WO2023223519A1 - Method for deactivating activated stellate cells - Google Patents

Method for deactivating activated stellate cells Download PDF

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WO2023223519A1
WO2023223519A1 PCT/JP2022/020894 JP2022020894W WO2023223519A1 WO 2023223519 A1 WO2023223519 A1 WO 2023223519A1 JP 2022020894 W JP2022020894 W JP 2022020894W WO 2023223519 A1 WO2023223519 A1 WO 2023223519A1
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hepatic stellate
stellate cells
cells
activated
inhibitor
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PCT/JP2022/020894
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French (fr)
Japanese (ja)
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篤 宮島
丈友 木戸
泰博 中野
みつる 宮戸
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Ｉｓｍ株式会社
国立大学法人 東京大学
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Publication of WO2023223519A1 publication Critical patent/WO2023223519A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present invention relates to a method for deactivating activated hepatic stellate cells.
  • Liver fibrosis is caused by various causes of liver diseases such as hepatitis virus infection, alcoholic fatty liver, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease (NAFLD). It occurs when the liver is repeatedly or persistently damaged by inflammatory diseases, and is characterized by excessive production and accumulation of extracellular matrix (also called extracellular matrix) in the connective tissue. It is known that when liver fibrosis increases, it causes serious liver diseases such as liver cirrhosis and liver cancer.
  • liver diseases such as hepatitis virus infection, alcoholic fatty liver, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease (NAFLD). It occurs when the liver is repeatedly or persistently damaged by inflammatory diseases, and is characterized by excessive production and accumulation of extracellular matrix (also called extracellular matrix) in the connective tissue. It is known that when liver fibrosis increases, it causes serious liver diseases such as liver cirrhosis and liver cancer.
  • extracellular matrix also called extracellular matrix
  • the liver consists of hepatic parenchymal cells and non-parenchymal cells (hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, Pit cells, etc.), and the connective tissue of the liver consists of extracellular matrix (collagen, etc.) and cells localized there. It consists of Hepatic stellate cells (HSC) are cells responsible for matrix production in connective tissue, and are known as a major source of extracellular matrix, which is the main cause of liver fibrosis. Hepatic stellate cells change their morphology into myofibroblast-like cells through a process called activation in the art, and begin to produce large amounts of extracellular matrix, which can promote liver fibrosis. Are known. Activation of hepatic stellate cells has been established as a major promoting factor in liver fibrosis, and a search for therapeutic agents for diseases based on liver fibrosis that targets this activation is underway.
  • HSC Hepatic stellate cells
  • Non-Patent Document 1 It has been reported that Y-27632 suppresses activation of hepatic stellate cells (Non-Patent Document 1). Furthermore, anti-fibrotic effects by SB431542 and dorsomorphin (Non-Patent Document 2), and suppression of proliferation and activation of human hepatic stellate cells by ranifibranol (Non-Patent Document 3) have also been reported.
  • Non-Patent Document 4 Although it has been reported that activated hepatic stellate cells may decline in connective tissue due to apoptosis, aging, or recovery (deactivation), the detailed mechanism has not been clarified. No (Non-Patent Document 5).
  • the problem to be solved by the present invention is to provide a method for deactivating activated hepatic stellate cells.
  • a transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor As a result of extensive studies by the present inventors, the use of at least three drugs in combination: a transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor has been shown to be effective.
  • the present invention was completed by discovering that it is possible to deactivate hepatic stellate cells.
  • the present invention includes the following.
  • Deactivators of activated hepatic stellate cells including transforming growth factor ⁇ /bone morphogenetic protein signal inhibitors, activin receptor-like kinase 2 inhibitors, and Rho-kinase inhibitors.
  • the transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor is selected from the group consisting of SB431542, A 83-01, EW-7195, IN-1130, TGFBR1-IN-1, bactosertib, Z12601011 and salts thereof.
  • the deactivating agent according to (1) which is at least three.
  • the activin receptor-like kinase 2 inhibitor is from the group consisting of dorsomorphin, ALK2-IN-2, ALK2-IN-4, DMH1, K02288, LDN193189, LDN-214117, LDN-212854, ML347, OD36 and salts thereof.
  • Rho-kinase inhibitors include Y27632, narciclasine, aflesertib, azaindole 1, AT13148, Belumosudil (KD025), BDP5290, Chroman 1, CMPD101, CRT0066854, emetine, fasudil, GSK-25, GSK180736A, GSK 269962A, GSK429286A, Glycyl H- 1152, H-1152, Hydroxyfasudil (HA-1100), HSD1590, Hu7691, Netarsudil (AR-13324), ROCK2-IN-2, ROCK2-IN-5, ROCK-IN-1, ROCK-IN-2, Ripasudil , RKI-1447, Sovesudil, SR-3677, SAR407899, Thiazovivine, Verosudil, Y-33075, Y-39983 and salts thereof, any one of (1) to (3) The deactivating agent described in Crab.
  • the peroxisome proliferator-activated receptor agonist is selected from the group consisting of ranifibranol, capric acid, EPI-001, fenofibric acid, FH535, GW9662, GSK3787, GSK0660, GW6471, harmine, T0070907 and salts thereof;
  • a method for deactivating activated hepatic stellate cells comprising: A method comprising the step of contacting activated hepatic stellate cells with the deactivating agent according to any one of (1) to (10).
  • a method for producing deactivated cells comprising: A method for producing cells, the method comprising the step of contacting activated hepatic stellate cells with the deactivating agent according to any one of (1) to (10).
  • the present invention includes the following.
  • A1 A method for deactivating activated hepatic stellate cells, the method comprising: A method comprising contacting activated hepatic stellate cells with a transforming growth factor beta/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor.
  • B1 Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells agents and Rho-kinase inhibitors.
  • C1 Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells
  • a composition comprising a Rho-kinase inhibitor and a Rho-kinase inhibitor.
  • D1 Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells Combination of agents and Rho-kinase inhibitors.
  • (E1) A method for producing deactivated cells, the method comprising: A method for producing a cell, comprising contacting an activated hepatic stellate cell with a transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor.
  • the transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibitor, and Rho-kinase inhibitor are any of the above (1) to (10). It may be replaced with the deactivating agent described in . Further, the deactivating agent may be any combination of the embodiments described below.
  • a method for deactivating activated hepatic stellate cells can be provided.
  • FIG. 1 shows the results of quantifying the expression level of the activation marker ACTA2 by quantitative RT-PCR (S: SB431542, D: dorsomorphin, Y: Y27632, L: lanifibranol, V: retinol and palmitic acid, respectively).
  • FIG. 2 shows the results of quantifying the expression level of the activation marker COL1A1 by quantitative RT-PCR.
  • FIG. 3 shows the results of quantifying the expression level of the deactivation marker TCF21 by quantitative RT-PCR.
  • FIG. 4 shows the results of cell morphology observation using a fluorescence microscope.
  • Day 1 indicates the results of the first day of culture of hepatic stellate cells induced to differentiate from iPS cells.
  • YLV had decreased amounts of ⁇ SMA and collagen.
  • SDYLV the amounts of ⁇ SMA and collagen were further decreased.
  • cell morphology similar to stationary hepatic stellate cells was confirmed.
  • Hepatic stellate cells typically refer to stellate fibroblast-like cells that exist in the liver and have vitamin A storage capacity, and are in a dormant state (quiescent state), It may be in an activated state or a deactivated state.
  • hepatic stellate cells in a dormant state resting phase
  • hepatic stellate cells in an activated state are referred to as "activated hepatic stellate cells.”
  • Hepatic stellate cells in the activated state are referred to as "deactivated hepatic stellate cells.”
  • activated hepatic stellate cells are not particularly limited as long as they are hepatic stellate cells in an activated state; for example, they may be derived from stationary hepatic stellate cells, or may be naturally occurring It may also be something that has arisen.
  • Activated hepatic stellate cells obtained by conventionally known methods may be used as activated hepatic stellate cells in the present invention. Even in a dormant state (resting phase), hepatic stellate cells are known to produce extracellular matrices such as collagen to form connective tissue, which is important for the normal organization of organ structures.
  • hepatic stellate cells when activated, they produce excessive amounts of collagen and inflammatory molecules.
  • deactivation refers to the state of activated hepatic stellate cells returning to a state similar to that of resting hepatic stellate cells.
  • the return of the cell state to a resting hepatic stellate cell state involves changes in the morphology, shape, and cell cycle of activated hepatic stellate cells.
  • the morphology, shape, and cell cycle of activated hepatic stellate cells can be determined by the cytoskeleton (CSK) and extracellular matrix (ECM).
  • CSK cytoskeleton
  • ECM extracellular matrix
  • the cytoskeleton includes actin filaments, intermediate filaments, microtubules, talin, and the like.
  • the extracellular matrix includes collagen, hyaluronic acid, proteoglycans, and the like. Return of the cell state to resting hepatic stellate cell-like state can also be determined by a decrease in connective tissue and/or a decrease in the amount accumulated. The reduction in connective tissue and/or the amount accumulated can be determined by the amount of extracellular matrix such as collagen.
  • the confirmation and differentiation of resting hepatic stellate cells, activated hepatic stellate cells, and deactivated hepatic stellate cells are as follows: hepatic stellate cells in a resting state, hepatic stellate cells in an activated state, and deactivated states. This can be done to confirm or differentiate each hepatic stellate cell, and these confirmations and distinctions are not particularly limited, but can be done, for example, by measuring protein production or gene expression that is mainly expressed in each hepatic stellate cell. .
  • each protein or gene is used as a marker and its mRNA expression is measured. Quantification and analysis of the amount of gene expression is performed by a commonly used method for quantifying gene expression, such as quantitative RT-PCR. Primers used in quantitative RT-PCR may be appropriately selected from those known in the art.
  • NGFR and/or genes that are expressed at the same time as NGFR can be used as an indicator of gene expression in stationary hepatic stellate cells. It can be fractionated or confirmed.
  • the above protein or gene may be referred to as a "stationary phase marker.”
  • NGFR is an abbreviation for Nerve Growth Factor Receptor
  • LRAT is a retinol esterification enzyme, lectin retinol acyltransferase.
  • Stationary phase markers may be used as indicators of gene expression in deactivated hepatic stellate cells when activated hepatic stellate cells are deactivated.
  • Activation of stationary hepatic stellate cells can be confirmed by using, but not limited to, ACTA2, COL1A1, etc. as indicators of gene expression when stationary hepatic stellate cells are activated.
  • the above protein or gene may be referred to as an "activation marker.”
  • ACTA2 is ⁇ -smooth muscle actin 2 ( ⁇ -smooth muscle actin)
  • COL1A1 is an abbreviation for type I collagen ⁇ 1 (Collagen, Type I, Alpha 1).
  • the activation marker may be used as an indicator of gene expression in deactivated hepatic stellate cells when activated hepatic stellate cells are deactivated.
  • TCF21 is an abbreviation for transcription factor 21.
  • a marker for confirming deactivation of activated hepatic stellate cells a stationary phase marker may be used, or a marker understood as a deactivation marker may be used.
  • the expression level of activation marker in deactivated hepatic stellate cells is the same as that in activated hepatic stellate cells. It may be low compared to the expression level, for example, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, etc. . Furthermore, the expression levels of stationary phase markers/deactivation markers in deactivated hepatic stellate cells are higher than the expression levels of stationary phase markers/deactivation markers in activated hepatic stellate cells; for example, 1.
  • 1x, 1.2x, 1.3x, 1.5x, 2x, 3x, 4x, 5x, 10x, 15x, 20x, 30x, 40x, 50x, 60x , 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 500 times, 100 times, etc. are preferable.
  • Observation of cell morphology is not particularly limited, and can be carried out appropriately by those skilled in the art using a known method, for example, using a fluorescence microscope. When using a fluorescence microscope, it is common to stain cytoskeletal molecules such as F-Actin.
  • Hepatic stellate cells may be appropriately produced by those skilled in the art using known methods, or commercially available ones may be used.
  • the method for producing stationary hepatic stellate cells is not particularly limited, but for example, a differentiation induction system from pluripotent stem cells to stationary hepatic stellate cells can be used (Koui Y, et al. Stem Cell Reports. 2021 ).
  • the method for producing activated hepatic stellate cells is not particularly limited, but, for example, activated hepatic stellate cells may be produced by culturing resting-phase hepatic stellate cells by appropriate methods known to those skilled in the art.
  • the deactivation agent of the present invention includes a transforming growth factor ⁇ /bone morphogenetic protein (TGF- ⁇ /BMP) signal inhibitor, an activin receptor-like kinase 2 (ALK2) inhibitor, and a Rho-kinase (ROCK ) Contains inhibitors.
  • the deactivator of the present invention contains at least one TGF- ⁇ /BMP signal inhibitor, at least one ALK2 inhibitor, and at least one Rho-kinase inhibitor.
  • "containing" a deactivating agent means that the deactivating agent is used for deactivating activated hepatic stellate cells, along with other similarly contained components. This means that each component may be contained in a mixed form, or may be unmixed and each may be in a different form.
  • Transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor Transforming growth factor ⁇ plays a role in various cellular functions such as cell proliferation, differentiation, and extracellular matrix remodeling. In mammals, there are three isoforms (TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3).
  • the structurally similar TGF- ⁇ superfamily includes activin, bone morphogenetic protein (BMP), and the like.
  • TGF- ⁇ in the liver is known as a potent fibrotic cytokine. Binding of TGF- ⁇ and phosphorylation of the receptor induces phosphorylation of downstream SMAD proteins. It is known that this promotes collagen transfer. It is also known that TGF- ⁇ activates the mitogen-activated protein kinase (MAPK) signal pathway and promotes the activation of hepatic stellate cells.
  • MAPK mitogen-activated protein kinase
  • BMPs are extracellular multifunctional signaling cytokines that belong to the TGF- ⁇ superfamily.
  • transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor refers to a “transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor” that inhibits the signal transduction function of transforming growth factor ⁇ and/or bone morphogenetic protein. It refers to a drug, and may be in the form of a low molecular compound, an antibody, an antisense compound, or the like.
  • TGF- ⁇ /BMP signal inhibitors include, but are not limited to, SB431542, A 83-01, EW-7195, IN-1130, TGFBR1-IN-1, Vactosertib, Z12601011, salts thereof, and the like. can be mentioned.
  • SB431542 A 83-01, more preferably SB431542.
  • SB431542 is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide and has the following structure.
  • a 83-01 is 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide and has the following structure.
  • the concentration of the TGF- ⁇ /BMP signal inhibitor added is not particularly limited, but for example, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0 Examples include .8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, and the like.
  • TGF- ⁇ /BMP signal inhibitor Only one TGF- ⁇ /BMP signal inhibitor may be used, or any two or more may be used in combination.
  • Activin receptor-like kinase 2 is a transmembrane receptor serine/threonine kinase that binds to bone morphogenetic proteins.
  • R-Smad is thought to be activated by a different type I receptor (ALK) depending on the cell tumor.
  • activin receptor-like kinase 2 inhibitor refers to a drug for inhibiting the function of ALK2, and is in the form of a low molecular compound, antibody, or antisense compound. It may be of.
  • ALK2 inhibitors include, but are not limited to, Dorsomorphin, ALK2-IN-2, ALK2-IN-4, DMH1, K02288, LDN193189, LDN-214117, LDN-212854, ML347, OD36, and these. salt, etc. Preferably it is dorsomorphin.
  • Dorsomorphin is 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine and has the following structure.
  • the concentration of the ALK2 inhibitor added is not particularly limited, but for example, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0 Examples include .9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, and the like.
  • Only one ALK2 inhibitor may be used, or any two or more may be used in combination.
  • Rho-kinase inhibitor Rho-kinase (iii) Rho-kinase inhibitor Rho-kinase (ROCK: Rho-associated protein kinase) is a serine-threonine protein kinase that has been identified as a target protein of the low molecular weight GTP-binding protein Rho, and is a protein kinase that inhibits smooth muscle contraction and cell It is known that it is involved in various physiological functions such as morphological changes.
  • Rho-kinase inhibitor refers to a drug for inhibiting the function of Rho-kinase, and is in the form of a low molecular compound, an antibody, or an antisense compound. It may be.
  • Rho-kinase inhibitors include, but are not limited to, Y27632, Narciclasine, Afuresertib, Azaindole 1, AT13148, Belumosudil (KD025), BDP5290, Chroman (C roman) 1, CMPD101 , CRT0066854, Emetine, Fasudil, GSK-25, GSK180736A, GSK269962A, GSK429286A, Glycyl H-1152, H-1152, Hydroxyfasudil ) (HA-1100), HSD1590, Hu7691, Netarsudil (AR-13324), ROCK2-IN-2, ROCK2-IN-5, ROCK-IN-1, ROCK-IN-2, Ripasudil, RKI-1447, Sovesudil, SR- 3677, SAR407899, Thiazovivin, Verosudil, Y-33075, Y-39983, and salts thereof.
  • Y27632 is R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide and has the following structure.
  • Y27632 may be its dihydrochloride.
  • the concentration of the Rho-kinase inhibitor to be added is not particularly limited, but for example, 1 ⁇ M or more, 2 ⁇ M or more, 3 ⁇ M or more, 4 ⁇ M or more, 5 ⁇ M or more, 6 ⁇ M or more, 7 ⁇ M or more, 8 ⁇ M or more, 9 ⁇ M or more, 10 ⁇ M or more, 11 ⁇ M or more. , 12 ⁇ M or more, 13 ⁇ M or more, 14 ⁇ M or more, 15 ⁇ M or more, and the like.
  • Rho-kinase inhibitor Only one Rho-kinase inhibitor may be used, or any two or more may be used in combination.
  • the deactivating agent of the present invention may further include a peroxisome proliferator-activated receptor agonist.
  • the deactivating agent of the present invention includes a peroxisome proliferator-activated receptor agonist (PPAR agonist)
  • the deactivating agent is a TGF- ⁇ /BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor. and PPAR agonists.
  • Peroxisome proliferator-activated receptors are proteins that function as nuclear receptor-type transcriptional regulators. Three subtypes of PPAR are known: ⁇ type, ⁇ type, and ⁇ / ⁇ type. Hepatic stellate cells express nuclear receptors and are known to control glucose and lipid metabolism and negatively regulate activation and the progression of fibrosis. PPAR agonists may be directed against any subtype.
  • a "peroxisome proliferator-activated receptor agonist" refers to an agent that activates a PPAR, such as a PPAR ⁇ receptor, a PPAR ⁇ receptor, a PPAR ⁇ receptor, or a combination thereof; It may be in the form of a low molecular compound, an antibody, an antisense compound, or the like.
  • PPAR agonists include, but are not particularly limited to, lanifibranor, capric acid, EPI-001, fenofibric acid, FH535, GW9662, GSK3787, GSK0660, GW6471. , harmine ( Hermine), T0070907 and salts thereof.
  • Preferred is lanifibranol.
  • Ranifibranol is 1-(6-benzothiazoylsulfonyl)-5-chloro-1H-indole-2-butanoic acid and has the following structure.
  • the concentration of the PPAR agonist added is not particularly limited, but for example, 1 ⁇ M or more, 2 ⁇ M or more, 3 ⁇ M or more, 4 ⁇ M or more, 5 ⁇ M or more, 6 ⁇ M or more, 7 ⁇ M or more, 8 ⁇ M or more, 9 ⁇ M or more, 10 ⁇ M or more, 11 ⁇ M or more, 12 ⁇ M or more. , 13 ⁇ M or more, 14 ⁇ M or more, 15 ⁇ M or more, 20 ⁇ M or more, 30 ⁇ M or more, 40 ⁇ M or more, and the like.
  • Only one PPAR agonist may be used, or any two or more may be used in combination.
  • the deactivator of the present invention may further contain vitamin A.
  • the deactivator of the present invention contains vitamin A
  • the deactivator contains a TGF- ⁇ /BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor, and vitamin A; Including BMP signal inhibitors, ALK2 inhibitors, Rho-kinase inhibitors, PPAR agonists and vitamin A.
  • the cytoplasmic droplets of hepatic stellate cells are known to contain retinyl esters, which are the main storage materials for retinoids in the body.
  • vitamin A examples include, but are not limited to, retinol, retinal, retinoic acid, or salts thereof.
  • the concentration of vitamin A added is not particularly limited, but for example, 1 ⁇ M or more, 2 ⁇ M or more, 3 ⁇ M or more, 4 ⁇ M or more, 5 ⁇ M or more, 6 ⁇ M or more, 7 ⁇ M or more, 8 ⁇ M or more, 9 ⁇ M or more, 10 ⁇ M or more, 11 ⁇ M or more, 12 ⁇ M or more. , 13 ⁇ M or more, 14 ⁇ M or more, 15 ⁇ M or more, and the like.
  • the deactivating agent of the present invention may further contain C 12 to C 18 fatty acids or salts thereof.
  • the deactivating agent of the present invention may include a TGF- ⁇ /BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor, and a C 12 -C 18 fatty acid or a salt thereof.
  • ⁇ C 18 fatty acids or salts thereof and may further contain a PPAR agonist and/or vitamin A.
  • the deactivator of the present invention may further contain any one of a PPAR agonist, vitamin A, and a C 12 to C 18 fatty acid or a salt thereof, but may contain two or more of them.
  • the activators include TGF- ⁇ /BMP signal inhibitors, ALK2 inhibitors, Rho-kinase inhibitors, PPAR agonists, vitamin A and C 12 to C 18 fatty acids or salts thereof.
  • Hepatic stellate cells express patatin-like phospholipase domain containing 3 (PNPLA3), which hydrolyzes retinyl palmitate into retinol and palmitic acid.
  • PNPLA3 is known to be influenced by available retinol in hepatic stellate cells.
  • the fatty acids used in the present invention are preferably C 12 to C 18 fatty acids or salts thereof.
  • Examples of the C 12 to C 18 fatty acids or their salts include, but are not limited to, palmitic acid, lauric acid, myristic acid, stearic acid, and the like.
  • Examples of the salts of C 12 to C 18 fatty acids include alkali salts, ammonium salts, and amino acid salts of C 12 to C 18 fatty acids.
  • the concentration of the C 12 to C 18 fatty acids or their salts is not particularly limited, but examples thereof include 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 60 ⁇ M, 70 ⁇ M, 80 ⁇ M, 90 ⁇ M, 100 ⁇ M, and the like.
  • the addition concentration may be changed as appropriate depending on the type of fatty acid to be added.
  • C 12 to C 18 fatty acids or their salts only one may be used, or any two or more may be used in combination.
  • Deactivators include TGF- ⁇ /BMP signal inhibitors, ALK2 inhibitors and Rho-kinase inhibitors, but in addition to these, PPAR agonists, vitamin A and/or C 12 -C 18 fatty acids or salts thereof. It may further include.
  • the combination of PPAR agonist, vitamin A and/or C 12 -C 18 fatty acids or their salts and their ratios are not particularly limited and may be any combination of the above-mentioned embodiments.
  • the deactivating agent may be a composition for deactivating activated hepatic stellate cells.
  • Such compositions may contain one or more other ingredients selected from, for example, physiologically acceptable carriers, excipients, stabilizers, or preservatives, etc.
  • physiologically acceptable carriers for example, physiologically acceptable carriers, excipients, stabilizers, or preservatives, etc.
  • the deactivating agent may be a combination for deactivating activated hepatic stellate cells.
  • it may be a combination of a transforming growth factor ⁇ /bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor to deactivate activated hepatic stellate cells, Transforming growth factor ⁇ /bone morphogenetic protein signal inhibitors, activin receptor-like kinase 2 inhibitors and Rho-kinase inhibitors may be substituted for the deactivating agents described above.
  • the deactivator may be any combination of the embodiments described above.
  • the deactivator may be in the form of a solution dissolved in a solvent.
  • Solvents that can be used are not particularly limited, but include substances that dissolve the components (i) to (vi) above, such as water, ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO), and formamide. (FA), N,N-dimethylformamide (DMFA), and ethyl acetate (EA).
  • EtOH ethanol
  • MeOH methanol
  • DMSO dimethyl sulfoxide
  • EA ethyl acetate
  • it is DMSO.
  • the deactivator can be produced by a method known to those skilled in the art. For example, although not particularly limited, it may be manufactured by mixing the above-mentioned constituents, or by preparing each of the above-mentioned constituents and preparing them for addition with other constituents.
  • the deactivating agent can be used to deactivate activated hepatic stellate cells.
  • the deactivating agent is not particularly limited, but can be used to deactivate activated hepatic stellate cells, for example, by adding it to a medium in which activated hepatic stellate cells are cultured.
  • "adding cells to the culture medium” includes attaching the cells to the culture bottom.
  • Activated hepatic stellate cells that are deactivated by a deactivating agent are not particularly limited as long as they are hepatic stellate cells that are in an activated state; for example, activated hepatic stellate cells that are activated resting phase hepatic stellate cells.
  • the cells may be commercially available activated hepatic stellate cells.
  • the present invention also relates to a method for deactivating activated hepatic stellate cells, comprising the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention. .
  • contact refers to components of the desired reaction, such as cells, and deactivated cells under conditions suitable for carrying out the desired reaction (e.g., uptake of substances, transformation, proliferation, etc.). For example, it includes, but is not limited to, adding a deactivating agent to a medium containing activated hepatic stellate cells. Methods and conditions for carrying out such reactions are well known in the art.
  • the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention is preferably performed in vitro.
  • the in vitro method those skilled in the art may appropriately use known techniques.
  • the method of deactivating activated hepatic stellate cells in the present invention may be an in vitro method or an in vitro method within cells.
  • the method for deactivating activated hepatic stellate cells may further include the step of culturing activated hepatic stellate cells.
  • cultivation is an operation performed in vitro, and “cultivation” includes maintaining hepatic stellate cells, proliferating hepatic stellate cells, and promoting the growth of hepatic stellate cells. This includes differentiating hepatic stellate cells, developing hepatic stellate cells, etc.
  • the components of the deactivating agent may be brought into contact with the activated hepatic stellate cells simultaneously or in combination. They may be brought into contact with each other independently. When contacting each independently, the order of contact is not particularly limited.
  • the deactivating agent of the present invention may also be in the form of a kit, which may include each component used at the same time, or may include each component used separately and, if desired, further include an instruction manual describing the protocol.
  • the deactivating agent may be brought into contact with activated hepatic stellate cells by adding it to a medium containing activated hepatic stellate cells.
  • the culture conditions after addition of the deactivating agent may be the same as those for culturing activated hepatic stellate cells, for example, culturing in a 37° C., 5% CO2 incubator for several days to about 30 days.
  • the culture period is not particularly limited, but for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 20 days, 3 weeks, 25 days, 30 days. etc.
  • the culture of hepatic stellate cells to which a deactivating agent has been added may be performed on a plate.
  • the plate used is not particularly limited, but for example, a plate with 4 wells, 6 wells, 8 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, or any number of wells can be used. .
  • the medium used for culturing hepatic stellate cells containing a deactivating agent is not particularly limited, but includes, for example, a medium used for stem cell culture such as StemPro34, Dulbecco's modified Eagle's medium, Alpha minimal essential medium, Alpha modified Eagle's medium. etc.
  • the volume of the medium containing cells per well can be appropriately set depending on the culture conditions and the like.
  • the volume of the medium is not particularly limited, but for example, per well, 1 ⁇ L, 2 ⁇ L, 3 ⁇ L, 4 ⁇ L, 5 ⁇ L, 6 ⁇ L, 7 ⁇ L, 8 ⁇ L, 9 ⁇ L, 10 ⁇ L, 11 ⁇ L, 12 ⁇ L, 13 ⁇ L, 14 ⁇ L, 15 ⁇ L, 16 ⁇ L, 17 ⁇ L.
  • the number of cells used for culture is not particularly limited, but for example, 1 or more, 10 or more, 100 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000. Examples include 7,000 or more, 8,000 or more, 9,000 or more, 10,000 or more.
  • the present invention also relates to a method for producing cells, which includes the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention.
  • the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention is performed in step 3 above. This may be carried out in the manner described in the method for deactivating activated hepatic stellate cells.
  • the present invention also relates to cells produced by a method for producing deactivated cells.
  • the cells produced by the method for producing deactivated cells are preferably in a deactivated state. Confirmation of whether cells produced by the method for producing deactivated cells are in a deactivated state is not particularly limited, but includes the following steps: 1. As described for hepatic stellate cells, for example, a method may be used in which a protein or gene that becomes an indicator when activated hepatic stellate cells are deactivated is used as a marker and the mRNA expression thereof is measured.
  • the storage period and storage container for cells can be set as appropriate depending on the usage, storage conditions, and other conditions.
  • Examples of storage containers include cell culture bags, tubes, flasks, infusion bags, and syringes.
  • the cells produced by the method for producing deactivated cells of the present invention may be in the form of a cell suspension suspended in a solution.
  • the method for suspending cells produced by the method for producing deactivated cells in a solution is not particularly limited, and can be carried out by a person skilled in the art using a known method as appropriate.
  • a cell suspension can be obtained by mixing and stirring cells produced by a method for producing deactivated cells and a solution.
  • the solution in which the cells are suspended is not particularly limited, and may be appropriately determined by those skilled in the art, such as the medium or preservation solution used for culture.
  • examples of such a solution include, but are not limited to, a medium used for stem cell culture such as StemPro34, Dulbecco's modified Eagle's medium, Alpha minimal essential medium, Alpha modified Eagle's medium, Japanese Pharmacy Ringer's solution, human serum, and the like.
  • the concentration of cells in a cell suspension containing cells produced by the method for producing deactivated cells of the present invention can be appropriately set depending on conditions such as usage and storage period.
  • the concentration of cells in the cell suspension is not particularly limited, but may be, for example, about 1.0 ⁇ 10 4 to 1.0 ⁇ 10 10 cells/mL.
  • the temperature at which the cell suspension of the present invention is stored may be appropriately set depending on the type of solution, the use of the cell suspension, the storage period, etc., and is not particularly limited, but is between 0°C and 50°C. There may be. In the case of cryopreservation, the temperature may be, for example, not less than -190°C and not more than -60°C, although there is no particular limitation.
  • the hepatic stellate cells were suspended in StemPro34 liquid medium (Gibco), seeded on a collagen-coated plate (VIOLAMO, VTC-P6), and cultured in a 37°C/5% CO2 incubator for 3 days. Thereafter, the medium was replaced with only StemPro34 liquid medium and cultured for 4 days to activate hepatic stellate cells to obtain activated hepatic stellate cells.
  • StemPro34 liquid medium Gibco
  • VIOLAMO collagen-coated plate
  • activation marker genes ACTA2, COL1A1, etc.

Abstract

The present invention relates to a deactivating agent for activated hepatic stellate cells, said deactivating agent including a transforming proliferative factor β/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 3 inhibitor, and a Rho kinase inhibitor.

Description

活性化星細胞を脱活性化する方法How to deactivate activated stellate cells
　本発明は、活性化肝星細胞を脱活性化する方法に関する。 The present invention relates to a method for deactivating activated hepatic stellate cells.
　肝臓の線維化は、肝炎ウイルス感染、アルコール性脂肪肝、非アルコール性脂肪肝炎（ＮＡＳＨ：ｎｏｎａｌｃｏｈｏｌｉｃ　ｓｔｅａｔｏｈｅｐａｔｉｔｉｓ）、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ：ｎｏｎａｌｃｏｈｏｌｉｃ　ｆａｔｔｙ　ｌｉｖｅｒ　ｄｉｓｅａｓｅ）等の肝疾患の種々の要因によって肝臓が反復的又は持続的に損傷すると生じ、結合組織における細胞外基質（細胞外マトリックスともいう）の過剰産生や蓄積を特徴とする。肝臓の線維化は亢進すると、肝硬変や肝臓がん等の重大な肝疾患を引き起こすことが知られている。 Liver fibrosis is caused by various causes of liver diseases such as hepatitis virus infection, alcoholic fatty liver, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease (NAFLD). It occurs when the liver is repeatedly or persistently damaged by inflammatory diseases, and is characterized by excessive production and accumulation of extracellular matrix (also called extracellular matrix) in the connective tissue. It is known that when liver fibrosis increases, it causes serious liver diseases such as liver cirrhosis and liver cancer.
　肝臓は、肝実質細胞と非実質細胞（肝星細胞、クッパ―細胞、類洞内皮細胞、Ｐｉｔ細胞等）からなり、肝臓の結合組織は細胞外基質（コラーゲン等）とそこに局在する細胞から構成されている。肝星細胞（ＨＳＣ：Ｈｅｐａｔｉｃ　Ｓｔｅｌｌａｔｅ　Ｃｅｌｌ）は、結合組織における基質産生を担う細胞であり、肝臓の線維化の主因である細胞外基質の主要な供給源として知られている。肝星細胞は、当業界において活性化と呼ばれるプロセスを介して筋線維芽細胞様細胞へと形態を変化させ、細胞外基質を多量に産生するようになり、肝臓の線維化を促進することが知られている。肝星細胞の活性化は、肝臓の線維化における主要な促進因子として確立されており、これを標的とした肝臓の線維化に基づく疾患の治療薬の探索が行われている。 The liver consists of hepatic parenchymal cells and non-parenchymal cells (hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, Pit cells, etc.), and the connective tissue of the liver consists of extracellular matrix (collagen, etc.) and cells localized there. It consists of Hepatic stellate cells (HSC) are cells responsible for matrix production in connective tissue, and are known as a major source of extracellular matrix, which is the main cause of liver fibrosis. Hepatic stellate cells change their morphology into myofibroblast-like cells through a process called activation in the art, and begin to produce large amounts of extracellular matrix, which can promote liver fibrosis. Are known. Activation of hepatic stellate cells has been established as a major promoting factor in liver fibrosis, and a search for therapeutic agents for diseases based on liver fibrosis that targets this activation is underway.
　Ｙ－２７６３２が、肝星細胞の活性化を抑制することが報告されている（非特許文献１）。また、例えば、ＳＢ４３１５４２とドルソモルフィンによる抗線維化や（非特許文献２）、ラニフィブラノールによるヒト肝星細胞の増殖及び活性化抑制（非特許文献３）等についても報告されている。 It has been reported that Y-27632 suppresses activation of hepatic stellate cells (Non-Patent Document 1). Furthermore, anti-fibrotic effects by SB431542 and dorsomorphin (Non-Patent Document 2), and suppression of proliferation and activation of human hepatic stellate cells by ranifibranol (Non-Patent Document 3) have also been reported.
　また、ＰＰＡＲγの強制発現により活性化肝星細胞の表現型変化が生じ得ることが報告されている（非特許文献４）。
　しかしながら、活性化した肝星細胞は、アポトーシス、老化あるいは回復（脱活性化）によって結合組織中で減退している可能性があることは報告されているものの、その詳細なメカニズムは明らかとなっていない（非特許文献５）。 Furthermore, it has been reported that forced expression of PPARγ can cause phenotypic changes in activated hepatic stellate cells (Non-Patent Document 4).
However, although it has been reported that activated hepatic stellate cells may decline in connective tissue due to apoptosis, aging, or recovery (deactivation), the detailed mechanism has not been clarified. No (Non-Patent Document 5).
　本発明が解決しようとする課題は、活性化肝星細胞を脱活性化する方法を提供することである。 The problem to be solved by the present invention is to provide a method for deactivating activated hepatic stellate cells.
　本発明者らが鋭意検討した結果、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤の少なくとも３剤を併用して用いることにより、活性化肝星細胞を脱活性化できることを見出し、本発明を完成した。 As a result of extensive studies by the present inventors, the use of at least three drugs in combination: a transforming growth factor β/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor has been shown to be effective. The present invention was completed by discovering that it is possible to deactivate hepatic stellate cells.
　すなわち、本発明は、以下を包含する。
（１）
　トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤を含む、活性化肝星細胞の脱活性化剤。
（２）
　トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤が、ＳＢ４３１５４２、Ａ　８３－０１、ＥＷ－７１９５、ＩＮ－１１３０、ＴＧＦＢＲ１－ＩＮ－１、バクトセルチブ、Ｚ１２６０１０１１及びこれらの塩からなる群から選択される１つ以上である、（１）に記載の脱活性化剤。
（３）
　アクチビン受容体様キナーゼ２阻害剤が、ドルソモルフィン、ＡＬＫ２－ＩＮ－２、ＡＬＫ２－ＩＮ－４、ＤＭＨ１、Ｋ０２２８８、ＬＤＮ１９３１８９、ＬＤＮ－２１４１１７、ＬＤＮ－２１２８５４、ＭＬ３４７、ＯＤ３６及びこれらの塩からなる群から選択される１つ以上である、（１）又は（２）に記載の脱活性化剤。
（４）
　Ｒｈｏ－キナーゼ阻害剤が、Ｙ２７６３２、ナルシクラシン、アフレセルチブ、アザインドール１、ＡＴ１３１４８、Ｂｅｌｕｍｏｓｕｄｉｌ（ＫＤ０２５）、ＢＤＰ５２９０、クロマン　１、ＣＭＰＤ１０１、ＣＲＴ００６６８５４、エメチン、ファスジル、ＧＳＫ－２５、ＧＳＫ１８０７３６Ａ、ＧＳＫ２６９９６２Ａ、ＧＳＫ４２９２８６Ａ、グリシル　Ｈ－１１５２、Ｈ－１１５２、ヒドロキシファスジル（ＨＡ－１１００）、ＨＳＤ１５９０、Ｈｕ７６９１、ネタルスジル（ＡＲ－１３３２４）、ＲＯＣＫ２－ＩＮ－２、ＲＯＣＫ２－ＩＮ－５、ＲＯＣＫ－ＩＮ－１、ＲＯＣＫ－ＩＮ－２、リパスジル、ＲＫＩ－１４４７、Ｓｏｖｅｓｕｄｉｌ、ＳＲ－３６７７、ＳＡＲ４０７８９９、チアゾビビン、Ｖｅｒｏｓｕｄｉｌ、Ｙ－３３０７５、Ｙ－３９９８３及びこれらの塩からなる群から選択される１つ以上である、（１）～（３）のいずれかに記載の脱活性化剤。
（５）
　ペルオキシソーム増殖剤活性化受容体アゴニストをさらに含む、（１）～（４）のいずれかに記載の脱活性化剤。
（６）
　ペルオキシソーム増殖剤活性化受容体アゴニストが、ラニフィブラノール、カプリン酸、ＥＰＩ－００１、フェノフィブリン酸、ＦＨ５３５、ＧＷ９６６２、ＧＳＫ３７８７、ＧＳＫ０６６０、ＧＷ６４７１、ハルミン、Ｔ００７０９０７及びこれらの塩からなる群から選択される１つ以上である、（１）～（５）のいずれかに記載の脱活性化剤。
（７）
　ビタミンＡをさらに含む、（１）～（６）のいずれかに記載の脱活性化剤。
（８）
　ビタミンＡが、レチノール、レチナール、レチノイン酸及びこれらの塩からなる群から選択される１つ以上である、（７）に記載の脱活性化剤。
（９）
　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩をさらに含む、（１）～（８）のいずれかに記載の脱活性化剤。
（１０）
　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩が、パルミチン酸、ラウリン酸、ミリスチン酸、ステアリン酸及びこれらの塩からなる群から選択される１つ以上である、（９）に記載の脱活性化剤。
（１１）
　活性化肝星細胞を脱活性化する方法であって、
　活性化肝星細胞を、（１）～（１０）のいずれかに記載の脱活性化剤と接触させる工程を含む、方法。
（１２）
　活性化肝星細胞が、ヒト活性化肝星細胞である、（１１）に記載の方法。
（１３）
　脱活性化された細胞の製造方法であって、
　活性化肝星細胞を、（１）～（１０）のいずれかに記載の脱活性化剤と接触させる工程を含む、細胞の製造方法。
（１４）
　（１３）に記載の方法で製造される、脱活性化肝星細胞。 That is, the present invention includes the following.
(1)
Deactivators of activated hepatic stellate cells, including transforming growth factor β/bone morphogenetic protein signal inhibitors, activin receptor-like kinase 2 inhibitors, and Rho-kinase inhibitors.
(2)
The transforming growth factor β/bone morphogenetic protein signal inhibitor is selected from the group consisting of SB431542, A 83-01, EW-7195, IN-1130, TGFBR1-IN-1, bactosertib, Z12601011 and salts thereof. The deactivating agent according to (1), which is at least three.
(3)
The activin receptor-like kinase 2 inhibitor is from the group consisting of dorsomorphin, ALK2-IN-2, ALK2-IN-4, DMH1, K02288, LDN193189, LDN-214117, LDN-212854, ML347, OD36 and salts thereof. The deactivating agent according to (1) or (2), which is one or more selected deactivating agents.
(4)
Rho-kinase inhibitors include Y27632, narciclasine, aflesertib, azaindole 1, AT13148, Belumosudil (KD025), BDP5290, Chroman 1, CMPD101, CRT0066854, emetine, fasudil, GSK-25, GSK180736A, GSK 269962A, GSK429286A, Glycyl H- 1152, H-1152, Hydroxyfasudil (HA-1100), HSD1590, Hu7691, Netarsudil (AR-13324), ROCK2-IN-2, ROCK2-IN-5, ROCK-IN-1, ROCK-IN-2, Ripasudil , RKI-1447, Sovesudil, SR-3677, SAR407899, Thiazovivine, Verosudil, Y-33075, Y-39983 and salts thereof, any one of (1) to (3) The deactivating agent described in Crab.
(5)
The deactivating agent according to any one of (1) to (4), further comprising a peroxisome proliferator-activated receptor agonist.
(6)
the peroxisome proliferator-activated receptor agonist is selected from the group consisting of ranifibranol, capric acid, EPI-001, fenofibric acid, FH535, GW9662, GSK3787, GSK0660, GW6471, harmine, T0070907 and salts thereof; The deactivating agent according to any one of (1) to (5), which is at least three.
(7)
The deactivator according to any one of (1) to (6), further comprising vitamin A.
(8)
The deactivator according to (7), wherein the vitamin A is one or more selected from the group consisting of retinol, retinal, retinoic acid, and salts thereof.
(9)
The deactivator according to any one of (1) to (8), further comprising a C 12 to C 18 fatty acid or a salt thereof.
(10)
The deactivator according to (9), wherein the C 12 to C 18 fatty acid or a salt thereof is one or more selected from the group consisting of palmitic acid, lauric acid, myristic acid, stearic acid, and salts thereof. .
(11)
A method for deactivating activated hepatic stellate cells, the method comprising:
A method comprising the step of contacting activated hepatic stellate cells with the deactivating agent according to any one of (1) to (10).
(12)
The method according to (11), wherein the activated hepatic stellate cells are human activated hepatic stellate cells.
(13)
A method for producing deactivated cells, the method comprising:
A method for producing cells, the method comprising the step of contacting activated hepatic stellate cells with the deactivating agent according to any one of (1) to (10).
(14)
Deactivated hepatic stellate cells produced by the method described in (13).
　また、本発明は、以下を包含する。
（Ａ１）
　活性化肝星細胞を脱活性化する方法であって、
　活性化肝星細胞を、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤と接触させる工程を含む、方法。
（Ｂ１）
　活性化肝星細胞を脱活性化するための、あるいは、活性化肝星細胞の脱活性化に使用するための、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤。
（Ｃ１）
　活性化肝星細胞を脱活性化するための、あるいは、活性化肝星細胞の脱活性化に使用するための、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤を含む組成物。
（Ｄ１）
　活性化肝星細胞を脱活性化するための、あるいは、活性化肝星細胞の脱活性化に使用するための、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤の組み合わせ。
（Ｅ１）
　脱活性化された細胞の製造方法であって、
　活性化肝星細胞を、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤と接触させる工程を含む、細胞の製造方法。
　（Ａ１）～（Ｅ１）においては、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤は、上記（１）～（１０）のいずれかに記載の脱活性化剤と置き換えてもよい。また、脱活性化剤については、以下記載する態様の任意の組み合わせであってよい。 Further, the present invention includes the following.
(A1)
A method for deactivating activated hepatic stellate cells, the method comprising:
A method comprising contacting activated hepatic stellate cells with a transforming growth factor beta/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor.
(B1)
Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells agents and Rho-kinase inhibitors.
(C1)
Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells A composition comprising a Rho-kinase inhibitor and a Rho-kinase inhibitor.
(D1)
Transforming growth factor beta/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibition for or for use in deactivating activated hepatic stellate cells Combination of agents and Rho-kinase inhibitors.
(E1)
A method for producing deactivated cells, the method comprising:
A method for producing a cell, comprising contacting an activated hepatic stellate cell with a transforming growth factor β/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor.
In (A1) to (E1), the transforming growth factor β/bone morphogenetic protein signal inhibitor, activin receptor-like kinase 2 inhibitor, and Rho-kinase inhibitor are any of the above (1) to (10). It may be replaced with the deactivating agent described in . Further, the deactivating agent may be any combination of the embodiments described below.
　本発明によれば、活性化肝星細胞を脱活性化する方法を提供することができる。 According to the present invention, a method for deactivating activated hepatic stellate cells can be provided.
図１は、定量ＲＴ－ＰＣＲによる活性化マーカーＡＣＴＡ２の発現量の定量結果を示す（Ｓ：ＳＢ４３１５４２、Ｄ：ドルソモルフィン、Ｙ：Ｙ２７６３２、Ｌ：ラニフィブラノール、Ｖ：レチノール及びパルミチン酸をそれぞれ意味する。以下の図において同様である。）。Figure 1 shows the results of quantifying the expression level of the activation marker ACTA2 by quantitative RT-PCR (S: SB431542, D: dorsomorphin, Y: Y27632, L: lanifibranol, V: retinol and palmitic acid, respectively). (The same applies in the following figures.) 図２は、定量ＲＴ－ＰＣＲによる活性化マーカーＣＯＬ１Ａ１の発現量の定量結果を示す。FIG. 2 shows the results of quantifying the expression level of the activation marker COL1A1 by quantitative RT-PCR. 図３は、定量ＲＴ－ＰＣＲによる脱活性化マーカーＴＣＦ２１の発現量の定量結果を示す。FIG. 3 shows the results of quantifying the expression level of the deactivation marker TCF21 by quantitative RT-PCR. 図４は、蛍光顕微鏡による細胞形態観察の結果を示す。「１日目」は、ｉＰＳ細胞から分化誘導した肝星細胞の培養１日目の結果を示す。ＤＭＳＯと比較して、ＹＬＶでは、αＳＭＡとコラーゲンの量が減少していた。ＳＤＹＬＶでは、αＳＭＡとコラーゲンの量がさらに減少していた。ＹＬＶ及びＳＤＹＬＶにおいては、静止期肝星細胞に類似した細胞形態が確認された。FIG. 4 shows the results of cell morphology observation using a fluorescence microscope. "Day 1" indicates the results of the first day of culture of hepatic stellate cells induced to differentiate from iPS cells. Compared to DMSO, YLV had decreased amounts of αSMA and collagen. In SDYLV, the amounts of αSMA and collagen were further decreased. In YLV and SDYLV, cell morphology similar to stationary hepatic stellate cells was confirmed.
　以下、本発明を実施するための形態について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではなく、その要旨の範囲内で種々変形して実施することができる。 Hereinafter, modes for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist.
１．肝星細胞
　本明細書において、「肝星細胞」とは、典型的には、肝臓に存在する、ビタミンＡ貯蔵能を有する星状の線維芽細胞様細胞を指し、休眠状態（静止期）、活性化状態又は脱活性化状態であってもよい。本明細書では、休眠状態（静止期）にある肝星細胞を「静止期肝星細胞」と記載し、活性化状態にある肝星細胞を「活性化肝星細胞」と記載し、脱活性化状態にある肝星細胞を「脱活性化肝星細胞」と記載する。 1. Hepatic stellate cells As used herein, "hepatic stellate cells" typically refer to stellate fibroblast-like cells that exist in the liver and have vitamin A storage capacity, and are in a dormant state (quiescent state), It may be in an activated state or a deactivated state. In this specification, hepatic stellate cells in a dormant state (resting phase) are referred to as "quiescent hepatic stellate cells," and hepatic stellate cells in an activated state are referred to as "activated hepatic stellate cells." Hepatic stellate cells in the activated state are referred to as "deactivated hepatic stellate cells."
　本明細書において、「活性化肝星細胞」は、活性化状態である肝星細胞であれば特に限定されず、例えば、静止期肝星細胞から誘導されたものであってもよく、自然発生的に生じたものであってもよい。従来公知の方法により得られる活性化肝星細胞を、本発明におる活性化肝星細胞として用いてよい。休眠状態（静止期）の肝星細胞であっても、臓器の構造の正常な組織化に重要な結合組織を形成するためにコラーゲン等の細胞外基質を産生することが知られている。一方、肝星細胞が活性化されると、肝星細胞は、多量で過剰のコラーゲンや炎症性分子を産生するようになる。 As used herein, "activated hepatic stellate cells" are not particularly limited as long as they are hepatic stellate cells in an activated state; for example, they may be derived from stationary hepatic stellate cells, or may be naturally occurring It may also be something that has arisen. Activated hepatic stellate cells obtained by conventionally known methods may be used as activated hepatic stellate cells in the present invention. Even in a dormant state (resting phase), hepatic stellate cells are known to produce extracellular matrices such as collagen to form connective tissue, which is important for the normal organization of organ structures. On the other hand, when hepatic stellate cells are activated, they produce excessive amounts of collagen and inflammatory molecules.
　本明細書において「脱活性化」とは、活性化肝星細胞が、静止期肝星細胞様へと細胞の状態が戻ることを指す。静止期肝星細胞様へと細胞の状態が戻ることは、活性化肝星細胞の形態、形状、細胞周期を変化させることを含む。活性化肝星細胞の形態、形状、細胞周期は、細胞骨格（ＣＳＫ：Ｃｙｔｏｓｋｅｌｔｏｎ）、細胞外基質（ＥＣＭ：Ｅｘｔｒａｃｅｌｌｕｌａｒ　ｍａｔｒｉｘ）によって判断され得る。細胞骨格は、アクチンフィラメント、中間径フィラメント、微小管及びタリン等を含む。細胞外基質は、コラーゲン、ヒアルロン酸及びプロテオグリカン等を含む。静止期肝星細胞様へと細胞の状態が戻ることは、結合組織の減少及び／又は蓄積量の減少によっても判断され得る。結合組織の減少及び／又は蓄積量の減少は、コラーゲン等の細胞外基質の量で判断され得る。 As used herein, "deactivation" refers to the state of activated hepatic stellate cells returning to a state similar to that of resting hepatic stellate cells. The return of the cell state to a resting hepatic stellate cell state involves changes in the morphology, shape, and cell cycle of activated hepatic stellate cells. The morphology, shape, and cell cycle of activated hepatic stellate cells can be determined by the cytoskeleton (CSK) and extracellular matrix (ECM). The cytoskeleton includes actin filaments, intermediate filaments, microtubules, talin, and the like. The extracellular matrix includes collagen, hyaluronic acid, proteoglycans, and the like. Return of the cell state to resting hepatic stellate cell-like state can also be determined by a decrease in connective tissue and/or a decrease in the amount accumulated. The reduction in connective tissue and/or the amount accumulated can be determined by the amount of extracellular matrix such as collagen.
　静止期肝星細胞、活性化肝星細胞及び脱活性化肝星細胞のそれぞれの確認や区別は、静止期状態にある肝星細胞、活性化状態にある肝星細胞及び脱活性化状態にある肝星細胞のそれぞれの確認や区別として行ってよく、それらの確認や区別は、特に限定されないが、例えば、各肝星細胞に主に発現するタンパク質産生や遺伝子発現を測定することにより可能である。各肝星細胞の遺伝子発現の有無の検討や比較検討をする場合、それぞれのタンパク質又は遺伝子をマーカーとして用いて、そのｍＲＮＡ発現を測定する方法が使用され得る。遺伝子発現量の定量及び分析は、通常用いられる遺伝子発現定量法によって行われ、例えば、定量ＲＴ－ＰＣＲ等によって行われる。定量ＲＴ－ＰＣＲにおいて用いられるプライマーは、当業界において知られているものを適宜用いてよい。 The confirmation and differentiation of resting hepatic stellate cells, activated hepatic stellate cells, and deactivated hepatic stellate cells are as follows: hepatic stellate cells in a resting state, hepatic stellate cells in an activated state, and deactivated states. This can be done to confirm or differentiate each hepatic stellate cell, and these confirmations and distinctions are not particularly limited, but can be done, for example, by measuring protein production or gene expression that is mainly expressed in each hepatic stellate cell. . When examining and comparing the presence or absence of gene expression in each hepatic stellate cell, a method may be used in which each protein or gene is used as a marker and its mRNA expression is measured. Quantification and analysis of the amount of gene expression is performed by a commonly used method for quantifying gene expression, such as quantitative RT-PCR. Primers used in quantitative RT-PCR may be appropriately selected from those known in the art.
　静止期肝星細胞の遺伝子発現の指標として、特に限定されないが、ＮＧＦＲ及び／又はＮＧＦＲと同時期に発現する遺伝子（例えば、ＬＲＡＴ、ＮＥＳ、ＬＨＸ２等）を用いることにより、静止期肝星細胞を分取又は確認することができる。本明細書において、上記のタンパク質又は遺伝子を「静止期マーカー」と称することがある。「ＮＧＦＲ」は、神経栄養因子受容体（Ｎｅｒｖｅ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ　Ｒｅｃｅｐｔｏｒ）の略であり、「ＬＲＡＴ」は、レチノールエステル化酵素であるレクチン　レチノール　アシルトランスフェラーゼ（Ｌｅｃｔｈｉｎ　Ｒｅｔｉｎｏｌ　Ａｃｙｌｔｒａｎｓｆｅｒａｓｅ）の略であり、「ＮＥＳ」は、ネスチン（Ｎｅｓｔｉｎ）の略であり、「ＬＨＸ２」は、ＬＩＭ　ホメオボックス　２（ＬＩＭ　Ｈｏｍｅｏｂｏｘ　２）の略である。静止期マーカーは、活性化肝星細胞が脱活性化された場合における脱活性化肝星細胞の遺伝子発現の指標として用いてもよい。 As an indicator of gene expression in stationary hepatic stellate cells, NGFR and/or genes that are expressed at the same time as NGFR (for example, LRAT, NES, LHX2, etc.) can be used as an indicator of gene expression in stationary hepatic stellate cells. It can be fractionated or confirmed. In this specification, the above protein or gene may be referred to as a "stationary phase marker." "NGFR" is an abbreviation for Nerve Growth Factor Receptor, and "LRAT" is a retinol esterification enzyme, lectin retinol acyltransferase. e), and “NES” is an abbreviation of , is an abbreviation for Nestin, and "LHX2" is an abbreviation for LIM Homeobox 2. Stationary phase markers may be used as indicators of gene expression in deactivated hepatic stellate cells when activated hepatic stellate cells are deactivated.
　静止期肝星細胞が活性化された場合における遺伝子発現の指標として、特に限定されないが、ＡＣＴＡ２、ＣＯＬ１Ａ１等を用いることにより、静止期肝星細胞の活性化を確認することができる。本明細書において、上記のタンパク質又は遺伝子を「活性化マーカー」と称することがある。「ＡＣＴＡ２」は、α－平滑筋アクチン２（α－ｓｍｏｏｔｈ　ｍｕｓｃｌｅ　ａｃｔｉｎ）であり、「ＣＯＬ１Ａ１」は、Ｉ型コラーゲンα１（Ｃｏｌｌａｇｅｎ，Ｔｙｐｅ　Ｉ，Ａｌｐｈａ　１）の略である。活性化マーカーは、活性化肝星細胞が脱活性化された場合における脱活性化肝星細胞の遺伝子発現の指標として用いてもよい。 Activation of stationary hepatic stellate cells can be confirmed by using, but not limited to, ACTA2, COL1A1, etc. as indicators of gene expression when stationary hepatic stellate cells are activated. In this specification, the above protein or gene may be referred to as an "activation marker." "ACTA2" is α-smooth muscle actin 2 (α-smooth muscle actin), and "COL1A1" is an abbreviation for type I collagen α1 (Collagen, Type I, Alpha 1). The activation marker may be used as an indicator of gene expression in deactivated hepatic stellate cells when activated hepatic stellate cells are deactivated.
　活性化肝星細胞が脱活性化された場合における遺伝子発現の指標として、特に限定されないが、ＴＣＦ２１等を用いることにより、活性化肝星細胞の脱活性化を確認することができる。「ＴＣＦ２１」は、転写因子２１（Ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｆａｃｔｏｒ　２１）の略である。活性化肝星細胞の脱活性化を確認するマーカーとしては、静止期マーカーを用いてもよく、脱活性化マーカーとして理解されるマーカーを用いてもよい。 Deactivation of activated hepatic stellate cells can be confirmed by using TCF21, etc., although not particularly limited, as an indicator of gene expression when activated hepatic stellate cells are deactivated. "TCF21" is an abbreviation for transcription factor 21. As a marker for confirming deactivation of activated hepatic stellate cells, a stationary phase marker may be used, or a marker understood as a deactivation marker may be used.
　活性化肝星細胞と脱活性化肝星細胞のそれぞれの遺伝子マーカーの発現量を比較した場合、脱活性化肝星細胞における活性化マーカーの発現量は、活性化肝星細胞における活性化マーカーの発現量と比較して低く、例えば、８０％以下、７０％以下、６０％以下、５０％以下、４０％以下、３０％以下、２０％以下、１０％以下、５％以下等であってよい。
　また、脱活性化肝星細胞における静止期マーカー／脱活性化マーカーの発現量は、活性化肝星細胞における静止期化マーカー／脱活性化マーカーの発現量と比較して高く、例えば、１．１倍、１．２倍、１．３倍、１．５倍、２倍、３倍、４倍、５倍、１０倍、１５倍、２０倍、３０倍、４０倍、５０倍、６０倍、７０倍、８０倍、９０倍、１００倍、２００倍、３００倍、５００倍、１００倍等であることが好ましい。 When comparing the expression level of each gene marker in activated hepatic stellate cells and deactivated hepatic stellate cells, the expression level of activation marker in deactivated hepatic stellate cells is the same as that in activated hepatic stellate cells. It may be low compared to the expression level, for example, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, etc. .
Furthermore, the expression levels of stationary phase markers/deactivation markers in deactivated hepatic stellate cells are higher than the expression levels of stationary phase markers/deactivation markers in activated hepatic stellate cells; for example, 1. 1x, 1.2x, 1.3x, 1.5x, 2x, 3x, 4x, 5x, 10x, 15x, 20x, 30x, 40x, 50x, 60x , 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 500 times, 100 times, etc. are preferable.
　静止期肝星細胞、活性化肝星細胞及び脱活性化肝星細胞のそれぞれの確認や区別は、細胞形態の観察によっても可能である。細胞形態の観察は、特に限定されず、当業者が適宜公知の手法により、例えば蛍光顕微鏡等を用いて行うことができる。蛍光顕微鏡を用いる場合には、Ｆ－Ａｃｔｉｎ等の細胞骨格分子を染色することが一般的である。 It is also possible to confirm and distinguish between resting hepatic stellate cells, activated hepatic stellate cells, and deactivated hepatic stellate cells by observing cell morphology. Observation of cell morphology is not particularly limited, and can be carried out appropriately by those skilled in the art using a known method, for example, using a fluorescence microscope. When using a fluorescence microscope, it is common to stain cytoskeletal molecules such as F-Actin.
　肝星細胞は、当業者が適宜公知の手法により作製してもよく、市販のものを用いてもよい。静止期肝星細胞の作製方法としては、特に限定されないが、例えば、多能性幹細胞から静止期肝星細胞への分化誘導系を用いることができる（Ｋｏｕｉ Ｙ，ｅｔ ａｌ．Ｓｔｅｍ Ｃｅｌｌ Ｒｅｐｏｒｔｓ．２０２１）。活性化肝星細胞の作製方法としては、特に限定されないが、例えば、当業者が適宜公知の手法により静止期肝星細胞を培養することにより作製してもよい。 Hepatic stellate cells may be appropriately produced by those skilled in the art using known methods, or commercially available ones may be used. The method for producing stationary hepatic stellate cells is not particularly limited, but for example, a differentiation induction system from pluripotent stem cells to stationary hepatic stellate cells can be used (Koui Y, et al. Stem Cell Reports. 2021 ). The method for producing activated hepatic stellate cells is not particularly limited, but, for example, activated hepatic stellate cells may be produced by culturing resting-phase hepatic stellate cells by appropriate methods known to those skilled in the art.
２．脱活性化剤
　本発明の脱活性化剤は、トランスフォーミング増殖因子β／骨形成タンパク質（ＴＧＦ－β／ＢＭＰ）シグナル阻害剤、アクチビン受容体様キナーゼ２（ＡＬＫ２）阻害剤及びＲｈｏ－キナーゼ（ＲＯＣＫ）阻害剤を含む。
　本発明の脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤を少なくとも１種含み、ＡＬＫ２阻害剤を少なくとも１種含み、Ｒｈｏ－キナーゼ阻害剤を少なくとも１種含む。
　本明細書において、脱活性化剤がある成分を「含む」とは、当該含有されるある成分を、同様に含有される他の成分とともに、活性化肝星細胞の脱活性化のために用いることを意味しており、含有される形態としては各成分が混合されていてもよく、混合されていない、それぞれが別の形態であってもよい。 2. Deactivation Agent The deactivation agent of the present invention includes a transforming growth factor β/bone morphogenetic protein (TGF-β/BMP) signal inhibitor, an activin receptor-like kinase 2 (ALK2) inhibitor, and a Rho-kinase (ROCK ) Contains inhibitors.
The deactivator of the present invention contains at least one TGF-β/BMP signal inhibitor, at least one ALK2 inhibitor, and at least one Rho-kinase inhibitor.
As used herein, "containing" a deactivating agent means that the deactivating agent is used for deactivating activated hepatic stellate cells, along with other similarly contained components. This means that each component may be contained in a mixed form, or may be unmixed and each may be in a different form.
（ｉ）トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤
　トランスフォーミング増殖因子β（ＴＧＦ－β：Ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β）は、細胞の増殖、分化、細胞外マトリクスの再構築等、細胞において様々な役割を担うタンパク質で、哺乳動物では３種類のアイソフォーム（ＴＧＦ－β１、ＴＧＦ－β２、ＴＧＦ－β３）が存在する。構造的に類似したＴＧＦ－βスーパーファミリーには、アクチビンや骨形成タンパク質（ＢＭＰ：Ｂｏｎｅ　ｍｏｒｐｈｏｇｅｎｅｔｉｃ　ｐｒｏｔｅｉｎ）等が含まれる。
　肝臓におけるＴＧＦ－βは、強力な線維化サイトカインとして知られている。ＴＧＦ－βの結合と受容体のリン酸化は、下流のＳＭＡＤタンパク質のリン酸化を誘導する。これにより、コラーゲンの転写が促進されることが知られている。また、ＴＧＦ－βは、マイトジェン活性化プロテインキナーゼ（ＭＡＰＫ：ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ）シグナル経路を活性化して、肝星細胞の活性化を促進することも知られている。 (i) Transforming growth factor β/bone morphogenetic protein signal inhibitor Transforming growth factor β (TGF-β) plays a role in various cellular functions such as cell proliferation, differentiation, and extracellular matrix remodeling. In mammals, there are three isoforms (TGF-β1, TGF-β2, and TGF-β3). The structurally similar TGF-β superfamily includes activin, bone morphogenetic protein (BMP), and the like.
TGF-β in the liver is known as a potent fibrotic cytokine. Binding of TGF-β and phosphorylation of the receptor induces phosphorylation of downstream SMAD proteins. It is known that this promotes collagen transfer. It is also known that TGF-β activates the mitogen-activated protein kinase (MAPK) signal pathway and promotes the activation of hepatic stellate cells.
　ＢＭＰは、ＴＧＦ－βスーパーファミリーに属する細胞外多機能シグナル伝達サイトカインである。 BMPs are extracellular multifunctional signaling cytokines that belong to the TGF-β superfamily.
　本明細書において、「トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤」（ＴＧＦ－β／ＢＭＰシグナル阻害剤）とは、トランスフォーミング増殖因子β及び／又は骨形成タンパク質のシグナル伝達機能を阻害する薬剤のことであり、低分子化合物、抗体、又はアンチセンス化合物等の形態のものであってもよい。 As used herein, “transforming growth factor β/bone morphogenetic protein signal inhibitor” (TGF-β/BMP signal inhibitor) refers to a “transforming growth factor β/bone morphogenetic protein signal inhibitor” that inhibits the signal transduction function of transforming growth factor β and/or bone morphogenetic protein. It refers to a drug, and may be in the form of a low molecular compound, an antibody, an antisense compound, or the like.
　ＴＧＦ－β／ＢＭＰシグナル阻害剤としては、特に限定されないが、例えば、ＳＢ４３１５４２、Ａ　８３－０１、ＥＷ－７１９５、ＩＮ－１１３０、ＴＧＦＢＲ１－ＩＮ－１、バクトセルチブ（Ｖａｃｔｏｓｅｒｔｉｂ）、Ｚ１２６０１０１１及びこれらの塩等が挙げられる。好ましくは、ＳＢ４３１５４２、Ａ　８３－０１であり、より好ましくは、ＳＢ４３１５４２である。
　ＳＢ４３１５４２は、４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］ベンズアミドであり、以下の構造を有する。
Figure JPOXMLDOC01-appb-C000001
 　Ａ　８３－０１は、３－（６－メチル－２－ピリジニル)－N－フェニル－４－（４―キノリニル）－１Ｈ－ピラゾール－１－カルボチオアミドであり、以下の構造を有する。
Figure JPOXMLDOC01-appb-C000002
 Examples of TGF-β/BMP signal inhibitors include, but are not limited to, SB431542, A 83-01, EW-7195, IN-1130, TGFBR1-IN-1, Vactosertib, Z12601011, salts thereof, and the like. can be mentioned. Preferably it is SB431542, A 83-01, more preferably SB431542.
SB431542 is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide and has the following structure.
Figure JPOXMLDOC01-appb-C000001
 A 83-01 is 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide and has the following structure.
Figure JPOXMLDOC01-appb-C000002
　ＴＧＦ－β／ＢＭＰシグナル阻害剤の添加濃度としては、特に限定されないが、例えば、０．１μＭ、０．２μＭ、０．３μＭ、０．４μＭ、０．５μＭ、０．６μＭ、０．７μＭ、０．８μＭ、０．９μＭ、１．０μＭ、１．１μＭ、１．２μＭ、１．３μＭ、１．４μＭ、１．５μＭ等が挙げられる。 The concentration of the TGF-β/BMP signal inhibitor added is not particularly limited, but for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0 Examples include .8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, and the like.
　ＴＧＦ－β／ＢＭＰシグナル阻害剤としては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 Only one TGF-β/BMP signal inhibitor may be used, or any two or more may be used in combination.
（ｉｉ）アクチビン受容体様キナーゼ２阻害剤
　アクチビン受容体様キナーゼ２は、骨形成タンパク質と結合する膜貫通型の受容体セリン／スレオニンキナーゼである。
　前述のＴＧＦ－βの作用を受けるＳＭＡＤタンパク質のうち、Ｒ－Ｓｍａｄは、細胞腫に依存した異なるタイプＩ受容体（ＡＬＫ）によって活性化されると考えられている。 (ii) Activin receptor-like kinase 2 inhibitor Activin receptor-like kinase 2 is a transmembrane receptor serine/threonine kinase that binds to bone morphogenetic proteins.
Among the SMAD proteins that are affected by TGF-β, R-Smad is thought to be activated by a different type I receptor (ALK) depending on the cell tumor.
　本明細書において、「アクチビン受容体様キナーゼ２阻害剤」（ＡＬＫ２阻害剤）とは、ＡＬＫ２の機能を阻害するための薬剤のことであり、低分子化合物、抗体、又はアンチセンス化合物等の形態のものであってもよい。 As used herein, "activin receptor-like kinase 2 inhibitor" (ALK2 inhibitor) refers to a drug for inhibiting the function of ALK2, and is in the form of a low molecular compound, antibody, or antisense compound. It may be of.
　ＡＬＫ２阻害剤としては、特に限定されないが、例えば、ドルソモルフィン（Ｄｏｒｓｏｍｏｒｐｈｉｎ）、ＡＬＫ２－ＩＮ－２、ＡＬＫ２－ＩＮ－４、ＤＭＨ１、Ｋ０２２８８、ＬＤＮ１９３１８９、ＬＤＮ－２１４１１７、ＬＤＮ－２１２８５４、ＭＬ３４７、ＯＤ３６及びこれらの塩等が挙げられる。好ましくは、ドルソモルフィンである。
　ドルソモルフィンは、６－［４－（２－ピペリジン－１－イルエトキシ）フェニル］－３－ピリジン－４－イルピラゾロ［１，５－ａ］ピリミジンであり、以下の構造を有する。
Figure JPOXMLDOC01-appb-C000003
 Examples of ALK2 inhibitors include, but are not limited to, Dorsomorphin, ALK2-IN-2, ALK2-IN-4, DMH1, K02288, LDN193189, LDN-214117, LDN-212854, ML347, OD36, and these. salt, etc. Preferably it is dorsomorphin.
Dorsomorphin is 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine and has the following structure.
Figure JPOXMLDOC01-appb-C000003
　ＡＬＫ２阻害剤の添加濃度としては、特に限定されないが、例えば、０．１μＭ、、０．２μＭ、０．３μＭ、０．４μＭ、０．５μＭ、０．６μＭ、０．７μＭ、０．８μＭ、０．９μＭ、１．０μＭ、１．１μＭ、１．２μＭ、１．３μＭ、１．４μＭ、１．５μＭ等が挙げられる。 The concentration of the ALK2 inhibitor added is not particularly limited, but for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0 Examples include .9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, and the like.
　ＡＬＫ２阻害剤としては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 Only one ALK2 inhibitor may be used, or any two or more may be used in combination.
（ｉｉｉ）Ｒｈｏ－キナーゼ阻害剤
　Ｒｈｏ－キナーゼ（ＲＯＣＫ：Ｒｈｏ－ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ）は、低分子量ＧＴＰ結合蛋白Ｒｈｏの標的蛋白質として同定されたセリン－スレオニン蛋白リン酸化酵素であり、平滑筋収縮や細胞の形態変化等、様々な生理機能に関与していることが知られている。 (iii) Rho-kinase inhibitor Rho-kinase (ROCK: Rho-associated protein kinase) is a serine-threonine protein kinase that has been identified as a target protein of the low molecular weight GTP-binding protein Rho, and is a protein kinase that inhibits smooth muscle contraction and cell It is known that it is involved in various physiological functions such as morphological changes.
本明細書において「Ｒｈｏ－キナーゼ阻害剤」（ＲＯＣＫ阻害剤）とは、Ｒｈｏ－キナーゼの機能を阻害するための薬剤のことであり、低分子化合物、抗体、又はアンチセンス化合物等の形態のものであってもよい。 As used herein, "Rho-kinase inhibitor" (ROCK inhibitor) refers to a drug for inhibiting the function of Rho-kinase, and is in the form of a low molecular compound, an antibody, or an antisense compound. It may be.
　Ｒｈｏ－キナーゼ阻害剤としては、特に限定されないが、例えば、Ｙ２７６３２、ナルシクラシン（Ｎａｒｃｉｃｌａｓｉｎｅ）、アフレセルチブ（Ａｆｕｒｅｓｅｒｔｉｂ）、アザインドール（Ａｚａｉｎｄｏｌｅ）１、ＡＴ１３１４８、Ｂｅｌｕｍｏｓｕｄｉｌ（ＫＤ０２５）、ＢＤＰ５２９０、クロマン（Ｃｈｒｏｍａｎ）１、ＣＭＰＤ１０１、ＣＲＴ００６６８５４、エメチン（Ｅｍｅｔｉｎｅ）、ファスジル（Ｆａｓｕｄｉｌ）、ＧＳＫ－２５、ＧＳＫ１８０７３６Ａ、ＧＳＫ２６９９６２Ａ、ＧＳＫ４２９２８６Ａ、グリシル（Ｇｌｙｃｙｌ）Ｈ－１１５２、Ｈ－１１５２、ヒドロキシファスジル（Ｈｙｄｒｏｘｙｆａｓｕｄｉｌ）（ＨＡ－１１００）、ＨＳＤ１５９０、Ｈｕ７６９１、ネタルスジル（Ｎｅｔａｒｓｕｄｉｌ）（ＡＲ－１３３２４）、ＲＯＣＫ２－ＩＮ－２、ＲＯＣＫ２－ＩＮ－５、ＲＯＣＫ－ＩＮ－１、ＲＯＣＫ－ＩＮ－２、リパスジル（Ｒｉｐａｓｕｄｉｌ）、ＲＫＩ－１４４７、ソヴェスジル（Ｓｏｖｅｓｕｄｉｌ）、ＳＲ－３６７７、ＳＡＲ４０７８９９、チアゾビビン（Ｔｈｉａｚｏｖｉｖｉｎ）、ヴェロスジル（Ｖｅｒｏｓｕｄｉｌ）、Ｙ－３３０７５、Ｙ－３９９８３及びこれらの塩等が挙げられる。好ましくは、Ｙ２７６３２である。
　Ｙ２７６３２は、Ｒ－（＋）－ｔｒａｎｓ－４－（１－アミノエチル）－Ｎ－（４－ピリジル）シクロヘキサンカルボキサミドであり、以下の構造を有する。
Figure JPOXMLDOC01-appb-C000004
 　Ｙ２７６３２は、その二塩酸塩であってもよい。 Examples of Rho-kinase inhibitors include, but are not limited to, Y27632, Narciclasine, Afuresertib, Azaindole 1, AT13148, Belumosudil (KD025), BDP5290, Chroman (C roman) 1, CMPD101 , CRT0066854, Emetine, Fasudil, GSK-25, GSK180736A, GSK269962A, GSK429286A, Glycyl H-1152, H-1152, Hydroxyfasudil ) (HA-1100), HSD1590, Hu7691, Netarsudil (AR-13324), ROCK2-IN-2, ROCK2-IN-5, ROCK-IN-1, ROCK-IN-2, Ripasudil, RKI-1447, Sovesudil, SR- 3677, SAR407899, Thiazovivin, Verosudil, Y-33075, Y-39983, and salts thereof. Preferably it is Y27632.
Y27632 is R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide and has the following structure.
Figure JPOXMLDOC01-appb-C000004
 Y27632 may be its dihydrochloride.
　Ｒｈｏ－キナーゼ阻害剤の添加濃度としては、特に限定されないが、例えば、１μＭ以上、２μＭ以上、３μＭ以上、４μＭ以上、５μＭ以上、６μＭ以上、７μＭ以上、８μＭ以上、９μＭ以上、１０μＭ以上、１１μＭ以上、１２μＭ以上、１３μＭ以上、１４μＭ以上、１５μＭ以上等が挙げられる。 The concentration of the Rho-kinase inhibitor to be added is not particularly limited, but for example, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, 5 μM or more, 6 μM or more, 7 μM or more, 8 μM or more, 9 μM or more, 10 μM or more, 11 μM or more. , 12 μM or more, 13 μM or more, 14 μM or more, 15 μM or more, and the like.
　Ｒｈｏ－キナーゼ阻害剤としては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 Only one Rho-kinase inhibitor may be used, or any two or more may be used in combination.
（ｉｖ）ペルオキシソーム増殖剤活性化受容体アゴニスト
　本発明の脱活性化剤は、ペルオキシソーム増殖剤活性化受容体アゴニストをさらに含んでもよい。
　本発明の脱活性化剤が、ペルオキシソーム増殖剤活性化受容体アゴニスト（ＰＰＡＲアアゴニスト）を含む時、脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤、Ｒｈｏ－キナーゼ阻害剤及びＰＰＡＲアゴニストを含む。
　ペルオキシソーム増殖剤活性化受容体（ＰＰＡＲ：ｐｅｒｏｘｉｓｏｍｅ　ｐｒｏｌｉｆｅｒａｔｏｒ－ａｃｔｉｖａｔｅｄ　ｒｅｃｅｐｔｏｒ）は、核内受容体型の転写調節因子として機能するタンパク質である。ＰＰＡＲには、α型、γ型及びβ／δ型の３種類のサブタイプが知られている。
　肝星細胞は、核内受容体を発現しており、グルコースと脂質の代謝を制御したり、活性化と線維化の進行を負に制御することが知られている。ＰＰＡＲアゴニストとしては、いずれのサブタイプに対するものであってもよい。 (iv) Peroxisome proliferator-activated receptor agonist The deactivating agent of the present invention may further include a peroxisome proliferator-activated receptor agonist.
When the deactivating agent of the present invention includes a peroxisome proliferator-activated receptor agonist (PPAR agonist), the deactivating agent is a TGF-β/BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor. and PPAR agonists.
Peroxisome proliferator-activated receptors (PPARs) are proteins that function as nuclear receptor-type transcriptional regulators. Three subtypes of PPAR are known: α type, γ type, and β/δ type.
Hepatic stellate cells express nuclear receptors and are known to control glucose and lipid metabolism and negatively regulate activation and the progression of fibrosis. PPAR agonists may be directed against any subtype.
　本明細書において、「ペルオキシソーム増殖剤活性化受容体アゴニスト」（ＰＰＡＲアゴニスト）とは、ＰＰＡＲγ受容体、ＰＰＡＲα受容体、ＰＰＡＲδ受容体又はそれらの組合せ等のＰＰＡＲを活性化する薬剤のことであり、低分子化合物、抗体、又はアンチセンス化合物等の形態のものであってもよい。 As used herein, a "peroxisome proliferator-activated receptor agonist" (PPAR agonist) refers to an agent that activates a PPAR, such as a PPARγ receptor, a PPARα receptor, a PPARδ receptor, or a combination thereof; It may be in the form of a low molecular compound, an antibody, an antisense compound, or the like.
　ＰＰＡＲアゴニストとしては、特に限定されないが、例えば、ラニフィブラノール（Ｌａｎｉｆｉｂｒａｎｏｒ）、カプリン酸（Ｃａｐｒｉｃ　ａｃｉｄ）、ＥＰＩ－００１、フェノフィブリン酸（Ｆｅｎｏｆｉｂｒｉｃ　ａｃｉｄ）、ＦＨ５３５、ＧＷ９６６２、ＧＳＫ３７８７、ＧＳＫ０６６０、ＧＷ６４７１、ハルミン（Ｈａｒｍｉｎｅ）、Ｔ００７０９０７及びこれらの塩等が挙げられる。好ましくは、ラニフィブラノールである。
　ラニフィブラノールは、１－（６－ベンゾチアゾイルスルホニル）－５－クロロー１Ｈ－インドール－２－ブタン酸であり、以下の構造を有する。
Figure JPOXMLDOC01-appb-C000005
 PPAR agonists include, but are not particularly limited to, lanifibranor, capric acid, EPI-001, fenofibric acid, FH535, GW9662, GSK3787, GSK0660, GW6471. , harmine ( Hermine), T0070907 and salts thereof. Preferred is lanifibranol.
Ranifibranol is 1-(6-benzothiazoylsulfonyl)-5-chloro-1H-indole-2-butanoic acid and has the following structure.
Figure JPOXMLDOC01-appb-C000005
　ＰＰＡＲアゴニストの添加濃度としては、特に限定されないが、例えば、１μＭ以上、２μＭ以上、３μＭ以上、４μＭ以上、５μＭ以上、６μＭ以上、７μＭ以上、８μＭ以上、９μＭ以上、１０μＭ以上、１１μＭ以上、１２μＭ以上、１３μＭ以上、１４μＭ以上、１５μＭ以上、２０μＭ以上、３０μＭ以上、４０μＭ以上等が挙げられる。 The concentration of the PPAR agonist added is not particularly limited, but for example, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, 5 μM or more, 6 μM or more, 7 μM or more, 8 μM or more, 9 μM or more, 10 μM or more, 11 μM or more, 12 μM or more. , 13 μM or more, 14 μM or more, 15 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, and the like.
　ＰＰＡＲアゴニストとしては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 Only one PPAR agonist may be used, or any two or more may be used in combination.
（ｖ）ビタミンＡ
　本発明の脱活性化剤は、ビタミンＡをさらに含んでもよい。
　本発明の脱活性化剤は、ビタミンＡを含む時、脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤、Ｒｈｏ－キナーゼ阻害剤及びビタミンＡを含むか、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤、Ｒｈｏ－キナーゼ阻害剤、ＰＰＡＲアゴニスト及びビタミンＡを含む。
　肝星細胞の細胞質液滴には、体内のレチノイドの主要な貯蔵物質であるレチニルエステルが含まれていることが知られている。活性化肝星細胞においては、これらの脂質液滴が喪失することが知られているが、貯蔵されたレチノイドの肝星細胞活性化への寄与は知られていない。レチノイドを含む肝細胞脂質液滴の喪失は、肝細胞の活性化を促進せず、むしろ肝発がんを減少させることが示唆されている。 (v) Vitamin A
The deactivator of the present invention may further contain vitamin A.
When the deactivator of the present invention contains vitamin A, the deactivator contains a TGF-β/BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor, and vitamin A; Including BMP signal inhibitors, ALK2 inhibitors, Rho-kinase inhibitors, PPAR agonists and vitamin A.
The cytoplasmic droplets of hepatic stellate cells are known to contain retinyl esters, which are the main storage materials for retinoids in the body. Although these lipid droplets are known to be lost in activated hepatic stellate cells, the contribution of stored retinoids to hepatic stellate cell activation is unknown. It has been suggested that loss of hepatocyte lipid droplets containing retinoids does not promote hepatocyte activation, but rather reduces hepatocarcinogenesis.
　ビタミンＡとしては、特に限定されないが、例えば、レチノール、レチナール、レチノイン酸、又はこれらの塩等が挙げられる。 Examples of vitamin A include, but are not limited to, retinol, retinal, retinoic acid, or salts thereof.
　ビタミンＡの添加濃度としては、特に限定されないが、例えば、１μＭ以上、２μＭ以上、３μＭ以上、４μＭ以上、５μＭ以上、６μＭ以上、７μＭ以上、８μＭ以上、９μＭ以上、１０μＭ以上、１１μＭ以上、１２μＭ以上、１３μＭ以上、１４μＭ以上、１５μＭ以上等が挙げられる。 The concentration of vitamin A added is not particularly limited, but for example, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, 5 μM or more, 6 μM or more, 7 μM or more, 8 μM or more, 9 μM or more, 10 μM or more, 11 μM or more, 12 μM or more. , 13 μM or more, 14 μM or more, 15 μM or more, and the like.
　ビタミンＡとしては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 Only one vitamin A may be used, or any two or more may be used in combination.
（ｖｉ）Ｃ１２～Ｃ１８脂肪酸又はそれらの塩
　本発明の脱活性化剤は、Ｃ１２～Ｃ１８脂肪酸又はそれらの塩をさらに含んでもよい。
　本発明の脱活性化剤は、Ｃ１２～Ｃ１８脂肪酸又はそれらの塩を含む時、脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤、Ｒｈｏ－キナーゼ阻害剤及びＣ１２～Ｃ１８脂肪酸又はそれらの塩を含み、さらに、ＰＰＡＲアゴニスト及び／又はビタミンＡを含んでもよい。
　本発明の脱活性化剤は、ＰＰＡＲアゴニスト、ビタミンＡ及びＣ１２～Ｃ１８脂肪酸又はそれらの塩のいずれか１種をさらに含んでいればよいが、２種以上を含んでいてもよく、脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤、Ｒｈｏ－キナーゼ阻害剤、ＰＰＡＲアゴニスト、ビタミンＡ及びＣ１２～Ｃ１８脂肪酸又はそれらの塩を含むことが好ましい。
　肝星細胞は、パタチン様ホスホリパーゼドメインを含むタンパク質３（ＰＮＰＬＡ３：ｐａｔａｉｎ－ｌｉｋｅ　ｐｈｏｓｐｈｏｌｉｐａｓｅ　ｄｏｍａｉｎ　ｃｏｎｔａｉｎｉｎｇ　３）を発現しており、レチニルパルミチン酸を、レチノールとパルミチン酸に加水分解する。ＰＮＰＬＡ３は、肝星細胞における利用可能なレチノールに影響を受けることが知られている。 (vi) C 12 to C 18 fatty acids or salts thereof The deactivating agent of the present invention may further contain C 12 to C 18 fatty acids or salts thereof.
When the deactivating agent of the present invention contains a C 12 to C 18 fatty acid or a salt thereof, the deactivating agent may include a TGF-β/BMP signal inhibitor, an ALK2 inhibitor, a Rho-kinase inhibitor, and a C 12 -C 18 fatty acid or a salt thereof. ~C 18 fatty acids or salts thereof, and may further contain a PPAR agonist and/or vitamin A.
The deactivator of the present invention may further contain any one of a PPAR agonist, vitamin A, and a C 12 to C 18 fatty acid or a salt thereof, but may contain two or more of them. Preferably, the activators include TGF-β/BMP signal inhibitors, ALK2 inhibitors, Rho-kinase inhibitors, PPAR agonists, vitamin A and C 12 to C 18 fatty acids or salts thereof.
Hepatic stellate cells express patatin-like phospholipase domain containing 3 (PNPLA3), which hydrolyzes retinyl palmitate into retinol and palmitic acid. PNPLA3 is known to be influenced by available retinol in hepatic stellate cells.
　本発明において用いられる脂肪酸は、好ましくは、Ｃ１２～Ｃ１８脂肪酸又はそれらの塩である。
　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩としては、特に限定されないが、例えば、パルミチン酸、ラウリン酸、ミリスチン酸、ステアリン酸等が挙げられる。
　Ｃ１２～Ｃ１８脂肪酸の塩としては、Ｃ１２～Ｃ１８脂肪酸のアルカリ塩、アンモニウム塩やアミノ酸塩が挙げられる。 The fatty acids used in the present invention are preferably C 12 to C 18 fatty acids or salts thereof.
Examples of the C 12 to C 18 fatty acids or their salts include, but are not limited to, palmitic acid, lauric acid, myristic acid, stearic acid, and the like.
Examples of the salts of C 12 to C 18 fatty acids include alkali salts, ammonium salts, and amino acid salts of C 12 to C 18 fatty acids.
　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩の添加濃度としては、特に限定されないが、例えば、２０μＭ、３０μＭ、４０μＭ、５０μＭ、６０μＭ、７０μＭ、８０μＭ、９０μＭ、１００μＭ等が挙げられる。添加濃度は、添加する脂肪酸の種類等により適宜変更してよい。 The concentration of the C 12 to C 18 fatty acids or their salts is not particularly limited, but examples thereof include 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, and the like. The addition concentration may be changed as appropriate depending on the type of fatty acid to be added.
　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩としては、１つのみを用いてもよく、任意の２つ以上を組み合わせて用いてもよい。 As the C 12 to C 18 fatty acids or their salts, only one may be used, or any two or more may be used in combination.
　脱活性化剤は、ＴＧＦ－β／ＢＭＰシグナル阻害剤、ＡＬＫ２阻害剤及びＲｈｏ－キナーゼ阻害剤を含むが、これらに加えてＰＰＡＲアゴニスト、ビタミンＡ及び／又はＣ１２～Ｃ１８脂肪酸若しくはそれらの塩をさらに含んでもよい。ＰＰＡＲアゴニスト、ビタミンＡ及び／又はＣ１２～Ｃ１８脂肪酸又はそれらの塩の組み合わせ及びそれらの比率は、特に限定されず、上述の態様の任意の組み合わせであってよい。 Deactivators include TGF-β/BMP signal inhibitors, ALK2 inhibitors and Rho-kinase inhibitors, but in addition to these, PPAR agonists, vitamin A and/or C 12 -C 18 fatty acids or salts thereof. It may further include. The combination of PPAR agonist, vitamin A and/or C 12 -C 18 fatty acids or their salts and their ratios are not particularly limited and may be any combination of the above-mentioned embodiments.
　脱活性化剤は、活性化肝星細胞を脱活性化するための組成物であってもよい。そのような組成物は、例えば、生理学的に許容される担体、賦形剤、安定剤、又は保存剤等から選択される１又は複数の他の成分を含んでよく、そのような組成物の製造方法は当業者が適宜公知の手法を使用することができる。
　また、脱活性化剤としては、活性化肝星細胞を脱活性化するための組み合わせであってもよい。すなわち、活性化肝星細胞を脱活性化するための、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤の組み合わせであってもよく、トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤は、上記で説明する脱活性化剤と置き換えてもよい。また、脱活性化剤については、上記で説明する態様の任意の組み合わせであってよい。 The deactivating agent may be a composition for deactivating activated hepatic stellate cells. Such compositions may contain one or more other ingredients selected from, for example, physiologically acceptable carriers, excipients, stabilizers, or preservatives, etc. For the manufacturing method, those skilled in the art can appropriately use known methods.
Further, the deactivating agent may be a combination for deactivating activated hepatic stellate cells. That is, it may be a combination of a transforming growth factor β/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor to deactivate activated hepatic stellate cells, Transforming growth factor β/bone morphogenetic protein signal inhibitors, activin receptor-like kinase 2 inhibitors and Rho-kinase inhibitors may be substituted for the deactivating agents described above. Furthermore, the deactivator may be any combination of the embodiments described above.
　脱活性化剤は、溶媒に溶解させた溶液の形態としてもよい。用いられ得る溶媒としては、特に限定されないが、上記（ｉ）～（ｖｉ）の成分を溶解する物質であり、例えば、水、エタノール（ＥｔＯＨ）、メタノール（ＭｅＯＨ）、ジメチルスルホキシド（ＤＭＳＯ）、ホルムアミド（ＦＡ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦＡ）及び酢酸エチル（ＥＡ）等が挙げられる。好ましくは、ＤＭＳＯである。 The deactivator may be in the form of a solution dissolved in a solvent. Solvents that can be used are not particularly limited, but include substances that dissolve the components (i) to (vi) above, such as water, ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO), and formamide. (FA), N,N-dimethylformamide (DMFA), and ethyl acetate (EA). Preferably it is DMSO.
　脱活性化剤は、当業者が適宜公知の手法により製造することができる。例えば、特に限定されないが、上述の構成成分を混合することにより製造してもよく、上述の構成成分をそれぞれ準備し他の構成成分とともに添加するために調製することにより製造してもよい。 The deactivator can be produced by a method known to those skilled in the art. For example, although not particularly limited, it may be manufactured by mixing the above-mentioned constituents, or by preparing each of the above-mentioned constituents and preparing them for addition with other constituents.
　脱活性化剤は、活性化肝星細胞の脱活性化に用いることができる。脱活性化剤は、特に限定されないが、例えば、活性化肝星細胞を培養する培地に添加することにより、活性化肝星細胞の脱活性化に用いることができる。本明細書において、細胞を「培地に添加」とは、細胞を培養底面に付すことを含む。 The deactivating agent can be used to deactivate activated hepatic stellate cells. The deactivating agent is not particularly limited, but can be used to deactivate activated hepatic stellate cells, for example, by adding it to a medium in which activated hepatic stellate cells are cultured. As used herein, "adding cells to the culture medium" includes attaching the cells to the culture bottom.
　脱活性化剤によって脱活性化される活性化肝星細胞は、活性化状態にある肝星細胞であれば特に限定されず、例えば、活性化静止期肝星細胞を活性化した活性化肝星細胞であってもよく、市販されている活性化肝星細胞であってもよい。 Activated hepatic stellate cells that are deactivated by a deactivating agent are not particularly limited as long as they are hepatic stellate cells that are in an activated state; for example, activated hepatic stellate cells that are activated resting phase hepatic stellate cells. The cells may be commercially available activated hepatic stellate cells.
３．活性化肝星細胞を脱活性化する方法
　本発明は、活性化肝星細胞を、本発明の脱活性化剤と接触させる工程を含む、活性化肝星細胞を脱活性化する方法にも関する。 3. Method for deactivating activated hepatic stellate cells The present invention also relates to a method for deactivating activated hepatic stellate cells, comprising the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention. .
　本明細書において、「接触」とは、所望の反応（例えば、物質の取り込み、形質転換、増殖等）を行うのに適切な条件下に、所望の反応の構成要素、例えば、細胞と脱活性化剤を一緒に置くことを意味し、特に限定されないが、例えば、活性化肝星細胞を含む培地に脱活性化剤を添加すること等を含む。そのような反応を行うための方法及び条件は、本技術分野でよく知られている。 As used herein, "contact" refers to components of the desired reaction, such as cells, and deactivated cells under conditions suitable for carrying out the desired reaction (e.g., uptake of substances, transformation, proliferation, etc.). For example, it includes, but is not limited to, adding a deactivating agent to a medium containing activated hepatic stellate cells. Methods and conditions for carrying out such reactions are well known in the art.
　活性化肝星細胞を、本発明の脱活性化剤と接触させる工程は、好ましくは、生体外で行われる。生体外で行う方法は、当業者が適宜公知の手法を用いてよい。
　本発明における活性化肝星細胞を脱活性化する方法は、インビトロにおける方法であってもよく、生体外の細胞内における方法であってもよい。 The step of contacting activated hepatic stellate cells with the deactivating agent of the present invention is preferably performed in vitro. For the in vitro method, those skilled in the art may appropriately use known techniques.
The method of deactivating activated hepatic stellate cells in the present invention may be an in vitro method or an in vitro method within cells.
　活性化肝星細胞を脱活性化する方法は、活性化肝星細胞を培養する工程をさらに含んでもよい。 The method for deactivating activated hepatic stellate cells may further include the step of culturing activated hepatic stellate cells.
　本明細書において、「培養」とは、生体外で行われる操作であり、「培養」には、肝星細胞を維持すること、肝星細胞を増殖すること、肝星細胞の成長を促進すること、肝星細胞を分化させること、肝星細胞を発達させること等を含む。 As used herein, "cultivation" is an operation performed in vitro, and "cultivation" includes maintaining hepatic stellate cells, proliferating hepatic stellate cells, and promoting the growth of hepatic stellate cells. This includes differentiating hepatic stellate cells, developing hepatic stellate cells, etc.
　活性化肝星細胞を、本発明の脱活性化剤と接触させる工程において、活性化肝星細胞に対して、脱活性化剤は、構成成分を同時に接触させてもよく、組み合わせて接触させてもよく、それぞれ独立に接触させてもよい。それぞれ独立に接触させる場合、接触させる順序は、特に限定されない。
　本発明の脱活性化剤も、各成分を同時に用いてもよく、別時に用いる各成分を含み、所望によりプロトコールを記載する説明書をさらに含む、キットのような形態であってよい。 In the step of contacting the activated hepatic stellate cells with the deactivating agent of the present invention, the components of the deactivating agent may be brought into contact with the activated hepatic stellate cells simultaneously or in combination. They may be brought into contact with each other independently. When contacting each independently, the order of contact is not particularly limited.
The deactivating agent of the present invention may also be in the form of a kit, which may include each component used at the same time, or may include each component used separately and, if desired, further include an instruction manual describing the protocol.
　脱活性化剤は、活性化肝星細胞を含む培地等に添加することによって、活性化肝星細胞と接触させてもよい。脱活性化剤添加後の培養条件は、通常、活性化肝星細胞を培養する場合と同様でよく、例えば、３７℃・５％ＣＯ２インキュベーター内で数日間～３０日間程度の培養でよい。培養期間としては、特に限定されないが、例えば、１日間、２日間、３日間、４日間、５日間、６日間、１週間、１０日間、２週間、２０日間、３週間、２５日間、３０日間等が挙げられる。 The deactivating agent may be brought into contact with activated hepatic stellate cells by adding it to a medium containing activated hepatic stellate cells. The culture conditions after addition of the deactivating agent may be the same as those for culturing activated hepatic stellate cells, for example, culturing in a 37° C., 5% CO2 incubator for several days to about 30 days. The culture period is not particularly limited, but for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 20 days, 3 weeks, 25 days, 30 days. etc.
　脱活性化剤を添加した肝星細胞の培養は、プレート上で行ってもよい。用いられるプレートとしては、特に限定されないが、例えば、４ウェル、６ウェル、８ウェル、１２ウェル、２４ウェル、４８ウェル、９６ウェル、３８４ウェル、１５３６ウェル、又は任意のウェル数のプレートを用い得る。 The culture of hepatic stellate cells to which a deactivating agent has been added may be performed on a plate. The plate used is not particularly limited, but for example, a plate with 4 wells, 6 wells, 8 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, or any number of wells can be used. .
　脱活性化剤を添加した肝星細胞の培養に用いられる培地としては、特に限定されないが、例えば、ＳｔｅｍＰｒｏ３４等の幹細胞培養に用いられる培地、ダルベッコ改変イーグル培地、アルファ最小必須培地、アルファ改変イーグル培地等が挙げられる。 The medium used for culturing hepatic stellate cells containing a deactivating agent is not particularly limited, but includes, for example, a medium used for stem cell culture such as StemPro34, Dulbecco's modified Eagle's medium, Alpha minimal essential medium, Alpha modified Eagle's medium. etc.
　１ウェル当たりの細胞を含む培地の容量は、培養条件等に応じて適宜設定することができる。培地の容量としては、特に限定されないが、例えば、１ウェル当たり、１μＬ、２μＬ、３μＬ、４μＬ、５μＬ、６μＬ、７μＬ、８μＬ、９μＬ、１０μＬ、１１μＬ、１２μＬ、１３μＬ、１４μＬ、１５μＬ、１６μＬ、１７μＬ、１８μＬ、１９μＬ、２０μＬ、２５μＬ、３０μＬ、３５μＬ、４０μＬ、５０μＬ、７５μＬ、１００μＬ、１５０μＬ、２００μＬ、２５０μＬ、５００μＬ、７００μＬ、１ｍＬ、１．５ｍＬ、２ｍＬ等が挙げられる。 The volume of the medium containing cells per well can be appropriately set depending on the culture conditions and the like. The volume of the medium is not particularly limited, but for example, per well, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL. , 18 μL, 19 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 50 μL, 75 μL, 100 μL, 150 μL, 200 μL, 250 μL, 500 μL, 700 μL, 1 mL, 1.5 mL, 2 mL, etc.
　培養に用いられる細胞数としては、特に限定されないが、例えば、１個以上、１０個以上、１００個以上、１０００個以上、２０００個以上、３０００個以上、４０００個以上、５０００個以上、６０００個以上、７０００個以上、８０００個以上、９０００個以上、１００００個以上等が挙げられる。 The number of cells used for culture is not particularly limited, but for example, 1 or more, 10 or more, 100 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000. Examples include 7,000 or more, 8,000 or more, 9,000 or more, 10,000 or more.
４．脱活性化された細胞を製造する方法
　本発明は、活性化肝星細胞を、本発明の脱活性化剤と接触させる工程を含む、細胞の製造方法にも関する。活性化肝星細胞を、本発明の脱活性化剤と接触させる工程は、上述の３．活性化肝星細胞を脱活性化する方法において説明した態様で行ってよい。 4. Method for Producing Deactivated Cells The present invention also relates to a method for producing cells, which includes the step of contacting activated hepatic stellate cells with the deactivating agent of the present invention. The step of contacting activated hepatic stellate cells with the deactivating agent of the present invention is performed in step 3 above. This may be carried out in the manner described in the method for deactivating activated hepatic stellate cells.
　本発明は、脱活性化された細胞を製造する方法によって製造された細胞にも関する。 The present invention also relates to cells produced by a method for producing deactivated cells.
　脱活性化された細胞を製造する方法によって製造された細胞は、脱活性化された状態にあることが好ましい。脱活性化された細胞を製造する方法によって製造された細胞が、脱活性化された状態にあるかどうかの確認は、特に限定されないが、１．肝星細胞において述べたように、例えば、活性化肝星細胞が脱活性化された場合に指標となるタンパク質又は遺伝子をマーカーとして用いて、そのｍＲＮＡ発現を測定する方法等が使用され得る。 The cells produced by the method for producing deactivated cells are preferably in a deactivated state. Confirmation of whether cells produced by the method for producing deactivated cells are in a deactivated state is not particularly limited, but includes the following steps: 1. As described for hepatic stellate cells, for example, a method may be used in which a protein or gene that becomes an indicator when activated hepatic stellate cells are deactivated is used as a marker and the mRNA expression thereof is measured.
　細胞の保存期間や保存容器は、用途や保存条件等の条件に応じて適宜設定することができる。保存容器としては、例えば、細胞培養用バッグ、チューブ、フラスコ、輸液バッグ、シリンジ等が挙げられる。 The storage period and storage container for cells can be set as appropriate depending on the usage, storage conditions, and other conditions. Examples of storage containers include cell culture bags, tubes, flasks, infusion bags, and syringes.
　本発明の脱活性化された細胞を製造する方法によって製造された細胞は、溶液中に懸濁させた細胞懸濁液の形態としてもよい。 The cells produced by the method for producing deactivated cells of the present invention may be in the form of a cell suspension suspended in a solution.
　脱活性化された細胞を製造する方法によって製造された細胞を、溶液に懸濁する方法は特に限定されず、当業者が適宜公知の手法により行うことができる。特に限定されないが、例えば、脱活性化された細胞を製造する方法によって製造された細胞と溶液とを混合して攪拌することにより、細胞懸濁液を得ることができる。 The method for suspending cells produced by the method for producing deactivated cells in a solution is not particularly limited, and can be carried out by a person skilled in the art using a known method as appropriate. Although not particularly limited, for example, a cell suspension can be obtained by mixing and stirring cells produced by a method for producing deactivated cells and a solution.
　細胞を懸濁する溶液としては、特に限定されず、培養に用いられる培地や保存液等、適宜当業者が決定してよい。そのような溶液としては、特に限定されないが、例えば、ＳｔｅｍＰｒｏ３４等の幹細胞培養に用いられる培地、ダルベッコ改変イーグル培地、アルファ最小必須培地、アルファ改変イーグル培地、日本薬局リンゲル液、ヒト血清等が挙げられる。 The solution in which the cells are suspended is not particularly limited, and may be appropriately determined by those skilled in the art, such as the medium or preservation solution used for culture. Examples of such a solution include, but are not limited to, a medium used for stem cell culture such as StemPro34, Dulbecco's modified Eagle's medium, Alpha minimal essential medium, Alpha modified Eagle's medium, Japanese Pharmacy Ringer's solution, human serum, and the like.
　本発明の脱活性化された細胞を製造する方法によって製造された細胞を含む細胞懸濁液における細胞の濃度は、用途や保存期間等の条件に応じて適宜設定することができる。細胞懸濁液の細胞の濃度は、特に限定されないが、例えば、１．０×１０4～１．０×１０10ｃｅｌｌｓ／ｍＬ程度が挙げられる。 The concentration of cells in a cell suspension containing cells produced by the method for producing deactivated cells of the present invention can be appropriately set depending on conditions such as usage and storage period. The concentration of cells in the cell suspension is not particularly limited, but may be, for example, about 1.0×10 4 to 1.0×10 10 cells/mL.
　本発明の細胞懸濁液を保存する際の温度は、溶液の種類、細胞懸濁液の用途、保存期間等に応じて適宜設定すればよく、特に限定されないが、０℃以上５０℃以下であってもよい。凍結保存する場合には、特に限定されないが、例えば－１９０℃以上－６０℃以下であってよい。 The temperature at which the cell suspension of the present invention is stored may be appropriately set depending on the type of solution, the use of the cell suspension, the storage period, etc., and is not particularly limited, but is between 0°C and 50°C. There may be. In the case of cryopreservation, the temperature may be, for example, not less than -190°C and not more than -60°C, although there is no particular limitation.
　以下、本発明を実施する形態を実施例によってさらに具体的に説明するが、本発明はこれらの実施例のみに限定されるものではない。 Hereinafter, the embodiments of the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.
（１）ｉＰＳ細胞由来活性化肝星細胞の調製
　肝星細胞の分化誘導法（Ｋｏｕｉ Ｙ，ｅｔ ａｌ．Ｓｔｅｍ Ｃｅｌｌ Ｒｅｐｏｒｔｓ．２０２１）に従い、野生型もしくはＡＣＴＡ２レポーター（ＡＣＴＡ２－ＲＦＰもしくはＡＣＴＡ２－ｆｉｒｅｆｌｙ ｌｕｃｉｆｅｒａｓｅ）ヒトｉＰＳ細胞（ＴｋＤＮ４－Ｍ株（ヒト線維芽細胞由来ｉＰＳ細胞）：東京大学医科学研究所）から肝星細胞を分化誘導した。この肝星細胞をＳｔｅｍＰｒｏ３４液体培地（Ｇｉｂｃｏ）に懸濁し、コラーゲンコートしたプレート（ＶＩＯＬＡＭＯ、ＶＴＣ－Ｐ６）上に播種、３７℃・５％ ＣＯ２インキュベーター内で３日間培養した。その後、培地をＳｔｅｍＰｒｏ３４液体培地のみのものに交換し、４日間培養することで肝星細胞を活性化させて、活性化肝星細胞を得た。 (1) Preparation of iPS cell-derived activated hepatic stellate cells Wild type or ACTA2 reporter (ACTA2-RFP or ACTA2-firefly luciferase) according to the hepatic stellate cell differentiation induction method (Koui Y, et al. Stem Cell Reports. 2021). Hepatic stellate cells were induced to differentiate from human iPS cells (TkDN4-M strain (human fibroblast-derived iPS cells): Institute of Medical Science, University of Tokyo). The hepatic stellate cells were suspended in StemPro34 liquid medium (Gibco), seeded on a collagen-coated plate (VIOLAMO, VTC-P6), and cultured in a 37°C/5% CO2 incubator for 3 days. Thereafter, the medium was replaced with only StemPro34 liquid medium and cultured for 4 days to activate hepatic stellate cells to obtain activated hepatic stellate cells.
（２）活性化肝星細胞の脱活性化誘導能の評価（遺伝子発現）
　（１）で得られた活性化肝星細胞を培養底面に付した培地に、ＳｔｅｍＰｒｏ３４液体培地に、ＳＢ４３１５４２（５μＭ）（Ｔｏｃｒｉｓ）、ドルソモルフィン二塩酸塩（０．５μＭ）（Ｔｏｃｒｉｓ）、Ｙ２７６３２（１０μＭ）（Ｗａｋｏ Ｐｕｒｅ Ｃｈｅｍｉｃａｌ Ｉｎｄｕｓｔｒｉｅｓ，Ｌｔｄ．）、ラニフィブラノール（３０μＭ）（Ｓｅｌｌｅｃｋ）、レチノール（１０μＭ）（Ｓｉｇｍａ）、パルミチン酸（１００μＭ）（Ｓｉｇｍａ）（上記６種の化合物を混合したものを以後「ＳＤＹＬＶ」と表記する）を添加し、（１）のプレート培養と同様の培養環境で３日間培養した。培養３日後に細胞をＰＢＳで洗浄し、ＲＮＡをＮｕｃｌｅｏｓｐｉｎ ＲＮＡ Ｐｌｕｓ（Ｔａｋａｒａ Ｂｉｏ）を用いて抽出・精製した。次に、このｍＲＮＡをＰｒｉｍｅ Ｓｃｒｉｐｔ ＲＴ Ｋｉｔ（Ｔａｋａｒａ Ｂｉｏ）を用いてｃＤＮＡ化し、ＴＢ Ｇｒｅｅｎ Ｐｒｅｍｉｘ ＥＸ ＴａｑＩＩ（Ｔａｋａｒａ Ｂｉｏ）を用いてｒｅａｌ－ｔｉｍｅ定量ＰＣＲ（ＬｉｇｈｔＣｙｃｌｅｒ　９６　Ｒｏｃｈｅ）を行った。活性化マーカーの遺伝子（ＡＣＴＡ２、ＣＯＬ１Ａ１等）の発現抑制に加えて、脱活性化マーカーの遺伝子であるＴＣＦ２１（Ｎａｋａｎｏ Ｙ，ｅｔ ａｌ．Ｈｅｐａｔｏｌｏｇｙ．２０２０）の発現を促進するかを検証した（図１～３）。 (2) Evaluation of ability to induce deactivation of activated hepatic stellate cells (gene expression)
The activated hepatic stellate cells obtained in (1) were added to the culture bottom, and the StemPro34 liquid medium was added to SB431542 (5 μM) (Tocris), dorsomorphin dihydrochloride (0.5 μM) (Tocris), Y27632 ( 10 μM) (Wako Pure Chemical Industries, Ltd.), ranifibranol (30 μM) (Selleck), retinol (10 μM) (Sigma), palmitic acid (100 μM) (Sigma) (hereinafter, a mixture of the above six compounds (referred to as "SDYLV") was added thereto, and cultured for 3 days in the same culture environment as the plate culture in (1). After 3 days of culture, the cells were washed with PBS, and RNA was extracted and purified using Nucleospin RNA Plus (Takara Bio). Next, this mRNA was converted into cDNA using Prime Script RT Kit (Takara Bio), and real-time quantitative PCR (LightCycler 96 Roc) was performed using TB Green Premix EX TaqII (Takara Bio). he) was performed. In addition to suppressing the expression of activation marker genes (ACTA2, COL1A1, etc.), we verified whether it promoted the expression of TCF21 (Nakano Y, et al. Hepatology. 2020), a deactivation marker gene (Figure 1 ~3).
（３）活性化肝星細胞の脱活性化誘導能の評価（細胞形態）
　上記（１）で３日間培養した後の細胞に対して、ＴｒｙｐＬＥ Ｅｘｐｒｅｓｓ／ＥＤＴＡ（Ｇｉｂｃｏ）を作用させ、培養底面より剥離・単一細胞化して回収した。回収したそれぞれの細胞を「剥離前と同じ成分の培養液」で懸濁し、コラーゲンコートしたプレートに播種し、２４時間培養した。その後、細胞骨格分子であるＦ－ＡｃｔｉｎをＰｈａｌｌｏｉｄｉｎ（Ａｌｅｘａ Ｆｌｕｏｒ（商標）６８０ Ｐｈａｌｌｏｉｄｉｎ： Ａ２２２８６）によって染色し、蛍光顕微鏡（Ｋｅｙｅｎｃｅ，ＢＺ－Ｘ８１０）を用いて撮影し、細胞形態を評価した（図４）。 (3) Evaluation of deactivation inducing ability of activated hepatic stellate cells (cell morphology)
After culturing for 3 days in the above (1), the cells were treated with TrypLE Express/EDTA (Gibco), detached from the bottom of the culture, and collected into single cells. Each of the collected cells was suspended in a "culture solution with the same components as before detachment", seeded on a collagen-coated plate, and cultured for 24 hours. Thereafter, F-Actin, a cytoskeletal molecule, was stained with Phalloidin (Alexa Fluor™ 680 Phalloidin: A22286), and the cells were photographed using a fluorescence microscope (Keyence, BZ-X810) to evaluate cell morphology (Figure 4). ).

Claims (14)

  1. 　トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤、アクチビン受容体様キナーゼ２阻害剤及びＲｈｏ－キナーゼ阻害剤を含む、活性化肝星細胞の脱活性化剤。 A deactivator for activated hepatic stellate cells, including a transforming growth factor β/bone morphogenetic protein signal inhibitor, an activin receptor-like kinase 2 inhibitor, and a Rho-kinase inhibitor.
  2. 　トランスフォーミング増殖因子β／骨形成タンパク質シグナル阻害剤が、ＳＢ４３１５４２、Ａ　８３－０１、ＥＷ－７１９５、ＩＮ－１１３０、ＴＧＦＢＲ１－ＩＮ－１、バクトセルチブ、Ｚ１２６０１０１１及びこれらの塩からなる群から選択される１つ以上である、請求項１に記載の脱活性化剤。 The transforming growth factor β/bone morphogenetic protein signal inhibitor is selected from the group consisting of SB431542, A83-01, EW-7195, IN-1130, TGFBR1-IN-1, bactosertib, Z12601011 and salts thereof. The deactivating agent according to claim 1, wherein the deactivating agent is one or more.
  3. 　アクチビン受容体様キナーゼ２阻害剤が、ドルソモルフィン、ＡＬＫ２－ＩＮ－２、ＡＬＫ２－ＩＮ－４、ＤＭＨ１、Ｋ０２２８８、ＬＤＮ１９３１８９、ＬＤＮ－２１４１１７、ＬＤＮ－２１２８５４、ＭＬ３４７、ＯＤ３６及びこれらの塩からなる群から選択される１つ以上である、請求項１又は２に記載の脱活性化剤。 The activin receptor-like kinase 2 inhibitor is from the group consisting of dorsomorphin, ALK2-IN-2, ALK2-IN-4, DMH1, K02288, LDN193189, LDN-214117, LDN-212854, ML347, OD36 and salts thereof. The deactivating agent according to claim 1 or 2, which is one or more selected deactivating agents.
  4. 　Ｒｈｏ－キナーゼ阻害剤が、Ｙ２７６３２、ナルシクラシン、アフレセルチブ、アザインドール１、ＡＴ１３１４８、Ｂｅｌｕｍｏｓｕｄｉｌ（ＫＤ０２５）、ＢＤＰ５２９０、クロマン　１、ＣＭＰＤ１０１、ＣＲＴ００６６８５４、エメチン、ファスジル、ＧＳＫ－２５、ＧＳＫ１８０７３６Ａ、ＧＳＫ２６９９６２Ａ、ＧＳＫ４２９２８６Ａ、グリシル　Ｈ－１１５２、Ｈ－１１５２、ヒドロキシファスジル（ＨＡ－１１００）、ＨＳＤ１５９０、Ｈｕ７６９１、ネタルスジル（ＡＲ－１３３２４）、ＲＯＣＫ２－ＩＮ－２、ＲＯＣＫ２－ＩＮ－５、ＲＯＣＫ－ＩＮ－１、ＲＯＣＫ－ＩＮ－２、リパスジル、ＲＫＩ－１４４７、Ｓｏｖｅｓｕｄｉｌ、ＳＲ－３６７７、ＳＡＲ４０７８９９、チアゾビビン、Ｖｅｒｏｓｕｄｉｌ、Ｙ－３３０７５、Ｙ－３９９８３及びこれらの塩からなる群から選択される１つ以上である、請求項１～３のいずれか１項に記載の脱活性化剤。 Rho-kinase inhibitors include Y27632, narciclasine, aflesertib, azaindole 1, AT13148, Belumosudil (KD025), BDP5290, Chroman 1, CMPD101, CRT0066854, emetine, fasudil, GSK-25, GSK180736A, G SK269962A, GSK429286A, Glycyl H- 1152, H-1152, Hydroxyfasudil (HA-1100), HSD1590, Hu7691, Netarsudil (AR-13324), ROCK2-IN-2, ROCK2-IN-5, ROCK-IN-1, ROCK-IN-2, Ripasudil , RKI-1447, Sovesudil, SR-3677, SAR407899, Thiazovivine, Verosudil, Y-33075, Y-39983, and salts thereof. The deactivating agent described in Section.
  5. 　ペルオキシソーム増殖剤活性化受容体アゴニストをさらに含む、請求項１～４のいずれか１項に記載の脱活性化剤。 The deactivation agent according to any one of claims 1 to 4, further comprising a peroxisome proliferator-activated receptor agonist.
  6. 　ペルオキシソーム増殖剤活性化受容体アゴニストが、ラニフィブラノール、カプリン酸、ＥＰＩ－００１、フェノフィブリン酸、ＦＨ５３５、ＧＷ９６６２、ＧＳＫ３７８７、ＧＳＫ０６６０、ＧＷ６４７１、ハルミン、Ｔ００７０９０７及びこれらの塩からなる群から選択される１つ以上である、請求項１～５のいずれか１項に記載の脱活性化剤。 the peroxisome proliferator-activated receptor agonist is selected from the group consisting of ranifibranol, capric acid, EPI-001, fenofibric acid, FH535, GW9662, GSK3787, GSK0660, GW6471, harmine, T0070907 and salts thereof; The deactivating agent according to any one of claims 1 to 5, wherein the deactivating agent is 3 or more.
  7. 　ビタミンＡをさらに含む、請求項１～６のいずれか１項に記載の脱活性化剤。 The deactivator according to any one of claims 1 to 6, further comprising vitamin A.
  8. 　ビタミンＡが、レチノール、レチナール、レチノイン酸及びこれらの塩からなる群から選択される１つ以上である、請求項７に記載の脱活性化剤。 The deactivator according to claim 7, wherein the vitamin A is one or more selected from the group consisting of retinol, retinal, retinoic acid, and salts thereof.
  9. 　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩をさらに含む、請求項１～８のいずれか１項に記載の脱活性化剤。 The deactivator according to any one of claims 1 to 8, further comprising a C 12 to C 18 fatty acid or a salt thereof.
  10. 　Ｃ１２～Ｃ１８脂肪酸又はそれらの塩が、パルミチン酸、ラウリン酸、ミリスチン酸、ステアリン酸及びこれらの塩からなる群から選択される１つ以上である、請求項９に記載の脱活性化剤。 The deactivator according to claim 9, wherein the C 12 to C 18 fatty acid or a salt thereof is one or more selected from the group consisting of palmitic acid, lauric acid, myristic acid, stearic acid, and salts thereof. .
  11. 　活性化肝星細胞を脱活性化する方法であって、
    　活性化肝星細胞を、請求項１～１０のいずれか１項に記載の脱活性化剤と接触させる工程を含む、方法。     A method for deactivating activated hepatic stellate cells, the method comprising:
    A method comprising the step of contacting activated hepatic stellate cells with a deactivating agent according to any one of claims 1 to 10.
  12. 　活性化肝星細胞が、ヒト活性化肝星細胞である、請求項１１に記載の方法。 The method according to claim 11, wherein the activated hepatic stellate cells are human activated hepatic stellate cells.
  13. 　脱活性化された細胞の製造方法であって、
    　活性化肝星細胞を、請求項１～１０のいずれか１項に記載の脱活性化剤と接触させる工程を含む、細胞の製造方法。     A method for producing deactivated cells, the method comprising:
    A method for producing cells, comprising the step of contacting activated hepatic stellate cells with the deactivating agent according to any one of claims 1 to 10.
  14. 　請求項１３に記載の方法で製造される、脱活性化肝星細胞。 Deactivated hepatic stellate cells produced by the method according to claim 13.
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Publication number Priority date Publication date Assignee Title
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020166726A1 (en) * 2019-02-15 2020-08-20 国立大学法人 東京大学 Method for preparing quiescent hepatic stellate cell, and model for evaluation of activation of quiescent hepatic stellate cell

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KOUI YUTA, HIMENO MISAO, MORI YUSUKE, NAKANO YASUHIRO, SAIJOU EIKO, TANIMIZU NAOKI, KAMIYA YOSHIKO, ANZAI HIROKO, MAEDA NATSUKI, W: "Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and in vitro disease modeling", STEM CELL REPORTS, CELL PRESS, UNITED STATES, vol. 16, no. 12, 1 December 2021 (2021-12-01), United States , pages 3050 - 3063, XP093109711, ISSN: 2213-6711, DOI: 10.1016/j.stemcr.2021.11.002 *
LEE TING FANG, MAK KI M., RACKOVSKY ORI, LIN YUN‐LIAN, KWONG ALLISON J., LOKE JOHNNY C., FRIEDMAN SCOTT L.: "Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation‐related protein (ADRP)", JOURNAL OF CELLULAR PHYSIOLOGY, WILEY SUBSCRIPTION SERVICES, INC., US, vol. 223, no. 3, 1 June 2010 (2010-06-01), US , pages 648 - 657, XP093109726, ISSN: 0021-9541, DOI: 10.1002/jcp.22063 *
PESCHL VANESSA, SEITZ TATJANA, SOMMER JUDITH, THASLER WOLFGANG, BOSSERHOFF ANJA, HELLERBRAND CLAUS: "Bone morphogenetic protein 13 in hepatic stellate cells and hepatic fibrosis", JOURNAL OF CELLULAR BIOCHEMISTRY, JOHN WILEY & SONS, INC. JOHN WILEY & SONS, INC., HOBOKEN, USA, vol. 123, no. 10, 1 October 2022 (2022-10-01), Hoboken, USA, pages 1544 - 1552, XP093109713, ISSN: 0730-2312, DOI: 10.1002/jcb.30248 *
WETTSTEIN GUILLAUME, LUCCARINI JEAN‐MICHEL, POEKES LAURENCE, FAYE PATRICK, KUPKOWSKI FRANCINE, ADARBES VANESSA, DEFRÊNE EVELYNE, E: "The new‐generation pan‐peroxisome proliferator‐activated receptor agonist IVA337 protects the liver from metabolic disorders and fibrosis", HEPATOLOGY COMMUNICATIONS, vol. 1, no. 6, 1 August 2017 (2017-08-01), pages 524 - 537, XP093109722, ISSN: 2471-254X, DOI: 10.1002/hep4.1057 *

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