Copolymer, antifibrotic agent, pharmaceutical composition for treating fibrosis, and method for inactivating myofibroblasts

Abstract

The present invention provides a copolymer which has a hydrophobic constituent unit (a), wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a C8-15 hydrocarbon group. The present invention also provides a copolymer which has a constituent unit (b) containing an N-acetylglucosamine group, wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a C8-15 hydrocarbon group.

Description

Copolymer, antifibrotic agent, pharmaceutical composition for treating fibrosis, and method for inactivating myofibroblasts

[0001] The present invention relates to a copolymer, an antifibrotic agent, a pharmaceutical composition for treating fibrosis, and a method for inactivating myofibroblasts.This application claims priority to Japanese Patent Application No. 2024-064915, filed April 12, 2024, the contents of which are incorporated herein by reference.

[0002] Tissue fibrosis is caused by chronic inflammation following repeated injury. Chronic inflammation transforms fibroblasts and astrocytes, maintaining tissue as myofibroblasts and activating astrocytes. This activation promotes the expansion of these cells and the abundant production of extracellular matrix such as collagen. Chronic inflammatory diseases such as tissue fibrosis, cancer, and autoimmunity are caused by continuous inflammation due to intermittent and repeated tissue injury.

[0003] Severe tissue damage caused by repeated tissue injury results in the generation of large amounts of cellular debris released from dying cells. Some intracellular molecules contained in the cellular debris released from damaged or dead cells serve to enable inflammatory cells to recognize tissue damage and are called damage-associated molecular patterns (DAMPs) (Non-Patent Document 1). DAMPs act as danger signals, inducing an inflammatory response to protect host tissues from harmful situations such as tissue injury and infection. High-mobility group box 1 (HMGB1), heat shock proteins (HSPs), and adenosine triphosphate (ATP) have clearly defined functions within cells, but these molecules leak from dying cells and act as DAMPs in the extracellular space.

[0004] The presence of abundant DAMPs after severe tissue injury induces the recruitment and activation of immune cells that secrete proinflammatory and profibrogenic cytokines (Non-Patent Document 2). Ultimately, these cytokines induce the differentiation of astrocytes and fibroblasts into activated astrocytes and myofibroblasts, promoting hyperplasia and fibrosis during tissue remodeling (Non-Patent Document 3). Thus, tissue fibrosis is caused by chronic inflammation associated with repeated injury. In chronic inflammation, abundant collagen deposition and expulsion of parenchymal cells ultimately lead to fibrotic tissue dysfunction.

[0005] The present inventors have proposed a polymer that functions in a similar manner to O-linked N-acetylglucosaminylated proteins, based on the fact that O-linked N-acetylglucosaminylated proteins released upon cell injury bind to vimentin on the surface of other cells, as well as an O-linked N-acetylglucosaminylated protein-like substance that efficiently binds to injured tissues or cells by using this polymer, thereby contributing to increased GDF15 expression, and a therapeutic agent for fibrosis containing the same (Patent Document 1).

[0006] International Publication No. 2021 / 095828

[0007] 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.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.An, P. et al., “Hepatocyte mitochondria-derived danger signals directly activate hepatic stellate cells and drive progression of liver fibrosis.”, Nat. Commun., Vol. 11, Article No. 2362, 2020.

[0008] Despite the above findings, there is a need for a more effective antifibrotic agent that can induce myofibroblast inactivation and suppress fibrosis. Therefore, an objective of the present invention is to provide a copolymer that can induce myofibroblast inactivation, an antifibrotic agent comprising the copolymer, a pharmaceutical composition for treating fibrosis containing the antifibrotic agent, and a method for inactivating myofibroblasts.

[0009] As a result of extensive research, the present inventors have found that a copolymer having a structure (T) at the polymer end containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms can suppress the expression of fibroblast-derived collagen and αSMA, which cause fibrosis, and can induce the inactivation of myofibroblasts, thereby completing the present invention.

[0010] That is, the present invention includes the following aspects: [1] A copolymer having a hydrophobic structural unit (a), wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. [2] A copolymer having a structural unit (b) containing an N-acetylglucosamine group, wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. [3] A copolymer having a hydrophobic structural unit (a) and a structural unit (b) containing an N-acetylglucosamine group, wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. [4] The copolymer according to [1] or [3], wherein the structural unit (a) is at least one structural unit selected from the group consisting of a structural unit derived from an α,β-unsaturated carboxylic acid monomer, a structural unit derived from a monomer having an acryloyl group or methacryloyl group attached to one end of a polyether, polyurethane, polyamino acid, or polyester, a structural unit derived from an acrylamide, and a structural unit derived from an ester of an alkoxy alcohol with acrylic acid or methacrylic acid. [5] The copolymer according to [3], wherein the molar ratio of the structural unit (a) to the structural unit (b) is 100:1 to 8:2. [6] The copolymer according to any one of [1] to [5], wherein the weight-average molecular weight is 3,000 to 20,000. [7] The copolymer according to any one of [1] to [6], wherein the number-average molecular weight is 2,000 to 15,000. [8] The copolymer according to any one of [1] to [7], wherein the ratio (Mw / Mn) of weight-average molecular weight (Mw) to number-average molecular weight (Mn) is 1.0 to 1.5. [9] An antifibrotic agent comprising the copolymer according to any one of [1] to [8].

[10] A pharmaceutical composition for treating fibrosis, comprising the antifibrotic agent according to [9] as an active ingredient.

[11] A method for inactivating myofibroblasts, comprising binding the antifibrotic agent according to [9] to myofibroblasts.

[0011] The present invention makes it possible to provide a copolymer capable of inducing the inactivation of myofibroblasts, an antifibrotic agent comprising the copolymer, a pharmaceutical composition for treating fibrosis containing the antifibrotic agent, and a method for inactivating the myofibroblasts.

[0012] 1 shows the results of Western blotting in Example 1. FIG. 2 shows the results of Western blotting in Example 2. FIG. 3 shows the results of Western blotting in Example 3.

[0013] In this specification, when a numerical range is described as, for example, "1 to 10," it means a range from 1 to 10, including the lower limit of 1 and the upper limit of 10.

[0014] <Copolymer> One embodiment of the copolymer of the present invention has a hydrophobic structural unit (a), and the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. This embodiment is referred to as "Copolymer 1." One embodiment of the copolymer of the present invention has a structural unit (b) containing an N-acetylglucosamine group, and the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. This embodiment is referred to as "Copolymer 2." One embodiment of the copolymer of the present invention has a hydrophobic structural unit (a) and a structural unit (b) containing an N-acetylglucosamine group, and the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. This embodiment is referred to as "Copolymer 3."

[0015] Copolymer 1 Copolymer 1 has a hydrophobic structural unit (a), and the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms.

[0016] [Structure (T)] Structure (T) contains a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms. The polymer end of the copolymer of this embodiment is Structure (T). Here, "polymer end" means one end of the polymer main chain. In this specification, one end of the main chain refers to either one of both ends located along the chain extension direction of the main chain.

[0017] The structure (T) is, for example, a group represented by the following general formula (T-1).

[0018] [In formula (T-1), R is a hydrocarbon group having 8 to 15 carbon atoms and is bonded to the main chain at the portion marked with "*."]

[0019] In General (T-1), R is a hydrocarbon group having 8 to 15 carbon atoms, and examples thereof include a linear or branched alkyl group or a group having a cyclic hydrocarbon group. In General (T-1), R is preferably a linear alkyl group, more preferably a linear alkyl group having 11 to 15 carbon atoms, more preferably a linear alkyl group having 12 to 14 carbon atoms, and particularly preferably a linear alkyl group having 12 carbon atoms. In General (T-1), when R is a branched alkyl group, it is preferably any of a butyloctyl group, an ethylhexyl group, a dihexyl group, an ethyloctyl group, and an ethyldecyl group. In General (T-1), R may also be a group having a cyclic hydrocarbon group. Examples of such groups include groups in which a cyclic aliphatic hydrocarbon group or a cyclic aromatic hydrocarbon group is bonded via a linking group. Examples of the linking group include an alkylene group having 1 to 4 carbon atoms. Examples of cyclic aliphatic hydrocarbon groups include cyclopentane and cyclohexane. Examples of cyclic aromatic hydrocarbon groups include benzene and naphthalene. General (T-1) is bonded to the main chain at the "*" portion, more specifically, to one end of the polymer main chain at the "*" portion.

[0020] When R has 8 or more carbon atoms, the hydrophobicity of copolymer 1 is increased, and copolymer 1 is more likely to interact with cell membranes.When R has 15 or less carbon atoms, the hydrophobicity of copolymer 1 is not too high, and copolymer 1 is soluble in water.

[0021] [Structural Unit (a)] The structural unit (a) is a hydrophobic structural unit. There are no particular restrictions on the structural unit (a), as long as it is hydrophobic.

[0022] In one embodiment of the present invention, the structural unit (a) is a structural unit derived from an α,β-unsaturated carboxylic acid monomer. Specific examples of α,β-unsaturated carboxylic acid monomers include acrylic acid, methacrylic acid, acryloxypropionic acid, citraconic acid, itaconic acid, crotonic acid, maleic acid, maleic anhydride, 2-carboxyethyl acrylate, and 2-carboxyethyl methacrylate. Of these, acrylic acid and methacrylic acid are preferred because of their good copolymerizability with other structural units.

[0023] In one embodiment of the present invention, the structural unit (a) is a structural unit derived from a monomer in which an acryloyl group or a methacryloyl group is added to one end of a polyether, polyurethane, polyamino acid, or polyester.

[0024] Examples of the monomer having an acryloyl group or methacryloyl group added to one end of a polyether, polyurethane, polyamino acid, or polyester include condensates of a polyether, polyurethane, polyamino acid, or polyester with acrylic acid or methacrylic acid. Among these, polyethylene glycol monomethyl ether methacrylate, polyethylene glycol monomethyl ether acrylate, etc. are preferred as the structural unit (a) because they have good copolymerizability with other structural units and good alignment properties.

[0025] In one embodiment of the present invention, the structural unit (a) is a structural unit derived from acrylamide. In one embodiment of the present invention, the structural unit (a) may be a structural unit derived from dimethylacrylamide.

[0026] In one embodiment of the present invention, the structural unit (a) is a structural unit derived from an ester of an alkoxy alcohol and an acrylic acid or methacrylic acid. Examples of the ester of an alkoxy alcohol and an acrylic acid or methacrylic acid include 2-methoxyethyl acrylate and 2-methoxyethyl methacrylate.

[0027] In one embodiment of the present invention, the structural unit (a) may be at least one unit selected from the group consisting of a styrene unit, a polyethyleneimine unit, a poly-L-lysine unit, and a biotin unit, which have been conventionally used in biocompatible materials.

[0028] The use of acrylamide, dimethylacrylamide, or the like as the structural unit (a) is particularly effective in improving the anti-fibrotic effect and improving shape retention and drug transportability in an aqueous system.

[0029] The compound from which the structural unit (a) is derived is preferably acrylamide, dimethylacrylamide, or a compound represented by the following general formula (a1-1). In the following formula (a1-1), k is preferably an integer of 1 to 10, and more preferably an integer of 1 to 9. k is, for example, 9. Furthermore, using a structural unit (a) with a small molecular weight is preferable because it reduces the dispersity ratio and allows for the production of a compound with a more uniform molecular weight. As the compound from which such structural unit (a) is derived, acrylamide or dimethylacrylamide is more preferable.

[0030] [In the formula, k represents an integer of 1 to 12.]

[0031] Specific examples of the structural unit (a) are shown below, but the structural unit (a) is preferably a structural unit represented by the following formula (a-1-1), (a-1-1-m), or (a-1-2). In the following formula (a-1-2), nb is preferably an integer of 1 to 10, and more preferably an integer of 1 to 9. The structural unit represented by (a-1-2) is preferably a structural unit represented by the following formula (a-1-2-1) or (a-1-2-2).

[0032] [In the formula, nb represents an integer of 1 to 12. α represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

[0033] The structural unit (a) may be used alone or in combination of two or more types.

[0034] Specific examples of copolymer 1 are shown below.

[0035] [In the formula, n represents an integer of 1 to 30.]

[0036] <Copolymer 2> Copolymer 2 has a structural unit (b) containing an N-acetylglucosamine group, and the polymer terminal has the above structure (T).

[0037] The explanation of the structure (T) in the copolymer 2 is the same as the explanation of the structure (T) in the copolymer 1 above.

[0038] [Structural Unit (b)] The structural unit (b) is a structural unit containing an N-acetylglucosamine group. Examples of the structural unit (b) include structural units containing N-acetylglucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, etc. Specific examples of the structural unit (b) include structural units derived from a monomer containing an N-acetylglucosamine group, and structural units derived from a monomer containing a chitopolyose group in which 2 to 6 N-acetylglucosamine rings are bonded.

[0039] Examples of the structural unit (b) include structural units represented by the following general formula (b-1).

[0040] [In the formula, Ra １ represents a hydrogen atom or an organic group; １ represents —C(═O)—O— or —C(═O)—NH—; and na represents an integer of 1 to 6.]

[0041] In the formula (b-1), Ra １ The organic group in Ra may be a hydrocarbon group which may have a substituent. １ The hydrocarbon group in the formula (I) preferably has 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, even more preferably 1 to 6 carbon atoms, still more 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 an aliphatic hydrocarbon group which may have a substituent, with an aliphatic hydrocarbon group which may have a substituent being preferred. The hydrocarbon group which may have a substituent may be saturated or unsaturated, with a saturated group being preferred.

[0042] Ra １ The aliphatic hydrocarbon group in Ra may be an alkyl group having 1 to 12 carbon atoms, which may have a substituent. The alkyl group may be linear or branched, but is preferably linear. １ The aliphatic hydrocarbon group in Ra may or may not have a substituent. １Examples of the substituent that the aliphatic hydrocarbon group may have include a carboxy group, an amino group, a hydroxy group, and an alkoxy group.

[0043] Ra １ is preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group.

[0044] In the formula (b-1), Ya １ In the formula (b-1), when —C(═O)—O— and —C(═O)—NH— are represented by *—C(═O)—O-** and *—C(═O)—NH-**, respectively, * represents Ra in formula (b-1). １ is bonded to the carbon atom to which is bonded, and ** is -(CH ２ ) ｎａ - binds to.

[0045] In the formula (b-1), na is preferably an integer of 1 to 4, more preferably an integer of 1 to 3, and even more preferably 1 or 2.

[0046] The structural unit (b) is preferably a structural unit represented by the following general formula (b-1-1).

[0047]

[0048] [Method for producing a compound from which structural unit (b) is derived] The compound from which structural unit (b) is derived can be produced by known methods. The monomer from which structural unit (b) is derived can be synthesized, for example, by binding an α,β-unsaturated carboxylic acid to the reducing end of N-acetylglucosamine.

[0049] 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. Among these, acrylic acid and methacrylic acid are preferred due to their excellent copolymerizability with other structural 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 α,β-unsaturated carboxylic acid monomers; and ammonium salts of β-unsaturated carboxylic acids.

[0050] A specific example of a method for producing a compound from which structural unit (b) is derived is a method in which an acrylic monomer having an N-acetylglucosamine group is obtained by condensing an α,β-unsaturated carboxylic acid with aminated N-acetylglucosamine using a condensing agent such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). For example, aminated N-acetylglucosamine and an α,β-unsaturated carboxylic acid are mixed in a 1:1 molar ratio and subjected to a condensation reaction in a solvent such as dimethyl sulfoxide (DMSO) or water in the presence of a condensing agent such as DMT-MM, thereby producing a monomer from which structural unit (b) is derived.

[0051] The method for amminating N-acetylglucosamine is not particularly limited, but examples include a method in which the amino group of a compound having an amino group is linked to the reducing end of N-acetylglucosamine by reductive amination. Alternatively, the hydroxy group of N-acetylglucosamine may be substituted with a carboxy group, and a compound having an amino group may be linked by a coupling method using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0052] The structural unit (b) may be used alone or in combination of two or more types.

[0053] Specific examples of copolymer 2 are shown below.

[0054] [In the formula, m represents an integer of 1 to 30.]

[0055] <Copolymer 3> Copolymer 3 has the structural unit (a) and the structural unit (b), and the polymer terminal has the structure (T).

[0056] The explanation of the structural unit (a) and the structure (T) in copolymer 3 is the same as that in copolymer 1. The explanation of the structural unit (b) in copolymer 3 is the same as that in copolymer 2.

[0057] Specific examples of copolymer 3 are shown below.

[0058] [In the formula, m and n each independently represent an integer of 1 to 30.]

[0059] Through studies by the present inventors, it has been found that copolymers 1 to 3, each having structure (T) at the polymer terminus, exhibit either or both of an inactivating effect and an activation-suppressing effect on myofibroblasts. When the copolymer contains structural unit (a), biocompatibility is likely to be improved. When the copolymer contains structural unit (b), myofibroblasts and activated stellate cells can be easily selected as targets.

[0060] Copolymers 1 to 3 further improve the inhibitory effect on the expression of collagen (Col1a2) and α-SMA, and the promoting effect on the expression of MMP1, HMOXA, and ANGPTL4. Therefore, the antifibrotic agent of the present embodiment, which contains any of Copolymers 1 to 3, has either or both of an excellent myofibroblast inactivation effect and an excellent myofibroblast activation inhibitory effect.

[0061] In copolymer 3, the molar ratio of the structural unit (a) to the structural unit (b) (structural unit (a):structural unit (b)) is preferably 100:1 to 8:2. When the molar ratio of the structural unit (a) to the structural unit (b) is within this range, the expression of α-SMA and collagen (Col1a2) in myofibroblasts becomes more difficult, and the expression levels of HMOX1, MMP, and ANGPTL4 can be further improved.

[0062] The molar ratio of structural unit (a):structural unit (b) is more preferably 80:1 to 7:3, even more preferably 60:1 to 6:4, and particularly preferably 50:1 to 5:5. From the viewpoint of promoting inactivation or suppressing activation of myofibroblasts, it is preferable that the proportion of structural unit (b) is smaller than the content proportion of structural unit (a).

[0063] The total number of structural units (a) and structural units (b) in copolymer 3 is, for example, 10 to 50, preferably 10 to 40, and more preferably 10 to 35. The total number of structural units (b) in copolymer 3 is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 13, even more preferably 1 to 12, and particularly preferably 1 to 10.

[0064] The number of structural units (a) in copolymer 3 can be, for example, 1 to 30, preferably 3 to 30, more preferably 4 to 30, even more preferably 8 to 30, even more preferably 10 to 26, and particularly preferably 15 to 26. From the viewpoint of promoting inactivation or inhibiting activation of myofibroblasts, the number of structural units (b) is preferably smaller than the number of structural units (a). In copolymer 3, for example, it is preferable that one structural unit (b) is introduced for every 2 to 5 structural units (a), and more preferably one structural unit (b) is introduced for every 3 to 4 structural units (a).

[0065] Copolymers 1 to 3 are preferably random copolymers.

[0066] The weight-average molecular weight (Mw) of Copolymers 1 to 3 is not particularly limited and can be appropriately set depending on the molecular weight of either or both of the structural unit (a) and the structural unit (b). The weight-average molecular weight (Mw) of Copolymers 1 to 3 is preferably, for example, within the range of 3,000 to 20,000. When the weight-average molecular weight (Mw) of Copolymers 1 to 3 is within the above preferred range, the expression levels of α-SMA and collagen (Col1a2) in myofibroblasts can be further suppressed, and the expression levels of HMOX1, MMP, and ANGPTL4 can be further improved.

[0067] The weight average molecular weight (Mw) of Copolymer 1 or 2 is preferably 3,000 to 6,000, more preferably 3,300 to 5,000. The weight average molecular weight (Mw) of Copolymer 3 is preferably 3,000 to 20,000, more preferably 3,500 to 16,000.

[0068] The number-average molecular weights (Mn) of copolymers 1 to 3 are not particularly limited and can be appropriately set depending on the molecular weight of either or both of the structural unit (a) and the structural unit (b). The number-average molecular weights (Mn) of copolymers 1 to 3 are preferably within the range of 200 to 15,000, for example. When the number-average molecular weights (Mn) of copolymers 1 to 3 are within the above preferred ranges, the expression levels of α-SMA and collagen (Col1a2) in myofibroblasts can be further suppressed, and the expression levels of HMOX1, MMP, and ANGPTL4 can be further improved.

[0069] The number average molecular weight (Mn) of Copolymer 1 or 2 is preferably 2,000 to 6,000, more preferably 2,300 to 5,000. The number average molecular weight (Mn) of Copolymer 3 is preferably 2,000 to 15,000, more preferably 3,000 to 12,000.

[0070] The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of each of the copolymers 1 to 3 is preferably within a range of 1.0 to 1.5, and more preferably 1.0 to 1.3.

[0071] The weight average molecular weight (Mw) and number average molecular weight (Mn) of copolymers 1 to 3 can be measured using a gel permeation chromatography (GPC) apparatus (product name: LC-9110G NEXT, manufactured by Japan Analytical Industry Co., Ltd.) under the following conditions. [Measurement conditions] The column used is a JAIGEL-GS510, and the eluent is 200 mM sodium nitrate / acetonitrile = 80 / 20. The flow rate is 1 mL / min, the detector is an RI detector, and the column temperature is 40°C. A molecular weight standard curve is performed using full run.

[0072] <<Method for Producing Copolymers 1 to 3>> The method for producing copolymers 1 to 3 is not particularly limited, and examples thereof include radical polymerization, living radical polymerization, and reversible addition-fragmentation chain transfer polymerization (RAFT polymerization) using a chain transfer agent (RAFT agent). The structure (T) is a residue of a RAFT agent, and copolymers 1 to 3 having structure (T) can be obtained by using a RAFT agent having an end group represented by -S-C(=S)-S-R (R is a hydrocarbon group having 8 to 15 carbon atoms) as the RAFT agent.

[0073] Examples of RAFT agents that can be used in this embodiment include 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid and 2-(hexylthiocarbonothioylthio)-2-methylpropanoic acid.

[0074] The monomer that derives the structural unit (a) is subjected to RAFT polymerization using the above-mentioned RAFT agent to obtain copolymer 1. The monomer that derives the structural unit (b) is subjected to RAFT polymerization using the above-mentioned RAFT agent to obtain copolymer 2. The monomer that derives the structural unit (a) and the monomer that derives the structural unit (b) are subjected to RAFT polymerization using the above-mentioned RAFT agent to obtain copolymer 3.

[0075] In the copolymerization reaction of copolymer 3, the mixing molar ratio of the monomer that derives structural unit (a) to the monomer that derives structural unit (b) is, for example, in the range of 100:1 to 8:2. The mixing molar ratio of the monomer that derives structural unit (a) to the monomer that derives structural unit (b) is preferably 100:1 to 8:2, more preferably 80:1 to 7:3, even more preferably 60:1 to 6:4, and particularly preferably 50:1 to 5:5.

[0076] <Antifibrotic Agent> The antifibrotic agent of this embodiment is made of the above-mentioned Copolymer 1. The antifibrotic agent of this embodiment is made of the above-mentioned Copolymer 2. The antifibrotic agent of this embodiment is made of the above-mentioned Copolymer 3.

[0077] <Pharmaceutical Composition for Treating Fibrosis> The pharmaceutical composition for treating fibrosis of this embodiment contains the above-mentioned antifibrotic agent as an active ingredient. The "active ingredient" refers to an ingredient that is effective in treating fibrosis, and more specifically, an ingredient that has the effect of improving or alleviating fibrosis and / or inhibiting the progression of fibrosis. The pharmaceutical composition for treating fibrosis of this embodiment contains a therapeutically effective amount of the copolymer as an active ingredient. The therapeutically effective amount refers to the amount of copolymer that can exert the above-mentioned therapeutic effect on the treatment of fibrosis.

[0078] The pharmaceutical composition for treating fibrosis of this embodiment can be administered orally or parenterally (intravenously, subcutaneously, transdermally, pulmonary, transmucosally, rectally, etc.) to humans or non-human mammals, such as rabbits, cats, dogs, cows, sheep, and monkeys.

[0079] The pharmaceutical composition for treating fibrosis of this embodiment can be prepared by mixing the copolymer with pharmaceutically acceptable carriers and additives typically used for oral or parenteral administration, and formulating the mixture into a desired form. Examples of carriers include excipients, binders, disintegrants, disintegration aids, lubricants, wetting agents, etc.

[0080] The pharmaceutical composition for treating fibrosis of this embodiment may be in the form of, for example, granules, powders, tablets, pills, buccal tablets, capsules, syrups, liquids, emulsions, suspensions, creams, ointments, eye drops, injections, drip infusions, nasal drops, patches, suppositories, etc. The above-mentioned tablets include sugar-coated tablets.

[0081] The content of the copolymer in the pharmaceutical composition for the treatment of fibrosis of this embodiment is not particularly limited as long as it suppresses the expression of α-SMA and collagen and provides an effect of improving fibrosis. The content of the copolymer is preferably 0.1% by mass or more, more preferably 1% by mass or more, and even more preferably 10% by mass or more.

[0082] [Fibrosis] As used herein, "fibrosis" refers to the loss of tissue parenchymal cells and the decline in tissue function caused by stresses such as chemical stimuli from drugs and the like, excessive pressure load, and inflammatory responses, followed by excessive migration and proliferation of fibroblasts in the process of compensating for this, and subsequent stiffening of the tissue accompanied by functional impairment due to the synthesis and deposition of extracellular matrix proteins, and is not particularly limited by the type of inducing stimulus or the site of onset. Examples of such tissue fibrosis diseases include fibrosis of visceral tissues such as the lung, intestinal prolapse, kidney, heart, and liver.

[0083] The fibrosis targeted by the pharmaceutical composition for treating fibrosis of this embodiment includes tissue fibrosis diseases caused by the administration of drugs such as antitumor agents, antibiotics, antibacterial agents, antiarrhythmic agents, anti-inflammatory agents, antirheumatic agents, interferon, or Sho-saiko-to; and tissue fibrosis diseases associated with diseases such as chronic nephritis, interstitial myocarditis, and interstitial intestinal prolapse. Specific examples include pulmonary fibrosis caused by a side effect of bleomycin administration; pulmonary fibrosis caused during or after interstitial pneumonia; fibrosis of intestinal prolapse and cervical sclerosis caused during interstitial intestinal prolapse; renal fibrosis and renal failure (nephrosclerosis) caused by genetic abnormalities, etc.; endocardial fibrosis caused by remodeling after myocardial infarction; hepatic fibrosis caused by damage to hepatocytes; non-alcoholic steatohepatitis (NASH) and associated portal hypertension and cirrhosis; keloids caused by excessive tissue repair; and sclerosing peritonitis, benign prostatic hyperplasia, scleroderma, uterine leiomyoma, retroperitoneal fibrosis, and myelofibrosis.

[0084] The pharmaceutical composition for treating fibrosis of this embodiment may inhibit STAT3 phosphorylation, thereby reducing the expression levels of αSMA and collagen, increasing the expression levels of MMP1, HMOX1, and ANGPTL4, thereby restoring myofibroblasts and activated stellate cells to normal fibroblasts or stellate cells and improving fibrosis. In the case of fibrosis caused by myofibroblasts and activated stellate cells, such as in the liver, activated stellate cells transform into myofibroblast-like cells and produce extracellular matrix, leading to the progression of fibrosis. Use of the pharmaceutical composition for treating fibrosis of this embodiment can improve liver fibrosis, improve liver function, and suppress the development of liver cancer. It can also be used to treat pancreatic fibrosis, etc.

[0085] <Method for inactivating myofibroblasts> The method for inactivating myofibroblasts of this embodiment includes binding the above-mentioned antifibrotic agent to myofibroblasts. In the method of this embodiment, a copolymer can be used as the antifibrotic agent.

[0086] "Myofibroblast inactivation" refers to the occurrence of at least one phenomenon selected from the group consisting of a decrease in collagen expression level, a decrease in α-SMA expression level, an increase in MMP1 expression level, an increase in HMOX1 expression level, and an increase in ANGPTL4 expression level in myofibroblasts. In myofibroblast inactivation, it is preferable that two or more of the above phenomena occur, more preferably three or more of the above phenomena occur, even more preferably four or more of the above phenomena occur, and it is particularly preferable that all of the above phenomena occur.

[0087] The binding of an antifibrotic agent to myofibroblasts can be achieved by contacting the antifibrotic agent with myofibroblasts. The contact of the antifibrotic agent with myofibroblasts can be achieved in vitro or in vivo. In vitro contact can be achieved by adding the antifibrotic agent to a culture medium of myofibroblasts. In vivo contact can be achieved by administering the antifibrotic agent to a living body.

[0088] The amount of anti-fibrotic agent added to myofibroblasts in vitro can 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. In vivo, the dosage to be administered to a living organism can be appropriately determined depending on the species, weight, sex, age, etc. of the living organism. The dosage of the anti-fibrotic agent to be administered to a living organism can be, for example, 10 to 1000 μg / kg.

[0089] In vitro, myofibroblasts can be cultured in an environment at about 37° C. (37±2° C.) and a carbon dioxide concentration of about 5% by volume (5±2% by volume).

[0090] (Other Embodiments) In one embodiment, the present invention provides a method for treating fibrosis, comprising administering an effective amount of the antifibrotic agent described above to a human or animal patient in need of treatment. The antifibrotic agent can be copolymers 1 to 3. Fibrosis to which the present invention can be applied includes those similar to those described above in the "Pharmaceutical composition for treating fibrosis."

[0091] In one embodiment, the present invention provides use of copolymers 1 to 3 for producing an antifibrotic agent. In one embodiment, the present invention provides use of copolymers 1 to 3 for producing a pharmaceutical composition for treating fibrosis. In one embodiment, the present invention provides use of copolymers 1 to 3 for use in treating fibrosis. In one embodiment, the present invention provides use of copolymers 1 to 3 for treating fibrosis.

[0092] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0093] (Preparation of fibroblasts) Primary cultured human dermal fibroblasts (Takara Bio Inc.) isolated from adult skin confirmed to be free of mycoplasma contamination were cultured in HFDM-1(+) medium (Cell Science and Technology Laboratories) supplemented with 2 mM L-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C and 5% v / v CO ２ The cells were cultured in a humidified incubator. Subsequently, 10 ng / mL of TGF-β was added to the medium to obtain myofibroblasts. Human dermal fibroblasts maintained for passages 3 to 6 were used to obtain the myofibroblasts.

[0094] (Confirmation of the Presence or Absence of Mycoplasma Contamination) It was confirmed that the human skin fibroblasts used were not contaminated with mycoplasma using Mycoplasma Detection Kit for Endpoint PCR, OneStep, VendorGeM (Minerva Biolabs GmbH).

[0095] (Synthesis of AC-GlcNAc Monomer) 5 g of GlcNAc was dissolved in 50 mL of water, and NH ４ CO ３The flask was stirred and incubated at 30-35°C for 4-5 days in an open system with the lid open. ４ CO ３ When the precipitate disappears, ４ CO ３ was added appropriately. Thin layer chromatography (TLC) confirmed that GlcNAc-NH ２ The synthesis was confirmed.

[0096] After 4 to 5 days, water was added to the reaction mixture and the mixture was evaporated (30°C) to remove excess NH ４ CO ３ This procedure was repeated until the odor disappeared. After evaporation, the mixture was freeze-dried. Next, GlcNAc-NH ２ (4.5 mol: approximately 1 g) was dissolved in dimethyl sulfoxide (DMSO) (10 mL), and 2-carboxyethyl acrylate (4.5 mmol) was added. After dissolution, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (6.8 mmol) was added, and the mixture was incubated at room temperature for 18 hours.

[0097] After incubation, the reaction mixture was added dropwise to 200-300 mL of chloroform. The resulting precipitate was collected using a Kiriyama funnel. The collected precipitate was dissolved in methanol, and the insoluble matter was removed using a Kiriyama funnel. The methanol-dissolved precipitate was collected. The mixture was evaporated to remove the methanol. The solid from which the methanol had been removed was dissolved in water, and then purified by preparative high-performance liquid chromatography (HPLC) (water / acetonitrile) to obtain the AC-GlcNAc monomer (molecular weight 346) represented by the following chemical formula (1).

[0098]

[0099] (Copolymer 1-1) 10.3 mg (0.145 mmol) of acrylamide monomer (molecular weight 71), 5.3 mg (0.0145 mmol) of 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (molecular weight 364), and 2,2′-azobis(2-methylpropionitrile) (AIBN; 1 mg) were dissolved in 250 μL of dimethyl sulfoxide (DMSO), placed in a shrink tube, and degassed by repeating freezing and thawing three times.

[0100] Thereafter, argon gas was sealed in and the solution was incubated at 65°C for 17 hours while stirring. After 17 hours, the reaction solution was added dropwise to 2-propanol to precipitate the produced polymer. The precipitated polymer was dissolved in water and dialyzed for 24 hours with a molecular weight cutoff of 1000, followed by freeze-drying to obtain Copolymer 1-1.

[0101]

[0102] Copolymer 1-1 had a weight average molecular weight (Mw) of 3,300, a number average molecular weight (Mn) of 2,400, and an Mw / Mn ratio of 1.4. These molecular weights were measured using a Gel Permeation Chromatography (GPC) apparatus (product name: LC-9110G NEXT, manufactured by Japan Analytical Industry Co., Ltd.) under the following conditions. A JAIGEL-GS510 column was used, and the eluent was 200 mM 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. A molecular weight standard curve was performed using full-flow chromatography. Copolymer 1-1 contained approximately 41 acrylamide monomers.

[0103] (Copolymer 1B) Copolymer 1B (formula 1B below) was obtained in the same manner as in the production method for copolymer 1-1, except that 2-[[(2-carboxyethyl)sulfanylthiocarbonyl]-sulfanyl]propanoic acid (molecular weight: 254) was used instead of 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (molecular weight: 364).

[0104] Copolymer 1B had a weight average molecular weight (Mw) of 3,300, a number average molecular weight (Mn) of 2,400, an Mw / Mn ratio of 1.4, and about 41 acrylamide monomers.

[0105]

[0106] (Copolymer 3-1) 5.0 mg (0.0145 mmol) of AC-GlcNAc monomer (molecular weight: 346), 9.3 mg (0.131 mmol) of acrylamide monomer (molecular weight: 71), 5.3 mg (0.0145 mmol) of 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (molecular weight: 364), and 2,2'-azobis(2-methylpropionitrile) (AIBN; 1 mg) were dissolved in 250 μL of dimethyl sulfoxide (DMSO), placed in a shrink tube, and degassed by repeating freeze-thaw cycles three times.

[0107] Thereafter, argon gas was sealed in and the solution was incubated at 65°C for 17 hours while stirring. After 17 hours, the reaction solution was added dropwise to 2-propanol to precipitate the produced polymer. The precipitated polymer was dissolved in water and dialyzed for 24 hours with a molecular weight cutoff of 3,500, followed by freeze-drying to obtain Copolymer 3-1 (formula 3-1 below).

[0108] This copolymer 3-1 had a weight average molecular weight (Mw) of 3,800, a number average molecular weight (Mn) of 3,300, Mw / Mn of 1.15, and a molar ratio of AC-GlcNAc monomer to acrylamide monomer of 1:43.

[0109]

[0110] (Copolymer 3B) Copolymer 3B (formula 3B below) was obtained in the same manner as in the production method of copolymer 3-1, except that 2-[[(2-carboxyethyl)sulfanylthiocarbonyl]-sulfanyl]propanoic acid (molecular weight: 254) was used instead of 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (molecular weight: 364).

[0111] Copolymer 3B had a weight average molecular weight (Mw) of 3,800, a number average molecular weight (Mn) of 3,300, Mw / Mn of 1.15, and a molar ratio of AC-GlcNAc monomer to acrylamide monomer of 1:43.

[0112]

[0113] (Copolymer 3B-2) 20 mg of copolymer 3-1 was dissolved in 1 mL of DMSO, 10 mg of aminoethanethiol was added, and the mixture was degassed and incubated with stirring for 20 minutes. Then, 10 mL of diethyl ether was added, and the mixture was vigorously stirred and centrifuged to obtain a precipitate of the polymer with the alkyl groups cleaved. The cleaved alkyl chains were removed by dissolving the mixture in diethyl ether.

[0114] Then, the mixture was dissolved in 1 mL of DMSO, and 5 mg of dodecyl acrylate and 2 mg of triethylamine were added thereto, followed by incubation with stirring for 18 hours at 45° C. After the 18-hour incubation, the mixture was precipitated with 2-propanol to obtain copolymer 3B-2 (formula 3B-2 below).

[0115] Copolymer 3B-2 was prepared by removing the trithiocarbonate structure and alkyl chain at the end of copolymer 3-1 to form an SH group, and then bonding dodecyl acrylate via Michael addition. Copolymer 3B-2 had a weight-average molecular weight (Mw) of 3,800, a number-average molecular weight (Mn) of 3,300, an Mw / Mn ratio of 1.15, and a molar ratio of AC-GlcNAc monomer to acrylamide monomer of 1:43.

[0116]

[0117] (Copolymer 3B-1) 5.0 mg (0.0145 mmol) of AC-GlcNAc monomer (molecular weight: 346), 9.3 mg (0.131 mmol) of acrylamide monomer (molecular weight: 71), 3.3 mg (0.0145 mmol) of 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid (molecular weight: 224), and 2,2′-azobis(2-methylpropionitrile) (AIBN; 5 mg) were dissolved in 250 μL of dimethyl sulfoxide (DMSO), placed in a shrink tube, and degassed by three repeated freeze-thaw cycles.

[0118] Thereafter, argon gas was sealed in and the solution was incubated at 65°C for 17 hours while stirring. After 17 hours, the reaction solution was added dropwise to 2-propanol to precipitate the produced polymer. The precipitated polymer was dissolved in water and dialyzed for 24 hours at a molecular weight cutoff of 3,500, followed by freeze-drying to obtain copolymer 3B-1 (formula 3B-1 below).

[0119] Copolymer 3B-1 was a copolymer polymerized with 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid having an ethyl group at the terminal structure. Copolymer 3B-1 had a weight-average molecular weight (Mw) of 3,800, a number-average molecular weight (Mn) of 3,300, an Mw / Mn ratio of 1.55, and a molar ratio of AC-GlcNAc monomer to acrylamide monomer of 1:43.

[0120]

[0121] (Copolymer 2B) 50 mg (0.145 mmol) of AC-GlcNAc monomer (molecular weight: 346), 3.3 mg (0.0145 mmol) of 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid (molecular weight: 224), and 2,2′-azobis(2-methylpropionitrile) (AIBN; 1 mg) were dissolved in 250 μL of dimethyl sulfoxide (DMSO), placed in a shrink tube, and degassed by repeating freeze-thaw cycles three times.

[0122] Thereafter, argon gas was sealed in and the solution was incubated at 65°C for 17 hours while stirring. After 17 hours, the reaction solution was added dropwise to 2-propanol to precipitate the produced polymer. The precipitated polymer was dissolved in water and dialyzed for 24 hours with a molecular weight cutoff of 3,500, followed by freeze-drying to obtain copolymer 2B (formula 2B below).

[0123] Copolymer 2B was obtained by replacing 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid, a component of copolymer 2-1, with 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid, and had a weight-average molecular weight (Mw) of 3,600, a number-average molecular weight (Mn) of 2,500, an Mw / Mn ratio of 1.44, and nine AC-GlcNAc monomers.

[0124]

[0125] (Copolymer 1B-1) 10.3 mg (0.145 mmol) of acrylamide monomer (molecular weight 71), 3.3 mg (0.0145 mmol) of 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid (molecular weight 224), and 2,2′-azobis(2-methylpropionitrile) (AIBN; 5 mg) were dissolved in 250 μL of dimethyl sulfoxide (DMSO), placed in a shrink tube, and degassed by repeating freeze-thaw cycles three times.

[0126] Thereafter, argon gas was sealed in and the solution was incubated at 65°C for 17 hours while stirring. After 17 hours, the reaction solution was added dropwise to 2-propanol to precipitate the produced polymer. The precipitated polymer was dissolved in water and dialyzed for 24 hours with a molecular weight cutoff of 3,500, followed by freeze-drying to obtain Copolymer 1B-1 (formula 1B-1 below).

[0127] Copolymer 1B-1 was obtained by replacing 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid, a component of copolymer 1-1, with 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid, and had a weight average molecular weight (Mw) of 3,600, a number average molecular weight (Mn) of 2,500, and an Mw / Mn ratio of 1.44.

[0128]

[0129] (Copolymer 2-1) Copolymer 2-1 (formula 2-1 below) was obtained in the same manner as in the production of copolymer 2B, except that 2-[[(ethylthio)carbonothioyl]thio]-2-methylpropanoic acid was replaced with 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (molecular weight 364). The weight-average molecular weight (Mw) of copolymer 2-1 was 3,600, the number-average molecular weight (Mn) was 2,500, and the Mw / Mn ratio was 1.44.

[0130]

[0131] [Example 1] Each test compound was added at various concentrations to the myofibroblasts prepared in the above (Preparation of myofibroblasts), and the cells were incubated at 37°C and 5% CO ２ The cells were cultured in a humidified incubator at 17 °C for 48 hours. The concentrations were 10 μg / mL, 50 μg / mL, or 100 μg / mL. The test compounds were Copolymer 1-1, Copolymer 3-1, Copolymer 1B, or Copolymer 3B.

[0132] After culturing, the cells were harvested. The expression of α-smooth muscle actin (αSMA), collagen (Col1a2), matrix metalloproteinase 1 (MMP1), heme oxygenase 1 (HMOX1), and β-actin in the harvested myofibroblasts was observed by Western blotting. As controls, human dermal fibroblasts cultured without any addition (Control) and myofibroblasts cultured without the addition of any test compound were used. The results are shown in Figure 1.

[0133] The results shown in FIG. 1 confirmed that the expression levels of αSMA and collagen (Col1a2) were significantly higher in myofibroblasts to which 10 ng / mL of TGF-β had been added.

[0134] In myofibroblasts to which either Copolymer 1-1 or Copolymer 3-1 was added, the expression levels of αSMA and collagen (Col1a2) were decreased and the expression levels of MMP1 and HMOX1 were increased, when compared with myofibroblasts to which either Copolymer 1B or Copolymer 3B was added at the same addition amount. In particular, it was confirmed that the expression level of collagen (Colla2) was more suppressed in myofibroblasts to which Copolymer 1-1 was added than in myofibroblasts to which Copolymer 3-1 was added.

[0135] It was also confirmed that the expression levels of αSMA and collagen (Col1a2) decreased and the expression level of HMOX1 increased significantly in myofibroblasts to which copolymer 3-1 was added.

[0136] Furthermore, in comparison between copolymer 3-1 and copolymer 3B, it was confirmed that the expression of MMP1 and HMOX1 was extremely low when copolymer 3B was added in an amount 10 times that of copolymer 3-1, but the expression of α-SMA and collagen (Col1a2) was extremely high when copolymer 3B was added in an amount 10 times that of copolymer 3-1.

[0137] HMOX1 is a protein that exerts a protective effect against various oxidative stresses, and increased expression of this gene alleviates fibrosis through its antioxidant effect. Therefore, it is thought that the decreased expression of α-SMA and collagen and the increased expression of HMOX1 further suppress the activation of myofibroblasts.

[0138] [Example 2] 100 μg / mL of a test compound was added to the myofibroblasts prepared in the above (Preparation of myofibroblasts), and the cells were incubated at 37°C and 5% CO ２ The cells were cultured for 48 hours in a humidified incubator. The test compound was copolymer 3-1 or copolymer 3B-2. After culture, the cells were recovered. The expression levels of α-smooth muscle actin (αSMA), collagen (Col1a2), matrix metalloproteinase 1 (MMP1), HMOX1, and β-actin in the recovered myofibroblasts were observed by Western blotting. As controls, human dermal fibroblasts cultured without any addition (Control) and myofibroblasts cultured without the addition of any test compound were used.

[0139] The results are shown in Figure 2. The numbers below each band in Figure 2 indicate the relative intensity of each band, with the signal intensity of the control band set at 100. The signal intensity of each band was normalized by the signal intensity of the β-actin band. The results shown in Figure 2 confirmed that in myofibroblasts treated with 10 ng / mL TGF-β, the expression levels of α-SMA and collagen (Col1a2) were very high, and the expression levels of MMP1 and HMOX1 were undetectable or very low.

[0140] It was confirmed that the expression levels of αSMA and collagen (Col1a2) were decreased and the expression levels of MMP1 and HMOX1 were increased in myofibroblasts to which copolymer 3-1 was added, compared to myofibroblasts to which copolymer 3B-2 was added.

[0141] Furthermore, it was confirmed that myofibroblasts to which copolymer 3B-2 was added at a concentration five times that of copolymer 3-1 showed very high expression levels of α-SMA and collagen (Col1a2), while the expression levels of MMP1 and HMOX1 were undetectable or very low.

[0142] MMP1 is an enzyme that breaks down collagen deposited in fibrous tissue. HMOX1 is a protein that exerts a protective effect against various oxidative stresses. The addition of copolymer 3-1 suppresses the expression levels of α-SMA and collagen in myofibroblasts, thereby inhibiting fibrosis. Furthermore, the increased expression level of MMP1 decomposes the deposited collagen. Furthermore, the increased expression level of HMOX1 promotes wound healing, and its anti-inflammatory and antioxidant effects alleviate fibrosis.

[0143] [Example 3] 100 μg / mL of a test compound was added to the myofibroblasts prepared in the above (Preparation of myofibroblasts), and the cells were incubated at 37°C and 5% CO ２The cells were cultured for 48 hours in a humidified incubator. The test compounds were copolymer 3-1, copolymer 3B-1, 2B, 1B-1, or 2-1. After culture, the cells were recovered. The expression levels of α-smooth muscle actin (αSMA), collagen (Col1a2), matrix metalloproteinase 1 (MMP1), HMOX1, and β-actin in the recovered myofibroblasts were observed by Western blotting. As controls, human dermal fibroblasts cultured without any addition (Control) and myofibroblasts cultured without the addition of any test compound were used.

[0144] The results are shown in Figure 3. The numbers below each band in Figure 3 indicate the relative intensity of each band, with the signal intensity of the control band set at 100. The signal intensity of each band was normalized by the signal intensity of the β-actin band. The results shown in Figure 3 confirmed that in myofibroblasts treated with 10 ng / mL TGF-β, the expression levels of αSMA and collagen (Col1a2) were very high, and the expression levels of MMP1 and HMOX1 were undetectable or very low. It was confirmed that in myofibroblasts treated with either copolymer 3-1 or copolymer 2-1, the expression levels of αSMA and collagen (Col1a2) were reduced and the expression levels of MMP1 and HMOX1 were increased, compared to myofibroblasts treated with either copolymer 3B-1, 2B, or 1B-1.

[0145] The addition of either copolymer 3-1 or 2-1 suppresses the expression of α-SMA and collagen in myofibroblasts, thereby inhibiting fibrosis. Furthermore, the increased expression of MMP1 degrades the attached collagen. Furthermore, the increased expression of HMOX1 promotes wound healing, and the anti-inflammatory and antioxidant effects alleviate fibrosis.

[0146] These findings indicate that the copolymer having a trithiocarbonate structure and a structural unit having 12 carbon atoms has the necessary anti-fibrotic properties and anti-inflammatory and antioxidant effects compared to the copolymer having a structural unit having 4 carbon atoms.

[0147] The anti-fibrotic agent of the embodiment can be used to suppress the expression levels of α-SMA and collagen in myofibroblasts and activated stellate cells, and to repair tissue.

Claims

1. A copolymer having a hydrophobic structural unit (a), wherein the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms.

2. A copolymer having a structural unit (b) containing an N-acetylglucosamine group, wherein the polymer terminal is a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms.

3. A copolymer having a hydrophobic structural unit (a) and a structural unit (b) containing an N-acetylglucosamine group, wherein the polymer terminal has a structure (T) containing a trithiocarbonate structure and a hydrocarbon group having 8 to 15 carbon atoms.

4. The copolymer according to claim 1 or 3, wherein the structural unit (a) is at least one structural unit selected from the group consisting of structural units derived from an α,β-unsaturated carboxylic acid monomer, structural units derived from a monomer having an acryloyl group or methacryloyl group attached to one end of polyether, polyurethane, polyamino acid, or polyester, structural units derived from acrylamide, and structural units derived from an ester of an alkoxy alcohol with acrylic acid or methacrylic acid.

5. The copolymer according to claim 3, wherein the molar ratio of the structural unit (a) to the structural unit (b) is 100:1 to 8:

2.

6. The copolymer according to claim 1 or 2, having a weight average molecular weight of 3,000 to 20,000.

7. The copolymer according to claim 1 or 2, having a number average molecular weight of 2,000 to 15,000.

8. The copolymer according to claim 1 or 2, wherein the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is 1.0 to 1.

5.

9. An anti-fibrotic agent comprising the copolymer according to claim 1 or 2.

10. A pharmaceutical composition for treating fibrosis, comprising the antifibrotic agent according to claim 9 as an active ingredient.

11. A method for inactivating myofibroblasts, comprising binding the antifibrotic agent according to claim 9 to myofibroblasts.

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