, antibiotics, trace metals, stem cell differentiation inducers, antioxidants, carbon sources, salts, sugars, sugar precursors, Plant-derived hydrolyzate, surfactant, ammonia, lipid, hormone, buffer, indicator, nucleoside, nucleotide, butyric acid, organic matter, DMSO, animal-derived product, gene inducer, intracellular pH regulator, betaine, osmotic pressure protection Substances such as agents and minerals may be added, but are not limited to these substances. The use concentration of these substances is not particularly limited, and can be used at a concentration used in a normal medium for mammalian cells.

Examples of the amino acid include glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, Examples include L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like.

Examples of the inorganic salts include calcium chloride, copper sulfate, iron (III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate. Etc.

Examples of the vitamins include choline, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, vitamin B7, vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin Bh, vitamin Examples include Bt, vitamin Bx, vitamin C, vitamin D, vitamin E, vitamin F, vitamin K, vitamin M, vitamin P and the like.

Other specific substances that can be added to the basal medium include basic fibroblast growth factor (bFGF), endothelial growth factor (EGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like Growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), hepatocyte growth factor (HGF) and other growth factors; penicillin, streptomycin, neomycin sulfate, amphotericin B, blasticidin Lambphenicol, Amoxicillin, Bacitracin, Bure Antibiotics such as mycin, cephalosporin, chlortetracycline, zeocin and puromycin; carbon sources such as glucose, galactose, fructose, sucrose; magnesium, iron, zinc, calcium, potassium, sodium, copper, selenium, cobalt, tin, Trace metals such as molybdenum, nickel, silicon; β-glycerophosphate, dexamethasone, rosiglitazone, isobutylmethylxanthine, 5-azacytidine and other stem cell differentiation inducers; 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc. Antioxidants: adenosine 5'-monophosphate, corticosterone, ethanolamine, insulin, reduced glutathione, lipoic acid, melatonin, hypoxanthine, phenol red, progestero , Putrescine, pyruvic acid, thymidine, triiodothyronine, transferrin, lactoferrin and the like.

A serum-free medium suitable for stromal cells in the present invention includes a commercially available serum-free medium. Examples thereof include those provided as media prepared in advance for stromal cells from PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, and the like.

Subsequently, the stromal cells are cultured on a solid surface such as a culture vessel without differentiating using the above-described appropriate medium at an appropriate cell density and culture conditions. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish or a flask is preferably used. The culture conditions for stromal cells in the present invention are not particularly limited as long as they are suitable for each stromal cell, and the same method as in the past can be used. Usually, it is performed at a temperature of 30 ° C. to 37 ° C., in a 2% to 7% CO ₂ environment, and in a 5% to 21% O ₂ environment. Further, the passage time and method of stromal cells are not particularly limited as long as they are suitable for each cell, and can be performed in the same manner as before while observing the state of the cells.

In step (c), non-adherent floating cells and cell debris are removed from the solid surface of the culture vessel, and the adherent cells are washed with physiological saline (for example, phosphate buffered saline (PBS)). . In the present invention, cells that finally remain attached to the solid surface of the culture vessel can be selected as a cell population of stromal cells.

In step (d), stromal cells are cultured. The culture method is not particularly limited as long as it is a method suitable for each cell, and a conventional method is used. Usually, it is performed at a temperature of 30 ° C. to 37 ° C., in a 2% to 7% CO ₂ environment, and in a 5% to 21% O ₂ environment. Further, the passage time and method of stromal cells are not particularly limited as long as they are suitable for each stromal cell, and can be performed in the same manner as before while observing the form of stromal cells. As the medium used for the culture, the same medium as in step (b) can be used. In addition, you may perform using a serum-free culture medium etc. over the whole culture | cultivation period of a cell.

<Small particles derived from stromal cells>
Stromal cell-derived microparticles in the present invention are microparticles obtained from stromal cells and are produced by stromal cells. The microparticles derived from stromal cells in the present invention may be contained in stromal cells, may be contained in stromal cell culture supernatants, or may be isolated from stromal cells or stromal cell culture supernatants. Separated microparticles may be used. That is, the stromal cell-derived microparticles in the present invention may be in any form, and may be in the form of stromal cells having the ability to contain or secrete microparticles as well as the microparticles themselves. Good. Examples of the method for isolating microparticles include ultracentrifugation, microfiltration, antibody capture, and use of a microfluidic system. The isolated microparticles may contain cells such as stromal cells, and may contain stromal cell culture medium.

The stromal cell-derived microparticles in the present invention can be recovered from the culture supernatant obtained by culturing the stromal cells using the above-mentioned medium. When recovering the culture supernatant, for example, the stromal cells are brought into a subconfluent or confluent state in a culture vessel, exchanged with a new medium, and further cultured for 1 to 5 days. Kiyo can be recovered. The culture supernatant can also be used as a therapeutic agent for liver disease of the present invention. Microparticles are separated by ultracentrifugation, density gradient centrifugation, various microparticle separation kits, etc. It can also be used as a material for a disease therapeutic agent.
The therapeutic agent for liver disease of the present invention may contain stromal cells themselves that include or secrete the microparticles in addition to the microparticles. In addition, when the therapeutic agent for liver disease includes the stromal cells including or secreting the microparticles, satisfying the requirement that the microparticles are included at the same time by including the stromal cells. It can be interpreted as well.

The stromal cell-derived microparticles in the present invention are vesicles of a size that can be confirmed with an electron microscope, typically released from stromal cells. As for the size of the fine particles, the average particle diameter is about 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 is an average value of the diameters of the respective particles measured by a dynamic light scattering method or an electron microscope. The microparticle can have a lipid bilayer surrounding a biomolecule. Examples of the microparticle include membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (microvesicles, average particle size 30 to 1,000 nm), exosome-like vesicles, exosomes, average Particle diameters of 30 to 200 nm), ectosome-like vesicles, ectosomes or exovesicles, and the like, preferably exosomes. Microparticles from different types of stromal cells can be derived from intracellular sources, density of microparticles in sucrose, shape, sedimentation rate, lipid composition, protein markers, and mode of secretion (ie after signal (inducible) or spontaneous (Construction)). The fine particles are fractionated to 1.0 to 1.5 g / mL, preferably 1.1 to 1.3 g / mL, for example, by density gradient centrifugation. Furthermore, the microparticle contains any one of phosphatidylserine, cholesterol, sphingomyelin and ceramide as its constituent lipid.

The stromal cell-derived microparticles in the present invention include nucleic acids such as proteins, fatty acids, and miRNA.

Examples of the protein and fatty acid include IL-10, HGF (Hepatocyte Growth Factor), Apolipoprotein A-2 (Apolipoprotein A-2), Pigment epithelium-derived factor (PEDF), SERPINF1 Gin, and SERPINF1 Gin. (Haptoglobin), pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, Isoheptadecanic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, nonadecylic acid , Isononadecylic acid, arachidic acid, 11Z-eicosenoic acid, Di omo-γ-linolenic acid, arachidonic acid, erucic acid, 13Z, 16Z-Docosadienic acid, 13Z, 16Z, 19Z-Docosadienic acid, adrenoic acid, Clupanodonic acid, etc., preferably IL-10, HGF-2, apolipoprotein Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid , Margaric acid, Isoheptadecanic acid, steari Acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, arachidic acid, 11Z-eicosenoic acid, arachidonic acid, erucic acid, 13Z, 16Z-Docosadienic acid, more preferably IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1, haptoglobin, myristic acid, pentadecanoic acid, palmitic acid, marcoic acid, isodeic steaic acid Linoleic acid, nonadecylic acid, erucic acid, 13Z, 16Z-Docosadienic cid, particularly preferably IL-10, HGF, palmitic acid, stearic acid, oleic acid, 13Z, include 16Z-Docosadienoic acid, preferably a microparticle containing them.

<Other ingredients>
The therapeutic agent for liver disease of the present invention may contain a pharmaceutically acceptable carrier or additive according to a conventional method according to its use or form as long as the effect of the present invention is not impaired. Examples of such carriers and additives include isotonic agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents. , Oily base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizer, dispersant, emulsifier, buffer, solubilizer , Antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances or refreshing agents, but are not limited thereto. Examples of typical components include the following carriers and additives.

Carrier: An aqueous carrier such as water or hydrous ethanol.
Isotonizing agents (inorganic salts): For example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like.
Polyhydric alcohol: For example, glycerin, propylene glycol, polyethylene glycol and the like.
Thickeners: for example, carboxyvinyl polymer, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, alginic acid, polyvinyl alcohol (completely or partially saponified product), polyvinylpyrrolidone, macrogol and the like.
Sugars: For example, cyclodextrin, glucose and the like.
Sugar alcohols: For example, xylitol, sorbitol, mannitol and the like. These may be d-form, l-form or dl-form.
Antiseptics, bactericides or antibacterials: for example, dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, sorbine Potassium acid, trometamol, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically polyhexanide hydrochloride ( Polyhexamethylene biguanide)), glow kill (trade name, manufactured by Rhodia), etc.
pH regulators: for example, hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, boro Sand, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, magnesium sulfate, phosphoric acid, polyphosphoric acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconolactone , Ammonium acetate and the like.
Stabilizers: for example, dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (Longalite), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glyceryl monostearate, sodium bisulfite, sodium sulfite and the like.
Oily base: for example, vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, cottonseed oil; medium chain fatty acid triglycerides and the like.
Aqueous base: for example, Macrogol 400
Gel base: For example, carboxyvinyl polymer, gum or the like.
Surfactant: For example, polysorbate 80, hydrogenated castor oil, glycerin fatty acid ester, sorbitan sesquioleate, and the like.
Suspending agent: For example, white beeswax, various surfactants, gum arabic, gum arabic powder, xanthan gum, soybean lecithin and the like.
Binder: For example, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like.
Excipients: For example, sucrose, lactose, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like.
Lubricant: For example, sucrose fatty acid ester, magnesium stearate, talc and the like.
Disintegrants: For example, low-substituted hydroxypropylcellulose, crospovidone, croscarmellose sodium and the like.
Foaming agent: For example, sodium bicarbonate.
Fluidizer: For example, sodium aluminate metasilicate, light anhydrous silicic acid and the like.

The therapeutic agent for liver disease of the present invention is obtained by suspending and mixing stromal cell-derived microparticles and / or stromal cells and the above-described components in a physiological saline or the like described later.

The liver disease therapeutic agent of the present invention can be provided in various forms according to the purpose, for example, various dosage forms such as a solid preparation, a semisolid preparation, and a liquid preparation. For example, solid preparations (tablets, powders, powders, granules, capsules, etc.), semi-solid preparations [ointment (hard ointment, ointment etc.), creams, etc.], liquid preparations (lotions, extracts, suspensions) Suspensions, emulsions, syrups, injections (including infusions, implants, continuous injections, injections prepared at the time of use), dialysis agents, aerosols, soft capsules, drinks, etc.], patches It can be used in the form of an agent, a poultice and the like. The therapeutic agent for liver disease 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 liver disease of the present invention can be applied to the affected area by spraying, and the therapeutic agent for liver disease of the present invention can also be used in the form of gelation or sheeting at the affected site after spraying. The therapeutic agent for liver disease of the present invention can also be applied to the affected area after the mesenchymal stem cells are made into a sheet or a three-dimensional structure.

The therapeutic agent for liver disease of the present invention may be used after suspending or diluting using a physiological saline solution, a Japanese saline solution, a 5% glucose solution, a glucose injection solution, or a cell culture medium such as DMEM. A physiological saline solution and a 5% glucose solution are preferable.

When the therapeutic agent for liver disease of the present invention is a liquid, the pH of the therapeutic agent for liver disease is not particularly limited as long as it is within a pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable range. 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.

When the therapeutic agent for liver disease of the present invention is a liquid, the osmotic pressure of the therapeutic agent for liver disease is not particularly limited as long as it is within a range acceptable for the living 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 particularly preferably 0.9 to 1.4. The osmotic pressure can be adjusted by a method known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, saccharides and the like. 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 15th revised Japanese Pharmacopoeia. Measure by referring to the freezing point method. The standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) was prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 ° C. for 40 to 50 minutes, and then in a desiccator (silica gel). The mixture is allowed to cool and 0.900 g is accurately weighed and dissolved in purified water to make exactly 100 mL, or a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) is used.

The administration route to the subject of the therapeutic agent for liver disease of the present invention includes oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, sublingual administration, transrectal administration, Examples include vaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implant, direct administration by spraying on the liver surface and sticking of a sheet, etc. The effectiveness of the therapeutic agent for liver disease of the present invention is exemplified. From the viewpoint, it is preferably direct administration by implant, spraying on the liver surface and sticking of a sheet, intrahepatic artery administration and intravenous administration, and more preferably intravenous administration from the viewpoint of reducing the burden on the subject. It is.

In the therapeutic agent for liver disease of the present invention, when it contains stromal cells that secrete microparticles, the dose (dosage) is the patient's condition (body weight, age, symptoms, physical condition, etc.) and the liver disease treatment of the present invention. The amount may vary depending on the dosage form of the agent, but from the viewpoint of achieving the therapeutic effect of a sufficient therapeutic agent for liver disease, a larger amount tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, the amount is A smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 1 × 10 ³ to 1 × 10 ¹² cells / time, preferably 1 × 10 ⁴ to 1 × 10 ¹¹ cells / time, more preferably 1 × 10 ⁵ to 1 × ^{10 10} cells / time, particularly preferably 5 × ¹⁰ 5. ⁶ to 1 × 10 ⁹ pieces / time. In addition, this dose may be administered once as a single dose, or the dose may be divided into multiple doses.

When the therapeutic agent for liver disease of the present invention contains stromal cells that secrete microparticles, the dose (dosage) depends on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the treatment of liver disease of the present invention. Usually, when administered to an adult, the number of cells is 1 × ¹⁰ to 5 × ^{10 10} cells / kg, preferably 1 × 10 ² to 5 × 10 ⁹ cells / kg, more preferably 1 × 10 ³ to 5 × 10. ⁸ pieces / kg, particularly preferably 1 × 10 ⁴ to 5 × 10 ⁷ pieces / kg. In addition, this dose may be administered once as a single dose, or the dose may be divided into multiple doses.

When the therapeutic agent for liver disease of the present invention contains isolated microparticles or a stromal cell culture supernatant containing microparticles, the dose (dosage) depends on the patient's condition (weight, age, symptoms, physical condition, etc.) ), And the dosage form of the therapeutic agent for liver disease of the present invention, etc. Usually, when administered to an adult, 1 × 10 ⁴ to 5 × 10 ¹³ particles / kg, preferably 1 × 10 ⁵ to 5 × 10 ¹² as fine particles. / Kg, more preferably 1 × 10 ⁶ to 5 × 10 ¹¹ pieces / kg, particularly preferably 1 × 10 ⁷ to 5 × ^{10 10} pieces / kg. In addition, this dose may be administered once as a single dose, or the dose may be divided into multiple doses.

The liver disease therapeutic agent of the present invention may be administered together with one or more other drugs. Examples of other drugs include drugs that can be used as therapeutic agents for liver, such as remedies for hepatitis B (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon α, interferon α, etc.) -2b, interferon β, peginterferon α-2a, peginterferon α-2b, etc.), hepatitis C drugs (ribavirin, telapyvir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), corticosteroids (prednisolone, methylprednisolone) Sodium succinate), anticoagulants (dry concentrated human antithrombin III, gabexate mesylate, thrombomodulin α, etc.), antidote (calcium disodium edetate hydrate, glutathione, Methicaprol, sodium thiosulfate hydrate, sugamadesk sodium, etc.), human serum albumin, liver extract, ursodeoxycholic acid, glycyrrhizic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine, L-isoleucine, L -Leucine, L-valine, L-threonine, L-serine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and their salts), vitamins (Tocopherol, flavin adenine dinucleotide, phosphate thiamine disulfide, pyridoxine, cyanocobalamin and their salts), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.) It is below.

The therapeutic agent for liver disease of the present invention can be used for autoimmune liver diseases such as autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary biliary cirrhosis (PSC).

The present invention also includes a method for treating liver disease by administering stromal cell-derived microparticles to a patient. According to the method of the present invention, liver diseases, particularly autoimmune liver diseases can be effectively treated. Specifically, the treatment method of the present invention is a method of administering the above-described therapeutic agent for liver disease of the present invention to a patient. For the liver disease therapeutic agent of the present invention, the contents described in the previous section can be applied as they are.

In the method of the present invention, the average particle diameter of the fine particles is preferably 1,000 nm or less. In addition, the method of the present invention is preferably a method in which stromal cells having the ability to contain or secrete the microparticles are administered to a patient. Furthermore, the microparticles are exosomes, IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1, and It is preferable to contain at least one factor selected from the group consisting of haptoglobin. The stromal cells are preferably derived from adipose tissue, and among autoimmune liver diseases, against autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), or primary biliary cirrhosis (PSC) Thus, the treatment method of the present invention has a remarkable effect.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[Preparation and culture of adipose-derived stromal cells]
Adipose-derived stromal cells were prepared using subcutaneous adipose tissue collected by a liposuction method from a healthy donor. Specifically, the subcutaneous adipose tissue was washed with a phosphate buffered saline solution, type II collagenase (solvent was a physiological saline solution (5 mg / mL)), and treated at 37 ° C. for 30 minutes. The collagenase was inactivated by adding DMEM-10% FBS, and the cell suspension was centrifuged to obtain a cell precipitate. Furthermore, the cell pellet was resuspended in DMEM-10% FBS containing 1% ampicillin-streptomycin, suspended at 2 × 10 ⁴ to 3 × 10 ⁴ cells / cm ² and plated. The cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ atmosphere to obtain adipose-derived stromal cells.

[Inhibition of microparticle formation by knockdown of nSMase2]
KD-hASC was prepared by knocking down neutral sphingomyelinase 2 (nSMase 2) of adipose-derived stromal cells by transfection of shRNA vector. As a control cell, mock-hASC obtained by transfecting a fat-derived stromal cell with a control vector was used. The secretion amount of microparticles from KD-hASC and mock-hASC was measured by a conventional method and compared (FIG. 1). The culture supernatants of KD-hASC and mock-hASC were collected. The collected culture supernatant is filtered with a filter (0.22 μm, Merck Millipore), and then microparticles are recovered by centrifugation (35,000 rpm, 70 minutes, 4 ° C., BECKMAN Optima XE-90). The amount of cytokine (IL-10 and HGF; Hepatocyte Growth Factor) was measured by ELISA (FIGS. 2 and 3).

As shown in FIG. 1, the generation of microparticles was suppressed in cells (KD-hASC) in which nSMase2 was knocked down, and the amount of secreted microparticles was significantly decreased compared to control cells (mock-hASC). It became clear. In addition, as shown in FIGS. 2 and 3, in the cells (KD-hASC) in which nSMase2 was knocked down, the microparticle-derived cytokines (IL-10 and HGF) in the culture supernatant were suppressed in the control cells (mock) by suppressing the production of microparticles. -Decreased compared to hASC). The average particle diameters of mock-hASC and KD-hASC-derived microparticles were 155 ± 3 and 155 ± 6 nm, respectively (mean ± standard error, n = 3).

[Analysis of microparticles (exosomes)]
To 100 mL of the stromal cell culture supernatant (serum-free medium), 50 mL of Total Exosome Isolation (Thermo Fisher Scientific Inc., product number: 4478359) was added and mixed by inversion. After storing overnight in a refrigerator (2-8 ° C.), it was centrifuged (10,000 × g, 1 hour, 2-8 ° C.), and the supernatant was removed. The precipitate generated at that time was suspended in 500 μL of PBS, and this suspension was used as a suspension containing fine particles (exosomes). 150 μL of 20% trichloroacetic acid (TCA) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 150 μL of the suspension containing fine particles (exosomes) to aggregate and precipitate the protein. 20 μL of Tris Hydrochloride Acid Buffer (Tris-HCl), pH 8.5) was added to the total amount of the resulting precipitate to dissolve the precipitate. Further, Tris buffer (Tris-HCl, pH 8.0) containing 0.01% trypsin (trypsin, manufactured by Apro Science) was added and reacted at 37 ° C. for 20 hours. Subsequently, the obtained sample solution was analyzed by LC-MS / MS (LC: manufactured by Michel BioResources, MS: manufactured by ThermoFisherScientific). As a result, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (Pigment epithelium-derived factor (PEDF), SERPINF1) and haptoglobin (Haptoglobin) were detected.

[Confirmation of therapeutic effect using autoimmune hepatitis model mice]
Autoimmune hepatitis was induced by a single administration of 20 mg / kg of concanavalin A (sigma-aldrich) to mice (BALB / cA, CLEA Japan). In mice induced with autoimmune hepatitis, adipose-derived stromal cells (Mock-hASC: 1 × 10 ⁶ cells / time) prepared in the same manner as described above, or adipose-derived stromal cells treated with microparticle generation inhibition (KD) -HASC; 1 × 10 ⁶ cells / dose) was administered intravenously once to evaluate liver damage. In addition, the animal (Control) and the healthy animal (Normal) which do not administer a cell were provided as a comparison object. The measurement results of the liver injury marker are shown in FIG. 4 (AST) and FIG. 5 (ALT). In addition, AST and ALT were each measured with the biochemical automatic analyzer. The results of H / E staining of the liver tissue of each mouse are shown in FIG.

Compared to healthy mice (Normal), AST and ALT, which are markers of liver damage, were increased by administration of Concanavalin A (Control), but the increase was significantly suppressed by administration of Mock-hASC. It was done. In contrast, in mice administered with stromal cells that had been treated to suppress the formation of fine particles (KD-hASC), the effect of suppressing the increase in AST and ALT was low. In addition, as shown in FIG. 6, lymphocyte infiltration was observed in the liver tissue of mice in which autoimmune hepatitis was induced with concanavalin A, whereas lymphocyte infiltration was suppressed in mice administered with CONT-hASC. It was. In contrast, in mice administered with mesenchymal stem cells that had been treated to suppress the production of fine particles (KD-hASC), the effect of suppressing lymphocyte infiltration was weak. As mentioned above, it turned out that the therapeutic effect with respect to autoimmune hepatitis by a fat-derived stromal cell reduces notably by suppressing the production | generation of a microparticle.

From the above results, adipose-derived stromal cells have a remarkable therapeutic effect on autoimmune hepatitis, and the therapeutic effect is reduced by suppressing the production of fine particles. It was suggested that it plays a central role in the therapeutic effect of cells.

The present invention provides a novel therapeutic agent for liver disease and a novel therapeutic method for liver disease.

Claims (16)

A therapeutic agent for liver disease containing microparticles derived from stromal cells. The therapeutic agent for liver disease according to claim 1, wherein the average particle size of the fine particles is 1,000 nm or less. The therapeutic agent for liver disease according to claim 1 or 2, comprising stromal cells having the ability to contain or secrete the microparticles. The therapeutic agent for liver disease according to any one of claims 1 to 3, wherein the fine particles are exosomes. The fine particles are selected from the group consisting of IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1), and haptoglobin (Haptoglobin). The therapeutic agent for liver disease according to any one of claims 1 to 4, comprising at least one factor selected from the above. The therapeutic agent for liver disease according to any one of claims 1 to 5, wherein the stromal cells are derived from adipose tissue. The liver disease therapeutic agent according to any one of claims 1 to 6, wherein the liver disease is an autoimmune liver disease. The therapeutic agent for liver disease according to claim 7, wherein the autoimmune liver disease is autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), or primary biliary cirrhosis (PSC). A method of treating liver disease by administering stromal cell-derived microparticles to a patient. The method according to claim 9, wherein the average particle size of the fine particles is 1,000 nm or less. The method according to claim 9 or 10, wherein stromal cells having the ability to contain or secrete the microparticles are administered to a patient. The method according to any one of claims 9 to 11, wherein the microparticle is an exosome. The fine particles are selected from the group consisting of IL-10, HGF, apolipoprotein A-2 (Apolipoprotein A-2), pigment epithelium-derived factor (PEDF), SERPINF1), and haptoglobin (Haptoglobin). 13. The method according to any one of claims 9 to 12, comprising at least one factor that is selected. The method according to any one of claims 9 to 13, wherein the stromal cells are derived from adipose tissue. The method according to any one of claims 9 to 14, wherein the liver disease is an autoimmune liver disease. The method according to any one of claims 9 to 15, wherein the autoimmune liver disease is autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), or primary biliary cirrhosis (PSC).

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