Pharmaceutical composition for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis

Abstract

In the present invention, the direct involvement of GaINAc-T12 in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA) are analyzed and the analysis results are used for drug discovery. Provided is a pharmaceutical composition for preventing and / or treating osteoarthritis and / or rheumatoid arthritis. The pharmaceutical composition contains GaINAc-T12.

Description

Technical Field

[0003]

[0001] The present invention relates to a pharmaceutical composition for preventing and / or treating osteoarthritis and / or rheumatoid arthritis.

Background Art

[0002] Cartilage tissue consists of chondrocytes as the only cells and a cartilage matrix synthesized by chondrocytes, and the chondrocytes are distributed in the cartilage matrix. Proteoglycan, which is the main component of the cartilage matrix, has a structure in which glycosaminoglycans (GAGs) such as chondroitin sulfate are bound to a core protein, and binds a large amount of water molecules within its molecule. By forming a flexible higher-order structure together with collagen fibers containing type II collagen, it retains water retention and elasticity, which plays an important role in the joint lubrication mechanism for maintaining smooth muscle and skeletal system motor functions.

[0003] It is becoming increasingly clear that modifications of proteoglycans with sugar chains such as GAGs not only confer structural functionality, but also contribute to various biological reactions such as cell morphology maintenance, proliferation and migration, differentiation, signal transduction, and metabolic activity by interacting with cells and physiologically active substances. Sugar chain modifications in proteins are broadly classified into two types: N-linked and O-linked. N-linked sugar chains are formed when N-acetylglucosamine (GlcNAc) is attached to asparagine residues of proteins, and have been reported to be involved in signal transduction by cytokines such as IL-6, TNFα, and IL-1β (Non-Patent Literature 1). On the other hand, O-linked sugar chains are formed when sugars such as GlcNAc or N-acetylgalactosamine (GalNAc) are attached to serine or threonine residues of proteins. O-linked GlcNAc (O-GlcNAc) has been reported to activate signal transduction pathways mediated by JNK, ERK, and p38 (Non-Patent Literature 2). Furthermore, O-linked GalNAc (O-GalNAc) is formed by GalNAc transferase (GalNAc-T), and 20 isozymes of this enzyme have been reported in humans (Non-Patent Documents 3, 4). In mice overexpressing GalNAc-T, an increase in O-GalNAc has been reported to suppress chondrocyte hypertrophy, decrease GAG, and cause developmental problems (Non-Patent Document 5). However, there have been few analytical reports on O-GalNAc and GalNAc-T, and their detailed functions remain unclear.

[0004] While glycosylation is suggested to be important in cartilage tissue and chondrocytes, recent reports indicate that changes in these glycosylation are observed in osteoarthritis (OA) and rheumatoid arthritis (RA), diseases involving the destruction of cartilage tissue. Regarding N-linked glycans, it has been reported that high-mannose N-linked glycans are increased in human OA and OA model mouse chondrocytes (Non-Patent Literature 6), and that N-linked glycans are involved in the development of RA in RA model mice (Non-Patent Literature 1). Regarding O-linked glycans, it has been reported that O-GlcNAc is increased in OA chondrocytes (Non-Patent Literature 7) and that it promotes chondrocyte hypertrophy (Non-Patent Literature 2). Regarding O-GalNAc, it has been reported that GalNAc-T expression is increased in OA chondrocytes (Non-Patent Literature 8). The association between GalNAc-T and RA has been suggested. Previous genome-wide association studies have reported that single nucleotide polymorphisms (SNPs) of GalNAc transferase 12 (GalNAc-T12) are significantly associated with the rapid progression of joint destruction in rheumatoid arthritis (RA) (Non-Patent Literature 9). However, the direct involvement of O-GalNAc and GalNAc-T in the pathogenesis of OA and RA has not been analyzed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Uehara I et al. 2-Deoxy-D-glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation. Pharmacol Res Perspect 10:e00940, 2022. [Non-Patent Document 2] Andres-Bergos J et al. The increase in O-linked N-acetylglucosamine protein modification stimulates chondrogenic differentiation both in vitro and in vivo. J Biol Chem 287:33615, 2012. [Non-Patent Document 3] Bennett EP et al. Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22:736, 2012. [Non-Patent Document 4] Wandall HH et al. Global functions of O-glycosylation: promises and challenges in O-glycobiology. FEBS J 288:7183, 2021. [Non-Patent Document 5] Yoshida CA et al. Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans. J Biol Chem 289:26584, 2014. [Non-Patent Document 6] Urita A et al. Alterations of high-mannose type N-glycosylation in human and mouse osteoarthritis cartilage. Arthritis Rhum 63:3428, 2011. [Non-Patent Document 7] Tardio L et al. O-linked N-acetylglucosamine (O-GlcNAc) protein modification is increased in the cartilage of patients with knee osteoarthritis. Osteoarthritis Cartilage 22:259, 2014. [Non-Patent Document 8] Yoshimoto M et al. Bioinformatic analysis reveals potential relationship between chondrocyte senescence and protein glycosylation in osteoarthritis pathogenesis. Front Endocrinol 14:1153689, 2023. [Non-Patent Document 9] Hayashi S et al. A genome-wide association study identifying the SNPs predictive of rapid joint destruction in patients with rheumatoid arthritis. Biomed Rep 14:31, 2021. [Overview of the project] [Problems that the invention aims to solve]

[0006] Thus, while the functions of N-linked glycans and O-GlcNAc in chondrocytes and their association with OA and RA are being demonstrated, the functions and disease involvement of O-GalNAc and GalNAc-T remain unclear.

[0007] This invention focuses on GalNAc-T, which is involved in the formation of O-GalNAc, a glycosylation that remains largely unknown compared to other glycosylation modifications. In particular, it aims to investigate the function of GalNAc-T12 in maintaining chondrocyte homeostasis, promoting hypertrophy, and stimulating inflammatory reactions in chondrocytes. Furthermore, it aims to analyze the direct involvement of GalNAc-T12 in the pathogenesis of OA and RA using model mice, and to utilize the results for drug discovery. [Means for solving the problem]

[0008] In cartilage tissue, glycosaminoglycans (GAGs) are responsible for maintaining water retention and elasticity, thereby creating a lubricating mechanism for joints. Thus, the importance of sugar chains like GAGs has been suggested in cartilage tissue and chondrocytes. The inventors have previously reported, based on genome-wide association studies, that a single nucleotide polymorphism (SNP) of GalNAc transferase 12 (GalNAc-T12), a glycosyltransferase that forms O-linked N-acetylgalactosamine (O-GalNAc), a type of glycosylation, is significantly associated with the rapid progression of joint destruction in rheumatoid arthritis (RA) (Non-Patent Literature 9). Therefore, we analyzed the function of GalNAc-T12 in chondrocytes. As a result, we found that GalNAc-T12 is involved in maintaining chondrocyte homeostasis and suppresses hypertrophy and cytokine-induced inflammation. Furthermore, in studies using model mice, GalNAc-T12 suppressed cartilage destruction in osteoarthritis (OA) and RA. These results demonstrate that GalNAc-T12 suppresses cartilage destruction in OA and RA by contributing to the characteristics of chondrocytes. This invention is completed based on these findings.

[0009] The gist of this invention is as follows: (1) A pharmaceutical composition comprising GalNAc-T12 for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis. (2) A method for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis, comprising administering GalNAc-T12 to a subject in a pharmaceutically effective amount. (3) Use of GalNAc-T12 for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis. (4) Use of GalNAc-T12 in the manufacture of pharmaceuticals for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis.

[0010] While numerous drugs have been developed to treat rheumatoid arthritis, these drugs are neither effective nor applicable to the treatment of osteoarthritis. For osteoarthritis, treatment options are limited to intra-articular injections of hyaluronic acid and the administration of supplements containing glucosamine, collagen, or mixtures thereof; no effective treatment has been found. Therefore, the provision of an effective treatment for osteoarthritis through this invention will bring great benefit to humanity. [Effects of the Invention]

[0011] This invention makes it possible to prevent and treat osteoarthritis and rheumatoid arthritis. This specification includes the content described in the specification and / or drawings of the Japanese Patent Application No. 2024-015445, which forms the basis of the priority of this application. [Brief explanation of the drawing]

[0012] [Figure 1] This graph shows the results of the WST assay, with the initial number of viable cells set to 1. [Figure 2] The graph shows the results of the WST assay, with the amount of viable cells set to 1 when neither GalNAc-T12 nor actinomycin D was added. [Figure 3] Results of detecting caspase 3, PARP, and cleaved PARP by Western blotting. [Figure 4] The graph shows the gene expression levels of each molecule, with the expression levels when GalNAc-T12, GalNAc, and GlcNAc are not added (culture medium only) set to 1. [Figure 5] This graph shows the gene expression levels of each molecule, with the expression level when no hypertrophy is induced set to 1. [Figure 6] Graph showing the gene expression of each molecule when stimulated with IL-6 + sIL-6R, with the expression level when GalNAc-T12, IL-6, and sIL-6R were not added (only medium) set to 1. [Figure 7] Graph showing the gene expression of each molecule when stimulated with TNFα, with the expression level when both GalNAc-T12 and TNFα were not added (only medium) set to 1. [Figure 8] Graph showing the gene expression of each molecule when stimulated with IL-1β, with the expression level when both GalNAc-T12 and IL-1β were not added (only medium) set to 1. [Figure 9] Graph showing the gene expression of each molecule, with the expression level when GalNAc-T12 was not added set to 1. [Figure 10] Histological image of the right hindlimb knee joint of mice administered with DMM surgery and PBS or GalNAc-T12, stained with Safranin O. [Figure 11] Graph of the OARSI score for the histological image in Figure 10. [Figure 12] Graph of the scoring results of limb swelling in CAIA model mice administered with PBS or GalNAc-T12. [Figure 13] Histological image of the right hindlimb knee joint of CAIA model mice administered with PBS or GalNAc-T12, stained with Safranin O. [Figure 14] Graph of the OARSI score for the histological image in Figure 13. [Figure 15] Graph showing the expression of p21, with the expression level when hypertrophy was not induced set to 1. [Figure 16] Results of detecting O-GalNAc by Western blot.

Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail.

[0014] The present invention provides a pharmaceutical composition comprising GalNAc-T12 for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis.

[0015] Furthermore, the present invention provides a method for preventing and / or treating osteoarthritis and / or rheumatoid arthritis, comprising administering GalNAc-T12 to a subject in a pharmaceutically effective amount.

[0016] Furthermore, the present invention provides for the use of GalNAc-T12 for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis.

[0017] Furthermore, the use of GalNAc-T12 in the manufacture of pharmaceuticals for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis is also provided.

[0018] GalNAc-T12 is a glycosyltransferase that initiates O-linked glycosylation, adding GalNAc to serine or threonine residues of proteins. GalNAc-T12 is widely expressed in the body, particularly in the large and small intestines, stomach, and pancreas, where it is highly expressed; in the thyroid gland, spleen, and testes where it is moderately expressed; and in the brain, bone marrow, thymus, heart, lungs, liver, kidneys, and esophagus where it is lowly expressed. NCBI Gene ID: 79695 (Information regarding the GalNAc-T12 gene) NCBI Reference Sequence No; NG_028218.1 (Full-length nucleotide sequence of the GalNAc-T12 gene) GenBank accession No; AB078146.1 (GalNAc-T12 cDNA base sequence) The amino acid sequence and nucleotide sequence of recombinant human GalNAc-T12 used in the examples described below are shown in Sequence ID No. 1 and No. 2 of the sequence listing, respectively. In the present invention, GalNAc-T12 may consist of the amino acid sequence of Sequence ID No. 1, but it may also be a protein consisting of an amino acid sequence having 90% or more but less than 100% identity with the amino acid sequence of Sequence ID No. 1. The identity of the two amino acid sequences can be determined using BLAST. Furthermore, in the present invention, GalNAc-T12 may have deletions, substitutions, or additions of 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, or 10 or fewer amino acid residues in the amino acid sequence of Sequence ID No. 1. These mutant polypeptides may exhibit preventive and / or therapeutic effects on osteoarthritis and / or rheumatoid arthritis.

[0019] GalNAc-T12 can be obtained, for example, by inducing its production in host cells using genetic engineering techniques, followed by isolation and purification. Alternatively, it can be manufactured by chemical synthesis.

[0020] To produce GalNAc-T12 in host cells using genetic engineering techniques, the DNA encoding GalNAc-T12 is incorporated into a vector, introduced into host cells, and then recombinant GalNAc-T12 is produced in the host cells. Plasmids are commonly used as vectors, and host cells such as E. coli, yeast, animal cells, and human cells are used. Methods for preparing GalNAc-T12 are described in Guo JM et al. Molecular cloning and characterization of a novel member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, pp-GalNAc-T12. FEBS Lett 524:211, 2002. The GalNAc-T12 produced by host cells can be recovered from the culture medium using known protein purification methods.

[0021] Furthermore, GalNAc-T12 is commercially available, and you may use a commercially available version.

[0022] To introduce a mutation into GalNAc-T12, which consists of the amino acid sequence of Sequence ID No. 1, site-directed mutagenesis is recommended using a CRISPR / Cas9 system and specific primers (containing the target mutation). Variants of GalNAc-T12 have been reported in publications such as Guda K et al. (Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers. Proc Natl Acad Sci USA 106:12921, 2009.), and these variants may also be used in the present invention.

[0023] GalNAc-T12 can be used for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis.

[0024] In the present invention, "treatment" means recovery, remission, alleviation, and / or delay of the worsening of the clinical symptoms of a disease in a patient who has developed such a disease.

[0025] In this invention, "prevention" means reducing the incidence of a disease. Prevention includes reducing the risk of disease progression or reducing the severity of those diseases, and also includes preventing recurrence.

[0026] GalNAc-T12 (hereinafter referred to as the "active ingredient") can be administered orally or parenterally to mammals (e.g., humans, rabbits, dogs, cats, rats, mice) as a pharmaceutical composition in a suitable dosage form, either alone or with pharmacologically acceptable carriers, diluents, or excipients. The dosage varies depending on the target organism, target disease, symptoms, and route of administration, but for example, when used for the prevention or treatment of osteoarthritis, a single dose of the active ingredient is usually about 0.01 to 10 mg / kg body weight, preferably about 1 to 2 mg / kg body weight, administered orally, intramuscularly, subcutaneously, or intravenously (preferably consecutively or every other day) at a frequency of once a month to three times a day, preferably once a week to once a day. When used for the prevention or treatment of rheumatoid arthritis, the active ingredient should be administered orally, intramuscularly, subcutaneously, intravenously, or intra-articularly (preferably continuously or every other day) at a frequency of once a month to three times a day, preferably once a week to once a day.

[0027] Compositions for oral administration may be in solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, and suspensions. Such compositions can be manufactured by conventional methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. For example, carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

[0028] Compositions for parenteral administration include, for example, injections and suppositories. Injections may be in the form of intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion injection, or intra-articular injection. Such injections are prepared by conventional methods, that is, by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily solution commonly used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, and may be used in combination with appropriate solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), or nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduc of hydrogenated castor oil)). Examples of oily solutions include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate or benzyl alcohol. The prepared injection solution is usually filled into a suitable ampoule. Suppositories used for rectal administration can be prepared by mixing the active ingredient with a standard suppository base.

[0029] The above-mentioned oral or parenteral pharmaceutical compositions are preferably prepared into dosage forms of medication units that are appropriate for the dosage of the active ingredient. Examples of such dosage forms of medication units include tablets, pills, capsules, injections (ampoules), and suppositories, and it is preferable that each dosage form of medication unit contains typically 0.5 to 500 mg of the active ingredient. [Examples]

[0030] The present invention will be described in more detail below with reference to examples.

[0031] [Example 1] Effects of GalNAc-T12 on chondrocyte proliferation and survival (method) Normal human knee joint chondrocytes (NHAC-Kn; Lonza) were cultured with GalNAc-T12 (R&D systems) at concentrations of 0-800 ng / ml. After 72 hours, a WST assay was performed using Cell Counting Kit-8 (Dojin Chemical Laboratories) to assess the number of viable cells. Culturing was performed in two types of media: Dulbecco's modified Eagle medium (DMEM; Nissui Pharmaceutical) containing 10% fetal bovine serum (Biowest), and serum-free DMEM. (result) Figure 1 shows a graph of the WST assay results, with the initial amount of viable cells set to 1 (n = 3). When cultured in a medium containing 10% serum, chondrocytes increased approximately 1.5 times compared to the initial level, and the addition of GalNAc-T12 further increased chondrocytes. In particular, GalNAc-T12 concentrations of 200 ng / ml or higher significantly increased chondrocytes. On the other hand, when cultured in serum-free medium, chondrocytes decreased to approximately 60% compared to the initial level, but this decrease was significantly suppressed by the addition of 200 ng / ml of GalNAc-T12. These results indicate that GalNAc-T12 is involved in the proliferation and survival of chondrocytes.

[0032] [Example 2] Effect of GalNAc-T12 on chondrocyte apoptosis (method) NHAC-Kn cells were cultured with 200 ng / ml of GalNAc-T12. Simultaneously, 5 μg / ml of actinomycin D (Sigma-Aldrich) was added to induce apoptosis. After 72 hours, a WST assay was performed using Cell Counting Kit-8 to assess the number of viable cells. Western blotting was also performed to examine the expression of caspase 3, PARP, and cleaved PARP. (result) Figure 2 shows a graph (n = 3) of the WST assay results, with the amount of viable cells set to 1 when neither GalNAc-T12 nor actinomycin D was added. The addition of GalNAc-T12 significantly increased chondrocytes. Conversely, the addition of actinomycin D reduced chondrocytes through apoptosis. However, the addition of GalNAc-T12 significantly suppressed the decrease in chondrocytes caused by actinomycin D. Figure 3 shows the results of Western blotting to detect caspase 3, PARP, and cleaved PARP. Actinomycin D-induced apoptosis reduced the expression levels of caspase 3 and PARP, while increasing the expression of cleaved PARP. However, these changes were suppressed by the addition of GalNAc-T12. These results indicate that GalNAc-T12 suppresses apoptosis in chondrocytes.

[0033] [Example 3] Effect of GalNAc-T12 on gene expression in chondrocytes (method) NHAC-Kn cells were cultured with 200 ng / ml GalNAc-T12, 200 μM N-acetylgalactosamine (GalNAc; Sigma-Aldrich), or 200 μM N-acetylglucosamine (GlcNAc; Sigma-Aldrich). After 72 hours, cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). 1 μg of RNA was used for reverse transcription using the GeneAmp RNA PCR kit (Applied Biosystems) to synthesize cDNA. Real-time PCR was performed using TB Green Premix Ex Taq II (Takara Bio) to examine changes in gene expression. The expression levels of each molecule were standardized by the GAPDH Ct value and then compared using the ΔΔCt method, with GalNAc-T12, GalNAc, and GlcNAc not added (culture medium only) as the baseline. (result) Figure 4 shows graphs (n = 3) of gene expression for each molecule, with the expression levels for the absence of GalNAc-T12, GalNAc, and GlcNAc (culture medium only) set to 1. The addition of GalNAc-T12 significantly increased the expression of aggrecan and type II collagen. On the other hand, no changes were observed for any molecule when GalNAc and GlcNAc were added. These results indicate that GalNAc-T12 increases the expression of cartilage matrix components.

[0034] [Example 4] Effect of GalNAc-T12 on chondrocyte hypertrophy (method) NHAC-Kn cells were cultured with 1x ITS supplement (R&D systems) and 50 μg / ml ascorbic acid (Wako Pure Chemical Industries) to induce hypertrophy in chondrocytes. Simultaneous addition of 200 ng / ml GalNAc-T12, 200 μM GalNAc, or 200 μM GlcNAc was also performed during culture. The culture medium was changed every 72 hours, and cells were harvested on day 15. RNA was extracted using the RNeasy Mini Kit. Reverse transcription was performed using 1 μg of RNA with the GeneAmp RNA PCR kit to synthesize cDNA. Real-time PCR was performed using TB Green Premix Ex Taq II to examine changes in gene expression of molecules related to hypertrophy. The expression levels of each molecule were standardized by the GAPDH Ct value and then compared using the ΔΔCt method, with the control group (without hypertrophy) as the baseline. (result) Figure 5 shows graphs of gene expression for each molecule, with the expression level without hypertrophy induction set to 1 (n = 3). The expression of type X collagen, MMP13, ADAMTS5, EPAS1 (HIF-2α), and RUNX2 increased upon induction of hypertrophy. However, the addition of GalNAc-T12 significantly suppressed these increases. Furthermore, the expression of aggrecan, type II collagen, and SOX9 decreased upon induction of hypertrophy, but the addition of GalNAc-T12 significantly suppressed these decreases. On the other hand, no changes were observed for any of the molecules when GalNAc and GlcNAc were added. These results indicate that GalNAc-T12 suppresses chondrocyte hypertrophy.

[0035] [Example 5] Effect of GalNAc-T12 on inflammatory stimulation of chondrocytes (method) NHAC-Kn cells were cultured with 200 ng / ml GalNAc-T12 for 72 hours, then stimulated with 100 ng / ml IL-6 (Peprotech) + 100 ng / ml soluble IL-6 receptor (sIL-6R; Peprotech), 10 ng / ml TNFα, or 10 ng / ml IL-1β. After 24 hours, cells were harvested and RNA was extracted using the RNeasy Mini Kit. 1 μg of RNA was used for reverse transcription using the GeneAmp RNA PCR kit to synthesize cDNA. Real-time PCR was performed using TB Green Premix Ex Taq II to examine changes in gene expression. The expression levels of each molecule were standardized by the Ct value of GAPDH, and then compared using the ΔΔCt method with the control group (culture medium only) without GalNAc-T12 or each cytokine as the baseline. (result) Figure 6 shows a graph (n = 3) of the gene expression of each molecule upon stimulation with IL-6 + sIL-6R, with the expression levels for when GalNAc-T12, IL-6, and sIL-6R were not added (culture medium only) set to 1. MMP3, RUNX2, and NOS2 (iNOS) showed a significant increase in expression upon IL-6 stimulation. However, this increase was significantly suppressed by culturing with GalNAc-T12 before IL-6 stimulation. Furthermore, aggrecan, type II collagen, and SOX9 showed a significant decrease in expression upon IL-6 stimulation, but this decrease was significantly suppressed by culturing with GalNAc-T12 before IL-6 stimulation. Figure 7 shows a graph of gene expression for each molecule upon TNFα stimulation, with the expression level when neither GalNAc-T12 nor TNFα was added (culture medium only) set to 1 (n = 3). The expression of type X collagen, MMP3, MMP13, RUNX2, NOS2 (iNOS), PTGS2 (COX-2), and NFKB1 significantly increased upon TNFα stimulation. However, this increase was significantly suppressed by culturing with GalNAc-T12 before TNFα stimulation. Furthermore, the expression of aggrecan, type II collagen, and SOX9 significantly decreased upon TNFα stimulation, but this decrease was significantly suppressed by culturing with GalNAc-T12 before TNFα stimulation. Figure 8 shows a graph of gene expression for each molecule upon stimulation with IL-1β, with the expression level when neither GalNAc-T12 nor IL-1β was added (culture medium only) set to 1 (n = 3). MMP3, MMP13, ADAMTS5, SPP1(OPN), type X collagen, and RUNX2 showed a significant increase in expression upon IL-1β stimulation. However, this increase was significantly suppressed by culturing with GalNAc-T12 before IL-1β stimulation. Furthermore, aggrecan, type II collagen, and SOX9 showed a significant decrease in expression upon IL-1β stimulation, but this decrease was significantly suppressed by culturing with GalNAc-T12 before IL-1β stimulation.

[0036] [Example 6] Effects of GalNAc-T12 on chondrocytes in osteoarthritis (OA) patients (method) Chondrocytes from OA patients were cultured with 200 ng / ml of GalNAc-T12. After 72 hours, the cells were harvested and RNA was extracted using the RNeasy Mini Kit. Using 1 μg of RNA, cDNA was synthesized by reverse transcription using the GeneAmp RNA PCR kit. Real-time PCR was performed using TB Green Premix Ex Taq II to examine changes in gene expression. The expression levels of each molecule were standardized by the Ct value of GAPDH and then compared using the ΔΔCt method with the control group without GalNAc-T12 as the baseline. (result) Figure 9 shows graphs of gene expression for each molecule, with the expression level without GalNAc-T12 added set to 1 (n = 3). The expression of MMP3, MMP13, SPP1 (OPN), NOS2 (iNOS), TNF (TNFα), IL1B, VEGFA, PTGS2 (COX-2), and NFKB1 decreased with the addition of GalNAc-T12. On the other hand, the expression of aggrecan, type II collagen, and SOX9 increased with the addition of GalNAc-T12. No statistically significant differences were observed for any of the molecules. However, GalNAc-T12 decreased the expression of molecules whose expression increases in an inflammatory state (MMP3, MMP13, SPP1 (OPN), NOS2 (iNOS), TNF (TNFα), IL1B, VEGFA, PTGS2 (COX-2), NFKB1), while conversely, GalNAc-T12 increased the expression of molecules whose expression decreases in an inflammatory state (agrecan, type II collagen, SOX9). The results from Examples 5 and 6 demonstrate that GalNAc-T12 suppresses inflammation.

[0037] [Example 7] Effects of GalNAc-T12 on chondrocytes in osteoarthritis using a mouse model. (method) As an osteoarthritis (OA) model, we used a medial meniscus instability (DMM) model for our study. Male C57BL / 6 mice at 10 weeks of age underwent DMM surgery on the right hindlimb knee joint, and were administered 5 μg of GalNAc-T12 or PBS (Nissui Pharmaceutical) to the same joint. After 8 weeks, safranin O staining was performed, and the cartilage tissue was pathologically evaluated using the OARSI score. (result) Figure 10 shows the histological images of the right hindlimb knee joint of mice that underwent DMM surgery and administration of PBS or GalNAc-T12, stained with safranin O (n = 2). Figure 11 shows the OARSI score graph (PBS group n = 5, GalNAc-T12 group n = 4). In the pathological evaluation of cartilage tissue using the OARSI score, the GalNAc-T12 group showed a significantly lower score compared to the PBS-administered control group. This indicates that GalNAc-T12 suppresses the destruction of cartilage tissue in osteoarthritis (OA).

[0038] [Example 8] The effect of GalNAc-T12 on chondrocytes in rheumatoid arthritis (RA) using a mouse model. (method) As a model for rheumatoid arthritis (RA), we used an anti-type II collagen antibody-induced arthritis (CAIA) model. Seven-week-old male DBA1 / J mice were intraperitoneally administered 1.5 mg of an anti-type II collagen antibody cocktail (Chondrex). Three days later, 50 μg of LPS was administered intraperitoneally to enhance the induction of arthritis, and at the same time, 5 μg of GalNAc-T12 or PBS (Nissui Pharmaceutical) was administered to the right hindlimb knee joint. Limb swelling was scored daily, and safranin O staining was performed 14 days later, followed by pathological evaluation of cartilage tissue using the OARSI score. (result) Figure 12 shows a graph of the scoring results for limb swelling (0-4 points / limbs, maximum 16 points / limbs; PBS administration group n = 6, GalNAc-T12 administration group n = 5). No difference in limb swelling was observed between the PBS-administered control group and the GalNAc-T12-administered group. Furthermore, when examining only the right hind limb administered with GalNAc-T12, no difference was observed between the two groups. Figure 13 shows the histological images of the right hindlimb knee joint of CAIA model mice administered with PBS or GalNAc-T12, stained with safranin O (n = 2). Figure 14 shows the OARSI score graph (PBS group n = 6, GalNAc-T12 group n = 5). In the pathological evaluation of cartilage tissue using the OARSI score, the GalNAc-T12 group showed a lower score compared to the PBS-administered control group. However, no statistically significant difference was observed. This suggests that GalNAc-T12 tends to suppress cartilage tissue destruction in rheumatoid arthritis (RA).

[0039] [Example 9] The effect of GalNAc-T12 on p21 expression during chondrocyte hypertrophy. (method) NHAC-Kn cells were cultured with 1x ITS supplement and 50 g / ml ascorbic acid to induce hypertrophy in chondrocytes. Simultaneously, 200 ng / ml of GalNAc-T12 was added during the culture process. The culture medium was changed every 72 hours, and cells were harvested on day 15. RNA was extracted using the RNeasy Mini Kit. 1 g of RNA was used for reverse transcription using the GeneAmp RNA PCR kit to synthesize cDNA. Real-time PCR was performed using TB Green Premix Ex Taq II to examine p21 expression. p21 expression levels were standardized by the GAPDH Ct value and then compared using the ΔΔCt method, with the control group (without hypertrophy) as the baseline. (result) Figure 15 shows a graph of p21 expression, with the expression level when hypertrophy was not induced set to 1 (n = 4). p21 expression increased upon induction of hypertrophy. However, this increase was significantly suppressed by the addition of GalNAc-T12. Previous reports have shown that suppressing p21 expression inhibits chondrocyte hypertrophy (Kikuchi K et al. P21 deficiency exhibits delayed endochondrocyte ossification during fracture healing. Bone 165:116572, 2022). Therefore, based on these reports and the results of this study, GalNAc-T12 suppresses chondrocyte hypertrophy by inhibiting p21 expression.

[0040] [Example 10] Effects of GalNAc-T12 on O-linked glycans in chondrocytes (method) To analyze the changes in O-linked glycans induced by GalNAc-T12, we focused on gp130, an IL-6 receptor, and investigated the changes in O-linked glycans at gp130 (Non-Patent Literature 1). NHAC-Kn cells were cultured with 200 ng / ml GalNAc-T12 for 5, 10, 30, 60, and 360 minutes. Cells were harvested, proteins were extracted, and gp130 was immunoprecipitated. Subsequently, O-GalNAc was detected by Western blotting. (result) Figure 16 shows the results of detecting O-GalNAc by Western blotting. The addition of GalNAc-T12 increased O-GalNAc in gp130. These results indicate that GalNAc-T12 increases O-linked glycans in chondrocytes. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0041] The present invention makes it possible to prevent and / or treat osteoarthritis and / or rheumatoid arthritis. [Sequence Listing Free Text]

[0042] <Sequence ID 1> shows the amino acid sequence of recombinant human GalNAc-T12. UniProt accession No; Q8IXK2 1 MWGRTARRRC PRELRRGREA LLVLLALLAL AGLGSVLRAQ RGAGAGAAEP GPPRTPRPGR 61 REPVMPRPPV PANALGARGE AVRLQLQGEE LRLQEESVRL HQINIYLSDR ISLHRRLPER 121 WNPLCKEKKY DYDNLPRTSV IIAFYNEAWS TLLRTVYSVL ETSPDILLEE VILVDDYSDR 181 EHLKERLANE LSGLPKVRLI RANKREGLVR ARLLGASAAR GDVLTFLDCH CECHEGWLEP 241 LLQRIHEEEES AVVCPVIDVI DWNTFEYLGN SGEPQIGGFD WRLVFTWHTV PERERIRMQS 301 PVDVIRSPTM AGGLFAVSKK YFEYLGSYDT GMEVWGGENL EFSFRIWQCG GVLETHPCSH 361 VGHVFPKQAP YSRNKALANS VRAAEVWMDE FKELYYHRNP RARLEPFGDV TERKQLRDKL 421 QCKDFKWFLE TVYPELHVPE DRPGFFGMLQ NKGLTDYCFD YNPPDENQIV GHQVILYLCH 481 GMGQNQFFEY TSQKEIRYNT HQPEGCIAVE AGMDTLIMHL CEETAPENQK FILQEDGSLF 541 HEQSKKCVQA ARKESSDSFV PLLRDCTNSD HQKWFFKERM L <SEQ ID NO:2> shows the nucleotide sequence of recombinant human GalNAc-T12. GenBank accession No; AB078146.1 1 atgtgggggc gcacggcgcg gcggcgctgc ccgcgggaac tgcggcgcgg ccgggaggcg 61 ctgttggtgc tcctggcgct actggcgttg gccgggctgg gctcggtgct gcgggcgcag 121 cgtggggccg gggccggggc tgccgagccg ggacccccgc gcaccccgcg ccccgggcgg 181 cgcgagccgg tcatgccgcg gccgccggtg ccggcgaacg cgctgggcgc gcggggcgag 241 gcggtgcggc tgcagctgca gggcgaggag ctgcggctgc aggaggagag cgtgcggctg 301 caccagatta acatctacct cagcgaccgc atctcactgc accgccgcct gcccgtgcgc 361 tggaacccgc tgtgcaaaga gaagaaatat gattatgata atttgcccag gacatctgtt 421 atcatagcat tttataatga agcctggtca actctccttc ggacagttta cagtgtcctt 481 gagacatccc cggatatcct gctagaagaa gtgatccttg tagatgacta cagtgataga 541 gagcacctga aggagcgctt ggccaatgag ctttcgggac tgcccaaggt gcgcctgatc 601 cgcgccaaca agagagaggg cctggtgcga gcccggctgc tgggggcgtc tgcggcgagg 661 ggcgatgttc tgaccttcct ggactgtcac tgtgagtgcc acgaagggtg gctggagccg 721 ctgctgcaga ggatccatga agaggagtcg gcagtggtgt gcccggtgat tgatgtgatc 781 gactggaaca ccttcgaata cctggggaac tccggggagc cccagatcgg cggtttcgac 841 tggaggctgg tgttcacgtg gcacacagtt cctgagaggg agaggatacg gatgcaatcc 901 cccgtcgatg tcatcaggtc tccaacaatg gctggtgggc tgtttgctgt gagtaagaaa 961 tattttgaat atctggggtc ttatgataca ggaatggaag tttggggagg agaaaacctc 1021 gaattttcct ttaggatctg gcagtgtggt ggggttctgg aaacacaccc atgttcccat 1081 gttggccatg ttttccccaa gcaagctccc tactcccgca acaaggctct ggccaacagt 1141 gttcgtgcag ctgaagtatg gatggatgaa tttaaagagc tctactacca tcgcaacccc 1201 cgtgcccgct tggaaccttt tggggatgtg acagagagga agcagctccg ggacaagctc 1261 cagtgtaaag acttcaagtg gttcttggag actgtgtatc cagaactgca tgtgcctgag 1321 gacaggcctg gcttcttcgg gatgctccag aacaaaggac taacagacta ctgctttgac 1381 tataccctc ccgatgaaaa ccagattgtg ggacaccagg tcattctgta cctctgtcat 1441 gggatgggcc agaatcagtt tttcgagtac acgtcccaga aagaatacg ctaacacc 1501 caccagcctg agggctgcat tgctgtggaa gcaggaatgg atacccttat catgcatctc 1561 tgcgaagaaa ctgccccaga gaatcagaag ttcatcttgc aggaggatgg atctttattt 1621 cacgaacagt ccaagaaatg tgtccaggct gcgaggaagg agtcgagtga cagtttcgtt 1681 ccactcttac gagactgcac caactcggat catcagaaat ggttcttcaa agagcgcatg 1741 today

Claims

1. A pharmaceutical composition containing GalNAc-T12 for the prevention and / or treatment of osteoarthritis and / or rheumatoid arthritis.

2. A pharmaceutical composition for the prevention and / or treatment of osteoarthritis, comprising GalNAc-T12.

3. A pharmaceutical composition for the prevention and / or treatment of rheumatoid arthritis, comprising GalNAc-T12.

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