Anti-fibrosis agent, pharmaceutical composition for treating fibrosis, and method for deactivating muscle fibroblasts

Abstract

An anti-fibrosis agent containing a copolymer having: a constituent unit (a) containing an N-acetylglucosamine group; and a constituent unit (b) that includes a structure represented by general formula (b), but that does not conform to the constituent unit (a). In general formula (b), Yb represents a divalent linking group containing an oxygen atom. And Rb represents a hydrogen atom or an organic group. * represents an atomic bond. *-Yb-Rb (b).

Description

Technical Field

[0001] The present invention relates to an anti-fibrotic agent, a pharmaceutical composition for treating fibrosis, and a method for deactivating myofibroblasts.

[0002] This application claims priority based on Japanese Patent Application No. 2022-165423 filed in Japan on October 14, 2022, and the contents of Japanese Patent Application No. 2022-165423 are cited in this specification. Background Art

[0003] Tissue fibrosis is caused by chronic inflammation due to repeated damage. Chronic inflammation changes fibroblasts and astrocytes, maintains tissues in myofibroblasts, and activates astrocytes. This activation promotes the strengthening of these cells and the production of abundant extracellular matrices such as collagen. Chronic inflammatory diseases such as tissue fibrosis, cancer, and autoimmunity that occur in this way are caused by persistent inflammation caused by intermittent and repeated tissue damage.

[0004] Severe tissue damage caused by repeated tissue damage can lead to the production of a large amount of cell fragments released by dying cells. Among the intracellular molecules contained in the cell fragments released by damaged or dead cells, there are molecules that can play a role in allowing inflammatory cells to recognize tissue damage, which are called damage-associated molecular patterns (DAMPs) (Non-patent literature 1). DAMPs act as danger signals to induce inflammatory responses to protect host tissues from harmful conditions such as tissue damage and infection. High-mobility group box 1 (HMGB1), heat shock protein (HSP), adenosine triphosphate (ATP), etc. have clearly defined functions in cells, but these molecules leak out from dying cells and act as DAMPs in the extracellular space.

[0005] The presence of abundant DAMPs after severe tissue damage can induce the recruitment and activation of immune cells, which can secrete inflammatory cytokines and cytokines that promote fiber formation (non-patent literature 2). These cytokines can eventually induce astrocytes and fibroblasts to differentiate into activated astrocytes and myofibroblasts, promoting hyperplasia or fibrosis during tissue remodeling (non-patent literature 3). The fibrosis of such tissues is caused by the chronic inflammation produced by repeated injuries. In chronic inflammation, due to abundant collagen deposition and parenchymal cells being discharged, the dysfunction of fibrous tissue is finally caused.

[0006] In order to recover from tissue fibrosis, it is necessary to selectively target myofibroblasts or activated stellate cells and inhibit the activation of each cell. For example, a method has been proposed in which an antagonist of SERPINE2 is administered to human lung fibroblasts to inhibit the expression level of collagen 1A1 and / or α-smooth muscle actin (αSMA) in human lung fibroblasts exposed to SERPINE2 (Patent Document 1). In addition, as a therapeutic drug targeting chronic inflammation or fibrotic sites, for example, a fibrosis therapeutic drug has been proposed, which contains a polypeptide composed of a specific amino acid sequence, or an amino acid sequence that shows 85% or more identity with the specific amino acid sequence and is selected from at least one of the group consisting of COL1A1, COL1A2, and αSMA (Patent Document 2).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application No. 2012-509941

[0010] Patent Document 2: Japanese Patent Application Publication No. 2022-66026

[0011] Non-patent literature

[0012] Non-patent document 1: Bianchi ME., “DAMPs, PAMPs and alarmins: all we need to know about danger.”, J Leukoc Biol., Vol. 81, Issue 1, pp. 1-5, 2007. doi: 10.1189 / jlb.0306164. Epub 2006 Oct 10. PMID: 17032697.

[0013] Non-patent document 2: Bolourani, S., “The interplay of DAMPs, TLR4, and proinflammatory cytokines in pulmonary fibrosis.”, J. Mol. Med. (Berl)., Vol. 99, Issue 10, pp. 1373-1384, 2021.

[0014] Non-patent document 3: An, P. et al., "Hepatocyte mitochondria-derived dangersignals directly activate hepatic stellate cells and drive progression ofliver fibrosis.", Nat. Commun., Vol. 11, Article No. 2362, 2020. Summary of the invention

[0015] Problems to be solved by the invention

[0016] Despite the above findings, there is still a demand for a more effective anti-fibrotic agent that can induce myofibroblast deactivation and inhibit fibrosis.

[0017] Therefore, an object of the present invention is to provide an antifibrotic agent, a pharmaceutical composition for treating fibrosis containing the antifibrotic agent, and a method for deactivating the myofibroblasts, which are capable of inducing the deactivation of myofibroblasts.

[0018] Technical means of solving problems

[0019] The present inventors have conducted intensive studies and, as a result, have found a copolymer having a structural unit specifically containing N-acetylglucosamine and other structural units, and that the copolymer targets myofibroblasts and inhibits their activation, thereby completing the present invention.

[0020] The present invention includes the following aspects.

[0021] [1] An anti-fibrosis agent comprising a copolymer having: a constituent unit (a) comprising an N-acetylglucosamine group; and a constituent unit (b) comprising a structure represented by the following general formula (b), except for constituent unit (a).

[0022] *—Yb——Rb (b)

[0023] In formula (b), Yb represents a divalent linking group including an oxygen atom; Rb represents a hydrogen atom or an organic group. * represents an atomic bond.

[0024] [2] The anti-fibrosis agent according to [1], wherein the structural unit (b) is a structural unit represented by the following general formula (b-1).

[0025]

[0026] In formula (b-1), Rb 1 represents a hydrogen atom or an organic group; Yb1 represents -C(=O)-O- or -C(=O)-NH-; Rb 2 represents a hydrogen atom or an organic group.

[0027] [3] The anti-fibrosis agent according to [1], wherein the copolymer is a compound represented by the following general formula (b-2).

[0028]

[0029] In formula (b-2), Lc 1 A linking group representing (a+b) valence; Ya 2 and Yb 2 Each independently represents a divalent linking group containing an oxygen atom; GlcNAC represents N-acetylglucosamine; Rb 3 represents an organic group; as long as the atomic valence permits, a and b each independently represent an integer greater than 1. When a is an integer greater than 2, two or more Ya 2 They may be the same or different from each other. When b is an integer greater than 2, two or more Yb 2 Can be the same or different, 2 or more Rb 3 They can be the same as each other or different.

[0030] [4] The anti-fibrosis agent according to any one of [1] to [3], wherein the molar ratio of the structural unit (a) to the structural unit (b) is 10:1 to 1:20.

[0031] [5] The anti-fibrosis agent according to [2], wherein Rb in the general formula (b-1) is 2 The invention comprises at least one structure selected from the group consisting of an ether bond, an ester bond, an amine ester bond, and an amide bond.

[0032] [6] The anti-fibrosis agent according to any one of [1] to [5], wherein the structural unit (a) is a structural unit comprising a structure represented by the following formula (a1-1-1).

[0033]

[0034] In formula (a1-1-1), * represents an atomic bond.

[0035] [7] The anti-fibrosis agent according to [1] or [2], wherein the structural unit (a) is a structural unit represented by the following formula (a-1-1).

[0036]

[0037] [8] The anti-fibrosis agent according to [2], wherein the structural unit (b) is a structural unit derived from acrylamide or a compound represented by the following general formula (b1-1).

[0038]

[0039] In the formula (b1-1), k represents an integer of 1-12.

[0040] [9] The anti-fibrosis agent according to any one of [1] to [8], wherein the copolymer is a copolymer represented by any one of the following general formulas (C-1) to (C-5).

[0041]

[0042] In formulae (C-1) to (C-4), R represents an alkyl group having 1 to 18 carbon atoms; and a and b each independently represent an integer of 1 to 30.

[0043]

[0044] In formula (C-5), Rx 1 ~Rx 4 Each independently represents a group represented by formula (RX-1) or (RX-2). 1 ~Rx 4 At least one of the groups is a group represented by formula (RX-1). n represents an integer greater than or equal to 1. * represents an atomic bond.

[0045]

[10] The anti-fibrosis agent according to any one of [1] to [9], wherein the weight average molecular weight of the copolymer is in the range of 2,000 to 20,000.

[0046]

[11] The anti-fibrosis agent according to any one of [1] to

[10] , wherein the number average molecular weight of the copolymer is in the range of 2,000 to 15,000.

[0047]

[12] The anti-fibrosis agent according to any one of [1] to

[11] , wherein the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the copolymer is in the range of 1.0 to 1.7.

[0048]

[13] A pharmaceutical composition for treating fibrosis, comprising the anti-fibrosis agent according to any one of [1] to

[12] as an active ingredient.

[0049]

[14] A method for inactivating myofibroblasts, comprising binding the anti-fibrotic agent according to any one of [1] to

[12] to myofibroblasts.

[0050] Effects of the Invention

[0051] According to the present invention, there can be provided an antifibrotic agent, a pharmaceutical composition for treating fibrosis containing the antifibrotic agent, and a method for deactivating the myofibroblasts, which can induce the deactivation of myofibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : This is a diagram showing the results of the protein blotting method in Example 1. Human skin fibroblasts were cultured for 48 hours in the presence of 10 ng / mL of TGF-β1 to make them into myofibroblasts, and the test compound (copolymer 1, copolymer 2, or polymer 1) was added (10 μg / mL, 50 μg / mL, or 100 μg / mL) to the myofibroblasts, and then cultured for 48 hours. Then, the expression levels of αSMA, collagen (col1a2), heme oxygenase 1 (HMOX1), and β-actin were confirmed by protein blotting. The "control group" represents the results of human skin fibroblasts after culture without any addition, and the "10 ng / mL TGFβ1" represents the results of myofibroblasts after culture without the addition of the test compound.

[0053] Figure 2 This is a diagram showing the results of the protein blotting method in Example 2. Human skin fibroblasts were cultured for 48 hours in the presence of 10 ng / mL TGF-β1 to convert them into myofibroblasts, and the test compound (copolymer 3, copolymer 4, copolymer 5, copolymer 6 or polymer 1) was added (100 μg / mL) to the myofibroblasts, and then cultured for 48 hours. Then, the expression levels of αSMA, collagen (col1a2), matrix metalloproteinase 1 (MMP1), HMOX1, angiopoietin-like protein 4 (ANGPTL4) and β-actin were confirmed by protein blotting. The "control group" represents the results of human skin fibroblasts cultured without any addition, and the "10 ng / mL TGFβ1" represents the results of myofibroblasts cultured without the addition of the test compound.

[0054] Figure 3 The structure of copolymer 8 used in Example 4 is shown.

[0055] Figure 4: This is a diagram showing the results of the protein blotting method in Example 3. Human skin fibroblasts were cultured for 48 hours in the presence of 10 ng / mL of TGF-β1 to convert them into myofibroblasts, and the test compound (copolymer 7) was added (10 μg / mL) to the myofibroblasts, and then cultured for 48 hours. Then, the expression levels of αSMA, collagen (col1a2), matrix metalloproteinase 1 (MMP1), HMOX1, ANGPTL4, superoxide dismutase 2 (SOD2) and β-actin were confirmed by protein blotting. The "control group" represents the results of human skin fibroblasts cultured without any addition, and the "10 ng / mL TGFβ1" represents the results of myofibroblasts cultured without adding the test compound.

[0056] Figure 5 This is a diagram showing the results of the protein blotting method in Example 4. Human skin fibroblasts were cultured for 48 hours in the presence of 10 ng / mL TGF-β1 to convert them into myofibroblasts, and the test compound (copolymer 8) was added (100 μg / mL) to the myofibroblasts, and then cultured for 48 hours. Then, the expression levels of αSMA, collagen (col1a2), and β-actin were confirmed by protein blotting. The "control group" represents the results of human skin fibroblasts cultured without any addition, and the "10 ng / mL TGFβ1" represents the results of myofibroblasts cultured without adding the test compound. DETAILED DESCRIPTION

[0057] (Antifibrotic Agent)

[0058] The anti-fibrosis agent of this embodiment contains a copolymer (hereinafter also referred to as "copolymer C") having: a constituent unit (a) containing an N-acetylglucosamine group; and a constituent unit (b) containing a structure represented by the following general formula (b), but excluding those that conform to the constituent unit (a).

[0059] *—Yb—Rb (b)

[0060] In formula (b), Yb represents a divalent linking group including an oxygen atom; and Rb represents a hydrogen atom or an organic group.

[0061] In one embodiment, the structural unit (b) is a structural unit represented by the following general formula (b-1).

[0062]

[0063] In formula (b-1), Rb 1 represents a hydrogen atom or an organic group; Yb 1represents C(=O)-O- or -C(=O)-NH-; Rb 2 represents a hydrogen atom or an organic group.

[0064] In one embodiment, the copolymer (C) is a compound represented by the following general formula (b-2).

[0065]

[0066] In formula (b-2), Lc 1 A linking group representing (a+b) valence; Ya 2 and Yb 2 Each independently represents a divalent linking group containing an oxygen atom; GlcNAC represents N-acetylglucosamine; Rb 3 represents an organic group; a and b represent integers greater than 1 as long as the atomic valence permits. When a is an integer greater than 2, more than 2 Ya 2 They may be the same or different from each other. When b is an integer greater than 2, two or more Yb 2 Can be the same or different, 2 or more Rb 3 They can be the same as each other or different.

[0067] <Copolymer (C)>

[0068] The copolymer (C) has a structural unit (a) and a structural unit (b).

[0069] 《Constituent unit (a)》

[0070] The constituent unit (a) is a constituent unit containing an N-acetylglucosamine group. As constituent unit (a), constituent units containing N-acetylglucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, or chitohexaose can be cited. Specific examples of constituent unit (a) include constituent units derived from monomers containing an N-acetylglucosamine group, constituent units derived from monomers containing a chitosan group bonded to 2 to 6 N-acetylglucosamine rings, and the like.

[0071] N-acetylglucosamine group is a group represented by the following formula (a1-1): In the following formula, * represents an atomic bond.

[0072]

[0073] The structural unit (a) preferably includes a structure represented by the following formula (a1-1-1): In the following formula (a1-1-1), * represents an atomic bond.

[0074]

[0075] The structural unit (a) further preferably includes a structure represented by the following formula (a1-1-11): In the following formula (a1-1-1), * represents an atomic bond.

[0076]

[0077] As the structural unit (a), there can be mentioned the structural unit represented by the following general formula (a-1).

[0078]

[0079] In formula (a-1), Ra 1 Represents a hydrogen atom or an organic group; Ya 1 represents -C(=O)-O- or -C(=O)-NH-; na represents an integer of 1 to 6.

[0080] In the above formula (a-1), Ra 1 The organic group in Ra includes a hydrocarbon group which may have a substituent. 1 The hydrocarbon group in the formula (a) preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, further preferably 1 to 6 carbon atoms, further preferably 1 to 4 carbon atoms, and particularly preferably 1 to 3 carbon atoms. The hydrocarbon group which may have a substituent may be an aromatic hydrocarbon group which may have a substituent, or may be an aliphatic hydrocarbon group which may have a substituent, and is preferably an aliphatic hydrocarbon group which may have a substituent. The hydrocarbon group which may have a substituent may be saturated or unsaturated, and is preferably saturated.

[0081] As Ra 1 The aliphatic hydrocarbon group in the formula (a) may be an alkyl group having 1 to 12 carbon atoms which may have a substituent. The alkyl group may be linear or branched, and is preferably linear.

[0082] Ra 1 The aliphatic hydrocarbon group in may or may not have a substituent. 1 The substituent that the aliphatic hydrocarbon group in may have includes a carboxyl group, an amino group, a hydroxyl group, an alkoxy group and the like.

[0083] As Ra 1 , preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group.

[0084] In the aforementioned formula (a-1), Ya 1 When -C(=O)-O- and -C(=O)-NH- in formula (a-1) are represented by *-C(=O)-O-** and *-C(=O)-NH-**, respectively, * and Ra in formula (a-1) are 1 The carbon atom to which it is bonded is bonded to -(CH2) in formula (a-1) na-Bonding.

[0085] In the above formula (a-1), na is preferably an integer of 1 to 4, more preferably an integer of 1 to 3, and further preferably 1 or 2.

[0086] As the structural unit (a), a structural unit represented by the following general formula (a-1-1) is preferred.

[0087]

[0088] When the copolymer (C) is a compound represented by the aforementioned general formula (b-2), -Ya in the aforementioned general formula (b-2) 2 The structure represented by GlcNAc is the constitutional unit (a).

[0089] In the above general formula (b-2), Ya 2 is a divalent linking group containing an oxygen atom. 2 , and may include at least one divalent linking group selected from the group consisting of an ether bond, an ester bond, an amide bond, an amine ester bond, and a thioester bond. 2 , the following divalent linking groups can be listed: a combination of at least one selected from the group consisting of an ether bond, an ester bond, an amide bond, an amine ester bond, and a thioester bond and an alkylene group.

[0090] Ya 2 The polyol may contain at least one selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkoxyalkylene group having 1 to 10 carbon atoms, a polyether group, a polyester group, a polyurethane group, and a polyamino acid group. The polyether group is preferably a polyethylene glycol group.

[0091] As Ya 2 , preferably the following divalent linking groups: a combination of at least one selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkoxyalkylene group, and a polyethylene glycol group and at least one selected from the group consisting of an ether bond, an ester bond, an amide bond, an amine ester bond, and a thioester bond.

[0092] In the above general formula (b-2), GlcNAc is an N-acetylglucosamine group and is a group represented by the above formula (a1-1).

[0093] Specific examples of the constituent unit (a) in the copolymer (C) represented by the general formula (b-2) include an example having a structure represented by the following formula (RX-1). 1 Bonded atoms are bonded.

[0094]

[0095] In formula (RX-1), n ​​represents an integer of 1 or more.

[0096] In the above formula (RX-1), n ​​may be an integer of 1 to 30, and an integer of 10 to 30 is preferred.

[0097] Method for producing a compound from which constituent unit (a) can be derived:

[0098] The compound from which the structural unit (a) can be derived can be produced by a known method. For example, a monomer from which the structural unit (a) can be derived can be synthesized by bonding an α,β-unsaturated carboxylic acid to the reducing end of N-acetylglucosamine.

[0099] Specific examples of α,β-unsaturated carboxylic acids include acrylic acid, methacrylic acid, acryloxypropionic acid, citraconic acid, itaconic acid, crotonic acid, maleic acid, and maleic anhydride. Acrylic acid and methacrylic acid are preferred because they have good copolymerizability with other constituent units. The α,β-unsaturated carboxylic acid may be in the form of a salt. Examples of salts of α,β-unsaturated carboxylic acids include metal salts such as sodium salts and potassium salts of the aforementioned α,β-unsaturated carboxylic acid monomers; ammonium salts of β-unsaturated carboxylic acids; and the like.

[0100] As a specific example of a method for producing a compound from which the constituent unit (a) can be derived, the following method can be mentioned: using 4-(4,6-dimethoxy-1,3,5-trichlorobenzene) The acrylic monomer into which the N-acetylglucosamine group is introduced is obtained by subjecting an α,β-unsaturated carboxylic acid to a condensation reaction with an aminated N-acetylglucosamine using a condensation agent such as methyl-2-yl)-4-methylmorpholinium (DMT-MM). For example, the monomer from which the constituent unit (a) can be derived can be produced by mixing aminated N-acetylglucosamine and an α,β-unsaturated carboxylic acid at a molar ratio of 1:1 and subjecting them to a condensation reaction in a solvent such as dimethyl sulfoxide (DMSO) or water in the presence of a condensation agent such as DMT-MM.

[0101] The method for aminated N-acetylglucosamine is not particularly limited, and examples thereof include a method in which an amine group of a compound having an amine group is bonded to the reducing end of N-acetylglucosamine by a reductive amination method. Alternatively, a hydroxyl group of N-acetylglucosamine may be substituted with a carboxyl group, and a compound having an amine group may be bonded by a coupling method using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0102] The structural unit (a) may be used alone or in combination of two or more.

[0103] 《Constituent unit (b)》

[0104] The structural unit (b) is a structural unit including a structure represented by the above-mentioned general formula (b).

[0105] In the above general formula (b), Yb represents a divalent linking group containing an oxygen atom. Examples of Yb include divalent linking groups containing at least one selected from the group consisting of an ether bond, an ester bond, an amide bond, an amine ester bond, and a thioester bond. Yb may be a combination of at least one selected from the group consisting of an ether bond, an ester bond, an amide bond, an amine ester bond, and a thioester bond and an alkylene group.

[0106] In the aforementioned general formula (b), Rb represents a hydrogen atom or an organic group (but excluding organic groups containing N-acetylglucosamine groups). The organic group in Rb is not particularly limited. Examples of the organic group in Rb include hydrocarbon groups that may have substituents. The aforementioned hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group. The aforementioned aliphatic hydrocarbon group may be saturated or unsaturated, preferably saturated. Examples of the organic group in Rb include hydrocarbon groups having 1 to 20 carbon atoms. The aforementioned hydrocarbon group may or may not have a substituent. Examples of the substituent include: carboxyl, amine, hydroxyl, alkoxy, and the like.

[0107] Examples of the structural unit (b) include structural units represented by the above-mentioned general formula (b-1).

[0108] In the above formula (b-1), Rb 1 The organic group in Rb may be a hydrocarbon group which may have a substituent. 1 The hydrocarbon group in is preferably 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, further preferably 1 to 6 carbon atoms, further preferably 1 to 4 carbon atoms, and particularly preferably 1 to 3 carbon atoms. The hydrocarbon group which may have a substituent may be an aromatic hydrocarbon group which may have a substituent, or may be an aliphatic hydrocarbon group which may have a substituent, and is preferably an aliphatic hydrocarbon group which may have a substituent. The hydrocarbon group which may have a substituent may be saturated or unsaturated, and is preferably saturated. Rb 1 The organic group in the formula (I) excludes an organic group containing N-acetylglucosamine.

[0109] As Rb 1 The aliphatic hydrocarbon group in the formula (a) may be an alkyl group having 1 to 12 carbon atoms which may have a substituent. The alkyl group may be linear or branched, and is preferably linear.

[0110] R 1 The aliphatic hydrocarbon group in may or may not have a substituent. 1 The substituent that the aliphatic hydrocarbon group in may have includes a carboxyl group, an amino group, a hydroxyl group, an alkoxy group and the like.

[0111] As Rb 1, preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group.

[0112] In the above formula (b-1), Yb 1 When -C(=O)-O- and -C(=O)-NH- in formula (b-1) are represented by *-C(=O)-O-** and *-C(=O)-NH-**, respectively, * and Rb in formula (b-1) are 1 The carbon atom to which it is bonded is bonded to Rb in formula (b-1) 2 Bonding.

[0113] In the above formula (b-1), Rb 2 The organic group in Rb may be a hydrocarbon group which may have a substituent. 2 The hydrocarbon group in is preferably 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 12 carbon atoms, and particularly preferably 1 to 10 carbon atoms. The hydrocarbon group which may have a substituent may be an aromatic hydrocarbon group which may have a substituent, or may be an aliphatic hydrocarbon group which may have a substituent, and is preferably an aliphatic hydrocarbon group which may have a substituent. The hydrocarbon group which may have a substituent may be saturated or unsaturated, and is preferably saturated. 2 The organic group in the formula (I) excludes an organic group containing N-acetylglucosamine.

[0114] As Rb 2 The aliphatic hydrocarbon group in the embodiment includes an alkyl group having 1 to 20 carbon atoms which may have a substituent. The alkyl group may be linear or branched, and is preferably linear.

[0115] R 2 The aliphatic hydrocarbon group in may or may not have a substituent. 2 The substituents that the aliphatic hydrocarbon group in Rb may have include a carboxyl group, an amino group, a hydroxyl group, and the like. 2 The substituent in may be a substituent that can replace the methylene group (-CH2-) constituting the hydrocarbon chain. Examples of the substituent that can replace the methylene group include an ester bond (-C(=O)-O-), an ether bond (-O-), an amide bond (-C(=O)-NH-), an amine ester bond (-NH-C(=O)-O-), and a thioester bond (-C(=O)-S-). 2 , may be a group containing at least one structure selected from the group consisting of an ether bond, an ester bond, an amine ester bond, and an amide bond.

[0116] As Rb 2 Specific examples of the organic group in include an alkyl group, an alkoxyalkylene group, a polyether group, a polyester group, a polyurethane group, and a polyamino acid group.

[0117] As Rb2 , preferably a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxyalkylene group, or a polyethylene glycol group.

[0118] Specific examples of the structural unit (b) are shown below, but the present invention is not limited to these examples. nb in the following formula (b-1-2) is preferably an integer of 1 to 10, more preferably an integer of 1 to 9. nb is 9, for example.

[0119]

[0120] In the formula (b-1-2), nb represents an integer of 1 to 12. α It represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

[0121] When the copolymer (C) is a compound represented by the general formula (b-2), -Yb in the general formula (b-2) 2 -Rb 3 The structure shown is the constitutional unit (b).

[0122] In the above general formula (b-2), Yb 2 is a divalent linking group containing an oxygen atom. 2 The divalent linking group containing an oxygen atom in the above-mentioned Ya can be exemplified. 2 The same divalent linking group.

[0123] In the above general formula (b-2), Rb 3 Represents an organic group. Examples of the organic group include a hydrocarbon group which may have a substituent. The hydrocarbon group preferably has 1 to 20 carbon atoms, more preferably 1 to 18 carbon atoms, further preferably 1 to 16 carbon atoms, and particularly preferably 1 to 12 carbon atoms. The hydrocarbon group which may have a substituent may be an aromatic hydrocarbon group which may have a substituent, or may be an aliphatic hydrocarbon group which may have a substituent, and is preferably an aliphatic hydrocarbon group which may have a substituent. The hydrocarbon group which may have a substituent may be saturated or unsaturated, and is preferably saturated. Rb 3 The organic group in the formula (I) excludes an organic group containing N-acetylglucosamine.

[0124] As Rb 3 The aliphatic hydrocarbon group in the embodiment includes an alkyl group having 1 to 20 carbon atoms which may have a substituent. The alkyl group may be linear or branched, and is preferably linear.

[0125] R 3 The aliphatic hydrocarbon group in may or may not have a substituent. 2 The substituent that the aliphatic hydrocarbon group in may have includes a carboxyl group, an amino group, a hydroxyl group, and the like.

[0126] Specific examples of the constituent unit (b) in the copolymer (C) represented by the general formula (b-2) include an example having a structure represented by the following formula (RX-2-1). 1 Bonded atoms are bonded.

[0127]

[0128] In formula (RX-2-1), m and n each independently represent an integer of 1 or more.

[0129] In the above formula (RX-2-1), m is preferably an integer of 1 to 20, more preferably an integer of 1 to 16, further preferably an integer of 1 to 14, and particularly preferably an integer of 1 to 12. Specific examples of m include 12.

[0130] In the above formula (RX-2-1), n ​​may be an integer of 1 to 30, and an integer of 10 to 30 is preferred.

[0131] Compounds from which constituent unit (b) can be derived:

[0132] Specific examples of compounds from which the constituent unit (b) can be derived include: α,β-unsaturated carboxylic acid monomers; monomers obtained by adding an acryloyl group or a methacryloyl group to one end of a polyether, polyurethane, polyamino acid, or polyester; acrylamide; esters of alkoxy alcohols and acrylic acid or methacrylic acid, etc.

[0133] Specific examples of the α,β-unsaturated carboxylic acid monomer include acrylic acid, methacrylic acid, acryloxypropionic acid, citraconic acid, itaconic acid, crotonic acid, maleic acid, and maleic anhydride. Acrylic acid and methacrylic acid are preferred because they have good copolymerizability with other constituent units.

[0134] As a monomer obtained by adding an acryloyl group or a methacryloyl group to one end of a polyether, polyurethane, polyamino acid, or polyester, condensates of the aforementioned compounds with acrylic acid or methacrylic acid can be cited. Among them, polyethylene glycol monomethyl ether methacrylate, polyethylene glycol monomethyl ether acrylate, etc. are preferred because of their good copolymerizability with other constituent units and good arrangement between constituent units (a).

[0135] Examples of the esters of alkoxy alcohol and acrylic acid or methacrylic acid include 2-methoxyethyl acrylate and 2-methoxyethyl methacrylate.

[0136] As the compound from which the constituent unit (b) can be derived, acrylamide or a compound represented by the following general formula (b1-1) is preferred. In the following formula (b1-1), k is preferably an integer of 1 to 10, and more preferably an integer of 1 to 9. k is, for example, 9. In addition, as the constituent unit (b), by using a smaller molecular weight, the dispersion ratio can be reduced, and a compound with a more uniform molecular weight can be obtained, which is preferred. As such a compound from which the constituent unit (b) can be derived, acrylamide is more preferred.

[0137]

[0138] In the formula (b1-1), k represents an integer of 1-12.

[0139] The structural unit (b) may be used alone or in combination of two or more.

[0140] When the copolymer (C) is a compound represented by the above general formula (b-2), Lc in the general formula (b-2) 1 Represents a (a+b)-valent linking group. 1 There are no particular limitations, and examples include (a+b)-valent hydrocarbon groups that may have substituents. The hydrocarbon group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, further preferably 5 carbon atoms, further preferably 1 to 3 carbon atoms, and particularly preferably 1 or 2 carbon atoms. The hydrocarbon group that may have substituents may be an aromatic hydrocarbon group that may have substituents, or an aliphatic hydrocarbon group that may have substituents, and is preferably an aliphatic hydrocarbon group that may have substituents. The hydrocarbon group that may have substituents may be saturated or unsaturated, and is preferably saturated.

[0141] Lc 1 The hydrocarbon group in may or may not have a substituent. 1 The substituent that the hydrocarbon group in may have includes a carboxyl group, an amino group, a hydroxyl group, an alkoxy group and the like.

[0142] As Lc 1 Specific examples of include carbon atoms.

[0143] In the general formula (b-2), a and b are each independently an integer greater than 1 as long as the atomic valence permits. a and b are preferably integers of 1 to 4, more preferably integers of 1 to 3. As a specific example, a is 3 and b is 1.

[0144] Copolymer (C) is a copolymer having a constituent unit (a) represented by the aforementioned general formula (a-1) and a constituent unit (b) represented by the aforementioned general formula (b-1) (hereinafter also referred to as "copolymer (C01)"), preferably a compound represented by general formula (b-2) (hereinafter also referred to as "copolymer (C02)").

[0145] The molar ratio of constituent unit (a) to constituent unit (b) (constituent unit (a): constituent unit (b)) is preferably 10:1 to 1:20, more preferably 10:1 to 1:10. If the molar ratio of constituent unit (a) to constituent unit (b) is within the above range, the expression levels of αSMA and collagen (Col1a2) in myofibroblasts can be further suppressed, and the expression levels of HMOX1, MMP, ANGPTL4, and SOD2 can be further increased.

[0146] When the copolymer (C) is the copolymer (C01), the molar ratio of the constituent unit (a) to the constituent unit (b) is preferably 1:20 to 8:2, more preferably 1:10 to 8:2, more preferably 1:9 to 6:4, further preferably 1:9 to 5:5, and particularly preferably 1:9 to 4:6. From the viewpoint of promoting the deactivation of myofibroblasts or inhibiting the activation of myofibroblasts, the proportion of the constituent unit (a) is preferably smaller than that of the constituent unit (b).

[0147] The total number of the constituent units (a) and the constituent units (b) in the copolymer (C01) is, for example, 10 to 50, preferably 10 to 40, and more preferably 10 to 35. The number of the constituent units (a) in the copolymer (C01) is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 13, further preferably 1 to 12, and particularly preferably 1 to 10. The number of the constituent units (b) in the copolymer (C01) is, for example, 1 to 30, preferably 3 to 30, more preferably 4 to 30, further preferably 8 to 30, further preferably 10 to 26, and particularly preferably 15 to 26. From the viewpoint of promoting the deactivation of myofibroblasts or inhibiting the activation of myofibroblasts, the number of constituent units (a) is preferably smaller than the number of constituent units (b). For example, the copolymer (C01) is preferably such that: 1 constituent unit (a) is introduced for every 1 to 10 (e.g. 2 to 5) constituent units (b); more preferably: 1 constituent unit (a) is introduced for every 3 to 10 (e.g. 3 to 4) constituent units (b); further preferably: 1 constituent unit (a) is introduced for every 5 to 10 (e.g. 5 to 9) constituent units (b).

[0148] When the copolymer (C) is the copolymer (C02), the molar ratio of the constituent unit (a) to the constituent unit (b) is preferably 1:5 to 5:1, preferably 1:4 to 4:1, and more preferably 1:3 to 3:1. From the viewpoint of promoting the deactivation of myofibroblasts or inhibiting the activation of myofibroblasts, the proportion of the constituent unit (a) is preferably smaller than that of the constituent unit (b).

[0149] The total number of the constituent units (a) and the constituent units (b) in the copolymer (C02) is, for example, 2 to 10, preferably 2 to 6, and more preferably 2 to 4. The number of the constituent units (a) in the copolymer (C02) is, for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, and further preferably 1 to 3. The number of the constituent units (b) in the copolymer (C02) is, for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, and further preferably 1 to 3. From the viewpoint of promoting the deactivation of myofibroblasts or inhibiting the activation of myofibroblasts, the number of constituent units (a) is preferably less than the number of constituent units (b).

[0150] Specific examples of the copolymer (C01) are shown below, but the copolymer is not limited to these examples.

[0151]

[0152]

[0153] In formulae (C-1) to (C-4), R represents an alkyl group having 1 to 18 carbon atoms; and a and b each independently represent an integer of 1 to 30.

[0154] Specific examples of the copolymer (C02) are shown below, but the copolymer is not limited to these examples.

[0155]

[0156] In formula (C-5), Rx 1 ~Rx 4 Each independently represents a group represented by formula (RX-1) or (RX-2). 1 ~Rx 4 At least one of the groups is a group represented by formula (RX-1). n represents an integer greater than or equal to 1. * represents an atomic bond.

[0157] Examples of n in the formulae (RX-1) and (RX-2) include integers of 1 to 30, and an integer of 10 to 30 is preferred.

[0158] The copolymer (C01) is preferably a random copolymer. The copolymers represented by the above formulae (C-1) to (C-4) are random copolymers.

[0159] The weight average molecular weight (Mw) of the copolymer (C) is not particularly limited and can be appropriately set according to the molecular weight of the constituent unit (a) and the constituent unit. For example, the weight average molecular weight (Mw) of the copolymer (C) is preferably in the range of 2000 to 20000. If the weight average molecular weight (Mw) of the copolymer (C) is within the above preferred range, the expression of αSMA and collagen (Col1a2) in myofibroblasts can be further suppressed, and the expression of HMOX1, MMP, ANGPTL4, and SOD2 can be further increased.

[0160] When the copolymer (C) is a copolymer represented by the aforementioned formula (C-1) or (C-2), the weight average molecular weight (Mw) is preferably 2000 to 6000, more preferably 3000 to 5000. When the copolymer (C) is a copolymer represented by the aforementioned formula (C-3) or (C-4), the weight average molecular weight (Mw) is preferably 7000 to 20000, more preferably 7000 to 16000. When the copolymer (C) is a copolymer represented by the aforementioned formula (C-5), the weight average molecular weight (Mw) is preferably 4000 to 10000, more preferably 5000 to 8000.

[0161] The number average molecular weight (Mn) of the copolymer (C) is not particularly limited and can be appropriately set according to the molecular weight of the constituent unit (a) and the constituent unit. For example, the number average molecular weight (Mn) of the copolymer (C) is preferably in the range of 2000 to 15000. If the number average molecular weight (Mn) of the copolymer (C) is within the above preferred range, the expression levels of αSMA and collagen (Col1a2) in myofibroblasts can be further suppressed, and the expression levels of HMOX1, MMP, ANGPTL4, and SOD2 can be further increased.

[0162] When the copolymer (C) is a copolymer represented by the aforementioned formula (C-1) or (C-2), the number average molecular weight (Mn) is preferably 2000 to 6000, more preferably 3000 to 5000. When the copolymer (C) is a copolymer represented by the aforementioned formula (C-3) or (C-4), the number average molecular weight (Mn) is preferably 4500 to 15000, more preferably 8000 to 12000. When the copolymer (C) is a copolymer represented by the aforementioned formula (C-5), the number average molecular weight (Mn) is preferably 4000 to 10000, more preferably 5000 to 8000.

[0163] The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the copolymer (C) is preferably in the range of 1.0 to 1.7, and more preferably 1.0 to 1.6.

[0164] 《Method for producing copolymer (C)》

[0165] The method for producing the copolymer (C) is not particularly limited, and free radical polymerization, living radical polymerization, reversible addition-fragmentation chain transfer polymerization (RAFT polymerization) and the like can be used, wherein a chain transfer agent (RAFT agent) is used in the RAFT polymerization. For example, the copolymer (C) can be produced by mixing a monomer from which the constituent unit (a) can be derived and a monomer from which the constituent unit (b) can be derived, and copolymerizing them by the method described above.

[0166] The mixing molar ratio of the monomer from which the constituent unit (a) can be derived and the monomer from which the constituent unit (b) can be derived in the copolymerization reaction is 1:20 to 10:1 or 1:10 to 10:1. The mixing molar ratio of the monomer from which the constituent unit (a) can be derived and the monomer from which the constituent unit (b) can be derived is preferably 1:20 to 8:2, more preferably 1:10 to 8:2, more preferably 1:9 to 6:4, further preferably 1:9 to 5:5, particularly preferably 2:9 to 4:6 or 2:8 to 4:6.

[0167] In the case of the copolymer (C01), it is preferred to use RAFT polymerization as the polymerization method because the molecular weight distribution of the copolymer (C01) can be controlled to be narrow. For example, the copolymer (C01) can be obtained by mixing a monomer from which the constituent unit (a) can be derived and a monomer from which the constituent unit (b) can be derived, and subjecting them to RAFT polymerization in a solvent such as DMSO in the presence of a RAFT agent.

[0168] After RAFT polymerization, the terminal group represented by -SC(=S)-Z (Z is an organic group) derived from the RAFT agent can be reduced using a reducing agent such as sodium borohydride to convert it into a thiol group. Furthermore, the thiol group can be reacted with an unsaturated group-containing compound reactive with the thiol group to introduce an arbitrary functional group. For example, a succinimide group can be introduced at the end of the copolymer (C01) by reacting the thiol group with maleimide.

[0169] The copolymer (C02) can be obtained by reacting a polyfunctional compound having multiple functional groups with a compound containing an N-acetylglucosamine group (hereinafter also referred to as a "GlcNac compound") and a compound not containing an N-acetylglucosamine group (hereinafter also referred to as a "non-GlcNac compound"). The GlcNac compound and the non-GlcNac compound can be compounds having functional groups reactive with the functional groups of the polyfunctional compound. For example, when the polyfunctional compound has a thiol group, a compound having a vinyl group can be used as the GlcNac compound and the non-GlcNac compound. When the polyfunctional compound has a thiol group, it is preferred to reduce the thiol group in advance before the reaction.

[0170] The anti-fibrotic agent of the present embodiment exhibits a deactivation effect and / or an activation inhibition effect on myofibroblasts by means of a copolymer (C) containing a constituent unit (a) containing an N-acetylglucosamine group and other constituent units (b). More specifically, the copolymer (C) inhibits tissue fibrosis by inhibiting the expression of collagen (Col1a2) and αSMA in myofibroblasts. Further, the copolymer (C) protects cells from oxidative stress and alleviates tissue fibrosis by increasing the expression of HMOX1 in myofibroblasts. The copolymer (C) promotes the decomposition of collagen deposited in fibrous tissue and inhibits tissue fibrosis by increasing the expression of MMP1. The copolymer (C) inhibits tissue fibrosis by increasing the expression of ANGPTL4, thereby utilizing wound healing and anti-inflammatory effects. The copolymer (C) inhibits tissue fibrosis by increasing the expression of SOD2, thereby utilizing an antioxidant effect.

[0171] By having the constituent units (a) and (b), the copolymer (C) can reduce the number of constituent units (a) contained in the copolymer 1 molecule. As a result, the N-acetylglucosamine groups contained in the copolymer 1 molecule can be reduced. Thereby, the expression inhibitory effect of the copolymer (C) on collagen (Col1a2) and αSMA, and the expression promoting effect on MMP1, HMOXA, ANGPTL4, and SOD2 are further enhanced.

[0172] Therefore, the anti-fibrosis agent of the present embodiment has a more excellent myofibroblast deactivation effect and / or activation inhibitory effect.

[0173] (Medical composition for treating fibrosis)

[0174] The pharmaceutical composition for treating fibrosis of the present embodiment contains the above-mentioned anti-fibrotic agent as an active ingredient. The so-called "active ingredient" is an ingredient effective for treating fibrosis, and more specifically, refers to an ingredient that has the effects of improving or alleviating fibrosis and / or inhibiting the progression of fibrosis. The pharmaceutical composition for treating fibrosis of the present embodiment contains a therapeutically effective amount of the above-mentioned copolymer (C) as an active ingredient. The therapeutically effective amount refers to the amount of the copolymer (C) that can exert the therapeutic effect as described above for the treatment of fibrosis.

[0175] The pharmaceutical composition for treating fibrosis of the present embodiment can be administered orally or parenterally (intravenously, subcutaneously, transdermally, transpulmonary, transmucosal, or rectal) to humans or mammals other than humans (e.g., mice, rats, guinea pigs, rabbits, cats, dogs, cattle, sheep, monkeys, etc.). The pharmaceutical composition for treating fibrosis of the present embodiment can be prepared by the following method: the copolymer (C) is mixed with pharmaceutically acceptable carriers (excipients, binders, disintegrants, disintegrant aids, lubricants, wetting agents, etc.) and additives generally used for oral or parenteral administration to prepare the composition in a desired form. The dosage form of the pharmaceutical composition for treating fibrosis according to the present embodiment includes, for example, granules, powders, tablets (including sugar-coated tablets), pills, buccal tablets, capsules, syrups, liquids, emulsions, suspensions, pastes, ointments, eye drops, injections, drops, nasal drops, patches, suppositories, and the like.

[0176] As long as the expression of αSMA and collagen can be inhibited and the effect of improving fibrosis can be obtained, the content of the copolymer (C) in the pharmaceutical composition for treating fibrosis of this embodiment is not particularly limited. The content of the copolymer (C) is preferably 0.1% by mass or more, more preferably 1% by mass or more, and further preferably 10% by mass or more.

[0177] <Fibrosis>

[0178] "Fibrosis" in this specification means: the migration and proliferation of excess fibroblasts in the process of compensating for the loss of tissue parenchymal cells and decreased tissue function caused by chemical stimulation such as drugs, excessive pressure load, inflammatory response, and subsequent hardening accompanied by tissue dysfunction, which is caused by protein synthesis and precipitation of the extracellular matrix; the difference in inducing stimulation and the site of onset are not particularly limited. Examples of such tissue fibrosis diseases include fibrosis of visceral tissues such as the lungs, bladder, kidneys, heart, and liver.

[0179] The fibrosis that is the subject of the fibrosis treatment pharmaceutical composition of this embodiment also includes: tissue fibrosis diseases caused by the administration of anti-tumor agents, antibiotics, bactericides, antiarrhythmic agents, anti-inflammatory agents, anti-rheumatic drugs, interferons or Xiao Chaihu Decoction and other drugs; and tissue fibrosis diseases accompanied by chronic nephritis, interstitial myocarditis and interstitial cystitis and other diseases. Specifically, the following can be cited: pulmonary fibrosis caused by the side effects of bleomycin administration, and pulmonary fibrosis caused during or after interstitial pneumonia; bladder fibrosis and bladder neck sclerosis caused by interstitial cystitis; renal fibrosis and renal failure (nephrosclerosis) caused by genetic abnormalities, etc.; intimal fibrosis caused by remodeling after myocardial infarction; liver fibrosis caused by damage to liver cells; non-alcoholic fatty liver disease (NASH) and portal hypertension and cirrhosis associated with NASH; scars caused by excessive tissue repair; and sclerosing peritonitis, prostatic hypertrophy, pachyderma, uterine leiomyoma, retroperitoneal fibrosis and myelofibrosis, etc.

[0180] The pharmaceutical composition for treating fibrosis of the present embodiment can be a pharmaceutical composition that reduces the expression of αSMA and collagen by inhibiting the phosphorylation of STAT3, increases the expression of MMP1, HMOX1 and ANGPTL4, repairs myofibroblasts and activated astrocytes into normal fibroblasts and astrocytes, and improves fibrosis. In fibrosis caused by myofibroblasts or activated astrocytes, for example in the liver, activated astrocytes transform into myofibroblasts and produce extracellular matrix, thereby causing fibrosis to develop. However, by using the pharmaceutical composition for treating fibrosis of the present embodiment, liver fibrosis can be improved, and liver function can be improved and liver cancer can be inhibited. Similarly, it can also be used for pancreatic fibrosis, etc.

[0181] (Method for Deactivating Myofibroblasts)

[0182] The method for inactivating myofibroblasts according to the present embodiment comprises binding the antifibrotic agent to myofibroblasts. In the method of the present embodiment, the copolymer (C) can be used as the antifibrotic agent.

[0183] "Deactivation of myofibroblasts" refers to the occurrence of at least one phenomenon selected from the group consisting of: decreased collagen expression, decreased αSMA expression, increased MMP1 expression, increased HMOX1 expression, and increased ANGPTL4 expression in myofibroblasts. In the deactivation of myofibroblasts, preferably two or more of the above phenomena occur, more preferably three or more of the above phenomena occur, further preferably four or more of the above phenomena occur, and particularly preferably all of the above phenomena occur.

[0184] The binding of the antifibrotic agent to the myofibroblasts can be achieved by bringing the antifibrotic agent into contact with the myofibroblasts. The contact of the antifibrotic agent to the myofibroblasts can be achieved in vitro or in vivo. The in vitro contact can be achieved by adding the antifibrotic agent to the culture medium of the myofibroblasts. The in vivo contact can be achieved by administering the antifibrotic agent to the living body.

[0185] The amount of the antifibrotic agent added to myofibroblasts in vitro may be, for example, 10 to 500 μg / mL, preferably 50 to 300 μg / mL, and more preferably 50 to 100 μg / mL relative to the culture medium of myofibroblasts.

[0186] In the case of in vivo administration, the dosage to the living body can be appropriately set according to the living body's biological species, body weight, sex, age, etc. Examples of the dosage of the antifibrotic agent to the living body include 10 to 1000 μg / kg.

[0187] In vitro, myofibroblasts can be cultured in an environment of about 37° C. (37±2° C.) and a carbon dioxide concentration of about 5% by volume (5±2% by volume).

[0188] (Other embodiments)

[0189] In one embodiment, the present invention provides a method for treating fibrosis, comprising administering an effective amount of the above-mentioned anti-fibrosis agent to a patient or animal in need of treatment. The anti-fibrosis agent can use a copolymer (C). The fibrosis as an applicable object can be the same as the fibrosis described in the above-mentioned "pharmaceutical composition for treating fibrosis".

[0190] In one embodiment, the present invention provides a use of a copolymer (C) for preparing an anti-fibrosis agent.

[0191] In one embodiment, the present invention provides a use of a copolymer (C) for preparing a pharmaceutical composition for treating fibrosis.

[0192] In one embodiment, the present invention provides a copolymer (C) for use in treating fibrosis.

[0193] In one embodiment, the present invention provides a use of a copolymer (C) for treating fibrosis.

[0194] [Example]

[0195] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the Examples.

[0196] (Myofibroblast Preparation)

[0197] Primary cultured human skin fibroblasts (manufactured by Takara Bio Co., Ltd.) isolated from adult skin confirmed to be free of Mycoplasma contamination were cultured in HFDM-1(+) medium (manufactured by the Institute of Cell Science and Technology) supplemented with 2 mM L-glutamic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at 37°C and 5 v / v% CO2 in a humidified incubator. Subsequently, 10 ng / mL of TGF-β was added to the medium to obtain myofibroblasts. Human skin fibroblasts used to obtain the myofibroblasts were cells maintained at 3 to 6 passages.

[0198] (Confirm whether there is mycoplasma contamination)

[0199] Mycoplasma Detection Kit for Endpoint PCR, OneStep, VendorGeM (manufactured by Minerva Biolabs Gmbh) was used to confirm that the human skin fibroblasts used were not contaminated by Mycoplasma.

[0200] (Synthesis of AC-GlcNAc Monomer)

[0201] Dissolve 5 g of GlcNAc in 50 mL of water, and add NH4HCO3 until saturated. Stir in a round-bottom flask and react at 30-35°C in an open system with the lid open for 4-5 days. If the precipitate of NH4HCO3 disappears, add an appropriate amount of NH4HCO3. Confirm the synthesis of GlcNAc-NH2 by thin film chromatography (TLC). After 4-5 days, add water to the reaction solution and perform distillation (30°C) to remove excess NH4HCO3. Repeat this operation until the odor disappears. After distillation, freeze-dry. Next, dissolve GlcNAc-NH2 (4.5 mol: about 1 g) in dimethyl sulfoxide (DMSO) (10 mL), and add 2-carboxyethyl acrylate (4.5 mmol). After dissolution, add 4-(4,6-dimethoxy-1,3,5-trimethoxy) chloride. -2-yl)-4-methylmorpholinium (DMT-MM) (6.8 mmol) was reacted at room temperature for 18 hours. After the reaction, the reactant was dropped into 200-300 mL of chloroform. The obtained precipitate was recovered using a Kiriyama funnel. The recovered precipitate was dissolved using methanol, and the insoluble matter was removed using a Kiriyama funnel, and then the components soluble in methanol were recovered. Methanol was removed by distillation. The solid matter after methanol was removed was dissolved in water. Then, it was purified using preparative high performance liquid chromatography (HPLC) (water / acetonitrile) to obtain the AC-GlcNAc monomer (molecular weight 346) represented by the following chemical formula (1).

[0202]

[0203] (Production of Copolymer 1)

[0204] 40 mg (0.116 mmol) of AC-GlcNAc monomer and 2.1 mg (0.029 mmol) of acrylamide (molecular weight 71) were dissolved in 250 μL of DMSO. 5.3 mg (0.0145 mmol) of RAFT agent (2-(dodecylthio(thiocarbonyl)thio)-2-methylpropionic acid) and 1 mg of azobisisobutyronitrile (AIBN) were mixed into this solution. The solution was degassed while repeating freeze-thawing for about 5 times to remove dissolved oxygen. Then, argon gas was sealed in, and the polymerization was carried out by heating at 65°C for about 17 hours while stirring. After the polymerization, the solution was suspended in isopropanol, and the polymer was recovered as a precipitate. The precipitate was dissolved in water, and dialyzed in water for 17 hours using a dialysis membrane (MWCO1000). After dialysis, freeze-drying was performed to obtain copolymer 1 (the following formula (C1-1)).

[0205]

[0206] The weight average molecular weight (Mw) of copolymer 1 is 5000, the number average molecular weight (Mn) is 4400, Mw / Mn is 1.14, and the molar ratio of AC-GlcNAc unit to acrylamide unit is 13:3. The average molecular weights were determined under the following conditions using a gel permeation chromatography (GPC) device (product name: LC-9110G NEXT, manufactured by Japan Analytical Industry Co., Ltd.). The column used was JAIGEL-GS510, and the eluent was 200mM sodium nitrate / acetonitrile = 80 / 20. The flow rate was 1 mL / min, the detector was an RI detector, and the column temperature was 40°C. The molecular weight standard curve was obtained based on polytriglucose. There are 13 AC-GlcNAc units and 3 acrylamide units in copolymer 1.

[0207] (Production of Copolymer 2)

[0208] Copolymer 2 was obtained by the same method as (Production of Copolymer 1), except that the amount of AC-GlcNAc monomer (molecular weight of 346) was 30 mg (0.087 mmol) and the amount of acrylamide (molecular weight of 71) was 4.2 mg (0.059 mmol). Copolymer 2 had a weight average molecular weight (Mw) of 4900, a number average molecular weight (Mn) of 4500, Mw / Mn of 1.09, and a molar ratio of AC-GlcNAc unit to acrylamide unit of 12:8.

[0209] (Production of Copolymer 3)

[0210] 40 mg (0.116 mmol) of AC-GlcNAc monomer (molecular weight 346) and 14.4 mg (0.029 mmol) of polyethylene glycol methyl ether methacrylate (molecular weight 500) were dissolved in 250 μL of DMSO. 5.3 mg (0.0145 mmol) of RAFT agent (4-cyano-4-[(dodecylthiothiocarbonyl)thio]pentanoic acid) and 1 mg of AIBN were mixed into this solution. The solution was degassed while repeatedly freezing and thawing for about 5 times to remove dissolved oxygen. Then, argon gas was sealed in, and the solution was heated at 65°C for about 17 hours while stirring to carry out polymerization. After polymerization, the solution was suspended in isopropanol, and the polymer was recovered as a precipitate. The precipitate was dissolved in water, and dialyzed in water for 17 hours using a dialysis membrane (MWCO1000). After dialysis, freeze-drying was performed to obtain copolymer 3 (the following formula (C1-3)).

[0211] The weight average molecular weight (Mw) of copolymer 3 is 7100, the number average molecular weight (Mn) is 4700, Mw / Mn is 1.51, and the molar ratio of AC-GlcNAc unit to polyethylene glycol methyl ether methacrylate unit is 15:4. The average molecular weights were determined under the following conditions using a gel permeation chromatography (GPC) device (product name: LC-9110G NEXT, manufactured by Japan Analytical Industry Co., Ltd.). The column used was JAIGEL-GS510, and the elution solution used was 200mM sodium nitrate / acetonitrile = 80 / 20. The flow rate was 1 mL / min, the detector was an RI detector, and the column temperature was 40°C. The molecular weight standard curve was obtained based on polytriglucose.

[0212]

[0213] (Production of Copolymer 4)

[0214] Copolymer 4 was obtained by the same method as (Production of Copolymer 3), except that the amount of AC-GlcNAc monomer (molecular weight of 346) was 30 mg (0.087 mmol) and the amount of polyethylene glycol methyl ether methacrylate (molecular weight of 500) was 28.8 mg (0.0576 mmol). Copolymer 4 had a weight average molecular weight (Mw) of 10,300, a number average molecular weight (Mn) of 7,400, Mw / Mn of 1.39, and a molar ratio of AC-GlcNAc unit to polyethylene glycol methyl ether methacrylate unit of 15:10.

[0215] (Production of Copolymer 5)

[0216] Copolymer 5 was obtained by the same method as (Production of Copolymer 3) except that the amount of AC-GlcNAc10 monomer (molecular weight of 346) was 20 mg (0.059 mmol) and the amount of polyethylene glycol methyl ether methacrylate (molecular weight of 500) was 43.5 mg (0.087 mmol). Copolymer 5 had a weight average molecular weight (Mw) of 13,000, a number average molecular weight (Mn) of 9,200, Mw / Mn of 1.41, and a molar ratio of AC-GlcNAc unit to polyethylene glycol methyl ether methacrylate unit of 12:18.

[0217] (Production of Copolymer 6)

[0218] Copolymer 6 was obtained by the same method as (Production of Copolymer 3) except that the amount of AC-GlcNAc monomer (molecular weight of 346) was 10 mg (0.029 mmol) and the amount of polyethylene glycol methyl ether methacrylate (molecular weight of 500) was 58 mg (0.116 mmol). Copolymer 6 had a weight average molecular weight (Mw) of 15100, a number average molecular weight (Mn) of 11800, Mw / Mn of 1.28, and a molar ratio of AC-GlcNAc unit to polyethylene glycol methyl ether methacrylate unit of 6:26.

[0219] (Production of Polymer 1)

[0220] (1) 50 mg of AC-GlcNAc monomer (molecular weight 346) was measured in a 1 mL microcentrifuge tube and dissolved in 250 μL of DMSO.

[0221] (2) About 5.26 mg of a RAFT agent (2-(dodecylthiocarbonylthiothio)-2-methylpropionic acid (DTMPA: manufactured by Sigma, 1 / 10 molar equivalent of the monomer) was weighed and dissolved in 200 μL of DMSO in a 1 mL microcentrifuge tube.

[0222] (3) Furthermore, 1 mg of azobisisobutyronitrile (AIBN, 2% relative to the monomer) was weighed in a microcentrifuge tube and dissolved in 50 μL of DMSO.

[0223] After mixing 200 μL of the aforementioned solution (2) with 250 μL of the aforementioned solution (1), 50 μL of the aforementioned solution (3) was mixed and degassed (freeze-thaw degassing was repeated about three times). Then, the reaction was carried out at 65°C for 18 hours to polymerize the AC-GlcNAc monomer. After polymerization, the polymer was precipitated with isopropanol, centrifuged and recovered. The recovered product was dissolved in water and dialyzed (MW100-500) for about 1 day to remove unreacted monomers. After dialysis, freeze-drying was carried out to obtain polymer 1 (the following formula (C2-1)).

[0224] The weight average molecular weight (Mw) of Polymer 1 was 4,000, the number average molecular weight (Mn) was 3,100, and Mw / Mn was 1.29. The average molecular weights were measured by the same method as that of Copolymer 1.

[0225]

[0226] The measurement results obtained by GPC of copolymers 1 to 6 and polymer 1 are summarized in Table 1. In Table 1, "a" in "mixing molar ratio of monomers" and "molar ratio of constituent units" represents the molar ratio of AC-GlcNAc monomers or AC-GlcNAc units, and "b" represents the molar ratio of acrylamide or polyethylene glycol methyl ether methacrylate, or constituent units derived therefrom.

[0227] [Table 1]

[0228]

[0229] [Example 1]

[0230] The test compound (copolymer 1, copolymer 2, or polymer 1) at various concentrations (10 μg / mL, 50 μg / mL, or 100 μg / mL) was added to the myofibroblasts prepared in (Preparation of myofibroblasts) and cultured in a humidified incubator at 37°C and 5% CO2 for 48 hours.

[0231] After culture, the cells were collected. The expression of collagen (Col1a2), α-smooth muscle actin (αSMA), heme oxygenase 1 (HMOX1), and β-actin in the collected myofibroblasts was observed by Western blotting. As controls, human skin fibroblasts cultured without any addition (control group) and myofibroblasts cultured without the addition of the test compound (10 ng / mL TGFβ1 (myofibroblasts)) were used.

[0232] The results are shown in Figure 1 . Figure 1 The numerical value below each band in the graph represents the relative intensity of each band when the signal intensity of the band in the control group is set to 100. The signal intensity of each band was normalized based on the signal intensity of the β-actin band. Figure 1 The results shown in the figure show that the expression levels of collagen (Col1a2) and αSMA in myofibroblasts cultured without adding the test compound became very high. Compared with myofibroblasts added with polymer 1, myofibroblasts added with either copolymer 1 or copolymer 2 showed that the expression levels of collagen (Col1a2) and αSMA decreased, and the expression level of HMOX1 increased when compared with the same addition amount. In particular, in fibroblasts added with copolymer 2, the expression levels of collagen (Col1a2) and αSMA decreased significantly, and the expression level of HMOX1 increased significantly.

[0233] HMOX1 is a protein that protects against various oxidative stresses, and by increasing the expression of this gene, fibrosis can be alleviated through antioxidant effects. Therefore, it is believed that by decreasing the expression of αSMA and collagen and increasing the expression of HMOX1, myofibroblast activation can be further suppressed.

[0234] [Example 2]

[0235] 100 μg / mL of the test compound (copolymer 3, copolymer 4, copolymer 5, copolymer 6, or polymer 1) was added to the myofibroblasts prepared in (Preparation of myofibroblasts) and cultured in a humidified incubator at 37°C and 5% CO2 for 48 hours. The cells were recovered after culture. The expression levels of αSMA, collagen (Col1a2), matrix metalloproteinase 1 (MMP1), HMOX1, angiopoietin-like protein 4 (ANGPTL4), and β-actin in the recovered myofibroblasts were observed by Western blotting. As controls, human skin fibroblasts (control group) cultured without any additions and myofibroblasts (10 ng / mL TGFβ1 (myofibroblasts)) cultured without the addition of the test compound were used.

[0236] The results are shown in Figure 2 . Figure 2 The numerical values ​​below each band in the figure represent the relative intensity of each band when the signal intensity of the band in the control group is set to 100. The signal intensity of each band was normalized based on the signal intensity of the β-actin band. Figure 2 The results shown show that in myofibroblasts cultured without adding the test compound, the expression levels of collagen (Col1a2) and αSMA were very high, and the expression levels of MMP1, HMOX1, and ANGPTL4 were not confirmed or were very low. Compared with the myofibroblasts added with polymer 1, the myofibroblasts added with any of copolymers 3 to 6 showed decreased expression levels of αSMA and collagen (Col1a2), and increased expression levels of MMP1, HMOX1, and ANGPTL4.

[0237] MMP1 is an enzyme that can decompose collagen deposited in fibrous tissue. HMOX1 is a protein that can protect against various oxidative stresses. ANGPTL4 is a protein that can promote wound healing. By adding any one of copolymers 3 to 5, the expression of collagen and αSMA in myofibroblasts can be suppressed, thereby inhibiting fibrosis. Furthermore, by increasing the expression of MMP1 and ANGPTL4, attached collagen can be decomposed. Furthermore, by increasing the expression of HMOX1 and ANGPTL4, wound healing can be promoted, and fibrosis can be alleviated by anti-inflammatory and antioxidant effects.

[0238] In comparison of copolymers 3 to 6, the one with a smaller ratio of AC-GlcNAc units showed a significant decrease in the expression levels of αSMA and collagen (Col1a2), and a significant increase in the expression levels of MMP1, HMOX1, and ANGPTL4.

[0239] (Production of Copolymer 7)

[0240] Copolymer 7 was obtained by the same method as (Production of Copolymer 1), except that the amount of AC-GlcNAc monomer (molecular weight of 346) was 10 mg (0.029 mmol) and the amount of acrylamide (molecular weight of 71) was 18.9 mg (0.261 mmol). Copolymer 7 had a weight average molecular weight (Mw) of 3800, a number average molecular weight (Mn) of 3300, Mw / Mn of 1.15, and a molar ratio of AC-GlcNAc unit to acrylamide unit of 4:35.

[0241] (Production of Copolymer 8)

[0242] <Reduction of four-arm polyethylene glycol thiol (4arm PEG-SH) (weight average molecular weight 5000)>

[0243] Dissolve 20 mg of 4-arm PEG-SH (weight average molecular weight 5000) (SUNBRIGHT (registered trademark) PTE-050SH; Petrochemical Industry Co., Ltd.) in 500 μL of DMSO. Add 1 M dithiothreitol (DTT) and allow it to react overnight to reduce the SH. Then, add 10 mL of ether, centrifuge and remove the ether. Repeat this step 3 times to precipitate the reduced 4-arm PEG-SH and remove DTT and DMSO.

[0244] <Production of 4arm-PEG-AC-GlcNAc:C12(3:1)>

[0245] The reduced 4arm PEG-SH (20 mg) was dissolved in 500 μL of DMSO (degassed and substituted with argon). 8.3 mg (2 equivalents relative to 3 molecules of SH groups of 4arm-PEG-SH) of AC-GlcNAc monomer and 1.92 mg (2.2 μL) (2 equivalents relative to 1 molecule of SH group of 4arm-PEG-SH) of dodecyl acrylate were added thereto, and 10 μL of triethylamine was further added, and the mixture was reacted overnight while stirring at 65°C. After reacting overnight, the reactant was precipitated with ether, and the unreacted product was removed by centrifugation, and then the precipitate was recovered. Then, the mixture was dialyzed overnight using a dialysis membrane with a molecular weight of 1000, and freeze-dried to obtain copolymer 8 (the following formula (C1-8)).

[0246] The weight average molecular weight (Mw) of copolymer 8 is 6300, and the molar ratio of AC-GlcNAc unit, 4arm PEG-SH unit and dodecyl acrylate unit is 3:1:1. In the following formula, n is about 10-30.

[0247]

[0248] In formula (C1-8), Rx1 to Rx3 represent a group represented by formula (RX-1); Rx4 represents a group represented by formula (RX-2). n is an integer of 1 or more.

[0249] The measurement results of copolymers 7 to 8 obtained by GPC are summarized in Table 2. In Table 2, "a" in "mixing molar ratio of monomers" and "molar ratio of constituent units" represents the molar ratio of AC-GlcNAc monomers or AC-GlcNAc units, "b" represents the molar ratio of 4arm PEG-SH or 4arm PEG-SH units, and "c" represents the molar ratio of dodecyl acrylate units.

[0250] [Table 2]

[0251]

[0252] [Example 3]

[0253] Myofibroblasts were cultured in the same manner as in Example 2 except that the test compound of 100 μg / mL was changed to the test compound of 10 μg / mL (copolymer 7). The cells were recovered after culture. The expression levels of αSMA, collagen (Col1a2), matrix metalloproteinase 1 (MMP1), angiopoietin-like protein 4 (ANGPTL4), HMOX1, superoxide dismutase 2 (SOD2), and β-actin in the recovered myofibroblasts were observed by Western blotting. As controls, human skin fibroblasts cultured without any addition (control group) and myofibroblasts cultured without adding the test compound (10 ng / mL TGF (myofibroblasts)) were used.

[0254] The results are shown in Figure 4 . Figure 4 The numerical values ​​below each band in the figure represent the relative intensity of each band when the signal intensity of the band in the control group is set to 100. Figure 4 The results shown show that in myofibroblasts cultured without adding the test compound, the expression levels of collagen (Col1a2) and αSMA were very high, while the expression levels of MMP1, HMOX1, ANGPTL4, and SOD2 were not confirmed or were very low. In myofibroblasts added with copolymer 7, the expression levels of αSMA and collagen (Col1a2) decreased, while the expression levels of MMP1, HMOX1, ANGPTL4, and SOD2 increased.

[0255] SOD2 is an enzyme that can remove active oxygen. Copolymer 7 can inhibit the expression of collagen and αSMA in myofibroblasts at a lower concentration than the copolymers added in Example 2, and the expression of MMP1, ANGPTL4 and SOD2 is increased. By inhibiting the expression of collagen and αSMA, fibrosis can be inhibited. Furthermore, by increasing the expression of MMP1 and ANGPTL4, attached collagen can be decomposed. Furthermore, by increasing the expression of HMOX1, ANGPTL4 and SOD2, wound healing can be promoted, and fibrosis can be alleviated by anti-inflammatory and antioxidant effects.

[0256] [Example 4]

[0257] 100 μg / mL of the test compound (copolymer 8) was added to the myofibroblasts prepared in (Preparation of myofibroblasts), and cultured in a humidified incubator at 37°C and 5% CO2 for 48 hours. The cells were recovered after culture. The expression levels of αSMA, collagen (Col1a2) and β-actin in the recovered myofibroblasts were observed by Western blotting. As controls, human skin fibroblasts (control group) cultured without any addition and myofibroblasts [10 ng / mL TGF (myofibroblasts)] cultured without adding the test compound were used.

[0258] The results are shown in Figure 5 . Figure 5 The numerical values ​​below each band in the figure represent the relative intensity of each band when the signal intensity of the band in the control group is set to 100. The signal intensity of each band was normalized based on the signal intensity of the β-actin band. Figure 5 The results shown show that the expression levels of collagen (Col1a2) and αSMA were significantly increased in myofibroblasts cultured without the addition of the test compound, while the expression levels of αSMA and collagen (Col1a2) were decreased in myofibroblasts to which copolymer 8 was added.

[0259] [Industrial Applicability]

[0260] The anti-fibrotic agent of the embodiment can suppress the expression levels of αSMA and collagen in myofibroblasts and activated astrocytes, and can be used to repair tissues.

[0261] The above describes the preferred embodiments of the present invention, but the present invention is not limited to these embodiments. Additions, omissions, substitutions, and other changes can be made to the structure without departing from the scope of the present invention. The present invention is not limited by the above description, but only by the scope of the attached claims.

Claims

1. An anti-fibrosis agent, comprising a copolymer having: A constituent unit (a) comprising an N-acetylglucosamine group; and A structural unit (b) comprising a structure represented by the following general formula (b), except for the structural unit (a), *——Yb——Rb (b) In formula (b), Yb represents a divalent linking group including an oxygen atom, Rb represents a hydrogen atom or an organic group, and * represents an atomic bond.

2. The anti-fibrosis agent according to claim 1, wherein The aforementioned structural unit (b) is a structural unit represented by the following general formula (b-1), In formula (b-1), Rb 1 represents a hydrogen atom or an organic group, Yb 1 represents -C(=O)-O- or -C(=O)-NH-, Rb 2 represents a hydrogen atom or an organic group.

3. The anti-fibrosis agent according to claim 1, wherein The aforementioned copolymer is a compound represented by the following general formula (b-2), In formula (b-2), Lc 1 Indicates a (a+b) valence linking group, Ya 2 and Yb 2 Each independently represents a divalent linking group containing an oxygen atom, GlcNAC represents N-acetylglucosamine, Rb 3 represents an organic group, and as long as the atomic valence permits, a and b each independently represent an integer greater than 1, and when a is an integer greater than 2, two or more Ya 2 They may be the same or different from each other. When b is an integer greater than 2, two or more Yb 2 Can be the same or different, more than 2 Rb 3 They can be the same as each other or different.

4. The anti-fibrosis agent according to any one of claims 1 to 3, wherein The molar ratio of the structural unit (a) to the structural unit (b) is 10:1 to 1:

20.

5. The anti-fibrosis agent according to claim 2, wherein Rb in the above general formula (b-1) 2 The invention comprises at least one structure selected from the group consisting of an ether bond, an ester bond, an amine ester bond, and an amide bond.

6. The anti-fibrosis agent according to any one of claims 1 to 3, wherein The aforementioned structural unit (a) is a structural unit comprising a structure represented by the following formula (a1-1-1), In formula (a1-1-1), * represents an atomic bond.

7. The anti-fibrosis agent according to claim 2, wherein The aforementioned structural unit (a) is a structural unit represented by the following formula (a-1-1), 8. The anti-fibrosis agent according to claim 2, wherein The structural unit (b) is a structural unit derived from acrylamide or a compound represented by the following general formula (b1-1), In the formula (b1-1), k represents an integer of 1-12.

9. The anti-fibrosis agent according to claim 1, wherein The aforementioned copolymer is a copolymer represented by any one of the following general formulae (C-1) to (C-5), In formulae (C-1) to (C-4), R represents an alkyl group having 1 to 18 carbon atoms, and a and b each independently represent an integer of 1 to 30. In formula (C-5), Rx 1 ~Rx 4 Each independently represents a group represented by formula (RX-1) or (RX-2), wherein Rx 1 ~Rx 4 At least one of them is a group represented by formula (RX-1), n ​​represents an integer greater than or equal to 1, and * represents an atomic bond.

10. The anti-fibrosis agent according to any one of claims 1 to 3, wherein The weight average molecular weight of the copolymer is in the range of 2,000 to 20,000.

11. The anti-fibrosis agent according to any one of claims 1 to 3, wherein The number average molecular weight of the copolymer is in the range of 2,000 to 15,000.

12. The anti-fibrosis agent according to any one of claims 1 to 3, wherein The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the copolymer is in the range of 1.0 to 1.

7.

13. A pharmaceutical composition for treating fibrosis, comprising the anti-fibrosis agent according to any one of claims 1 to 3 as an active ingredient. 14 . A method for inactivating myofibroblasts, comprising binding the anti-fibrotic agent according to claim 1 to myofibroblasts.

Images