O-linked N-acetylglucosaminylated protein-like substance and fibrosis treatment drug containing the same

Abstract

This O-GlcNAcylated protein-like substance contains at least one unit selected from the group consisting of an N-acetylglucosamine unit, carboxy group-containing radical polymerizable units, a styrene unit, a polyethyleneimine unit, a poly-L-lysine unit, and a biotin unit. This fibrosis therapeutic drug contains the O-GlcNAcylated protein-like substance.

Description

[Technical field]

[0001] The present invention relates to an O-linked N-acetylglucosaminylated proteinaceous substance and a therapeutic agent for fibrosis containing the same. This application claims priority based on Japanese Patent Application No. 2019-206265, filed on November 14, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] Connective tissue, which constitutes the internal organs, grows abnormally, and activated fibroblasts accumulate at the fibrotic site, producing large amounts of collagen and other proteins that attach to the tissue, causing fibrosis of the tissue. Fibrosis can occur in various tissues. For example, hepatic fibrosis, which is fibrosis of the liver, is a common pathology caused by various factors, including persistent infection with hepatitis viruses, excessive alcohol intake, nonalcoholic steatohepatitis, autoimmune mechanisms, intrahepatic cholestasis, drug-induced, metal metabolic disorders, and hepatic congestion. Activated stellate cells transform into myofibroblast-like cells and produce extracellular matrix, which progresses. As fibrosis progresses, collagen and other extracellular matrix are excessively deposited in the liver tissue, and as a result, hepatocellular dysfunction and portal hypertension progress in cirrhosis. It is known that in tissues other than the liver, such as the kidney, lung, heart, pancreas, and other organs, fibrosis caused by myofibroblasts leads to various diseases. Since the fibrotic state of various organs progresses to further diseases, it is expected that preventing fibrosis or improving fibrosis will lead to effective treatment of liver cirrhosis, liver cancer, and various other diseases associated with fibrosis of organs, and several improvement methods have been proposed. For example, Patent Document 1 proposes a preventive or therapeutic agent for tissue fibrosis diseases, which contains a metal chelate compound or a pharma- ceutical acceptable salt thereof as an active ingredient. In addition, Patent Document 2 proposes a fibrosis inhibitor containing as an active ingredient a polynucleotide encoding a protein belonging to the Sema6 family or a partial fragment thereof; a protein belonging to the Sema6 family or a partial fragment thereof; or an agonist for a receptor that uses the protein or partial fragment thereof as a ligand, in order to prevent the onset of fibrosis, slow the progression of fibrosis, or improve fibrosis. Although many anti-fibrotic substances have been identified and reported through cell culture and animal experiments, no specific and effective therapeutic drug for liver fibrosis has yet been proposed for clinical use. Recently, it has been known that the blood concentration of growth and differentiation factor 15 (hereinafter, simply referred to as GDF15) is increased in patients with various malignant tumors. GDF15 is a cytokine that belongs to the TGF-β superfamily and exhibits diverse biological properties, and is significantly expressed in the placenta, kidney, intestine, prostate, choroid plexus, etc. In addition to these tissues or cells, gene expression of GDF15 is induced in response to stresses such as hypoxia, inflammation, UV exposure, and tissue damage (Non-Patent Document 1). Furthermore, GDF15 is known to be involved in the proliferation, metastasis, and treatment resistance of cancer cells, in addition to anti-inflammatory, myocardial protection, neuroprotection, and appetite / metabolism regulation effects (Non-Patent Document 2). For example, it has been reported that mesenchymal stem cells differentiated from iPS cells ameliorate doxorubicin-induced cardiac damage by secreting GDF15 (Non-Patent Document 3). It has also been reported that in mouse models of alcohol- and carbon tetrachloride-induced liver injury, GDF15 deficiency worsens the pathological condition, but that administration of GDF15 improves it (Non-Patent Document 4), that GDF15 improves non-alcoholic steatohepatitis (hereinafter referred to as NASH) (Non-Patent Document 5), that the progression of rheumatoid arthritis is suppressed in transgenic mice that highly express GDF15 (Non-Patent Document 6), and that pancreatic cancer secretes GDF15 by activating NF-κB, which acts on surrounding macrophages to suppress NF-κB in the macrophages and stop the production of TNFα, and that by suppressing TNFα secretion by the macrophages, the pancreatic cancer itself avoids macrophage attack and proliferates (Non-Patent Document 7). Thus, GDF15 is known to increase in the blood in myocardial infarction, fibrosis, and autoimmune diseases, and to have an injury-protecting effect against pathological conditions through its anti-inflammatory properties. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2011-190257 A [Patent Document 2] JP 2018-21026 A

Outdoor Tools

[0004]

Linked Book1

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0005] Despite the above findings, an effective preventive or therapeutic agent for tissue fibrosis has not yet been developed. In addition, although it is known that GDF15 has an injury-protecting effect through its anti-inflammatory effect against pathological conditions, there is currently no method to increase GDF15 expression at or near the diseased area to effectively exert anti-inflammatory effects or inhibit proliferation.

[0006] Therefore, an object of the present invention is to provide an O-linked N-acetylglucosaminylated protein-like substance that contributes to increased GDF15 expression, and a therapeutic agent for fibrosis containing the same. [Means for solving the problem]

[0007] Since O-linked N-acetylglucosaminylated proteins released upon cell injury bind to vimentin on the surface of other cells, the inventors have discovered a polymer that functions in a similar manner to O-linked N-acetylglucosaminylated proteins, and that by using this polymer, it is possible to efficiently bind the polymer to injured tissue or cells, thereby achieving anti-inflammatory effects and proliferation inhibition by increasing GDF15 expression, and have thus completed the present invention.

[0008] That is, the present invention relates to an O-linked N-acetylglucosaminylated proteinaceous substance and a therapeutic agent for fibrosis containing the same, as described below in [1] to [5]. [1] An O-linked N-acetylglucosaminylated protein-like substance having an N-acetylglucosamine unit and at least one unit selected from the group consisting of a carboxyl group-containing radically polymerizable unit, a styrene unit, a polyethyleneimine unit, a poly-L-lysine unit and a biotin unit. [2] The O-linked N-acetylglucosaminylated protein-like substance according to [1], wherein the carboxy group-containing radically polymerizable unit is at least one selected from the group consisting of acrylic acid, methacrylic acid, itaconic acid, maleic acid, crotonic acid, 2-carboxyethyl acrylate, and 2-carboxyethyl methacrylate. [3] An O-linked N-acetylglucosaminylated protein-like substance according to [1] or [2], characterized in that the O-linked N-acetylglucosaminylated protein-like substance does not have an alkyl chain. [4] The O-linked N-acetylglucosaminylated protein-like substance according to any one of [1] to [3] above, which is a compound represented by the following chemical formula (1) or (2):

[0009] [ka] In the formula (1), n ​​is an integer of 3 to 100.

[0010] [ka] In the formula (2), n is an integer of 3 to 100. [5] A therapeutic agent for fibrosis, comprising as an active ingredient the O-linked N-acetylglucosaminylated protein-like substance according to any one of [1] to [4] above. Effect of the Invention

[0011] INDUSTRIAL APPLICABILITY The present invention makes it possible to provide an O-linked N-acetylglucosaminylated proteinaceous substance and a therapeutic agent for fibrosis containing the same. [Brief description of the drawings]

[0012] [Figure 1] The graph shows the results of a DNA microarray study on the effect of AC-GlcNAc (10-mer) on human fibroblasts (hereinafter referred to as NHDF) using an O-linked N-acetylglucosaminylated protein-like substance. [Diagram 2] The microarray results shown in Figure 1 are shown for each protein. [Figure 3A] The upper figure is a Western blot showing the increase in GDF15 expression at the protein level and β-actin expression after 24 hours of culture in NHDF with the addition of O-linked N-acetylglucosaminylated protein-like substances, AC-GlcNAc (10-mer), AC-GlcNAc (40-mer), AC-GlcNAc (monomer), and GlcNAc. The control was a sample without the addition of O-linked N-acetylglucosaminylated protein-like substances. The lower figure is a graph showing the ratio of GDF15 to β-actin (GDF15 / β-actin). [Figure 3B] The upper figure is a Western blot showing the increase in GDF15 expression at the protein level and β-actin expression after 24 hours of culture in NHDF with the addition of O-linked N-acetylglucosaminylated protein-like substances AC-GlcNAc (10-mer), AC-GlcNAc (62-mer), and AC-GlcNAc (69-mer). The control was a sample without the addition of O-linked N-acetylglucosaminylated protein-like substances. The lower figure is a graph showing the ratio of GDF15 to β-actin (GDF15 / β-actin). [Figure 3C]The upper figure is a Western blot showing the increase in GDF15 protein expression and β-actin expression after 24 hours of culture in NHDF cultured with 10μg / mL, 50μg / mL, and 100μg / mL AC-GlcNAc (10-mer) as an O-linked N-acetylglucosaminylated protein-like substance. The lower figure is a graph showing the ratio of GDF15 to β-actin (GDF15 / β-actin). [Figure 4] The results of analyzing the pathways of altered genes based on the microarray results shown in Figure 1 are shown below. [Diagram 5] The figure shows the growth rate of NHDF when 50 mL of O-linked N-acetylglucosaminylated protein-like substances (AC-GlcNAc (10-mer) and AC-GlcNAc (40-mer)) and other components, AC-GlcNAc monomer and N-acetylglucosamine, were added to NHDF and cultured for 6 days. The control was one without the addition of O-linked N-acetylglucosaminylated protein-like substances. [Figure 6A] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance, and 50 μg / mL of AC-GlcNAc (10-mer) was added to NHDF. 24 hours later, 5 μg / mL of lipopolysaccharide (hereinafter, also referred to as LPS) was added. The results shown here show the production of TNFα in the cells 8 hours later detected by Western blotting. [Figure 6B] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance, and 50 μg / mL of AC-GlcNAc (10-mer) was added to NHDF. The results of detecting the production of GDF15, survivin, and β-actin in the cells 24 hours later by Western blotting are shown. [Figure 6C] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance, and 50 μg / mL of AC-GlcNAc (10-mer) was added to NHDF. 24 hours later, 5 μg / mL of LPS was added, and the ratio of intracellular TNFα to β-actin (TNFα / β-actin) after 8 hours was shown in the graph. [Figure 7] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance, and 50 μg / mL of AC-GlcNAc (10-mer) was added to NHDF. 24 hours later, 5 μg / mL of LPS was added, and the amount of TNFα secreted in the medium after 24 hours was detected by ELISA. [Figure 8] A glass plate was placed in a petri dish, and agarose gel mixed with AC-GlcNAc (10-mer) was dropped onto it. NHDF was then seeded over the entire glass plate, and the state of NHDF after one day of incubation was observed under a phase contrast microscope, and the state of invasion into cells was observed on the bottom of the agarose gel. The control shows the results of using PBS instead of AC-GlcNAc (10-mer). [Figure 9] The state of NHDF when O-linked N-acetylglucosaminylated proteinaceous substances were mixed into agarose gel at a concentration of 50 μg / mL, 100 μg / mL, or 250 μg / mL was observed using a phase contrast microscope, and the state of penetration into cells was observed on the bottom of the agarose gel. [Figure 10] The state of NHDF when the O-linked N-acetylglucosaminylated proteinaceous substance prepared in Polymerization Example 1, the AC-GlcNAc monomer prepared in Polymerization Example 5, N-acetylglucosamine (GlcNAc) used in Example 2, or PBS was used was observed under a phase contrast microscope, and the state of penetration into cells was observed on the bottom of the agarose gel. [Figure 11] The state of NHDF when the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1 and the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 2 were used was observed with a phase contrast microscope, and the state of penetration into cells was observed on the bottom of the agarose gel. [Figure 12] The results of evaluating the chemotaxis of NHDFs using a petri dish in which a gel containing the O-linked N-acetylglucosaminylated protein-like substance of Polymerization Example 1 was dropped onto an agarose gel and the medium contained 10 μg / mL of the O-linked N-acetylglucosaminylated protein-like substance of Polymerization Example 1. are shown. [Figure 13]The graph shows the results of vimentin phosphorylation detected by Western blotting with anti-phosphorylated vimentin serine 38 antibody 10, 30, 60, and 120 minutes after adding 50 μg / mL of O-linked N-acetylglucosaminylated protein-like substance to NHDF. [Figure 14A] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance. 50 μg / mL AC-GlcNAc (10-mer) was added to osteoarthritis-derived synovial fibroblasts and rheumatoid arthritis-derived synovial fibroblasts and cultured for 24 hours. The results of detecting intracellular production of GDF15 and β-actin by Western blotting are shown. [Figure 14B] AC-GlcNAc (10-mer) was used as an O-linked N-acetylglucosaminylated protein-like substance, and 10 ng / mL of TNFα was added to osteoarthritis-derived synovial fibroblasts and rheumatoid arthritis-derived synovial fibroblasts. The results shown here are the results of Western blotting to detect the expression of receptor activator of NF-κB ligand (hereinafter referred to as RANKL), survivin, and β-actin 6 hours later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] O-linked N-acetylglucosaminylated proteins are a type of post-translational modification in which O-linked N-acetylglucosamine is attached to the hydroxyl groups of serine and threonine residues of proteins. They are intracellular proteins that are O-linked N-acetylglucosamine-modified, such as those represented by the following general formula. It is expected that these O-linked N-acetylglucosamine-modified proteins leak from injured cells and act like alarmin molecules. That is, more than 4,000 types of proteins, such as nuclear proteins and cytoskeleton proteins, are O-linked N-acetylglucosamine-modified, and O-linked N-acetylglucosamine-modified proteins are constantly present in large amounts in cells. When injury to cells occurs, such as fibrosis, these O-linked N-acetylglucosamine-modified proteins are released in large amounts outside the cells, bind to and act on vimentin on the cell surface of other cells, affecting the intracellular variable genes and expressing GDF15.

[0014] [ka]

[0015] The O-linked N-acetylglucosaminylated protein-like substance of the present invention is a compound that functions in the same manner as the above-mentioned O-linked N-acetylglucosaminylated protein, and is composed of a specific N-acetylglucosamine group-containing polymer. A therapeutic agent for fibrosis contains this O-linked N-acetylglucosaminylated protein-like substance, and can improve fibrosis by binding and acting on the affected area of ​​diseased tissues, cells, etc.

[0016] The O-linked N-acetylglucosaminylated proteinaceous substance of the present invention and the therapeutic agent for fibrosis containing the same are described in detail below.

[0017] [O-linked N-acetylglucosaminylated protein-like substances] In the present invention, the O-linked N-acetylglucosaminylated proteinaceous substance is a polymer having an N-acetylglucosamine unit and at least one unit selected from the group consisting of a carboxyl group-containing radically polymerizable unit, a styrene unit, a polyethyleneimine unit, a poly-L-lysine unit, and a biotin unit. The mass average molecular weight of these N-acetylglucosamine group-containing polymers is preferably in the range of 3,000 to 30,000 and the number average molecular weight is preferably in the range of 2,500 to 25,000. It is more preferable that the mass average molecular weight of the N-acetylglucosamine group-containing polymer is 3,000 or more and 30,000 or less, or the number average molecular weight is 2,500 or more and 25,000 or less, because the effect on cells is improved. For the N-acetylglucosamine group-containing polymer used as an O-linked N-acetylglucosaminylated protein-like substance, in terms of the yield of the compound by polymerization, the mass average molecular weight is preferably in the range of 4,000 to 20,000 and the number average molecular weight is preferably in the range of 3,000 to 10,000, and in terms of the expressibility of GDF15, the mass average molecular weight is preferably in the range of 4,000 to 15,000 and the number average molecular weight is preferably in the range of 3,000 to 8,000. The ratio (Mw / Mn) of the mass average molecular weight (Mw) to the number average molecular weight (Mn) is preferably in the range of 1.0 to 2.5, and more preferably in the range of 1.2 to 2.2 from the viewpoint of the expression of GDF15 and the inhibition of the expression of RANKL and Survivin.

[0018] Examples of the N-acetylglucosamine unit include N-acetylglucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, etc. Examples of the N-acetylglucosamine sugar chain group-containing organic resin compound include compounds containing, as the N-acetylglucosamine sugar chain group, an N-acetylglucosamine group or a chitopolyose group to which 2 to 6 N-acetylglucosamine groups are bonded. Examples of the carboxy group-containing radically polymerizable unit include acrylic acid, methacrylic acid, itaconic acid, maleic acid, crotonic acid, 2-carboxyethyl acrylate, and 2-carboxyethyl methacrylate. The method for producing the N-acetylglucosamine group-containing polymer is not particularly limited, and radical polymerization, living radical polymerization, reversible addition-fragmentation chain transfer polymerization (RAFT polymerization) using a chain transfer agent (RAFT agent), and the like can be used. For example, a method for producing a polymer obtained by polymerizing a monomer having an N-acetylglucosamine group includes a method for polymerizing a monomer having a compound having a hydrophobic group, such as 2-carboxyethyl acrylate, bound to the reducing end of N-acetylglucosamine. A more specific production method includes a method in which 2-carboxyethyl acrylate, a compound having a hydrophobic group, and aminated N-acetylglucosamine are condensed with DMT-MM to obtain an acrylic monomer having an N-acetylglucosamine group, and then the monomer is polymerized to produce the polymer. In addition, a method for producing a polymer in which N-acetylglucosamine is bound to the polymer compound includes a method in which a polymer compound having a hydrophobic group, such as polyethyleneimine or poly-L-lysine, which is a cationic polymer, is bound to the reducing end of the N-acetylglucosamine sugar chain. Here, the method for binding the N-acetylglucosamine sugar chain to the compound is not particularly limited, but for example, the amino group of the compound having an amino group and the reducing end of the N-acetylglucosamine sugar chain can be bound by the reductive amination method. In addition, the hydroxyl group of the N-acetylglucosamine sugar chain may be substituted with a carboxyl group, and then bound to the amino group of the compound having an amino group by the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling method or the like. In addition, it is preferable to use the RAFT polymerization method to control the molecular weight, since it can narrow the molecular weight distribution. In controlling the molecular weight, it is preferable to set the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) to 1.0 or more and less than 1.5, since this allows a large amount of N-acetylglucosamine to be present on the surface of the cell culture material, and it is possible to capture more mesenchymal stem cells in the cell population. As a specific example of the polymerization method, N-acetylglucosamine and 2-carboxyethyl acrylate as a compound having a hydrophobic group are mixed in a molar ratio of 1:1, and condensed in a solution such as DMT-MM, DMSO, or water to prepare an AC-GlcNAc monomer. Poly(N-ethylacrylic acid-O-2-acetamide-2-deoxy-β-D-glucopyranosylamine) (hereinafter referred to as AC-GlcNAc polymer) can be obtained by the RAFT polymerization method. In addition, it is preferable to remove the alkyl chain derived from the RAFT polymerization method from the O-linked N-acetylglucosaminylated proteinaceous substance of the present invention in order to improve its activity with cells. As a method for removing the alkyl chain, a method in which the terminal of the polymer obtained by the RAFT polymerization method is thiolated using a reducing agent or the like, and then reacted with maleimide can be mentioned.

[0019] Furthermore, the O-linked N-acetylglucosaminylated proteinaceous substance of the present invention preferably contains 3 to 100 N-acetylglucosamine groups per molecule. If the number is less than this range, the binding affinity to vimentin tends to decrease, and if the number is more than this range, the amount of N-acetylglucosamine per unit area decreases, so that the binding affinity to vimentin tends to decrease. Therefore, the more preferred number is in the range of 8 to 80, the even more preferred number is in the range of 10 to 70, the particularly preferred number is in the range of 10 to 69, and the most preferred number is in the range of 10 to 50.

[0020] <Fibrosis treatment drug> The therapeutic agent for fibrosis of the present invention contains the above-mentioned O-linked N-acetylglucosaminylated proteinaceous substance, and can be administered orally or parenterally (intravenously, subcutaneously, transdermally, pulmonary, mucosally, rectally, etc.) to humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.). The therapeutic agent for fibrosis of the present invention can be prepared by mixing the above-mentioned O-linked N-acetylglucosaminylated proteinaceous substance with a pharma- ceutically acceptable carrier (excipient, binder, disintegrant, disintegration aid, lubricant, wetting agent, etc.) or additive that is usually used for oral or parenteral administration, and formulating it into a desired form such as granules, powders, tablets (including sugar-coated tablets), pills, buccal tablets, capsules, syrups, liquids, emulsions, suspensions, creams, ointments, eye drops, injections, drops, nasal drops, patches, suppositories, etc. The content of O-linked N-acetylglucosaminylated protein-like substances in fibrosis therapeutic agents is not particularly limited as long as anti-inflammatory effects and inhibition of cell proliferation due to increased expression of GDF15 are obtained, but it is preferably 0.1% or more, more preferably 1% or more, and even more preferably 10% or more.

[0021] <Fibrosis> In the present invention, fibrosis refers to the loss of tissue parenchymal cells and the functional decline of tissues caused by stresses such as chemical stimuli such as drugs, excessive pressure load, and inflammatory reactions, followed by the migration and proliferation of excessive fibroblasts in the process of compensating for the loss, and the stiffening of tissues accompanied by functional impairment due to the subsequent synthesis and deposition of intercellular 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, bladder, kidney, heart, and liver. The fibrosis targeted by the present invention also includes tissue fibrosis diseases caused by the administration of drugs such as antitumor agents, antibiotics, antibacterial agents, antiarrhythmic agents, anti-inflammatory agents, antirheumatic agents, interferons, and Shosaikoto, as well as tissue fibrosis diseases associated with diseases such as chronic nephritis, interstitial myocarditis, and interstitial cystitis. Specific examples include pulmonary fibrosis caused by side effects of bleomycin administration, and pulmonary fibrosis occurring 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.; endocardial fibrosis caused by remodeling after myocardial infarction; hepatic fibrosis and non-alcoholic steatohepatitis (NASH) caused by damage to hepatocytes, and the associated portal hypertension and cirrhosis; keloids caused by excessive tissue repair, and other conditions such as sclerosing peritonitis, benign prostatic hyperplasia, scleroderma, uterine leiomyoma, retroperitoneal fibrosis, and myelofibrosis. Among the fibrosis therapeutic drugs of the present invention, the fibrosis inhibitor improves fibrosis by inhibiting the fibrosis signal that activates stellate cells and transforms them into myofibroblast-like cells. Therefore, in the case of fibrosis in which stellate cells are activated to become myofibroblast-like cells, for example in the case of the liver, the activated stellate cells transform into myofibroblast-like cells and produce extracellular matrix, which progresses fibrosis, but by using the fibrosis therapeutic drug of the present invention, it is possible to improve liver fibrosis, improve liver function, and suppress the occurrence of liver cancer. It can also be used for pancreatic fibrosis, etc.

[0022] <Treatment method for fibrosis> The method for treating fibrosis of the present invention is characterized by binding the above-mentioned therapeutic agent of the present invention to fibrotic tissues and cells. The method of binding is not particularly limited, and examples thereof include directly adding the therapeutic agent to fibrotic tissues and cells, orally or parenterally administering the formulated therapeutic agent for fibrosis, and allowing the O-linked N-acetylglucosaminylated protein-like substance to reach the affected area and its vicinity.

Examples

[0023] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples. In the following, "% " means by mass unless otherwise specified.

[0024] [Polymerization of O-linked N-acetylglucosaminylated protein-like substance] [Polymerization Example 1] [Preparation of GlcNAc-NH2] 5 g of GlcNAc was dissolved in 50 mL of water, and NH4HCO3 was added until saturation. The solution was stirred in an eggplant flask and incubated in an open system with the lid open at 30 - 37 °C for 4 - 5 days. When the precipitation of NH4HCO3 disappeared, it was added as appropriate. The synthesis of GlcNAc-NH2 was confirmed by thin layer chromatography (TLC) using ethyl acetate / acetic acid / methanol / water = 4:3:3:1 (v / v / v / v) with a GlcNAc Rf value of 0.71. After 4 - 5 days, water was added to the reaction solution and an evaporator (30 °C) was used to remove the excess NH4HCO3. This operation was repeated several times until no odor remained. After the evaporator, freeze-drying was performed. [Preparation of AC-GlcNAc monomer] GlcNAc-NH2 (4.5 mmol: approximately 1 g) was dissolved in DMSO (10 mL), and 2-carboxyethyl acrylate (4.5 mmol: approximately 0.65 g) was added. After dissolution, DMT-MM (6.8 mmol) was added and incubated at room temperature for 18 hours. After incubation, the reaction mixture was dropped into 200 - 300 mL of chloroform. Since a precipitate was obtained, it was collected using a Kiriyama funnel. The collected precipitate was dissolved in methanol, the insoluble matter was removed using a Kiriyama funnel, and the solution dissolved in methanol was collected. The methanol was removed using an evaporator. The solid residue after removing methanol was dissolved in water, then purified by preparative HPLC (water / acetonitrile), and after removing acetonitrile using an evaporator, lyophilization was performed to obtain the AC-GlcNAc monomer of the following chemical formula.

[0025]

Chemical formula

[0026] [Polymerization Example 2] In the <Polymerization of AC-GlcNAc Polymer> of Polymerization Example 1, an O-linked N-acetylglucosaminylated protein-like substance (referred to as AC-GlcNAc40) was prepared in the same manner as Polymerization Example 1 except that the blending ratio of the RAFT agent was about 1 mg (1 / 50 molar equivalent of the monomer). The mass average molecular weight of this was 14,000, and the number average molecular weight was 6800.

[0027] [Polymerization Example 3] In the <Polymerization of AC-GlcNAc polymer> of Polymerization Example 1, an O-linked N-acetylglucosaminylated protein-like substance (referred to as AC-GlcNAc62) was prepared in the same manner as Polymerization Example 1, except that the blending ratio of the RAFT agent was about 0.75 mg (1 / 70 molar equivalent of the monomer). The mass average molecular weight of this substance was 21,800, and the number average molecular weight was 12,500.

[0028] [Polymerization Example 4] In the <Polymerization of AC-GlcNAc polymer> of Polymerization Example 1, an O-linked N-acetylglucosaminylated protein-like substance (referred to as AC-GlcNAc69) was prepared in the same manner as Polymerization Example 1, except that the blending ratio of the RAFT agent was about 0.52 mg (1 / 100 molar equivalent of the monomer). The mass average molecular weight of this substance was 24,450, and the number average molecular weight was 12,800.

[0029] [Polymerization Example 5] Polymerization was carried out up to the <Production of AC-GlcNAc monomer> of Polymerization Example 1 to produce an AC-GlcNAc monomer. Both the mass average molecular weight and the number average molecular weight of this substance were 346.

[0030] [Example 1] The effect of the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) obtained in Polymerization Example 1 on human fibroblasts (NHDF) was confirmed using a DNA microarray. 50 μg / mL of the O-linked N-acetylglucosaminylated protein-like substance obtained in Polymerization Example 1 was added to NHDF, and incubation was carried out for 24 hours. After incubation, mRNA was recovered and a DNA microarray was performed to confirm the gene expression changes of the O-linked N-acetylglucosaminylated protein-like substance. The DNA microarray was performed using the microarray system of Agilent Technologies. The results are shown in FIGS. 1 and 2. Figure 1 plots gene expression levels of NHDF stimulated with O-linked N-acetylglucosaminylated protein-like substances (vertical axis) and unstimulated NHDF (horizontal axis). The left panel shows all the genes analyzed. The right panel plots the gene groups with significantly different expression levels (those with a difference in expression levels between the two of 1.5-fold or more and 0.66-fold or less). As shown in Figure 2, stimulation with O-linked N-acetylglucosaminylated protein-like substances increased the expression of GDF15 and adiponectin (ADIPOQ). These results indicate that O-linked N-acetylglucosaminylated protein-like substances increase the expression of GDF15 and adiponectin at the protein level by acting on NHDF.

[0031] [Example 2] NHDF was added with 50 μg / mL of the O-linked N-acetylglucosaminylated protein-like substances prepared in Polymerization Examples 1 to 5, AC-GlcNAc monomer, or N-acetylglucosamine (GlcNAc), and incubated for 24 hours. After incubation, the expression of GDF15 in NHDF was examined by Western blotting. The results are shown in Figures 3A and 3B. The control was one to which nothing was added. These results show that the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1 (AC-GlcNAc10, mass average molecular weight 4000, number average molecular weight 3100), the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 2 (AC-GlcNAc40, mass average molecular weight 14,000, number average molecular weight 6800), the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 3 (AC-GlcNAc62, mass average molecular weight 21,800, number average molecular weight 12,500), and the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 4 (AC-GlcNAc69, mass average molecular weight 24,450, number average molecular weight 12,800) increased the expression of GDF15, but the AC-GlcNAc monomer and GlcNAc prepared in Polymerization Example 5 had almost the same effect as the control. Furthermore, 10 μg / mL, 50 μg / mL, and 100 μg / mL of the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10, mass average molecular weight 4000, number average molecular weight 3100) prepared in Polymerization Example 1 was added to the NHDF, and the mixture was incubated for 24 hours. After incubation, the expression of GDF15 in the NHDF was observed by Western blotting. The results are shown in FIG. 3C. The control was one to which nothing was added. These results show that the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1 (AC-GlcNAc10, mass average molecular weight 4000, number average molecular weight 3100) increases the expression of GDF15 in proportion to the amount added.

[0032] [Example 3] Based on the results of the microarray in Example 1, pathway analysis was performed on genes whose expression was changed by the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1. The results are shown in Figure 4. All genes whose expression was significantly changed (shown in the right panel of Figure 1) were input into pathway analysis software DAVID Bioinformatics Resources 6.8, and pathway analysis was performed. From these results, it is understood that the arrest of cell proliferation is caused by the O-linked N-acetylglucosaminylated protein-like substance of Polymerization Example 1. In particular, a decrease in genes involved in the progression of the G2 and M phases of the cell cycle was observed. This is because O-linked N-acetylglucosaminylated protein-like substances interact with vimentin on the cell surface, suppressing the cell division function of vimentin and causing cell cycle arrest. However, this did not occur in the control where nothing was added, and therefore it is believed that the proliferation rate was higher than that of NHDF exposed to O-linked N-acetylglucosaminylated protein-like substances.

[0033] [Example 4] NHDF were seeded on a 48-well plate, and 50 μg / mL of the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1, the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc40) prepared in Polymerization Example 2, and the AC-GlcNAc monomer and N-acetylglucosamine prepared in Polymerization Example 5 were added. After six days of culture, the cell viability was measured each day using Cell Counting Kit-8 (CCK-8) (Dojindo Chemical Industries, Ltd.). The results are shown in FIG. 5. These results demonstrate that the proliferation of NHDF treated with O-linked N-acetylglucosaminylated protein-like substances (AC-GlcNAc10 and AC-GlcNAc40) was reduced.

[0034] [Example 5] The anti-inflammatory effect of O-linked N-acetylglucosaminylated protein-like substances was examined as follows. NHDF was added with 50 μg / mL of O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1, and cultured for 24 hours. Lipopolysaccharide (LPS) was added to this NHDF, and changes in TNFα expression were observed. Furthermore, the expression of TNFα in the cells 8 hours after the addition of LPS was examined by Western blotting. The results are shown in Figure 6A. Furthermore, the expression of GDF15, survivin, and β-actin in the cells 8 hours after the addition of LPS was examined by Western blotting. The results are shown in Figure 6B. Furthermore, the ratio of TNFα to β-actin in the cells 8 hours after the addition of LPS (TNFα / β-actin) was examined. The results are shown in Figure 6C. As shown in Figures 6A and 6C, when LPS was applied to NHDF treated with the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1, the increase in TNFα expression due to LPS stimulation was suppressed. In addition, as shown in Figure 6B, the expression of GDF15 was increased and the expression of survivin was suppressed in NHDF treated with the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1. GDF15 is a member of the TGFβ superfamily and is known to inhibit the transcription activity of NF-κB. Therefore, it is presumed that the activation of NF-κB was suppressed by the increase in the expression of GDF15 due to the treatment of NHDF with the O-linked N-acetylglucosaminylated protein-like substance, and the expression of TNFα was reduced. Next, the amount of TNF-α secreted into the medium 24 hours after the addition of LPS to NHDF was measured by ELISA. The medium of NHDF was replaced with DMEM (serum-free) and O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) was added at 50 μg / mL. 24 hours after the addition of AC-GlcNAc10, LPS was added at 5 μg / mL, and the culture supernatant was collected after another 24 hours, and the TNF-α contained in the culture supernatant was measured by ELISA. At this time, the TNFα contained in the DMEM culture supernatant of NHDF (control), the culture supernatant (AC-GlcNAc10) 48 hours after the addition of 50 μg / mL of AC-GlcMAc10 to NHDF, and the culture supernatant 24 hours after the addition of 5 μg / mL of LPS to NHDF were also measured. The results are shown in Figure 7. As shown in Figure 7, there was no change in the amount of TNFα secreted in the control group compared to the group to which O-linked N-acetylglucosaminylated protein-like substances were added, whereas an increase in the amount of TNFα secreted was observed when LPS was added to the control group, and an inhibition of the LPS-induced increase in the amount of TNFα secreted was observed when LPS was added to the group to which O-linked N-acetylglucosaminylated protein-like substances were added.

[0035] [Example 6] We examined whether the stimulation of NHDF with O-linked N-acetylglucosaminylated protein-like substances affects the cell migration ability as follows. AC-GlcNAc (10-mer) was used as the O-linked N-acetylglucosaminylated protein-like substance, and 250 μg / mL of AC-GlcNAc (10-mer) was mixed with the agarose gel to prepare an O-linked N-acetylglucosaminylated protein-like substance-containing agarose gel. In addition, PBS was used instead of the O-linked N-acetylglucosaminylated protein-like substance to prepare a PBS-containing agarose gel. A glass plate was placed in a petri dish, and each agarose gel was dropped onto it. NHDF was then seeded over the entire glass plate, and the plate was incubated for one day. The state of the NHDF at this time was observed under a phase contrast microscope, and the results of observing the state of penetration into cells at the bottom of the agarose gel are shown in Figure 8. The results in Figure 8 show that NHDF penetrates under the agarose gel containing the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) of Polymerization Example 1, indicating that the O-linked N-acetylglucosaminylated protein-like substance confers chemotaxis to NHDF. Next, the O-linked N-acetylglucosaminylated protein-like substance was mixed in agarose gel at 50 μg / mL, 100 μg / mL, and 250 μg / mL, and the chemotaxis of NHDF was evaluated in the same manner as above. The results are shown in Figure 9. From the results in Figure 9, the chemotaxis of NHDF changed depending on the concentration of the O-linked N-acetylglucosaminylated protein-like substance. Specifically, when the concentration of the O-linked N-acetylglucosaminylated protein-like substance was 250 μg / mL, the distance that the cells penetrated into the gel was 750 μm, and the number of cells that penetrated into the gel was about 60. When the concentration of the O-linked N-acetylglucosaminylated protein-like substance was 100 μg / mL, the distance was 450 μm, and the number of cells was about 12. When the concentration of O-linked N-acetylglucosaminylated protein-like substance was 50 μg / mL, the distance was 280 μm and the number of cells was about 10, whereas in the agarose gel containing PBS, the distance and the number of cells were both 0. This indicates that the chemotaxis of NHDF depends on the concentration of O-linked N-acetylglucosaminylated protein-like substance. Next, the chemotaxis of NHDF by the O-linked N-acetylglucosaminylated proteinaceous substance was evaluated. The materials used were the O-linked N-acetylglucosaminylated proteinaceous substance prepared in Polymerization Example 1, the AC-GlcNAc monomer prepared in Polymerization Example 5, N-acetylglucosamine (GlcNAc) used in Example 2, and PBS, and the chemotaxis of NHDF was evaluated in the same manner as above. The results are shown in FIG. 10. As shown in FIG. 10, cells invaded under the agarose gel only with the O-linked N-acetylglucosaminylated proteinaceous substance of Polymerization Example 1, which is a polymer, the distance that NHDF invaded was 420 μm, and the number of NHDF invaded was 29. This shows that the O-linked N-acetylglucosaminylated proteinaceous substance acts on NHDF because it is a polymer. Next, we evaluated whether there was a difference in the chemotaxis of NHDF due to differences in the degree of polymerization of O-linked N-acetylglucosaminylated protein-like substances using the same method as above. In the evaluation, we used O-linked N-acetylglucosaminylated protein-like substances from Polymerization Example 1 (AC-GlcNAc10) and Polymerization Example 2 (AC-GlcNAc40). The results are shown in Figure 11. The results in Figure 11 show that cells penetrated beneath the agarose gel for both O-linked N-acetylglucosaminylated protein-like substances, but the distance that NHDF penetrated was 780 μm for the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1 and 600 μm for the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 2, and the number of NHDF that penetrated into the gel was 60 for the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 1 and 50 for the O-linked N-acetylglucosaminylated protein-like substance prepared in Polymerization Example 2. In addition, two petri dishes were prepared in which gel containing the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1 was dropped onto the agarose gel, and one of the dishes was made to contain 10 μg / mL of the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1 in the medium, and the chemotaxis of NHDF was evaluated. The results are shown in FIG. 12. From the results in FIG. 12, it can be seen that when the medium contained an O-linked N-acetylglucosaminylated protein-like substance, the amount of NHDF penetrating under the agarose gel was reduced. Specifically, the distance that NHDF penetrated was 1000 μm when the medium contained the O-linked N-acetylglucosaminylated protein-like substance, while it was 1250 μm when the medium did not contain the O-linked N-acetylglucosaminylated protein-like substance. The number of NHDFs that penetrated into the gel was 80 in the medium containing O-linked N-acetylglucosaminylated protein-like substances, and 140 in the medium not containing O-linked N-acetylglucosaminylated protein-like substances. In the agarose gel containing PBS, NHDFs did not penetrate into the gel. Furthermore, the stimulation of NHDF migration by the O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1 was examined by detecting the phosphorylation of vimentin. Since the O-linked N-acetylglucosaminylated protein-like substance binds to vimentin that appears on the cell surface, the signal transmission to vimentin by the addition of the O-linked N-acetylglucosaminylated protein-like substance was examined by the phosphorylation of serine 38 of the amino acid sequence of vimentin. It has been revealed that cell migration is stimulated by phosphorylation of serine 38 of vimentin. Therefore, the phosphorylation of vimentin 10, 30, 60, and 120 minutes after the addition of the O-linked N-acetylglucosaminylated protein-like substance to NHDF was detected by Western blotting using an anti-phosphorylated vimentin serine 38 antibody. The results are shown in FIG. 13. As shown in Figure 13, phosphorylation of vimentin was observed 10 minutes after the addition of the O-linked N-acetylglucosaminylated protein-like substance, and a decrease was observed 120 minutes later. This demonstrated that the O-linked N-acetylglucosaminylated protein-like substance binds to vimentin on the cell surface of NHDF and stimulates GDF15 expression and cell migration. These results confirmed that NHDFs treated with O-linked N-acetylglucosaminylated protein-like substances had high cell migration ability. As shown in Figure 13, phosphorylation of vimentin was observed in association with stimulation of cell surface vimentin by O-linked N-acetylglucosaminylated protein-like substances (AC-GlcNAc10), suggesting that cell migration ability was stimulated by depolymerization of vimentin and the associated signal transduction.

[0036] [Example 7] 50 μg / mL of O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) prepared in Polymerization Example 1 was added to osteoarthritis-derived synovial fibroblasts and rheumatoid arthritis-derived synovial fibroblasts, and the cells were cultured for 24 hours. The expression of GDF15 in the cells was examined by Western blotting. The results are shown in Figure 14A. Furthermore, 10 ng / mL of TNFα was added to the osteoarthritis-derived synovial fibroblasts and rheumatoid arthritis-derived synovial fibroblasts, and the expression of RANKL, survivin, and β-actin 6 hours after the addition of TNFα was examined by Western blotting. The results are shown in Figure 14B. As shown in Figure 14A, increased expression of GDF15 was observed in both osteoarthritis-derived synovial fibroblasts and rheumatoid arthritis-derived synovial fibroblasts treated with the O-linked N-acetylglucosaminylated proteinaceous substance prepared in Polymerization Example 1, and as shown in Figure 14B, inhibition of increased expression of RANKL and survivin due to TNFα stimulation was observed. In the diseased area of ​​rheumatoid arthritis, a chronic inflammatory disease, synovial fibroblasts increase in number, express RANKL, and activate osteoclasts, which leads to the progression of the disease. In addition, RANKL is secreted from synovial fibroblasts stimulated by TNFα or interleukin-17 (IL-17) during chronic inflammation, and differentiates osteoclast precursor cells into osteoclasts, which cause bone destruction and bone deformation in rheumatoid arthritis. As shown in Figures 14A and 14B, when rheumatoid arthritis-derived synovial fibroblasts were treated with O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) and TNFα, the expression of GDF15 was increased and the expression of RANKL and survivin was suppressed. From this, it is inferred that periosteal fibroblasts and rheumatoid arthritis-derived synovial fibroblasts treated with O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) suppressed the increase in RANKL expression, and as a result, their differentiation into osteoclasts can also be suppressed, thereby suppressing the onset of rheumatoid arthritis. Furthermore, survivin is known to be a molecule that inhibits cell death and is involved in the activation of synovial fibroblasts. Since O-linked N-acetylglucosaminylated protein-like substances (AC-GlcNAc10) were observed to inhibit the increased expression of RANKL and survivin in these cells, it is speculated that O-linked N-acetylglucosaminylated protein-like substances may also be effective in treating rheumatoid arthritis and suppressing drug resistance to various drugs, including cancer treatment drugs. From the results of Examples 1 to 6, it was found that stimulation of cell surface vimentin in myofibroblasts (NHDF) by O-linked N-acetylglucosaminylated protein-like substances increases GDF15 production, anti-inflammatory effects, proliferation inhibition, and cell migration ability. This suggests that O-linked N-acetylglucosaminylated protein-like substances have a therapeutic effect against fibrosis. Furthermore, the results of Example 7 showed that periosteal fibroblasts and rheumatoid arthritis-derived synovial fibroblasts treated with O-linked N-acetylglucosaminylated protein-like substance (AC-GlcNAc10) suppressed increased expression of RANKL and survivin even when treated with TNFα, suggesting that O-linked N-acetylglucosaminylated protein-like substance has therapeutic effects against rheumatoid arthritis and suppressive effects against drug resistance.

Claims

1. A method for using a polymer having an N-acetylglucosamine unit and a carboxy group-containing radically polymerizable unit, and containing 3 to 100 N-acetylglucosamine groups per molecule, as an O-linked N-acetylglucosaminylated protein-like substance.

2. The method for use of an O-linked N-acetylglucosaminylated protein-like substance according to claim 1, wherein the carboxy group-containing radically polymerizable unit is at least one selected from the group consisting of acrylic acid, methacrylic acid, itaconic acid, maleic acid, crotonic acid, 2-carboxyethyl acrylate, and 2-carboxyethyl methacrylate.

3. 3. The method for use as an O-linked N-acetylglucosaminylated protein-like substance according to claim 1 or 2, characterized in that the polymer does not have an alkyl chain.

4. A method for use as an O-linked N-acetylglucosaminylated protein-like substance described in any one of claims 1 to 3, characterized in that the polymer is a compound represented by the following chemical formula (1) or (2): 【Chemistry 1】 In formula (1), n ​​is an integer of 3 to 100. 【Chemistry 2】 In formula (2), n is an integer of 3 to 100.

5. A therapeutic agent for fibrosis comprising as an active ingredient a polymer having an N-acetylglucosamine unit and a carboxy group-containing radically polymerizable unit, the polymer containing 3 to 100 N-acetylglucosamine groups per molecule.

6. The fibrosis treatment agent according to claim 5, wherein the carboxy group-containing radically polymerizable unit is at least one selected from the group consisting of acrylic acid, methacrylic acid, itaconic acid, maleic acid, crotonic acid, 2-carboxyethyl acrylate, and 2-carboxyethyl methacrylate.

7. The fibrosis treatment drug described in claim 5 or 6, characterized in that the polymer does not have an alkyl chain.

8. A drug for treating fibrosis described in any one of claims 5 to 7, characterized in that the polymer is a compound represented by the following chemical formula (1) or (2). 【Chemistry 3】 In formula (1), n ​​is an integer of 3 to 100. 【Chemistry 4】 In formula (2), n is an integer of 3 to 100.

9. An O-linked N-acetylglucosamine protein-like substance having an N-acetylglucosamine unit and a carboxy group-containing radically polymerizable unit, characterized in that the O-linked N-acetylglucosamine protein-like substance is a compound represented by the following chemical formula (1) or (2), and containing 3 to 100 N-acetylglucosamine groups per molecule. 【Chemistry 5】 In formula (1), n ​​is an integer of 3 to 100. 【Chemistry 6】 In formula (2), n is an integer of 3 to 100.

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