Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)

Definitions

• the present invention relates to a pharmaceutical composition for the prevention and/or treatment of osteoarthritis and/or rheumatoid arthritis.
• Cartilage tissue consists of chondrocytes, the only cells present, and a cartilage matrix synthesized by the chondrocytes, with the chondrocytes scattered throughout the cartilage matrix.
• Proteoglycans the main component of the cartilage matrix, have a structure in which glycosaminoglycans (GAGs) such as chondroitin sulfate are bound to a core protein, with a large amount of water bound to them within the molecule.
• GAGs glycosaminoglycans
• Proteoglycans form a flexible higher-order structure together with collagen fibers, including type II collagen, which maintains water retention and elasticity, and this plays an important role in the joint lubrication mechanism that maintains smooth musculoskeletal movement.
• N-linked glycans are formed by the addition of N-acetylglucosamine (GlcNAc) to asparagine residues in proteins and have been reported to be involved in signal transduction by cytokines such as IL-6, TNF ⁇ , and IL-1 ⁇ (Non-Patent Document 1).
• O-linked glycans are formed by the addition of sugars such as GlcNAc or N-acetylgalactosamine (GalNAc) to serine or threonine residues in proteins.
• O-linked GlcNAc O-GlcNAc
• GalNAc-T GalNAc transferase
• Non-Patent Document 5 O-GalNAc and GalNAc-T, and their detailed functions have not been elucidated.
• Non-Patent Document 6 it has been reported that high-mannose N-linked glycans are increased in chondrocytes of human OA and OA model mice (Non-Patent Document 6), and that N-linked glycans are involved in the development of RA in RA model mice (Non-Patent Document 1).
• O-linked glycans it has been reported that O-GlcNAc is increased in OA chondrocytes (Non-Patent Document 7) and promotes chondrocyte hypertrophy (Non-Patent Document 2).
• O-GalNAc it has been reported that GalNAc-T expression is increased in OA chondrocytes (Non-Patent Document 8).
• SNPs single nucleotide polymorphisms
• GalNAc-T12 single nucleotide polymorphisms
• O-linked N-acetylglucosamine (O-GlcNAc) protein modification is increased in the cartilage of patients with knee osteoarthritis.
• Yoshimoto M et al. Bioinformatic analysis reveals potential relationship between chondrocyte senescence and protein glycosylation in osteoarthritis pathogenesis.
• 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.
• the present invention focuses on GalNAc-T, which is involved in the formation of O-GalNAc, which has many unknowns compared to other sugar chain modifications, and aims to investigate the function of GalNAc-T12 in particular in maintaining chondrocyte homeostasis, hypertrophy, and inflammatory stimuli to chondrocytes. It also aims to use model mice to analyze the direct involvement of GalNAc-T12 in the pathogenesis of OA and RA, with the results being used for drug discovery.
• glycosaminoglycans are responsible for maintaining water retention and elasticity, thereby creating a lubrication mechanism in joints.
• GAGs glycosaminoglycans
• GalNAc-T12 GalNAc transferase 12
• O-GalNAc O-linked N-acetylgalactosamine
• RA rheumatoid arthritis
• GalNAc-T12 suppressed cartilage destruction in osteoarthritis (OA) and RA.
• OA osteoarthritis
• RA osteoarthritis
• the gist of the present invention is as follows. (1) A pharmaceutical composition for preventing and/or treating osteoarthritis and/or rheumatoid arthritis, comprising GalNAc-T12. (2) A method for preventing and/or treating osteoarthritis and/or rheumatoid arthritis, comprising administering a pharmaceutically effective amount of GalNAc-T12 to a subject. (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 a medicament for the prevention and/or treatment of osteoarthritis and/or rheumatoid arthritis.
• Treatments for osteoarthritis include intra-articular injections of hyaluronic acid, as well as oral supplements consisting of glucosamine, collagen, and mixtures thereof, but no effective treatment has yet been found. Therefore, the provision of an effective treatment for osteoarthritis by this invention will be a great benefit to civilization.
• the present invention makes it possible to prevent and treat osteoarthritis and rheumatoid arthritis.
• This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 2024-015445, which is a priority document of the present application.
• This graph shows the results of the WST assay, with the number of live cells at the start of culture set at 1.
• This graph shows the results of the WST assay, with the amount of viable cells when neither GalNAc-T12 nor actinomycin D was added set at 1.
• Western blot results showed the detection of caspase 3, PARP and cleaved PARP.
• This graph shows the gene expression of each molecule, with the expression level when GalNAc-T12, GalNAc, and GlcNAc were not added (medium only) set at 1.
• This graph shows the gene expression of each molecule, with the expression level set to 1 when hypertrophy is not induced.
• This graph shows the gene expression of each molecule when stimulated with IL-6 + sIL-6R, with the expression level set to 1 when GalNAc-T12, IL-6, and sIL-6R were not added (medium only).
• This graph shows the gene expression of each molecule upon stimulation with TNF ⁇ , with the expression level set to 1 when neither GalNAc-T12 nor TNF ⁇ was added (medium only).
• This graph shows the gene expression of each molecule upon stimulation with IL-1 ⁇ , with the expression level set to 1 when neither GalNAc-T12 nor IL-1 ⁇ was added (medium alone).
• This graph shows the gene expression of each molecule, with the expression level when GalNAc-T12 was not added set at 1.
• the present invention provides a pharmaceutical composition containing GalNAc-T12 for the prevention and/or treatment of osteoarthritis and/or rheumatoid arthritis.
• the present invention also provides a method for preventing and/or treating osteoarthritis and/or rheumatoid arthritis, which comprises administering a pharmaceutically effective amount of GalNAc-T12 to a subject.
• the present invention provides the use of GalNAc-T12 for the prevention and/or treatment of osteoarthritis and/or rheumatoid arthritis.
• GalNAc-T12 in the manufacture of a medicament for the prevention and/or treatment of osteoarthritis and/or rheumatoid arthritis.
• GalNAc-transferase 12 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, with particularly high expression in the large intestine, small intestine, stomach, and pancreas, moderate expression in the thyroid, spleen, and testis, and low expression in the brain, bone marrow, thymus, heart, lung, liver, kidney, esophagus, etc.
• NCBI Gene ID: 79695 (information about the GalNAc-T12 gene) NCBI Reference Sequence No.
• GalNAc-T12 preferably consists of the amino acid sequence of SEQ ID NO: 1. However, it may also be a protein consisting of an amino acid sequence that is 90% to less than 100% identical to the amino acid sequence of SEQ ID NO: 1. The identity of two amino acid sequences can be determined using BLAST.
• GalNAc-T12 may have one, two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid residues deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 1. These mutant polypeptides can be effective in preventing and/or treating osteoarthritis and/or rheumatoid arthritis.
• GalNAc-T12 can be obtained, for example, by producing it in host cells using genetic engineering techniques, followed by separation and purification. It can also be produced by chemical synthesis.
• GalNAc-T12 To produce GalNAc-T12 in host cells using genetic engineering techniques, DNA encoding GalNAc-T12 is inserted into a vector and introduced into host cells, causing the host cells to produce recombinant GalNAc-T12. Plasmids are often used as vectors, and host cells include E. coli, yeast, animal cells, and human cells. Methods for preparing GalNAc-T12 are described in, for example, 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. GalNAc-T12 produced by host cells can be recovered from the culture medium using known protein purification methods.
• GalNAc-T12 is also commercially available, so commercially available products may also be used.
• GalNAc-T12 which consists of the amino acid sequence of SEQ ID NO: 1
• site-specific mutagenesis can be performed using the CRISPR/Cas9 system or specific primers (containing the desired mutation).
• GalNAc-T12 mutants have been reported, for example, in 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. These mutants may also be used in the present invention.
• GalNAc-T12 can be used to prevent and/or treat osteoarthritis and/or rheumatoid arthritis.
• treatment means recovery, remission, alleviation, and/or delay in the worsening of clinical symptoms of a disease in a patient who has developed the disease.
• prevention means reducing the incidence of a disease. Prevention includes reducing the risk of disease progression or reducing the severity of the disease, and also includes preventing recurrence.
• GalNAc-T12 (hereinafter referred to as the "active ingredient") can be administered orally or parenterally to mammals (e.g., humans, rabbits, dogs, cats, rats, and mice) either alone or together with pharmacologically acceptable carriers, diluents, or excipients in an appropriate pharmaceutical composition.
• mammals e.g., humans, rabbits, dogs, cats, rats, and mice
• pharmacologically acceptable carriers, diluents, or excipients in an appropriate pharmaceutical composition.
• the dosage varies depending on the recipient, target disease, symptoms, and route of administration.
• a single dose of the active ingredient is typically approximately 0.01-10 mg/kg body weight, preferably approximately 1-2 mg/kg body weight, administered orally, intramuscularly, subcutaneously, or intravenously (preferably continuously or every other day) at a frequency of approximately once a month to three times a day, preferably once a week to once a day.
• the single dose of active ingredient is typically about 0.01 to 10 mg/kg body weight, preferably about 1 to 2 mg/kg body weight, and is administered orally, intramuscularly, subcutaneously, intravenously, or intra-articularly (preferably continuously or every other day) at a frequency of about once a month to three times a day, preferably about once a week to once a day.
• compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc.
• Such compositions can be manufactured by conventional methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field.
• carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate, etc.
• Aqueous liquids for injection include saline, isotonic solutions containing glucose or other adjuvants, and may be used in combination with an appropriate solubilizing agent, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil))).
• solubilizing agent such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil))
• oily liquids include sesame oil and soybean oil, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination.
• the prepared injection solution is usually filled
• the oral or parenteral pharmaceutical composition may be prepared in a dosage unit form suitable for the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc., each of which preferably contains 0.5 to 500 mg of the active ingredient.
• Example 1 Effect of GalNAc-T12 on chondrocyte proliferation and survival (Method)
• Normal human knee articular chondrocytes (NHAC-Kn; Lonza) were cultured with 0-800 ng/ml GalNAc-T12 (R&D Systems). After 72 hours, a WST assay was performed using Cell Counting Kit-8 (Dojindo Laboratories) to assess the viability of the cells.
• Cells were cultured in two media: Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) containing 10% fetal bovine serum (Biowest), and serum-free DMEM.
• DMEM Dulbecco's modified Eagle's medium
• GalNAc-T12 significantly increased the number of chondrocytes.
• actinomycin D caused a decrease in the number of chondrocytes due to apoptosis.
• GalNAc-T12 significantly inhibited the decrease in chondrocytes caused by actinomycin D.
• the results of Western blot analysis of caspase 3, PARP, and cleaved PARP are shown in Figure 3. Induction of apoptosis by actinomycin D decreased the expression of caspase 3 and PARP, and increased the expression of cleaved PARP. However, these changes were suppressed by the addition of GalNAc-T12. These results indicated that GalNAc-T12 suppresses apoptosis in chondrocytes.
• Example 3 Effect of GalNAc-T12 on gene expression in chondrocytes (Method)
• NHAC-Kn was cultured with 200 ng/ml GalNAc-T12, 200 ⁇ M N-acetylgalactosamine (GalNAc; Sigma-Aldrich), or 200 ⁇ M N-acetylglucosamine (GlcNAc; Sigma-Aldrich).
• GalNAc N-acetylgalactosamine
• GlcNAc N-acetylglucosamine
• NHAC-Kn was cultured with 1x ITS supplement (R&D systems) and 50 ⁇ g/ml ascorbic acid (Wako Pure Chemical Industries) to induce hypertrophy in chondrocytes.
• the culture medium was simultaneously supplemented with 200 ng/ml GalNAc-T12, 200 ⁇ M GalNAc, or 200 ⁇ M GlcNAc.
• the medium was changed every 72 hours, and on day 15, cells were harvested and RNA was extracted using the RNeasy Mini Kit. 1 ⁇ g of RNA was reverse-transcribed using the GeneAmp RNA PCR kit to synthesize cDNA.
• GalNAc-T12 inhibits chondrocyte hypertrophy.
• NHAC-Kn cells were cultured with 200 ng/ml GalNAc-T12 for 72 hours and then stimulated with 100 ng/ml IL-6 (Peprotech) plus 100 ng/ml soluble IL-6 receptor (sIL-6R; Peprotech), 10 ng/ml TNF ⁇ , or 10 ng/ml IL-1 ⁇ . After 24 hours, cells were harvested and RNA was extracted using the RNeasy Mini Kit. 1 ⁇ g of RNA was reverse-transcribed to cDNA using the GeneAmp RNA PCR kit.
• Example 6 Effect of GalNAc-T12 on chondrocytes from osteoarthritis (OA) patients (Methods) Chondrocytes from OA patients were cultured with 200 ng/ml GalNAc-T12. After 72 hours, the cells were harvested and RNA was extracted using the RNeasy Mini Kit. 1 ⁇ g of RNA was reverse-transcribed 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. Expression levels of each molecule were normalized by the Ct value of GAPDH and compared using the ⁇ Ct method with the control group without GalNAc-T12.
• Expression of MMP3, MMP13, SPP1 (OPN), NOS2 (iNOS), TNF (TNF ⁇ ), IL1B, VEGFA, PTGS2 (COX-2), and NFKB1 decreased with the addition of GalNAc-T12.
• 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.
• GalNAc-T12 decreased the expression of molecules whose expression is increased under inflammatory conditions (MMP3, MMP13, SPP1 (OPN), NOS2 (iNOS), TNF (TNF ⁇ ), IL1B, VEGFA, PTGS2 (COX-2), NFKB1), while conversely, GalNAc-T12 increased the expression of molecules whose expression is decreased under inflammatory conditions (aggrecan, type II collagen, SOX9).
• the results of Examples 5 and 6 demonstrated that GalNAc-T12 suppresses inflammation.
• Example 7 Effect of GalNAc-T12 on chondrocytes in OA model mice (Methods)
• DMM medial meniscus destabilization
• the OARSI score for the pathological evaluation of cartilage tissue was significantly lower in the GalNAc-T12-administered group compared to the PBS-administered control group. This indicates that GalNAc-T12 suppresses the destruction of cartilage tissue in OA.
• Example 8 Effect of GalNAc-T12 on chondrocytes in a mouse model of rheumatoid arthritis (RA) (Methods)
• RA rheumatoid arthritis
• CAIA anti-type II collagen antibody-induced arthritis
• the OARSI score for the cartilage tissue pathology evaluation was lower in the GalNAc-T12-administered group compared to the PBS-administered control group. However, no statistically significant difference was observed. This indicates that GalNAc-T12 tends to suppress the destruction of cartilage tissue in RA.
• NHAC-Kn was cultured with 1x ITS supplement and 50 ⁇ g/ml ascorbic acid to induce hypertrophy in chondrocytes. 200 ng/ml GalNAc-T12 was also added during the culture. The medium was changed every 72 hours. Cells were harvested on day 15, and RNA was extracted using the RNeasy Mini Kit. One gram of RNA was reverse-transcribed 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 normalized by the Ct value of GAPDH and compared using the ⁇ Ct method relative to the control without hypertrophy induction. (result)
• p21 expression increased with hypertrophy induction, but this increase was significantly suppressed by the addition of GalNAc-T12.
• Previous reports have shown that suppression of p21 expression inhibits chondrocyte hypertrophy (Kikuchi K et al. P21 deficiency exhibits delayed endochondral ossification during fracture healing. Bone 165:116572, 2022.) Therefore, this report and the present results indicate that GalNAc-T12 inhibits chondrocyte hypertrophy by suppressing p21 expression.
• Example 10 Effect of GalNAc-T12 on O-linked glycans in chondrocytes (Methods)
• Method 1 To analyze the effects of GalNAc-T12 on O-linked glycans, we focused on the IL-6 receptor gp130 and examined the changes in O-linked glycans on gp130 (Non-Patent Document 1).
• NHAC-Kn cells were incubated with 200 ng/ml GalNAc-T12 for 5, 10, 30, 60, and 360 minutes. Cells were harvested, proteins were extracted, and gp130 was immunoprecipitated. O-GalNAc was then detected by Western blotting.
• the present invention makes it possible to prevent and/or treat osteoarthritis and/or rheumatoid arthritis.
• ⁇ SEQ ID NO: 1> Shows the amino acid sequence of recombinant human GalNAc-T12.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Genetics & Genomics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Rheumatology (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Biotechnology (AREA)
• Physical Education & Sports Medicine (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Pain & Pain Management (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=96699915

Family Applications (1)

Country Status (2)

Citations (3)

• 2025
  • 2025-02-03 WO PCT/JP2025/003340 patent/WO2025169865A1/ja active Pending
  • 2025-02-03 JP JP2025568955A patent/JP7840505B2/ja active Active

Patent Citations (3)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events