WO2020149593A2 - Composition for preventing, alleviating or treating rheumatoid arthritis, containing tetraspanin-2 inhibitor as active ingredient - Google Patents

Composition for preventing, alleviating or treating rheumatoid arthritis, containing tetraspanin-2 inhibitor as active ingredient Download PDF

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WO2020149593A2
WO2020149593A2 PCT/KR2020/000580 KR2020000580W WO2020149593A2 WO 2020149593 A2 WO2020149593 A2 WO 2020149593A2 KR 2020000580 W KR2020000580 W KR 2020000580W WO 2020149593 A2 WO2020149593 A2 WO 2020149593A2
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rheumatoid arthritis
tspan
expression
protein
group
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PCT/KR2020/000580
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French (fr)
Korean (ko)
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WO2020149593A3 (en
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황인후
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황인후
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention prevents any one or more diseases selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient,
  • the present invention relates to a method for selecting a therapeutic agent for rheumatoid arthritis by measuring an improvement or treatment composition and an expression level of tetraspanin-2 (Tetraspanin-2, TSPAN-2).
  • Rheumatoid arthritis is a chronic inflammatory disease characterized by inflammation and proliferation of synovial cells, and unlike osteoarthritis, osteoporosis and bone erosion of the bones around the joints occur.
  • Rheumatoid arthritis is an inflammation of the synovial membrane that spreads to the joint capsule, ligament, and tendon (step 1), and the joint gap narrows due to gradual destruction of the joint cartilage.
  • the tension of the joint membrane and ligament is lost (step 2), inflammation invades the bone, and partial erosion of the bone occurs (step 3), and the joint function is lost (step 4).
  • non-steroidal anti-inflammatory agents such as NSAID, salicylate, and COX-2 inhibitors; Adrenal cortical hormone preparations such as prednisolone and triamcinolone; Disease-Modifying Anti-rheumatic Drugs (DMARDs) such as methotrexate and sulfasalazine; And drugs such as biological agents such as etanercept, infliximab, and adalimumab.
  • DMARDs Disease-Modifying Anti-rheumatic Drugs
  • drugs such as biological agents such as etanercept, infliximab, and adalimumab.
  • nonsteroidal anti-inflammatory drugs and adrenal cortical hormone preparations are used in the early stages of symptom expression, antirheumatic drugs are used in consideration of symptoms and disease activity, and in severe cases, biological agents or combination therapy are used in clinical practice.
  • Nonsteroidal anti-inflammatory drugs such as NSAID, which are relatively inexpensive to treat, are drugs that cause typical gastrointestinal side effects, and antirheumatic drugs such as methotrexate and sulfasalazine are also noted as gastrointestinal disorders.
  • tetraspanin-2 (Tetraspanin-2, TSPAN-2) is a protein encoded by the TSPAN2 gene in humans.
  • the protein encoded by this gene is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members are membrane proteins composed of four hydrophobic domains, transmembrane domains, and are found in almost all animal cells.
  • the TSPAN-2 protein is involved in fundamental cytological processes including cell growth, adhesion and differentiation (Todd, S. C et al., Biochim. Biophys. Acta. 1399, 101104, 1998).
  • the tetraspanin family members tend to have a highly conserved amino acid sequence (Hemler, M. E et al., J. Cell Biol. 155, 11031107. 2001).
  • the inventors of the present invention searched for an agent capable of effectively treating rheumatoid arthritis, and when suppressing the expression of TSPAN-2, the swelling caused by rheumatoid arthritis was alleviated and the symptoms were treated within two weeks. Completed.
  • an object of the present invention is a type 1 rheumatoid arthritis, a type 2 rheumatoid arthritis, gestational activity, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding the same. It is to provide a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
  • another object of the present invention comprises the step of administering a pharmaceutical composition containing an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than humans, rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational It is to provide a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
  • another object of the present invention includes an expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, children It is to provide a food composition for preventing or improving any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid multiple muscle pain.
  • Another object of the present invention is to provide a method for screening a preparation for the prevention or treatment of rheumatoid arthritis.
  • another object of the present invention is a type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis, and rheumatoid polymyalgia selected from the group consisting of a prophylactic or therapeutic agent for the preparation of any one or more diseases It is to provide the use of an inhibitor of the expression or activity of a Spanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it.
  • Spanin-2 Tetraspanin-2, TSPAN-2
  • another object of the present invention is to administer an effective amount of a composition comprising an inhibitor or expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it to an individual in need thereof It is to provide a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia.
  • the present invention is a type 1 rheumatoid arthritis, comprising as an active ingredient an inhibitor of the expression or activity of a protein that encodes a tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or the same.
  • the present invention comprises the step of administering a pharmaceutical composition comprising an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than humans, rheumatoid arthritis type 1, It provides a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple myalgia.
  • the present invention includes an expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, rheumatoid arthritis type 1, rheumatoid type 2
  • tetraspanin-2 Tetraspanin-2, TSPAN-2
  • rheumatoid arthritis type 1 rheumatoid type 2
  • a food composition for preventing or ameliorating any one or more diseases selected from the group consisting of arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
  • the present invention comprises the steps of: (a) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal with rheumatoid arthritis as a control; (b) administering a candidate substance expected to be able to treat rheumatoid arthritis in the experimental animal; (c) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal to which the candidate substance, an experimental group, is administered; And (d) comparing the expression level of the TSPAN-2 protein or mRNA encoding the same measured in the control group with that measured in the experimental group, when the measured level in the experimental group is lower than that measured in the control group.
  • a method for screening a preparation for preventing or treating rheumatoid arthritis comprising determining that it can be used as a preventive agent for preventing the development of rheumatoid arthritis or a therapeutic agent for treating rheumatoid arthritis.
  • the present invention comprises the steps of (a) treating a candidate substance for the treatment of rheumatoid arthritis in isolated synovial cells expressing TSPAN-2 protein or mRNA encoding the same; (b) measuring the expression level of TSPAN-2 protein or mRNA encoding it in isolated synovial cells treated with the candidate substance; And (c) when the level of TSPAN-2 protein or mRNA expression encoding it measured in step (b) is lower than that of isolated synovial cells in which the candidate substance is not treated, the candidate substance develops rheumatoid arthritis.
  • It provides a screening method for preventing or treating rheumatoid arthritis comprising the step of determining that it can be used as a prophylactic agent for preventing or a therapeutic agent for treating rheumatoid arthritis.
  • the present invention is to prevent or treat any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia. It provides a use of an inhibitor of the expression or activity of a tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding the same to prepare a drug preparation.
  • Tetraspanin-2 TSPAN-2
  • the present invention provides an effective amount of a composition comprising an inhibitor of the expression or activity of an mRNA encoding the tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or the same as an active ingredient.
  • a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia comprising administering to the individual in need. .
  • the present invention is a type of rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it,
  • a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid multiple muscle pain.
  • the pharmaceutical composition of the present invention may be a composition comprising an inhibitor of expression or activity of TSPAN-2 protein or mRNA encoding it as an active ingredient, and an inhibitor of expression or activity of TSPAN-2 protein or mRNA encoding it as an active ingredient It may be a composition consisting of, or may be a composition consisting essentially of TSPAN-2 protein or an mRNA that encodes or inhibits the activity or activity as an active ingredient.
  • the term'comprising' is used in the same sense as'including' or'characterized by', and in the composition or method according to the present invention, specifically It does not exclude additional components or method steps not mentioned.
  • the term'consisting of' means excluding additional elements, steps, or ingredients not separately described.
  • the term'consisting essentially of' means that in the scope of the composition or method, it may include the substance or step described in addition to the substance or step which does not substantially affect its basic properties. .
  • 'Treatment' refers to suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving the condition of the disease, improving, alleviating or improving the prognosis. do.
  • the term'prevention' as used in the present invention means all actions that suppress the onset of disease or delay the progression.
  • TSPAN-2 is used interchangeably with'polypeptide' or'peptide' and refers to a polymer of amino acid residues, such as is commonly found in natural proteins.
  • The'fragment' of the TSPAN-2 protein refers to a peptide of a portion of the TSPAN-2 protein.
  • mRNA messenger RNA or messenger RNA
  • RNA messenger RNA or messenger RNA
  • TSPAN-2 used in the present invention is a cell surface glycoprotein receptor of about 24 to 27 kDa belonging to a member of the tetraspanin family, and plays an important role in regulating cell development, activity, growth and mobility. It is known to modulate signal transduction events. In addition, it is possible to regulate cell adhesion and cell migration, and induce platelet activation and aggregation involved in platelet-induced endothelial cell proliferation. In addition, it is involved in various phenomena in cells, such as promoting muscle cell fusion and contributing to myotube maintenance.
  • the TSPAN-2 protein in the present invention may be derived from mammals, and is preferably derived from humans. Most preferably, the TSPAN-2 protein in the present invention is human TSPAN-2 isoform 1 represented by SEQ ID NO: 1 (NP_001760.1) or human TSPAN-2 isoform 2 represented by SEQ ID NO: 2 (NP_001317241.1) It is characterized by including the amino acid sequence of (NCBI Genbank accession number in parentheses).
  • the TSPAN-2 expression or activity inhibitor is selected from the group consisting of antibodies, compounds, peptides, peptide mimetics, aptamers, natural extracts and synthetic compounds that specifically bind to TSPAN-2 protein or mRNA encoding the same. It is characterized by any one, but is not limited thereto.
  • the inhibitor of expression or activity of TSPAN-2 according to the present invention is preferably an antibody against TSPAN-2, but is not limited thereto.
  • the term "antibody” refers to a substance that reacts when an antigen that is an external substance invades while circulating blood or lymph in the immune system of a living body, and is also called an immunoglobulin as a globulin-based protein formed in lymphoid tissue.
  • Antibodies are proteins produced by B cells and released into body fluids. They bind specifically to antigens.
  • One antibody molecule has two heavy chains and two light chains, each of which is a heavy chain. And the light chain have a variable region at the end of its N-terminal. Each variable region is composed of three complementarity determining regions (CDRs) and four framework regions (FRs).
  • the complementarity determining regions determine the antigen binding specificity of the antibody, and the structure of the variable region It exists as a relatively short peptide sequence maintained by the formation sites.
  • the antibody may be an antibody capable of inhibiting the activity of TSPAN-2 by binding to TSPAN-2.
  • rheumatic arthritis is a different disease from “steoarthritis”. In the case of osteoarthritis, it usually starts after age 40, but rheumatoid arthritis starts between 25 and 50 years of age. In addition, osteoarthritis progresses slowly over the years and initially develops on one side of the body, but rheumatoid arthritis progresses within weeks or months, usually on both sides of the same joint.
  • osteoarthritis affects some joints and rarely occurs in the elbows and wrists, but rheumatoid arthritis affects many joints, including the elbows and wrists, and even during the pain period, osteoarthritis intensifies the pain after using the joint in the evening, but rheumatoid arthritis The pain intensifies in the morning.
  • rheumatoid arthritis is a typical'autoimmune disease' in which the genetic predisposition and environmental effects are complicated and cause abnormalities of the immune system and inflammation of the synovial membrane.
  • Rheumatoid arthritis is a tissue-type gene that is well known, and it is known that it occurs better in women than in men under the influence of female hormones, and a viral or bacterial infection may be a trigger.
  • osteoarthritis is a disease in which the bones and ligaments forming the joints are damaged by inflammation or degenerative changes of cartilage protecting the joints, and inflammation and pain are commonly called'degenerative arthritis'. Genetic predisposition, metabolic factors, biochemical factors, epidemiological factors, joint shape, and local inflammation affect each other and occur when the cartilage of the joint is damaged.
  • Osteoarthritis is a type of joint disease caused by the destruction of articular cartilage and the underlying bone. The most common symptoms are joint pain and stiffness. Although this disease does not affect other parts of the body, rheumatoid arthritis can affect other parts of the body as a systemic disease in other parts of the body.
  • the TSPAN-2 protein expression inhibitor is selected from the group consisting of antisense oligonucleotide (antisense oligonucleotide), siRNA, shRNA, miRNA, ribozyme, DNAzyme and protein nucleic acid (PNA) that complementarily bind to TSPAN-2 mRNA. It can be one.
  • the TSPAN-2 mRNA is most preferably human TSPAN-2 mRNA transcript variant 1 (NM_001769.3) represented by SEQ ID NO: 3 or human TSPAN-2 mRNA transcript variant 2 (NM_001330312.1) represented by SEQ ID NO: 4 It contains the base sequence of (NCBI Genbank accession number in parentheses). The characteristics of the coding region (exon) of the human TSPAN-2 mRNA transcript variants described above are confirmed in sequence information obtained by searching the NCBI database with the Genbank accession number in parentheses.
  • the TSPAN2 protein expression inhibitor according to the present invention may preferably be shRNA that binds complementarily to TSPAN2 mRNA and induces degradation of the mRNA.
  • the siRNA for TSPAN2 may specifically include any one nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 31.
  • The'siRNA small interfering RNA or short interfering RNA or silencing RNA
  • RNA It is a short double-stranded RNA that causes interference) and is composed of 20 to 25 nucleotides complementary to a specific region of the target mRNA.
  • an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex to bind to the target mRNA, and the argonaute protein in the RISC complex cuts and degrades the target mRNA Or inhibits the binding of proteins and ribosomes, which are important for protein translation, to mRNA, to suppress the expression of specific genes.
  • RISC RNA-induced silencing complex
  • the shRNA according to the present invention is a substance that induces RNA interference, and is a molecule of about 20 bases or more having a double-stranded structure within a molecule and a hairpin-like structure by including a partially sequential base sequence in a single-stranded RNA.
  • a siRNA of 21 to 23 base pairs is generated by an RNase III (ribonuclease III) enzyme called a dicer in the cell to induce RNAi.
  • the siRNA or shRNA according to the present invention may be added with various modifications to improve the stability of the oligonucleotide in vivo, to impart nucleic acid degrading enzyme resistance, and to reduce non-specific immune responses.
  • the modification of the oligonucleotide is that the OH group at the 2'carbon position of the sugar structure in one or more nucleotides is -CH3(methyl), -OCH3(methoxy), -NH2, -F, -O-2-methoxyethyl, -O- Propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl Modification by substitution with; Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from the modification of nucleotide
  • composition of the present invention can be applied in vivo through various transport methods such as liposomes and polymers, which are commonly known in the field of gene therapy.
  • the nucleic acid molecule of the present invention is included in a gene delivery system, or is naked DNA.
  • a gene delivery system comprising a nucleic acid molecule encoding a reprogramming factor is used as an active ingredient, it is preferred that the reprogramming factor-encoding nucleic acid molecule is present in a suitable expression construct.
  • gene transfer means that the gene is carried into a cell and has the same meaning as the gene's intracellular transduction. At the tissue level, the term gene transfer has the same meaning as the spread of the gene.
  • the reprogramming factor-encoding nucleic acid molecule can be operably linked to a promoter.
  • operably linked refers to a functional binding between a nucleic acid expression regulatory sequence (eg, a promoter, signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is It controls the transcription and/or translation of the other nucleic acid sequence.
  • a promoter coupled to a reprogramming factor-encoding nucleic acid molecule is capable of controlling transcription of a reprogramming factor-encoding nucleic acid molecule by operating in an animal cell (eg, a mammalian cell), derived from a mammalian virus.
  • an animal cell eg, a mammalian cell
  • a promoter and a promoter derived from the genome of a mammalian cell such as the cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha Promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and promoter of human GM-CSF gene Including, but not limited to.
  • CMV cytomegalo virus
  • adenovirus late promoter vaccinia virus 7.5K promoter
  • SV40 promoter HSV tk promoter
  • RSV promoter EF1 alpha Promoter
  • beta-actin promoter promoter of human IL-2 gene
  • promoter of human IFN gene promoter of human IL-4 gene
  • the nucleic acid molecule of the present invention can be produced in various forms, for example, (i) a naked recombinant DNA molecule using a plasmid as a carrier, (ii) a viral vector, (iii) liposomes or niosomes, (iv) It can be produced in the form of a lipid-gene complex (LipidDNA complex) or (v) a polymer-gene complex (Polymer-DNA complex).
  • Reprogramming factor-encoding nucleic acid molecules can be applied to all gene delivery systems used in conventional gene therapy, such as plasmids, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, It can be applied to liposomes or niosomes.
  • the gene delivery system of the present invention may be implemented as a viral vector.
  • the method for introducing the gene delivery system of the present invention into cells described above may be carried out through various methods known in the art.
  • T-cells As an effective platform for targeted delivery of an inhibitor to TSPAN2 to a target site, T-cells, B-cells, macrophage-cells, NK-cells and stem cells can be used.
  • the pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid multiple myalgia is a tetraspa according to the present invention
  • Nin-2 Tetraspanin-2, TSPAN-2
  • TSPAN-2 may contain at least one active ingredient that exhibits a function similar to the expression or activity inhibitor.
  • additional ingredients will further enhance the therapeutic effect of the compositions according to the invention.
  • the pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple muscle pain is a pharmaceutically acceptable carrier It may further include.
  • Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration.
  • Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.
  • carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like.
  • stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those known in the art.
  • the pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method.
  • it can be administered orally or parenterally, and the parenteral administration method is not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration.
  • the pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.
  • one or more buffers e.g. saline or PBS
  • antioxidants e.g. bacteriostats
  • chelating agents e.g. EDTA or glutathione
  • fillers e.g. extenders, binders, adjuvants (e.g. aluminum hydroxide) Side)
  • a suspending agent e.g. saline or PBS
  • a thickening agent wetting agent e.g. a disintegrating agent or a surfactant
  • a diluent or an excipient e.g. aluminum hydroxide
  • Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, capsules, etc., and these solid preparations include, for example, at least one excipient in the pharmaceutical composition of the present invention.
  • starch including corn starch, wheat starch, rice starch, potato starch, etc.
  • calcium carbonate sucrose, lactose, dextrose, sorbitol, mannitol (mannitol), xylitol, erythritol maltitol, cellulose, methyl celulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin
  • It can be prepared by mixing the back.
  • tablets or tablets of sugar can be obtained by blending the active ingredient with a solid excipient and then grinding it and adding a suitable adjuvant to the granule mixture.
  • Liquid preparations for oral use include suspensions, intravenous solutions, emulsions or syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances or preservatives, in addition to water or liquid paraffin, a simple diluent commonly used. .
  • cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and a preservative. .
  • the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations, and nasal inhalants with suitable parenteral carriers.
  • suitable parenteral carriers include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and/or vegetable oils.
  • solvents or dispersion media comprising water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and/or vegetable oils.
  • suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with ethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose.
  • Isotonic solutions such as can be used.
  • various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be additionally included.
  • the injection may further include isotonic agents such as sugars or sodium chloride in most cases.
  • transdermal dosage forms In the case of transdermal dosage forms, ointments, creams, lotions, gels, external solutions, pasta, linen agents, aerosols, and the like are included.
  • transdermal administration means that the pharmaceutical composition is topically applied to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.
  • suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable TSPAN-2 proteins of the invention or mRNA expression or activity inhibitors encoding them
  • a suitable gas it can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer.
  • the dosage unit can be determined by providing a valve that delivers a metered amount.
  • gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mixture of a compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in formulas generally known in all pharmaceutical chemistry.
  • the pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia is the expression of TSPAN-2 or
  • the active inhibitor is included in an effective amount, it is possible to provide a desirable disease prevention and treatment effect.
  • the term'effective amount' refers to an amount that exhibits a higher response than a negative control group, and preferably refers to an amount sufficient to relieve symptoms of rheumatoid arthritis, especially edema.
  • an inhibitor of TSPAN-2 expression or activity may be included in an amount of 0.01 to 99.99%, and the remaining amount may be occupied by a pharmaceutically acceptable carrier.
  • the effective amount of the TSPAN-2 expression or activity inhibitor contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.
  • the total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. .
  • the pharmaceutical composition of the present invention can vary the content of the active ingredient according to the degree of disease. When administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day, based on the inhibitor of expression or activity of TSPAN-2, and when administered orally, TSPAN- Based on the expression or activity inhibitor of 2, it can be divided into 1 to several times to be administered in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day.
  • the dose of the TSPAN-2 expression or activity inhibitor is considered by considering various factors such as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, as well as the route and frequency of administration of the pharmaceutical composition. Since the effective dosage for the drug is determined, those who have ordinary knowledge in the art in consideration of this point may express the TSPAN-2 expression or activity inhibitor of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, It may be possible to determine an appropriate effective dosage according to a specific use for the prevention and treatment of any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid polymyalgia.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effect of the present invention.
  • the pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia is also expressed by TSPAN-2 Or it can be provided as a formulation of an external preparation containing an active inhibitor as an active ingredient.
  • the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is used as an external preparation for skin, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Skin such as activators, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion blockers, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic actives, lipophilic actives or lipid vesicles It may contain adjuvants commonly used in the field of dermatology such as any other ingredients commonly used in external preparations. In addition, the ingredients may be introduced in an amount commonly used in the field of skin science.
  • a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia is provided as an external preparation for skin , but is not limited thereto, may be a formulation such as ointment, patch, gel, cream or spray.
  • the present invention comprises the step of administering a pharmaceutical composition comprising an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than a human, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis It provides a method for treating any one or more diseases selected from the group consisting of pediatric rheumatoid arthritis and rheumatoid multiple muscle pain.
  • the term "individual" in the present invention has or develops one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia of the present invention.
  • any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia can be alleviated or treated.
  • the alleviation refers to all actions in which the disease is improved or beneficial by administration of the composition according to the present invention.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a subject in any suitable way, and the route of administration can be administered through various routes, oral or parenteral, as long as the target tissue can be reached. .
  • the pharmaceutical composition may be appropriately administered to a subject according to the purpose or need, depending on the conventional method, route of administration, and dosage used in the art.
  • routes of administration may be oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • an appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention to be actually administered is the type of symptom to be treated, the route of administration, sex, and health status. , Diet, the age and weight of the individual, and the severity of the disease.
  • composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, and can be easily determined by those skilled in the art.
  • the present invention includes the expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis And it provides a food composition for preventing or improving any one or more diseases selected from the group consisting of rheumatoid multiple muscle pain.
  • the food composition according to the present invention can be used for the prevention or improvement of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia.
  • the food composition of the present invention includes all forms of functional foods, nutritional supplements, health foods, food additives, etc., and targets animals including humans or livestock. Is done. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
  • Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
  • Beverages including alcoholic beverages
  • fruits and processed foods thereof e.g. canned fruits, canned foods, jams, marmalades, etc.
  • fish e.g. ham, sausages
  • breads and noodles e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.
  • juice various drinks, cookies, syrup, dairy products (e.g.
  • TSPAN-2 can be prepared by adding the inhibitor of the expression or activity of TSPAN-2.
  • it is not limited to the nutritional supplement, but may be prepared by adding an inhibitor of TSPAN-2 expression or activity to capsules, tablets, pills, and the like.
  • the expression or activity inhibitor itself of TSPAN-2 is prepared in the form of tea, juice, and drink to liquefy, granulate, and encapsulate for drinking (health drink). And powdered.
  • TSPAN-2 expression or activity inhibitor in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.
  • an inhibitor of the expression or activity of TSPAN-2 and the improvement effect of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia It can be prepared in the form of a composition by mixing with known active ingredients.
  • the health drink composition may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, such as conventional beverages.
  • the natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol.
  • Sweeteners include natural sweeteners such as thaumatin and stevia extracts; Synthetic sweeteners such as saccharin and aspartame can be used.
  • the proportion of the natural carbohydrate is generally about 0.01 to 0.04 g per 100 mL of the composition of the present invention, preferably about 0.02 to 0.03 g.
  • the inhibitor of expression or activity of TSPAN-2 When contained as an active ingredient of a food composition, the amount is not particularly limited to an amount effective to achieve a symptom improvement effect, but is preferably 0.01 to 100% by weight relative to the total weight of the total composition.
  • the food composition of the present invention improves any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia with an inhibitor of expression or activity of TSPAN-2 It can be prepared by mixing with other active ingredients known to be effective.
  • the food composition of the present invention When the food composition of the present invention is used as a health food in addition to the above, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH It may contain a regulator, stabilizer, preservative, glycerin, alcohol or carbonic acid.
  • the health food of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage or vegetable beverage. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides a method for screening a formulation for the treatment of rheumatoid arthritis.
  • the screening method comprises the steps of (a) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal with rheumatoid arthritis as a control;
  • the candidate substance is rheumatoid when the level measured in the experimental group is lower than that measured in the control group. And determining that it can be used as a prophylactic agent for preventing the development of arthritis or a therapeutic agent for treating rheumatoid arthritis.
  • the screening method may include (a) processing a candidate substance for treating rheumatoid arthritis in isolated synovial cells expressing TSPAN-2 protein or mRNA encoding the same;
  • step (c) When the level of TSPAN-2 protein or mRNA expression encoding it measured in step (b) is lower than that of isolated synovial cells in which the candidate substance is not treated, the candidate substance is prevented from developing rheumatoid arthritis. And determining that it can be used as a preventive agent for preventing or a therapeutic agent for treating rheumatoid arthritis.
  • candidate refers to a substance to test the activity of alleviating or treating rheumatoid arthritis symptoms, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide range of compounds, etc. Any molecule can be included. These candidate substances also include natural as well as synthetic substances.
  • RT-PCR Reverse Transcription-Polymerase chain Reaction
  • Enzyme Immunity It can be measured by any one method selected from the group consisting of an assay (ELISA), immunohistochemistry, Western blotting and flow cytometry (FACS), but is not limited thereto.
  • ELISA Reverse Transcription-Polymerase chain Reaction
  • FACS flow cytometry
  • the candidate materials screened by the screening method of the present invention can be used for the relief or treatment of rheumatoid arthritis symptoms that can inhibit the expression or activity of TSPAN-2 protein or mRNA encoding it.
  • the present invention is a type of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid arthritis and tetraspanin-2 for preparing an agent for the prevention or treatment of any one or more diseases selected from the group consisting of rheumatoid arthritis (Tetraspanin-2, TSPAN-2) provides the use of inhibitors for the expression or activity of proteins or mRNAs encoding them.
  • the present invention comprises the step of administering an effective amount of a composition comprising an inhibitor or an expression or activity inhibitor of a protein or mRNA encoding tetraspanin-2 (Tetraspanin-2, TSPAN-2) to an individual in need thereof It provides a method of treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple muscle pain.
  • the'individual' may be an animal, preferably a mammal, particularly an animal including a human, or may be cells, tissues, organs, etc. derived from an animal. .
  • the subject may be a patient in need of the effect.
  • The'treatment' of the present invention collectively refers to improving diseases or symptoms of such diseases, such as rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia, Healing, substantially preventing, or improving the condition, including, but not limited to, alleviating, healing, or preventing one symptom or most symptoms resulting from the disease no.
  • diseases or symptoms of such diseases such as rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia, Healing, substantially preventing, or improving the condition, including, but not limited to, alleviating, healing, or preventing one symptom or most symptoms resulting from the disease no.
  • the present invention confirmed that swelling due to rheumatoid arthritis is alleviated when the expression or activity of TSPAN-2 is suppressed, and the TSPAN-2 protein or mRNA expression or activity inhibitor encoding the same is used for rheumatoid arthritis type 1, type 2 Rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid arthritis can be utilized as an agent for the prevention, improvement or treatment of any one or more diseases selected from the group consisting of, and administering a candidate substance to encode TSPAN-2 protein or the same
  • a candidate substance to encode TSPAN-2 protein or the same
  • 1 is a result of confirming the therapeutic effect by inducing rheumatoid arthritis in an animal model, and then administering an anti-TSPAN2 antibody at intervals of one week.
  • 2A to 2C are results confirming the expression of TSPAN2 in human fibroblast-like synoviocytes (hFLS).
  • 4A to 4C are results of confirming the expression of factors having a major influence on rheumatoid arthritis after treatment with an anti-TSPAN2 antibody.
  • FIG. 5A to 5C show results of synovial tissue analysis according to suppression of TSPAN2 expression.
  • FIG. 5A shows normal synovial tissue
  • FIG. 5B shows synovial tissue in a rheumatoid arthritis model
  • FIG. 5C shows optical micrographs after H&E staining of synovial tissue during anti-TSPAN2 antibody treatment.
  • FIG. 5A the synovial membrane layer is well preserved, and inflammatory infiltration is not observed much
  • FIG. 5B tissue is significantly damaged by inflammation, and the result of inflammatory infiltration is clearly shown.
  • Fig. 5c the synovial lining cell layer is well preserved, and inflammatory infiltration is rarely observed.
  • the left, middle, and right photographs respectively show magnifications of x40, x200, and x400.
  • FIGS. 6A to 6B show the therapeutic effect of anti-TSPAN2 antibody treatment in a rat rheumatoid arthritis model.
  • Figure 6a is a weight bearing test to check the balance and sense of movement of the rat
  • Figure 6b shows the results of measuring the thickness of the sole of the rats induced rheumatism.
  • TSPAN-2 expression or activity according to the present invention was confirmed in vivo.
  • Sprague dooli rats were obtained from Harlan Laboratories, Charles River Laboratories or Janvier Labs. All rats were bred in a pathogen-free environment, and were allowed to ingest sterile standard feed (Ralston Purina, St. Louis, MO) and water freely. 100 ⁇ l pristane (2,6,10,14-tetramethylpentadecane, 95%, Acros Organics, Morris Plains, NJ, USA) on the dorsal side of the tail base of the 8-11 week old rat.
  • Rheumatoid arthritis was induced by intradermal injection. Or oil-induced and collagen by intradermally injecting 100 ⁇ l IFA (incomplete freunds adjuvant, Difco Laboratories, Detroit, MI, USA) containing 0.4 mg of mycobacterium butyricum (Difco), 300 ⁇ l of pure IFA and 0.3 mg of pepsin digestion Induced arthritis.
  • Type II collagen (CII) purified from rat cartilage sarcoma was dissolved in 150 ⁇ L of 0.1M acetic acid and emulsified with the same amount of IFA, respectively.
  • Synthetic pristane was purchased from Sigma-Aldrich (P2870, St. Louis, MO, USA). Treated and untreated rats carrying out these different immunization protocols were housed together in cages. ELISA was used to detect various cytokines in serum and joint tissues, and the severity of arthritis and hind paw thickness were quantified.
  • Example 2 Confirmation of the therapeutic effect of rheumatoid arthritis according to TSPAN-2 expression or activity inhibition
  • Example 3 Analysis of TSPAN2 expression in human fibroblast-like synovial cells
  • hFLS human fibroblast-like synoviocytes
  • a mAb hybridoma (RA1Ab, RA2Ab, RA3Ab) culture medium for TSPAN2 was used. At this time, normal fibroblasts were used as negative control. Goat anti-mouse IgG (H+L) F(ab')2 fragment labeled with FITC (Fluorescein isothiocyanate) after washing with FACS buffer (PBS containing 0.1% BSA and 0.05% sodium azide) (Jackson ImmunoResearch) and reacted at 4°C for 15 minutes. After washing with FACS buffer, hFLS was analyzed by BD FACS Calibur.
  • FACS buffer PBS containing 0.1% BSA and 0.05% sodium azide
  • TSPAN2 was expressed in human rheumatoid arthritis hFLS as shown in FIGS. 2A to 2C.
  • RA1Ab binds most strongly, and the binding between RA2Ab and RA3Ab is relatively weak.
  • the hFLS obtained in three patients with rheumatoid arthritis showed a similar trend in the binding response to these three antibodies, suggesting the possibility that TSPAN2 does not show a variation among patients and is generally expressed in rheumatoid arthritis hFLS.
  • TSPAN2 expression was confirmed in rheumatoid arthritis synovial cells, and an effect of suppressing TSPAN2 expression was also confirmed using an antibody.
  • Human monoclonal TSPAN2 manufactured by Applon
  • goat polyclonal actin antibodies and secondary antibodies were prepared for this purpose.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNF- ⁇ tumor necrosis factor-alpha
  • IFN- ⁇ interferon-gamma
  • anti-TSPAN2 anti-TSPAN2
  • NHDF-adult Amaxa Human Dermal Fibroblast Nucleofector Kit
  • hFLSs were then incubated in DMEM with 10% FCS in 6-well plates for a suitable time and synchronized in DMEM with 0.1% FCS. hFLSs were treated with culture medium for 15 minutes and cell lysates were obtained as previously described.
  • Total cell lysate (50 ⁇ g) was fractionated on 10% SDS-PAGE buffered with Tris-glycine and transferred to a nitrocellulose membrane (Biorad, Hercules, CA, USA). The membrane was blocked with 5% skim milk in 0.05% Tween 20/Tris-buffered saline (TBS) for 1 hour at room temperature and then incubated with primary antibody (1:1000). The blot was then incubated for 2 hours at room temperature in the secondary antibody. Immunoreactive proteins were detected by enhanced chemiluminescence (Perkin Elmer, Waltham, MA, USA) and autoradiography, analyzed using NIH Image (version 1.63) and normalized to actin expression.
  • IL-1 ⁇ a causative agent of cartilage and bone erosion of rheumatoid arthritis
  • IL-1 ⁇ a causative agent of cartilage and bone erosion of rheumatoid arthritis
  • Example 5 Confirmation of changes in synovial tissue in rheumatoid arthritis animal model
  • Antibodies were injected subcutaneously once a week, and rats were sacrificed using CO 2 after weight bearing results and behavior observation experiments, and the lower leg including the joint was cut and fixed after deliming. After that, the synovial tissue was stained with H&E (hematoxylin & eosin) staining for comparative analysis.
  • H&E hematoxylin & eosin
  • FIGS. 5A to 5C in the normal synovial tissue (FIG. 5A), the synovial membrane layer was well preserved, and inflammatory infiltration was not observed much, and when inducing rheumatoid arthritis (FIG. 5B) ) It was confirmed that the tissue was significantly damaged by inflammation, and the inflammatory infiltration proceeded significantly. In contrast, when the anti-TSPAN2 antibody was treated against the rheumatoid arthritis model (FIG. 5C), it was confirmed that the synovial membrane layer was well preserved, and inflammatory infiltration was not observed much, similar to normal synovial tissue. It was confirmed that it was excellent.
  • TSPAN-2 protein or an mRNA expression or activity inhibitor encoding the same It can be used as a preparation for improvement or treatment, and by measuring the expression level of TSPAN-2 protein or mRNA encoding it by administering a candidate substance, a substance having a therapeutic effect on rheumatoid arthritis can be selected.

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Abstract

The present invention relates to a composition for preventing, alleviating or treating any one disease selected from the group consisting of type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and polymyalgia rheumatica, containing an agent that inhibits the expression or activity of a TSPAN-2 protein or an mRNA enconding same.

Description

테트라스파닌-2 억제제를 유효성분으로 포함하는 류마티스 관절염 예방, 개선 또는 치료용 조성물A composition for preventing, improving or treating rheumatoid arthritis comprising a tetraspanin-2 inhibitor as an active ingredient
본 출원은 2019년 1월 14일에 출원된 대한민국 특허출원 제10-2019-0004869호, 2019년 2월 20일에 출원된 대한민국 특허출원 제10-2019-0020148호를 우선권으로 주장하고, 상기 명세서들 전체는 본 출원의 참고문헌이다.This application claims priority to Korean Patent Application No. 10-2019-0004869 filed on January 14, 2019, and Korean Patent Application No. 10-2019-0020148 filed on February 20, 2019 as priority, All of these are references of this application.
본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방, 개선 또는 치료용 조성물 및 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 수준을 측정하여 류마티스 관절염 치료 물질을 선별하는 방법에 관한 것이다.The present invention prevents any one or more diseases selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, The present invention relates to a method for selecting a therapeutic agent for rheumatoid arthritis by measuring an improvement or treatment composition and an expression level of tetraspanin-2 (Tetraspanin-2, TSPAN-2).
류마티스 관절염(rheumatoid arthritis)은 활막 세포의 염증과 증식을 특징으로 하는, 만성 염증성 질환으로서, 골관절염과 달리 관절 주위 뼈의 골다공증 및 골미란 등이 발생한다. 류마티스 관절염은 활막(synovial membrane)의 염증이 관절막(joint capsule)과 인대(ligament), 건(tendon)으로 퍼지는 단계(1 단계), 관절연골(joint cartilage)의 점차적인 파괴로 관절 간격이 좁아지고 관절막과 인대의 장력이 소실되는 단계(2 단계), 염증이 뼈로 침범하여 뼈의 부분적 침식이 발생하는 단계(3 단계), 및 관절기능이 소실되는 단계(4 단계)로 진행되게 된다.Rheumatoid arthritis (rheumatoid arthritis) is a chronic inflammatory disease characterized by inflammation and proliferation of synovial cells, and unlike osteoarthritis, osteoporosis and bone erosion of the bones around the joints occur. Rheumatoid arthritis is an inflammation of the synovial membrane that spreads to the joint capsule, ligament, and tendon (step 1), and the joint gap narrows due to gradual destruction of the joint cartilage. The tension of the joint membrane and ligament is lost (step 2), inflammation invades the bone, and partial erosion of the bone occurs (step 3), and the joint function is lost (step 4).
종래에 류마티스 관절염의 치료를 위해서는, NSAID, 살리실레이트, COX-2 저해제 등의 비스테로이드 항염증제; 프레드니솔론, 트리암시놀론 등의 부신피질호르몬 제제; 메토트렉세이트(methotrexate), 설파살라진(sulfasalazine) 등의 항류마티스 약제(Disease-Modifying Anti-rheumatic Drugs, DMARDs); 및 에타너셉트(etanercept), 인플릭시맵(infliximab), 아달리무맵(adalimumab) 등의 생물학적 제제 등의 약물이 사용되고 있다. 통상, 증상 발현 초기에는 비스테로이드 항염증제 및 부신피질호르몬 제제가 사용되며, 증상 및 질병 활성도를 고려하여 항류마티스 약제가 사용되고, 심한 경우에는 생물학적 제제를 사용하거나 병합 요법 등이 임상에서 사용되고 있다.Conventionally, for the treatment of rheumatoid arthritis, non-steroidal anti-inflammatory agents such as NSAID, salicylate, and COX-2 inhibitors; Adrenal cortical hormone preparations such as prednisolone and triamcinolone; Disease-Modifying Anti-rheumatic Drugs (DMARDs) such as methotrexate and sulfasalazine; And drugs such as biological agents such as etanercept, infliximab, and adalimumab. Normally, nonsteroidal anti-inflammatory drugs and adrenal cortical hormone preparations are used in the early stages of symptom expression, antirheumatic drugs are used in consideration of symptoms and disease activity, and in severe cases, biological agents or combination therapy are used in clinical practice.
그러나, 생물학적 제제는 기존 치료제들에 비해 고가의 치료 비용이 발생하여 의료 현장에서 적용이 곤란한 문제점이 있다. 상대적으로 치료비용이 저렴한 NSAID 등의 비스테로이드 항염증제는 대표적인 위장관 부작용을 유발하는 약물이며, 메토트렉세이트, 설파살라진 등의 항류마티스 약제 또한 위장 장애가 주요 부작용으로 지적되고 있다. However, the biological agent has a problem in that it is difficult to apply in the medical field due to an expensive treatment cost compared to existing therapeutic agents. Nonsteroidal anti-inflammatory drugs, such as NSAID, which are relatively inexpensive to treat, are drugs that cause typical gastrointestinal side effects, and antirheumatic drugs such as methotrexate and sulfasalazine are also noted as gastrointestinal disorders.
한편, 테트라스파닌-2(Tetraspanin-2, TSPAN-2)는 인간에서 TSPAN2 유전자에 의해 암호화되는 단백질이다. 이 유전자에 의해 코딩되는 단백질은 tetraspanin family라고도 알려진 transmembrane 4 superfamily의 구성원이다. 이들 구성원의 대부분은 4개의 소수성 도메인인, 막관통(transmembrane) 도메인으로 구성된 막 단백질이며 거의 모든 동물 세포에서 발견된다. 상기 TSPAN-2 단백질은 세포 성장, 접착 및 분화를 포함하는 근본적인 세포학적 과정에 관련되어 있다(Todd, S. C et al., Biochim. Biophys. Acta. 1399, 101104, 1998). 테트라스파닌 패밀리 멤버는 고도로 보존된 아미노산 서열을 갖는 경향이 있다(Hemler, M. E et al., J. Cell Biol. 155, 11031107. 2001).Meanwhile, tetraspanin-2 (Tetraspanin-2, TSPAN-2) is a protein encoded by the TSPAN2 gene in humans. The protein encoded by this gene is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members are membrane proteins composed of four hydrophobic domains, transmembrane domains, and are found in almost all animal cells. The TSPAN-2 protein is involved in fundamental cytological processes including cell growth, adhesion and differentiation (Todd, S. C et al., Biochim. Biophys. Acta. 1399, 101104, 1998). The tetraspanin family members tend to have a highly conserved amino acid sequence (Hemler, M. E et al., J. Cell Biol. 155, 11031107. 2001).
그러나 본 발명의 이전에는 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성을 타겟으로 하는 류마티스 관절염 치료제에 관해서 보고된 바 없었다.However, prior to the present invention, there has been no report on a therapeutic agent for rheumatoid arthritis targeting the expression or activity of TSPAN-2 protein or mRNA encoding it.
이에 본 발명의 발명자들은 류마티스 관절염을 효과적으로 치료할 수 있는 제제를 탐색한 결과, TSPAN-2의 발현을 억제할 시 류마티스 관절염에 의한 붓기가 완화되고 2주 만에 증상이 치료되는 것을 확인하여 본 발명을 완성하였다.Accordingly, the inventors of the present invention searched for an agent capable of effectively treating rheumatoid arthritis, and when suppressing the expression of TSPAN-2, the swelling caused by rheumatoid arthritis was alleviated and the symptoms were treated within two weeks. Completed.
따라서 본 발명의 목적은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is a type 1 rheumatoid arthritis, a type 2 rheumatoid arthritis, gestational activity, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding the same. It is to provide a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
또한 본 발명의 다른 목적은 TSPAN-2의 발현 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 치료 방법을 제공하는 것이다.In addition, another object of the present invention comprises the step of administering a pharmaceutical composition containing an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than humans, rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational It is to provide a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
또한 본 발명의 다른 목적은 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제를 유효성분으로 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 개선용 식품 조성물을 제공하는 것이다.In addition, another object of the present invention includes an expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, children It is to provide a food composition for preventing or improving any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid multiple muscle pain.
또한 본 발명의 다른 목적은 류마티스 관절염의 예방 또는 치료용 제제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a preparation for the prevention or treatment of rheumatoid arthritis.
또한 본 발명의 다른 목적은 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 제제를 제조하기 위한 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제의 용도를 제공하는 것이다.In addition, another object of the present invention is a type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis, and rheumatoid polymyalgia selected from the group consisting of a prophylactic or therapeutic agent for the preparation of any one or more diseases It is to provide the use of an inhibitor of the expression or activity of a Spanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it.
또한 본 발명의 다른 목적은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 치료 방법을 제공하는 것이다.In addition, another object of the present invention is to administer an effective amount of a composition comprising an inhibitor or expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it to an individual in need thereof It is to provide a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia.
상기와 같은 목적을 달성하기 위하여, 본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention is a type 1 rheumatoid arthritis, comprising as an active ingredient an inhibitor of the expression or activity of a protein that encodes a tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or the same. Provided is a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 2, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid multiple myalgia.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 TSPAN-2의 발현 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 치료 방법을 제공한다.In order to achieve another object of the present invention, the present invention comprises the step of administering a pharmaceutical composition comprising an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than humans, rheumatoid arthritis type 1, It provides a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple myalgia.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제를 유효성분으로 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 개선용 식품 조성물을 제공한다.In order to achieve another object of the present invention, the present invention includes an expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, rheumatoid arthritis type 1, rheumatoid type 2 Provided is a food composition for preventing or ameliorating any one or more diseases selected from the group consisting of arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 (a) 대조군인 류마티스 관절염이 발병된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; (b) 상기 실험동물에서 류마티스 관절염을 치료할 수 있을 것으로 예상되는 후보물질을 투여하는 단계; (c) 실험군인 상기 후보물질이 투여된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및 (d) 대조군에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 실험군에서 측정된 것과 비교하여, 대조군에서 측정된 것보다도 실험군에서 측정된 것이 낮은 수준을 나타내는 경우, 상기 후보물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는, 류마티스 관절염의 예방 또는 치료용 제제의 스크리닝 방법을 제공한다.In order to achieve another object of the present invention, the present invention comprises the steps of: (a) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal with rheumatoid arthritis as a control; (b) administering a candidate substance expected to be able to treat rheumatoid arthritis in the experimental animal; (c) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal to which the candidate substance, an experimental group, is administered; And (d) comparing the expression level of the TSPAN-2 protein or mRNA encoding the same measured in the control group with that measured in the experimental group, when the measured level in the experimental group is lower than that measured in the control group. Provided is a method for screening a preparation for preventing or treating rheumatoid arthritis, comprising determining that it can be used as a preventive agent for preventing the development of rheumatoid arthritis or a therapeutic agent for treating rheumatoid arthritis.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 (a) TSPAN-2 단백질 또는 이를 암호화하는 mRNA를 발현하는 분리된 활막 세포에 류마티스 관절염 치료 후보 물질을 처리하는 단계; (b) 상기 후보 물질이 처리된 분리된 활막 세포에서 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및 (c) 상기 (b) 단계에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 수준이 후보 물질이 처리되지 않은 분리된 활막 세포에 비해 낮은 수준을 나타내는 경우, 상기 후보 물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는, 류마티스 관절염의 예방 또는 치료용 제제의 스크리닝 방법을 제공한다.In order to achieve another object of the present invention, the present invention comprises the steps of (a) treating a candidate substance for the treatment of rheumatoid arthritis in isolated synovial cells expressing TSPAN-2 protein or mRNA encoding the same; (b) measuring the expression level of TSPAN-2 protein or mRNA encoding it in isolated synovial cells treated with the candidate substance; And (c) when the level of TSPAN-2 protein or mRNA expression encoding it measured in step (b) is lower than that of isolated synovial cells in which the candidate substance is not treated, the candidate substance develops rheumatoid arthritis. It provides a screening method for preventing or treating rheumatoid arthritis comprising the step of determining that it can be used as a prophylactic agent for preventing or a therapeutic agent for treating rheumatoid arthritis.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 제제를 제조하기 위한 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제의 용도를 제공한다.In order to achieve another object of the present invention, the present invention is to prevent or treat any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia. It provides a use of an inhibitor of the expression or activity of a tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding the same to prepare a drug preparation.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 치료 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides an effective amount of a composition comprising an inhibitor of the expression or activity of an mRNA encoding the tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or the same as an active ingredient. Provides a method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia, comprising administering to the individual in need. .
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention is a type of rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, which includes an inhibitor of the expression or activity of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it, Provided is a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid multiple muscle pain.
본 발명의 상기 약학적 조성물은 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물일 수도 있고, 유효성분으로서 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제로 구성되는 조성물일 수도 있으며, 또는 유효성분으로서 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제가 필수적으로 구성되는 조성물일 수도 있다.The pharmaceutical composition of the present invention may be a composition comprising an inhibitor of expression or activity of TSPAN-2 protein or mRNA encoding it as an active ingredient, and an inhibitor of expression or activity of TSPAN-2 protein or mRNA encoding it as an active ingredient It may be a composition consisting of, or may be a composition consisting essentially of TSPAN-2 protein or an mRNA that encodes or inhibits the activity or activity as an active ingredient.
본 명세서에서 용어 ‘~을 포함하는(comprising)’이란 ‘함유하는(including)’ 또는 ‘특징으로 하는(characterized by)’과 동일한 의미로 사용되며, 본 발명에 따른 조성물 또는 방법에 있어서, 구체적으로 언급되지 않은 추가적인 구성 성분 또는 방법의 단계 등을 배제하지 않는다. 또한 용어 ‘로 구성되는(consisting of)’이란 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외하는 것을 의미한다. 용어 ‘필수적으로 구성되는(consisting essentially of)’이란, 조성물 또는 방법의 범위에 있어서, 기재된 물질 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 물질 또는 단계 등을 포함할 수 있는 것을 의미한다.In the present specification, the term'comprising' is used in the same sense as'including' or'characterized by', and in the composition or method according to the present invention, specifically It does not exclude additional components or method steps not mentioned. In addition, the term'consisting of' means excluding additional elements, steps, or ingredients not separately described. The term'consisting essentially of' means that in the scope of the composition or method, it may include the substance or step described in addition to the substance or step which does not substantially affect its basic properties. .
본 명세서에서 ‘치료’는 질환의 발생 또는 재발 억제, 증상의 완화, 질병의 직접 또는 간접적인 병리학적 결과의 감소, 질병 진행 속도의 감소, 질병 상태의 개선, 호전, 완화 또는 개선된 예후를 의미한다. 본 발명에서 사용되는 용어 ‘예방’은 질환의 발병을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다. 'Treatment' as used herein refers to suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving the condition of the disease, improving, alleviating or improving the prognosis. do. The term'prevention' as used in the present invention means all actions that suppress the onset of disease or delay the progression.
'단백질'은 '폴리펩타이드(polypeptide)' 또는 '펩타이드(peptide)'와 호환성 있게 사용되며, 예컨대, 자연 상태의 단백질에서 일반적으로 발견되는 바와 같이 아미노산 잔기의 중합체를 말한다. TSPAN-2 단백질의 '단편(fragment)'은 TSPAN-2 단백질의 일부분의 펩타이드를 말한다. 'Protein' is used interchangeably with'polypeptide' or'peptide' and refers to a polymer of amino acid residues, such as is commonly found in natural proteins. The'fragment' of the TSPAN-2 protein refers to a peptide of a portion of the TSPAN-2 protein.
한편, 'mRNA(messenger RNA 또는 전령 RNA)'는 단백질 합성 과정에서 특정 유전자의 염기서열의 유전 정보를 리보솜(ribosome)으로 전달하여 폴리펩티드 합성(단백질 번역, translation)의 청사진 역할을 하는 RNA이다. 유전자를 주형(template)으로 하여 단일 가닥의 mRNA가 전사(transcription) 과정을 통하여 합성된다.On the other hand,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the base sequence of a specific gene to a ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.
본 발명에서 사용된 용어 “TSPAN-2”는 테트라스파닌계(tetraspanin family)의 멤버에 속하는 약 24~27 kDa의 세포표면 당단백질 수용체로서, 세포 발달, 활성, 성장 및 운동성을 조절하는데 중요한 역할을 하는 신호전달 작용(signal transduction events)을 조절한다고 알려져 있다. 또한, 세포 부착 및 세포 이동을 조절할 수 있고, 혈소판-유발 내피세포 증식(platelet-induced endothelial cell proliferation)에 관여하는 혈소판활성화(platelet activation)와 집성(aggregation)을 유도한다. 아울러, 근육세포 융합(muscle cell fusion)을 촉진하고 근관 유지(myotube maintenance)에 기여하는 등 세포 내의 다양한 현상에 관여한다.The term “TSPAN-2” used in the present invention is a cell surface glycoprotein receptor of about 24 to 27 kDa belonging to a member of the tetraspanin family, and plays an important role in regulating cell development, activity, growth and mobility. It is known to modulate signal transduction events. In addition, it is possible to regulate cell adhesion and cell migration, and induce platelet activation and aggregation involved in platelet-induced endothelial cell proliferation. In addition, it is involved in various phenomena in cells, such as promoting muscle cell fusion and contributing to myotube maintenance.
본 발명에서의 TSPAN-2 단백질은 포유류에서 유래한 것일 수 있으며, 바람직하게는 인간에서 유래한 것이다. 가장 바람직하게, 본 발명에서의 TSPAN-2 단백질은 서열번호 1로 표시되는 인간의 TSPAN-2 isoform 1(NP_001760.1) 또는 서열번호 2로 표시되는 인간의 TSPAN-2 isoform 2(NP_001317241.1)의 아미노산 서열을 포함하는 것을 특징으로 한다(괄호 안은 NCBI Genbank accession number).The TSPAN-2 protein in the present invention may be derived from mammals, and is preferably derived from humans. Most preferably, the TSPAN-2 protein in the present invention is human TSPAN-2 isoform 1 represented by SEQ ID NO: 1 (NP_001760.1) or human TSPAN-2 isoform 2 represented by SEQ ID NO: 2 (NP_001317241.1) It is characterized by including the amino acid sequence of (NCBI Genbank accession number in parentheses).
상기 TSPAN-2의 발현 또는 활성 억제제는 TSPAN-2 단백질 또는 이를 암호화하는 mRNA에 특이적으로 결합하는 항체, 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 천연추출물 및 합성화합물로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하나, 이에 제한되는 것은 아니다.The TSPAN-2 expression or activity inhibitor is selected from the group consisting of antibodies, compounds, peptides, peptide mimetics, aptamers, natural extracts and synthetic compounds that specifically bind to TSPAN-2 protein or mRNA encoding the same. It is characterized by any one, but is not limited thereto.
본 발명의 일 실시예에서 류마티스 관절염을 유도한 동물 모델에 항-TSPAN-2 항체를 투여하였을 때 류마티스 관절염에 의한 붓기가 정상 실험군과 비슷할 정도로 완화되었고 2주만에 증상이 회복된 것을 확인하였다.When an anti-TSPAN-2 antibody was administered to an animal model inducing rheumatoid arthritis in one embodiment of the present invention, it was confirmed that swelling due to rheumatoid arthritis was relieved to be similar to that of the normal experimental group and symptoms were recovered within 2 weeks.
따라서 본 발명에 따른 TSPAN-2의 발현 또는 활성 억제제는 TSPAN-2에 대한 항체인 것이 바람직하나, 이에 제한되는 것은 아니다.Therefore, the inhibitor of expression or activity of TSPAN-2 according to the present invention is preferably an antibody against TSPAN-2, but is not limited thereto.
본 발명에서 용어, "항체"는 생체의 면역계에서 혈액이나 림프를 순환하면서 외부 물질인 항원이 침입한 경우 이에 반응하는 물질을 말하며, 림프조직에서 형성되는 글로불린계 단백질로 면역글로불린이라고도 불린다. 항체는 B세포가 생산해서 체액으로 흘려보내는 단백질로 항원과 특이적으로 결합하며, 하나의 항체 분자에는 두 개의 중사슬(heavy chain)과 두 개의 경사슬(light chain)이 있으며, 각각의 중사슬과 경사슬은 그의 N-터미널 말단에 가변영역을 갖고 있다. 각각의 가변영역은 3개의 상보성 결정부위(Complementarity determining region:CDR)와 4개의 구조형성부위(framework regions: FRs)로 구성되는데, 상보성 결정 부위들은 항체의 항원 결합 특이성을 결정하고, 가변영역의 구조형성 부위들로 유지되는 비교적 짧은 펩티드 서열로 존재한다. 본 발명의 목적상 상기 항체는 TSPAN-2과 결합하여 TSPAN-2의 활성을 억제할 수 있는 항체일 수 있다.In the present invention, the term "antibody" refers to a substance that reacts when an antigen that is an external substance invades while circulating blood or lymph in the immune system of a living body, and is also called an immunoglobulin as a globulin-based protein formed in lymphoid tissue. Antibodies are proteins produced by B cells and released into body fluids. They bind specifically to antigens. One antibody molecule has two heavy chains and two light chains, each of which is a heavy chain. And the light chain have a variable region at the end of its N-terminal. Each variable region is composed of three complementarity determining regions (CDRs) and four framework regions (FRs). The complementarity determining regions determine the antigen binding specificity of the antibody, and the structure of the variable region It exists as a relatively short peptide sequence maintained by the formation sites. For the purposes of the present invention, the antibody may be an antibody capable of inhibiting the activity of TSPAN-2 by binding to TSPAN-2.
상기 “류마티스 관절염”은 “골관절염(steoarthritis)”과 상이한 질환이다. 골관절염의 경우, 대개 40세 이후에 시작하나, 류마티스 관절염은 25세 내지 50세에 시작한다. 또한 골관절염은 수년에 걸쳐 서서히 진행되고, 초기에 몸의 한쪽 면에 발병하지만, 류마티스 관절염은 수주나 수개월 내에 진행되며 대게 같은 관절의 양쪽 면에 발병하게 된다. 또한 골관절염은 일부 관절에 발병하고 팔꿈치와 손목에는 거의 생기지 않으나, 류마티스 관절염은 팔꿈치와 손목을 포함하는 많은 관절에 발병하며, 통증 기간도 골관절염은 저녁에 관절을 사용한 후, 통증이 심화되지만 류마티스 관절염은 아침에 통증이 심화된다.The “rheumatic arthritis” is a different disease from “steoarthritis”. In the case of osteoarthritis, it usually starts after age 40, but rheumatoid arthritis starts between 25 and 50 years of age. In addition, osteoarthritis progresses slowly over the years and initially develops on one side of the body, but rheumatoid arthritis progresses within weeks or months, usually on both sides of the same joint. In addition, osteoarthritis affects some joints and rarely occurs in the elbows and wrists, but rheumatoid arthritis affects many joints, including the elbows and wrists, and even during the pain period, osteoarthritis intensifies the pain after using the joint in the evening, but rheumatoid arthritis The pain intensifies in the morning.
또한 류마티스 관절염은 유전적 소인과 환경적 영향이 복잡하게 작용해 면역 체계의 이상을 일으키고, 활막의 염증을 일으키게 되는 대표적인 '자가면역 질환'이다. 류마티스 관절염이 잘 발생하는 조직형 유전자가 알려져 있으며, 여성 호르몬의 영향으로 여성에서 남성보다 더 잘 발생하며, 바이러스나 세균 감염이 유발 요인일 수 있다고 알려져 있다.In addition, rheumatoid arthritis is a typical'autoimmune disease' in which the genetic predisposition and environmental effects are complicated and cause abnormalities of the immune system and inflammation of the synovial membrane. Rheumatoid arthritis is a tissue-type gene that is well known, and it is known that it occurs better in women than in men under the influence of female hormones, and a viral or bacterial infection may be a trigger.
이와는 다르게 골관절염은 관절을 보호하고 있는 연골의 손상 또는 퇴행성 변화로 관절을 이루는 뼈와 인대 등에 손상이 일어나고 염증과 통증이 생기는 질환으로 흔히 '퇴행성 관절염'이라 불렸다. 유전적인 소인, 대사 요인, 생화학적 요인, 역학적 요인, 관절의 모양, 국소 염증 등이 서로 영향을 미치며 관절의 연골이 손상되면서 발생한다. On the contrary, osteoarthritis is a disease in which the bones and ligaments forming the joints are damaged by inflammation or degenerative changes of cartilage protecting the joints, and inflammation and pain are commonly called'degenerative arthritis'. Genetic predisposition, metabolic factors, biochemical factors, epidemiological factors, joint shape, and local inflammation affect each other and occur when the cartilage of the joint is damaged.
골관절염(OA)은 관절 연골과 그 밑에 있는 뼈의 파괴로 인하여 발생하는 관절 질환의 한 유형으로 가장 흔한 증상은 관절 통증과 뻣뻣함이 있다. 이 질병의 경우 신체의 다른 부위에는 영향을 주지 않지만, 류마티스 관절염은 신체의 다른 부위에 전신질환으로 신체의 다른 부위에 영향을 줄 수 있다.Osteoarthritis (OA) is a type of joint disease caused by the destruction of articular cartilage and the underlying bone. The most common symptoms are joint pain and stiffness. Although this disease does not affect other parts of the body, rheumatoid arthritis can affect other parts of the body as a systemic disease in other parts of the body.
또는 상기 TSPAN-2 단백질의 발현 억제제는 TSPAN-2 mRNA에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide), siRNA, shRNA, miRNA, ribozyme, DNAzyme 및 PNA(protein nucleic acid)로 이루어진 군에서 선택된 어느 하나인 것일 수 있다. 또한 상기 TSPAN-2 mRNA는 가장 바람직하게는 서열번호 3으로 표시되는 인간 TSPAN-2 mRNA transcript variant 1(NM_001769.3) 또는 서열번호 4로 표시되는 인간 TSPAN-2 mRNA transcript variant 2(NM_001330312.1)의 염기서열을 포함하는 것이다(괄호 안은 NCBI Genbank accession number). 상기한 인간의 TSPAN-2 mRNA transcript variant들의 coding region(exon) 등의 특징 사항은 괄호 안에 기재된 Genbank accession number로 NCBI 데이터베이스를 검색하여 나오는 서열정보에서 확인된다.Or, the TSPAN-2 protein expression inhibitor is selected from the group consisting of antisense oligonucleotide (antisense oligonucleotide), siRNA, shRNA, miRNA, ribozyme, DNAzyme and protein nucleic acid (PNA) that complementarily bind to TSPAN-2 mRNA. It can be one. In addition, the TSPAN-2 mRNA is most preferably human TSPAN-2 mRNA transcript variant 1 (NM_001769.3) represented by SEQ ID NO: 3 or human TSPAN-2 mRNA transcript variant 2 (NM_001330312.1) represented by SEQ ID NO: 4 It contains the base sequence of (NCBI Genbank accession number in parentheses). The characteristics of the coding region (exon) of the human TSPAN-2 mRNA transcript variants described above are confirmed in sequence information obtained by searching the NCBI database with the Genbank accession number in parentheses.
나아가, 본 발명에 따른 TSPAN2 단백질 발현 억제제는 바람직하게는 TSPAN2 mRNA에 상보적으로 결합하여 mRNA의 분해를 유발하는 shRNA일 수 있다. 상기 TSPAN2에 대한 siRNA는 구체적으로는 서열번호 5 내지 서열번호 31로 이루어진 군에서 선택되는 어느 한 염기서열을 포함하는 것일 수 있다. Furthermore, the TSPAN2 protein expression inhibitor according to the present invention may preferably be shRNA that binds complementarily to TSPAN2 mRNA and induces degradation of the mRNA. The siRNA for TSPAN2 may specifically include any one nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 31.
상기 ‘siRNA(small interfering RNA 또는 short interfering RNA 또는 silencing RNA)'는 세포 안에 인위적으로 도입되어 특정 유전자의 mRNA의 분해를 유도하여 단백질 번역(translation)이 나지 않도록 하여 유전자 발현을 억제하는 RNA 간섭(RNA interference) 현상을 일으키는 짧은 이중가닥의 RNA로, 표적 mRNA의 특정 부위에 상보적인 20 내지 25개의 뉴클레오티드로 구성되어 있다. 세포 내에서 siRNA의 이중 가닥 중 표적 mRNA에 상보적인 가닥(antisense strand)이 RISC(RNA-induced silencing complex) 단백질 복합체와 결합하여 표적 mRNA에 결합하고, RISC 복합체 안의 argonaute 단백질이 표적 mRNA를 절단하여 분해시키거나, 단백질 번역에 중요한 단백질과 리보솜이 mRNA와 결합하는 것을 억제하는 등의 기작으로 특정 유전자의 발현을 억제한다. The'siRNA (small interfering RNA or short interfering RNA or silencing RNA)' is artificially introduced into a cell to induce the degradation of a specific gene mRNA, thereby preventing protein translation and RNA interference to suppress gene expression (RNA It is a short double-stranded RNA that causes interference) and is composed of 20 to 25 nucleotides complementary to a specific region of the target mRNA. Among the double strands of siRNA in the cell, an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex to bind to the target mRNA, and the argonaute protein in the RISC complex cuts and degrades the target mRNA Or inhibits the binding of proteins and ribosomes, which are important for protein translation, to mRNA, to suppress the expression of specific genes.
본 발명에 따른 shRNA는 RNA 간섭을 유도하는 물질로서, 1본쇄 RNA에서 부분적으로 회문상의 염기서열을 포함함으로써, 분자 내에서 2본쇄 구조를 가지고 헤어핀과 같은 구조가 되는 약 20염기 이상의 분자이다. 또한, shRNA 발현용 플라스미드(plasmid)를 세포 내로 도입시켜 발현하게 되면, 세포 내의 다이서(dicer)라는 RNaseⅢ(ribonucleaseⅢ) 효소에 의해 21~23개 염기쌍의 siRNA가생성되어 RNAi를 유도한다.The shRNA according to the present invention is a substance that induces RNA interference, and is a molecule of about 20 bases or more having a double-stranded structure within a molecule and a hairpin-like structure by including a partially sequential base sequence in a single-stranded RNA. In addition, when a plasmid for shRNA expression is introduced into a cell and expressed, a siRNA of 21 to 23 base pairs is generated by an RNase III (ribonuclease III) enzyme called a dicer in the cell to induce RNAi.
본 발명에 따른 siRNA 또는 shRNA는 올리고뉴클레오티드의 생체 내 안정성 향상, 핵산 분해효소 저항성 부여 및 비특이적 면역반응 감소를 위한 다양한 변형(modification)을 가한 것일 수 있다. 상기 올리고뉴클레오티드의 변형은 하나 이상의 뉴클레오티드 내 당 구조의 2´ 탄소 위치에서 OH기가 -CH3(메틸), -OCH3(methoxy), -NH2, -F, -O-2-메톡시에틸, -O-프로필(propyl), -O-2-메틸티오에틸(methylthioethyl), -O-3-아미노프로필, -O-3-디메틸아미노프로필, -O-N-메틸아세트아미도 또는 -O-디메틸아미도옥시에틸로의 치환에 의한 변형; 뉴클레오티드 내 당(sugar) 구조 내의 산소가 황으로 치환된 변형; 또는 뉴클레오티드결합의 포스포로티오에이트(phosphorothioate) 또는 보라노포스페이트(boranophosphate), 메틸포스포네이트(methyl phosphonate) 결합으로의 변형에서 선택된 하나 이상의 변형이 조합되어 사용될 수 있으며, PNA(peptide nucleic acid), LNA(locked nucleic acid) 또는 UNA(unlocked nucleic acid) 형태로의 변형도 사용이 가능하다.The siRNA or shRNA according to the present invention may be added with various modifications to improve the stability of the oligonucleotide in vivo, to impart nucleic acid degrading enzyme resistance, and to reduce non-specific immune responses. The modification of the oligonucleotide is that the OH group at the 2'carbon position of the sugar structure in one or more nucleotides is -CH3(methyl), -OCH3(methoxy), -NH2, -F, -O-2-methoxyethyl, -O- Propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl Modification by substitution with; Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from the modification of nucleotide bonds to phosphorothioate or boranophosphate or methyl phosphonate bonds may be used in combination, and peptide nucleic acid (PNA), Modification to the form of a locked nucleic acid (LNA) or an unlocked nucleic acid (UNA) is also possible.
본 발명의 조성물은 유전자 치료 분야에서 통상적으로 공지된, 리포좀(liposome), 폴리머(polymer)와 같은 다양한 운반 방법을 통하여 생체 내에 적용될 수 있다. The composition of the present invention can be applied in vivo through various transport methods such as liposomes and polymers, which are commonly known in the field of gene therapy.
본 발명의 일구현예에 따르면, 본 발명의 핵산 분자는 유전자 전달체에 포함되어 있거나, 또는 네이키드 DNA(naked DNA)이다. 리프로그래밍 인자를 암호화하는 핵산 분자를 포함하는 유전자 전달체를 유효성분으로 이용하는 경우, 리프로그래밍 인자-코딩 핵산 분자는 적합한 발현 컨스트럭트(expression construct) 내에 존재하는 것이 바람직하다.According to one embodiment of the present invention, the nucleic acid molecule of the present invention is included in a gene delivery system, or is naked DNA. When a gene delivery system comprising a nucleic acid molecule encoding a reprogramming factor is used as an active ingredient, it is preferred that the reprogramming factor-encoding nucleic acid molecule is present in a suitable expression construct.
본 명세서에서 사용된, 용어 "유전자 전달"은 유전자가 세포 내로 운반되는 것을 의미하며, 유전자의 세포 내 침투(transduction)와 동일한 의미를 가진다. 조직 수준에서, 상기 용어 유전자 전달은 유전자의 확산(spread)과 동일한 의미를 가진다.As used herein, the term "gene transfer" means that the gene is carried into a cell and has the same meaning as the gene's intracellular transduction. At the tissue level, the term gene transfer has the same meaning as the spread of the gene.
상기 발현 컨스트럭트에서, 리프로그래밍 인자-코딩 핵산 분자는 프로모터에 작동적으로 연결될 수 있다. 상기 용어, "작동적으로 연결된"은 핵산 발현조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열 사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다.In the expression construct, the reprogramming factor-encoding nucleic acid molecule can be operably linked to a promoter. The term, “operably linked” refers to a functional binding between a nucleic acid expression regulatory sequence (eg, a promoter, signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is It controls the transcription and/or translation of the other nucleic acid sequence.
본 발명에 있어서, 리프로그래밍 인자-코딩 핵산 분자에 결합된 프로모터는, 동물세포(예컨대, 포유동물 세포)에서 작동하여 리프로그래밍 인자-코딩 핵산 분자의 전사를 조절할 수 있는 것으로서, 포유동물 바이러스로부터 유래된 프로모터 및 포유동물 세포의 게놈으로부터 유래된 프로모터를 포함하며, 예컨대, CMV(cytomegalo virus) 프로모터, 아데노 바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk프로모터, RSV 프로모터, EF1 알파 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터 및 인간 GM-CSF 유전자의 프로모터를 포함하나, 이에 한정되는 것은 아니다.In the present invention, a promoter coupled to a reprogramming factor-encoding nucleic acid molecule is capable of controlling transcription of a reprogramming factor-encoding nucleic acid molecule by operating in an animal cell (eg, a mammalian cell), derived from a mammalian virus. Includes a promoter and a promoter derived from the genome of a mammalian cell, such as the cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha Promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and promoter of human GM-CSF gene Including, but not limited to.
본 발명의 핵산 분자는 다양한 형태로 제작할 수 있으며, 예를 들어 (i) 플라스미드를 전달체로 이용한 네이키드(naked) 재조합 DNA 분자, (ii) 바이러스 벡터, (iii) 리포좀 또는 니오좀, (iv) 지질-유전자 결합체(LipidDNA complex) 또는 (v) 폴리머-유전자 결합체(Polymer-DNA complex) 등의 형태로 제작할 수 있다.The nucleic acid molecule of the present invention can be produced in various forms, for example, (i) a naked recombinant DNA molecule using a plasmid as a carrier, (ii) a viral vector, (iii) liposomes or niosomes, (iv) It can be produced in the form of a lipid-gene complex (LipidDNA complex) or (v) a polymer-gene complex (Polymer-DNA complex).
리프로그래밍 인자-코딩 핵산 분자는 통상적인 유전자 치료에 이용되는 모든 유전자 전달 시스템에 적용될 수 있으며, 예컨대 플라스미드, 아데노 바이러스, 아데노-관련 바이러스, 레트로 바이러스, 렌티 바이러스, 헤르페스 심플렉스 바이러스, 배시니아 바이러스, 리포좀 또는 니오좀에 적용될 수 있다.Reprogramming factor-encoding nucleic acid molecules can be applied to all gene delivery systems used in conventional gene therapy, such as plasmids, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, It can be applied to liposomes or niosomes.
본 발명의 일구현예에 따르면, 본 발명의 유전자 전달체는 바이러스 벡터로 구현될 수 있다. 상술한 본 발명의 유전자 전달 시스템을 세포 내로 도입하는 방법은 당업계에 공지된 다양한 방법을 통해 실시될 수 있다.According to one embodiment of the present invention, the gene delivery system of the present invention may be implemented as a viral vector. The method for introducing the gene delivery system of the present invention into cells described above may be carried out through various methods known in the art.
아울러, TSPAN2에 대한 저해제의 표적 부위로의 표적화된 전달을 위한 효과적인 플랫폼으로서 예를 들어, T-세포, B-세포, 마크로파지(Macrophage)-세포, NK-세포 및 줄기세포 등을 이용할 수 있다.In addition, as an effective platform for targeted delivery of an inhibitor to TSPAN2 to a target site, T-cells, B-cells, macrophage-cells, NK-cells and stem cells can be used.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물은 본 발명에 따른 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제와 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 추가적인 성분을 포함하게 되면 본 발명에 따른 조성물의 치료 효과가 더욱 증진될 수 있을 것이다. 상기 성분 추가 시에는 복합 사용에 따른 피부 안전성, 제형화의 용이성, 유효성분들의 안정성을 고려할 수 있다.The pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid multiple myalgia is a tetraspa according to the present invention Nin-2 (Tetraspanin-2, TSPAN-2) may contain at least one active ingredient that exhibits a function similar to the expression or activity inhibitor. The inclusion of additional ingredients will further enhance the therapeutic effect of the compositions according to the invention. When adding the above components, skin safety, ease of formulation, and stability of active ingredients according to the combined use may be considered.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물은 약학적으로 허용 가능한 담체를 더 포함할 수 있다.The pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple muscle pain is a pharmaceutically acceptable carrier It may further include.
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 또한 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등을 포함할 수 있다. 또한, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 당업계에 공지되어 있는 것을 참고로 할 수 있다.Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like. In addition, stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those known in the art.
본 발명의 약학적 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들어, 경구 또는 비경구로 투여할 수 있으며, 비경구적인 투여방법으로는 이에 제한되는 것은 아니나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다.The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and the parenteral administration method is not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다. 제형화할 경우에는 하나 이상의 완충제(예를 들어, 식염수 또는 PBS), 항산화제, 정균제, 킬레이트화제(예를 들어, EDTA 또는 글루타치온), 충진제, 증량제, 결합제, 아쥬반트(예를 들어, 알루미늄 하이드록사이드), 현탁제, 농후제 습윤제, 붕해제 또는 계면활성제, 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above. When formulated, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostats, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g. aluminum hydroxide) Side), a suspending agent, a thickening agent wetting agent, a disintegrating agent or a surfactant, a diluent or an excipient.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 또는 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 약학적 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분(옥수수 전분, 밀 전분, 쌀 전분, 감자 전분 등 포함), 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 덱스트로오스(dextrose), 솔비톨(sorbitol), 만니톨(mannitol), 자일리톨(xylitol), 에리스리톨 말티톨(Erythritol maltitol), 셀룰로즈(celulose), 메틸 셀룰로즈(methyl celulose), 나트륨 카르복시메틸셀룰로오스(sodium carboxymethyl cellulose) 및 하이드록시프로필메틸-셀룰로스(Hydroxypropyl methylCellulose) 또는 젤라틴 등을 섞어 조제될 수 있다. 예컨대, 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의 정제를 수득할 수 있다. Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, capsules, etc., and these solid preparations include, for example, at least one excipient in the pharmaceutical composition of the present invention. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol (mannitol), xylitol, erythritol maltitol, cellulose, methyl celulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin It can be prepared by mixing the back. For example, tablets or tablets of sugar can be obtained by blending the active ingredient with a solid excipient and then grinding it and adding a suitable adjuvant to the granule mixture.
단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물 또는 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다.In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions or syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances or preservatives, in addition to water or liquid paraffin, a simple diluent commonly used. .
또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있으며, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and a preservative. .
비경구적으로 투여하는 경우 본 발명의 약학적 조성물은 적합한 비경구용 담체와 함께 주사제, 경피 투여제 및 비강 흡입제의 형태로 당 업계에 공지된 방법에 따라 제형화될 수 있다. 상기 주사제의 경우에는 반드시 멸균되어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올, 폴리올(예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등), 이들의 혼합물 및/또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는, 적합한 담체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS(phosphate buffered saline) 또는 주사용 멸균수, 10% 에탄올, 40% 프로필렌 글리콜(propylene glycol) 및 5% 덱스트로오스와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤(paraben), 클로로부탄올(Chlorobutanol), 페놀(phenol), 소르빈산(Sorbic acid), 티메로살(thimerosal) 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있다.When administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations, and nasal inhalants with suitable parenteral carriers. The injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and/or vegetable oils. Can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with ethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Isotonic solutions such as can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be additionally included. . In addition, the injection may further include isotonic agents such as sugars or sodium chloride in most cases.
경피 투여제의 경우 연고제, 크림제, 로션제, 겔제, 외용액제, 파스타제, 리니멘트제, 에어롤제 등의 형태가 포함된다. 상기에서 ‘경피 투여’는 약학적 조성물을 국소적으로 피부에 도포하여 약학적 조성물에 함유된 유효한 양의 활성성분이 피부 내로 전달되는 것을 의미한다. In the case of transdermal dosage forms, ointments, creams, lotions, gels, external solutions, pasta, linen agents, aerosols, and the like are included. In the above,'transdermal administration' means that the pharmaceutical composition is topically applied to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.
흡입 투여제의 경우, 본 발명의 TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 또는 활성 억제제를 적합한 추진제, 예를 들면, 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 또는 다른 적합한 기체를 사용하여, 가압 팩 또는 연무기로부터 에어로졸 스프레이 형태로 편리하게 전달할 수 있다. 가압 에어로졸의 경우, 투약 단위는 계량된 양을 전달하는 밸브를 제공하여 결정할 수 있다. 예를 들면, 흡입기 또는 취입기에 사용되는 젤라틴 캡슐 및 카트리지는 화합물, 및 락토오스(lactose) 또는 전분과 같은 적합한 분말 기제의 분말 혼합물을 함유하도록 제형화할 수 있다. 비경구 투여용 제형은 모든 제약 화학에 일반적으로 공지된 처방서에 기재되어 있다.For inhalation dosages, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable TSPAN-2 proteins of the invention or mRNA expression or activity inhibitors encoding them Using a suitable gas, it can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mixture of a compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in formulas generally known in all pharmaceutical chemistry.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물은 TSPAN-2의 발현 또는 활성 억제제를 유효량으로 포함할 때 바람직한 질환 예방 및 치료 효과를 제공할 수 있다. The pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia is the expression of TSPAN-2 or When the active inhibitor is included in an effective amount, it is possible to provide a desirable disease prevention and treatment effect.
본 명세서에서, '유효량'이라 함은 음성 대조군에 비해 그 이상의 반응을 나타내는 양을 말하며 바람직하게는 류마티스 관절염의 증상, 특히 부종 완화에 충분한 양을 말한다. 본 발명의 약학적 조성물에 TSPAN-2의 발현 또는 활성 억제제가 0.01 내지 99.99% 포함될 수 있으며, 잔량은 약학적으로 허용 가능한 담체가 차지할 수 있다. 본 발명의 약학적 조성물에 포함되는 TSPAN-2의 발현 또는 활성 억제제의 유효량은 조성물이 제품화되는 형태 등에 따라 달라질 것이다.In the present specification, the term'effective amount' refers to an amount that exhibits a higher response than a negative control group, and preferably refers to an amount sufficient to relieve symptoms of rheumatoid arthritis, especially edema. In the pharmaceutical composition of the present invention, an inhibitor of TSPAN-2 expression or activity may be included in an amount of 0.01 to 99.99%, and the remaining amount may be occupied by a pharmaceutically acceptable carrier. The effective amount of the TSPAN-2 expression or activity inhibitor contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.
본 발명의 약학적 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 비경구 투여시는 TSPAN-2의 발현 또는 활성 억제제를 기준으로 하루에 체중 1 kg당 바람직하게 0.01 내지 50 mg, 더 바람직하게는 0.1 내지 30 mg의 양으로 투여되도록, 그리고 경구 투여시는 TSPAN-2의 발현 또는 활성 억제제를 기준으로 하루에 체중 1 kg당 바람직하게 0.01 내지 100 mg, 더 바람직하게는 0.01 내지 10 mg의 양으로 투여되도록 1 내지 수회에 나누어 투여할 수 있다. 그러나 상기 TSPAN-2의 발현 또는 활성 억제제의 용량은 약학적 조성물의 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율 등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 상기 TSPAN-2의 발현 또는 활성 억제제를 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 및 치료를 위한 특정한 용도에 따른 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다.The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. . The pharmaceutical composition of the present invention can vary the content of the active ingredient according to the degree of disease. When administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day, based on the inhibitor of expression or activity of TSPAN-2, and when administered orally, TSPAN- Based on the expression or activity inhibitor of 2, it can be divided into 1 to several times to be administered in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day. However, the dose of the TSPAN-2 expression or activity inhibitor is considered by considering various factors such as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, as well as the route and frequency of administration of the pharmaceutical composition. Since the effective dosage for the drug is determined, those who have ordinary knowledge in the art in consideration of this point may express the TSPAN-2 expression or activity inhibitor of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, It may be possible to determine an appropriate effective dosage according to a specific use for the prevention and treatment of any one or more diseases selected from the group consisting of rheumatoid arthritis and rheumatoid polymyalgia. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effect of the present invention.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물은 단독으로 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 또는 생물학적 반응조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia, alone or by surgery, radiation It can be used in combination with treatment, hormone therapy, chemotherapy, or methods using biological response modifiers.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물은 또한 TSPAN-2의 발현 또는 활성 억제제를 유효성분으로 포함하는 외용제의 제형으로 제공할 수 있다.The pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, juvenile rheumatoid arthritis and rheumatoid polymyalgia is also expressed by TSPAN-2 Or it can be provided as a formulation of an external preparation containing an active inhibitor as an active ingredient.
본 발명의 약학적 조성물을 피부외용제로 사용하는 경우, 추가로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 유화제, 비이온형 유화제, 충전제, 금속이온봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 활성제, 친유성 활성제 또는 지질 소낭 등 피부 외용제에 통상적으로 사용되는 임의의 다른 성분과 같은 피부 과학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 또한 상기 성분들은 피부 과학 분야에서 일반적으로 사용되는 양으로 도입될 수 있다.When the pharmaceutical composition of the present invention is used as an external preparation for skin, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Skin such as activators, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion blockers, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic actives, lipophilic actives or lipid vesicles It may contain adjuvants commonly used in the field of dermatology such as any other ingredients commonly used in external preparations. In addition, the ingredients may be introduced in an amount commonly used in the field of skin science.
본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물이 피부 외용제로 제공될 경우, 이에 제한되는 것은 아니나, 연고, 패취, 겔, 크림 또는 분무제 등의 제형일 수 있다.When a pharmaceutical composition for preventing or treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia is provided as an external preparation for skin , But is not limited thereto, may be a formulation such as ointment, patch, gel, cream or spray.
또한, 본 발명은 TSPAN-2의 발현 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 치료 방법을 제공한다. In addition, the present invention comprises the step of administering a pharmaceutical composition comprising an inhibitor of expression or activity of TSPAN-2 as an active ingredient to an individual other than a human, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis It provides a method for treating any one or more diseases selected from the group consisting of pediatric rheumatoid arthritis and rheumatoid multiple muscle pain.
본 발명에서 용어, "개체"는 본 발명의 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환을 보유하거나 또는 발병한, 인간을 포함한 모든 동물을 의미한다. 본 발명의 약학적 조성물을 개체에 투여함으로써, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환을 완화 또는 치료할 수 있다. 상기 완화는 본 발명에 따른 조성물의 투여로 질환이 호전되거나 이롭게 되는 모든 행위를 말한다.The term "individual" in the present invention has or develops one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia of the present invention. One means all animals, including humans. By administering the pharmaceutical composition of the present invention to an individual, any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia can be alleviated or treated. have. The alleviation refers to all actions in which the disease is improved or beneficial by administration of the composition according to the present invention.
상기 본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 대상에게 본 발명의 약학적 조성물을 도입하는 것을 말하며, 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used herein, the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a subject in any suitable way, and the route of administration can be administered through various routes, oral or parenteral, as long as the target tissue can be reached. .
상기 약학적 조성물은 목적 또는 필요에 따라 당업계에서 사용되는 통상적인 방법, 투여 경로, 투여량에 따라 적절하게 개체에 투여될 수 있다. 투여 경로의 예로는 경구, 비경구, 피하, 복강 내, 폐 내, 및 비강 내로 투여될 수 있으며, 비경구 주입에는 근육 내, 정맥 내, 동맥 내, 복강 내 또는 피하투여가 포함된다. 또한 당업계에 공지된 방법에 따라 적절한 투여량 및 투여 횟수가 선택될 수 있으며, 실제로 투여되는 본 발명의 약학적 조성물의 양 및 투여 횟수는 치료하고자 하는 증상의 종류, 투여 경로, 성별, 건강 상태, 식이, 개체의 연령 및 체중, 및 질환의 중증도와 같은 다양한 인자에 의해 적절하게 결정될 수 있다.The pharmaceutical composition may be appropriately administered to a subject according to the purpose or need, depending on the conventional method, route of administration, and dosage used in the art. Examples of routes of administration may be oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, an appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention to be actually administered is the type of symptom to be treated, the route of administration, sex, and health status. , Diet, the age and weight of the individual, and the severity of the disease.
본 발명에서의 용어 "약학적으로 유효한 양"은 의학적 용도에 적용 가능한 합리적인 수혜/위험 비율로 혈관 투과성 증가를 억제 또는 완화하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The term "pharmaceutically effective amount" in the present invention means an amount sufficient to suppress or alleviate an increase in vascular permeability at a reasonable benefit/risk ratio applicable to medical use, and the effective dose level is the individual type and severity, age, It can be determined by gender, drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, and can be easily determined by those skilled in the art.
또한, 본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제를 유효성분으로 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention includes the expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) as an active ingredient, type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis And it provides a food composition for preventing or improving any one or more diseases selected from the group consisting of rheumatoid multiple muscle pain.
본 발명에 따른 식품 조성물은 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 예방 또는 개선에 사용될 수 있다. The food composition according to the present invention can be used for the prevention or improvement of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia.
본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food), 식품 첨가제(food additives) 등의 모든 형태를 포함하며, 인간 또는 가축을 비롯한 동물을 취식대상으로 한다. 상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition of the present invention includes all forms of functional foods, nutritional supplements, health foods, food additives, etc., and targets animals including humans or livestock. Is done. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 일반 식품으로는 이에 한정되지 않지만 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지 콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게이트, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물 유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 상기 TSPAN-2의 발현 또는 활성 억제제를 첨가하여 제조할 수 있다. 또한, 영양보조제로는 이에 한정되지 않지만 캡슐, 타블렛, 환 등에 TSPAN-2의 발현 또는 활성 억제제를 첨가하여 제조할 수 있다. 또한, 건강기능식품으로는 이에 한정되지 않지만 예를 들면, 상기 TSPAN-2의 발현 또는 활성 억제제 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용(건강음료)할 수 있도록 액상화, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 상기 TSPAN-2의 발현 또는 활성 억제제를 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다. 또한, TSPAN-2의 발현 또는 활성 억제제와 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 개선 효과가 있다고 알려진 공지의 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.Food compositions of this type can be prepared in various forms according to conventional methods known in the art. Beverages (including alcoholic beverages), fruits and processed foods thereof (e.g. canned fruits, canned foods, jams, marmalades, etc.), fish, meat and processed foods (e.g. ham, sausages) Corn beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible plant maintenance, margarine , Plant protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the inhibitor of the expression or activity of TSPAN-2. In addition, it is not limited to the nutritional supplement, but may be prepared by adding an inhibitor of TSPAN-2 expression or activity to capsules, tablets, pills, and the like. In addition, as a health functional food, but not limited thereto, for example, the expression or activity inhibitor itself of TSPAN-2 is prepared in the form of tea, juice, and drink to liquefy, granulate, and encapsulate for drinking (health drink). And powdered. In addition, in order to use the TSPAN-2 expression or activity inhibitor in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. In addition, it is known that an inhibitor of the expression or activity of TSPAN-2 and the improvement effect of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia It can be prepared in the form of a composition by mixing with known active ingredients.
본 발명의 식품 조성물이 건강음료 조성물로 이용되는 경우, 상기 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드(monosaccharide); 말토스, 수크로스와 같은 디사카라이드(disaccharide); 덱스트린(dextrin), 사이클로덱스트린(cyclodextrin)과 같은 폴리사카라이드(polysaccharide); 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제는 타우마틴(thaumatin), 스테비아(stevia) 추출물과 같은 천연 감미제; 사카린, 아스파르탐(aspartame)과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL 당 일반적으로 약 0.01 ∼ 0.04 g, 바람직하게는 약 0.02 ∼ 0.03 g 이다.When the food composition of the present invention is used as a health drink composition, the health drink composition may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, such as conventional beverages. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extracts; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g per 100 mL of the composition of the present invention, preferably about 0.02 to 0.03 g.
본 발명에 따른 TSPAN-2의 발현 또는 활성 억제제가 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 개선용 식품 조성물의 유효성분으로 함유될 때, 그 양은 증상 개선 작용을 달성하기에 유효한 양으로 특별히 한정되는 것은 아니나, 전체 조성물 총 중량에 대하여 0.01 내지 100 중량%인 것이 바람직하다. 본 발명의 식품 조성물은TSPAN-2의 발현 또는 활성 억제제와 함께 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환개선 효과가 있는 것으로 알려진 다른 활성 성분과 함께 혼합하여 제조될 수 있다.For preventing or improving any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia according to the present invention, the inhibitor of expression or activity of TSPAN-2 When contained as an active ingredient of a food composition, the amount is not particularly limited to an amount effective to achieve a symptom improvement effect, but is preferably 0.01 to 100% by weight relative to the total weight of the total composition. The food composition of the present invention improves any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia with an inhibitor of expression or activity of TSPAN-2 It can be prepared by mixing with other active ingredients known to be effective.
상기 외에 본 발명의 식품 조성물이 건강식품으로 이용되는 경우, 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강식품은 천연 과일주스, 과일주스 음료 또는 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.When the food composition of the present invention is used as a health food in addition to the above, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH It may contain a regulator, stabilizer, preservative, glycerin, alcohol or carbonic acid. In addition, the health food of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage or vegetable beverage. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명은 류마티스 관절염 치료용 제제의 스크리닝 방법을 제공한다. In addition, the present invention provides a method for screening a formulation for the treatment of rheumatoid arthritis.
상기 스크리닝 방법은 (a) 대조군인 류마티스 관절염이 발병된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계;The screening method comprises the steps of (a) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal with rheumatoid arthritis as a control;
(b) 상기 실험동물에서 류마티스 관절염을 치료할 수 있을 것으로 예상되는 후보물질을 투여하는 단계;(b) administering a candidate substance expected to be able to treat rheumatoid arthritis in the experimental animal;
(c) 실험군인 상기 후보물질이 투여된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및(c) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal to which the candidate substance, an experimental group, is administered; And
(d) 대조군에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 실험군에서 측정된 것과 비교하여, 대조군에서 측정된 것보다도 실험군에서 측정된 것이 낮은 수준을 나타내는 경우, 상기 후보물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는 것을 특징으로 한다.(d) When the expression level of the TSPAN-2 protein or mRNA encoding it measured in the control group is compared with that measured in the experimental group, the candidate substance is rheumatoid when the level measured in the experimental group is lower than that measured in the control group. And determining that it can be used as a prophylactic agent for preventing the development of arthritis or a therapeutic agent for treating rheumatoid arthritis.
또는 상기 스크리닝 방법은 (a) TSPAN-2 단백질 또는 이를 암호화하는 mRNA를 발현하는 분리된 활막 세포에 류마티스 관절염 치료 후보 물질을 처리하는 단계;Alternatively, the screening method may include (a) processing a candidate substance for treating rheumatoid arthritis in isolated synovial cells expressing TSPAN-2 protein or mRNA encoding the same;
(b) 상기 후보 물질이 처리된 분리된 활막 세포에서 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및(b) measuring the expression level of TSPAN-2 protein or mRNA encoding it in isolated synovial cells treated with the candidate substance; And
(c) 상기 (b) 단계에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 수준이 후보 물질이 처리되지 않은 분리된 활막 세포에 비해 낮은 수준을 나타내는 경우, 상기 후보 물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는 것을 특징으로 한다.(c) When the level of TSPAN-2 protein or mRNA expression encoding it measured in step (b) is lower than that of isolated synovial cells in which the candidate substance is not treated, the candidate substance is prevented from developing rheumatoid arthritis. And determining that it can be used as a preventive agent for preventing or a therapeutic agent for treating rheumatoid arthritis.
본 발명에서 사용된 용어, "후보 물질"이란 류마티스 관절염 증상을 완화 또는 치료하는 활성을 테스트할 물질을 의미하며, 예컨대 추출물, 단백질, 올리고펩티드, 소형 유기 분자, 다당류, 폴리뉴클레오티드 및 광범위한 화합물 등의 임의 분자를 포함할 수 있다. 이러한 후보 물질은 또한 천연물질뿐 아니라, 합성물질도 포함한다.As used herein, the term "candidate" refers to a substance to test the activity of alleviating or treating rheumatoid arthritis symptoms, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide range of compounds, etc. Any molecule can be included. These candidate substances also include natural as well as synthetic substances.
TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 수준을 측정하는 방법은 당업계에 공지된 방법을 제한 없이 사용할 수 있으며, 예컨대 역전사 중합효소 연쇄반응(Reverse Transcription-Polymerase chain Reaction, RT-PCR), 효소면역분석법(ELISA), 면역조직화학, 웨스턴 블랏(Western Blotting) 및 유세포 분석법(FACS)으로 구성된 군으로부터 선택된 어느 하나의 방법으로 측정할 수 있으나, 이에 제한되는 것은 아니다.As a method for measuring the TSPAN-2 protein or mRNA expression level encoding the same, methods known in the art can be used without limitation, for example, Reverse Transcription-Polymerase chain Reaction (RT-PCR), Enzyme Immunity It can be measured by any one method selected from the group consisting of an assay (ELISA), immunohistochemistry, Western blotting and flow cytometry (FACS), but is not limited thereto.
본 발명의 스크리닝 방법에 의하여 스크리닝된 후보 물질은 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성을 억제할 수 있는 바 류마티스 관절염 증상의 완화 또는 치료를 위하여 사용될 수 있다.The candidate materials screened by the screening method of the present invention can be used for the relief or treatment of rheumatoid arthritis symptoms that can inhibit the expression or activity of TSPAN-2 protein or mRNA encoding it.
본 발명은 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 제제를 제조하기 위한 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제의 용도를 제공한다.The present invention is a type of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid arthritis and tetraspanin-2 for preparing an agent for the prevention or treatment of any one or more diseases selected from the group consisting of rheumatoid arthritis (Tetraspanin-2, TSPAN-2) provides the use of inhibitors for the expression or activity of proteins or mRNAs encoding them.
본 발명은 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 치료 방법을 제공한다.The present invention comprises the step of administering an effective amount of a composition comprising an inhibitor or an expression or activity inhibitor of a protein or mRNA encoding tetraspanin-2 (Tetraspanin-2, TSPAN-2) to an individual in need thereof It provides a method of treating any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple muscle pain.
본 발명의 상기 ‘유효량’이란 개체에게 투여하였을 때, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증과 같은 질환의 개선, 치료, 예방, 검출, 진단 또는 상기 질환의 억제 또는 감소 효과를 나타내는 양을 말하며, 상기 ‘개체’란 동물, 바람직하게는 포유동물, 특히 인간을 포함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직, 기관 등일 수도 있다. 상기 개체는 상기 효과가 필요한 환자(patient) 일 수 있다.Improvement, treatment, prevention, detection of diseases such as rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia when administered to an individual with the'effective amount' of the present invention, Refers to an amount that shows a diagnosis or suppression or reduction effect of the disease, and the'individual' may be an animal, preferably a mammal, particularly an animal including a human, or may be cells, tissues, organs, etc. derived from an animal. . The subject may be a patient in need of the effect.
본 발명의 상기 ‘치료’는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증과 같은 질환 또는 상기 질환의 증상을 개선시키는 것을 포괄적으로 지칭하고, 이는 상기 질환을 치유하거나, 실질적으로 예방하거나, 또는 상태를 개선시키는 것을 포함할 수 있으며, 상기 질환으로부터 비롯된 한 가지 증상 또는 대부분의 증상을 완화시키거나, 치유하거나 예방하는 것을 포함하나, 이에 제한되는 것은 아니다.The'treatment' of the present invention collectively refers to improving diseases or symptoms of such diseases, such as rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, rheumatoid arthritis and rheumatoid polymyalgia, Healing, substantially preventing, or improving the condition, including, but not limited to, alleviating, healing, or preventing one symptom or most symptoms resulting from the disease no.
따라서 본 발명은 TSPAN-2의 발현 또는 활성을 억제할 시 류마티스 관절염에 의한 붓기가 완화되는 것을 확인한 것으로, TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 또는 활성 억제제를 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 예방, 개선 또는 치료용 제제로 활용할 수 있으며, 후보물질을 투여하여 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정함으로써 류마티스 관절염에 치료 효과가 있는 물질을 선별할 수 있다.Therefore, the present invention confirmed that swelling due to rheumatoid arthritis is alleviated when the expression or activity of TSPAN-2 is suppressed, and the TSPAN-2 protein or mRNA expression or activity inhibitor encoding the same is used for rheumatoid arthritis type 1, type 2 Rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid arthritis can be utilized as an agent for the prevention, improvement or treatment of any one or more diseases selected from the group consisting of, and administering a candidate substance to encode TSPAN-2 protein or the same By measuring the expression level of mRNA, it is possible to select a substance having a therapeutic effect on rheumatoid arthritis.
도 1은 동물모델에 류마티스 관절염을 유도한 후, 1주 간격으로 항-TSPAN2 항체를 투여하여 치료 효과를 확인한 결과이다.1 is a result of confirming the therapeutic effect by inducing rheumatoid arthritis in an animal model, and then administering an anti-TSPAN2 antibody at intervals of one week.
도 2a 내지 도 2c는 인간 섬유아세포 유사 활막 세포(Human fibroblast-like synoviocyte, hFLS)에서 TSPAN2의 발현을 확인한 결과이다.2A to 2C are results confirming the expression of TSPAN2 in human fibroblast-like synoviocytes (hFLS).
도 3은 류마티스 관절염 환자로부터 유래한 활막 세포에서 TSPAN2 발현을 확인하고, 항체를 이용하였을 때 이의 발현이 억제되는지 확인한 결과이다.3 is a result confirming TSPAN2 expression in synovial cells derived from a patient with rheumatoid arthritis, and confirming that its expression is suppressed when an antibody is used.
도 4a 내지 도 4c는 항-TSPAN2 항체를 처리한 후, 류마티스 관절염에 주요한 영향을 미치는 인자들의 발현을 확인한 결과이다.4A to 4C are results of confirming the expression of factors having a major influence on rheumatoid arthritis after treatment with an anti-TSPAN2 antibody.
도 5a 내지 도 5c는 TSPAN2 발현 억제에 따른 활액 조직(synovial tissue) 분석결과를 나타낸 것이다. 도 5a는 정상 활액 조직, 도 5b는 류마티스 관절염 모델에서의 활액 조직, 도 5c는 항-TSPAN2 항체 처리시의 활액 조직에 대한 H&E 염색 후 광학현미경 사진을 나타낸다. 도 5a에서는 활막 내막 세포층이 잘 보존되어 있고, 염증성 침윤이 그다지 관찰되지 않고, 도 5b에서는 염증에 의해서 조직이 상당히 손상되었고, 염증성 침윤의 결과가 명확히 나타나 있다. 도 5c에서는 활막 내막 세포층이 잘 보존되어 있고, 염증성 침윤은 그다지 관찰되지 않습니다. 각각 좌측, 중간, 우측 사진은 x40, x200, x400의 배율을 나타낸다.5A to 5C show results of synovial tissue analysis according to suppression of TSPAN2 expression. FIG. 5A shows normal synovial tissue, FIG. 5B shows synovial tissue in a rheumatoid arthritis model, and FIG. 5C shows optical micrographs after H&E staining of synovial tissue during anti-TSPAN2 antibody treatment. In FIG. 5A, the synovial membrane layer is well preserved, and inflammatory infiltration is not observed much, and in FIG. 5B, tissue is significantly damaged by inflammation, and the result of inflammatory infiltration is clearly shown. In Fig. 5c, the synovial lining cell layer is well preserved, and inflammatory infiltration is rarely observed. The left, middle, and right photographs respectively show magnifications of x40, x200, and x400.
도 6a 내지 도 6b는 랫트 류마티스 관절염 모델에서 항-TSPAN2 항체 처리시의 치료 효과를 나타낸 것이다. 도 6a는 Rat의 운동 균형감각과 평형감각을 확인하는 weight bearing 시험이며, 도 6b는 류마티스가 유발된 쥐의 발바닥 두께를 측정한 결과를 나타낸다.6A to 6B show the therapeutic effect of anti-TSPAN2 antibody treatment in a rat rheumatoid arthritis model. Figure 6a is a weight bearing test to check the balance and sense of movement of the rat, Figure 6b shows the results of measuring the thickness of the sole of the rats induced rheumatism.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 이에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited thereby.
실시예 1 : 류마티스 관절염 동물 모델 제작Example 1: Rheumatoid arthritis animal model production
본 발명에 따라 TSPAN-2 발현 또는 활성 억제가 류마티스 관절염에 미치는 영향을 in vivo 상에서 확인하였다. 이를 위하여, 스프라그 도올리 랫트를 Harlan Laboratories, Charles River Laboratories 또는 Janvier Labs로부터 입수하였다. 모든 랫트는 병원균이 없는 환경에서 사육되었고, 멸균된 표준 사료(Ralston Purina, St. Louis, MO)와 물을 자유롭게 섭취하도록 하였다. 8-11 주령 쥐의 tail base(꼬리가 시작되는 부분)의 등 쪽에서 100 μl 프리스탄(pristane (2,6,10,14-tetramethylpentadecane, 95 %, Acros Organics, Morris Plains, NJ, 미국))를 피내 주사하여 류마티스 관절염(PIA)을 유도하였다. 또는 부갑상선 호르몬(Mycoobacterium butyricum) (Difco) 0.4 mg, 순수 IFA 300 μl 및 펩신 소화 0.3 mg을 함유한 100 μl IFA(incomplete freunds adjuvant, Difco Laboratories, Detroit, MI, USA)를 피내주사하여 오일 유발 및 콜라겐 유도 관절염을 유도하였다. 쥐의 연골 육종에서 정제된 II 형 콜라겐 (CII)을 150 μL의 0.1M 아세트산에 용해시키고 동량의 IFA로 각각 유화시켰다. 합성 프리스탄은 Sigma-Aldrich (P2870, St. Louis, MO, USA)에서 구입하였다. 상기 상이한 면역화 프로토콜을 수행한 처리 및 비 처리된 랫트를 우리 내에 함께 수용하였다. ELISA는 혈청 및 관절 조직에서 다양한 사이토카인의 검출에 사용하였고, 관절염의 중증도 및 뒷발 두께를 정량화했다.The effect of suppressing TSPAN-2 expression or activity according to the present invention on rheumatoid arthritis was confirmed in vivo. To this end, Sprague dooli rats were obtained from Harlan Laboratories, Charles River Laboratories or Janvier Labs. All rats were bred in a pathogen-free environment, and were allowed to ingest sterile standard feed (Ralston Purina, St. Louis, MO) and water freely. 100 μl pristane (2,6,10,14-tetramethylpentadecane, 95%, Acros Organics, Morris Plains, NJ, USA) on the dorsal side of the tail base of the 8-11 week old rat. Rheumatoid arthritis (PIA) was induced by intradermal injection. Or oil-induced and collagen by intradermally injecting 100 μl IFA (incomplete freunds adjuvant, Difco Laboratories, Detroit, MI, USA) containing 0.4 mg of mycobacterium butyricum (Difco), 300 μl of pure IFA and 0.3 mg of pepsin digestion Induced arthritis. Type II collagen (CII) purified from rat cartilage sarcoma was dissolved in 150 μL of 0.1M acetic acid and emulsified with the same amount of IFA, respectively. Synthetic pristane was purchased from Sigma-Aldrich (P2870, St. Louis, MO, USA). Treated and untreated rats carrying out these different immunization protocols were housed together in cages. ELISA was used to detect various cytokines in serum and joint tissues, and the severity of arthritis and hind paw thickness were quantified.
실시예 2 : TSPAN-2 발현 또는 활성 억제에 따른 류마티스 관절염 치료 효과 확인Example 2: Confirmation of the therapeutic effect of rheumatoid arthritis according to TSPAN-2 expression or activity inhibition
상기에서 제조한 류마티스 관절염을 유도한 동물 모델에 항-TSPAN-2 항체(앱클론에서 제작됨)를 투여한 후, 류마티스 관절염 치료 효과를 확인하였다.After administering an anti-TSPAN-2 antibody (manufactured by Abclone) to the animal model inducing rheumatoid arthritis prepared above, the therapeutic effect of rheumatoid arthritis was confirmed.
류마티스 관절염을 유도한 후 1주 후부터 1주 간격으로 항-TSPAN-2 항체 20μg을 피하주사하였다. 그 결과 도 1과 같이, 항체 투여 1주 후에 발의 두께가 정상화되어 완전관해(complete remission, 병의 징후가 완전히 사라짐)가 된 것을 확인하였다.After inducing rheumatoid arthritis, 20 μg of anti-TSPAN-2 antibody was injected subcutaneously at 1 week intervals. As a result, as shown in FIG. 1, it was confirmed that the thickness of the foot was normalized 1 week after administration of the antibody, resulting in complete remission (complete signs of disease).
실시예 3 : 인간 섬유아세포 유사 활막 세포에서의 TSPAN2 발현 분석Example 3: Analysis of TSPAN2 expression in human fibroblast-like synovial cells
인간 섬유아세포 유사 활막 세포(Human fibroblast-like synoviocyte, hFLS)로부터 TSPAN2의 발현을 분석하기 위하여, 강원대학교 의과대학교 실험실에 보관되어 있던 류마티스 관절염 환자에서 얻은 3종류의 세포를 사용하였다. 상기 hFLS를 파라포름알데히드(paraformaldehyde)로 상온에서 15분 동안 고정하고, 0.1% BSA(bovine serum albumin)가 포함된 PBS로 세척한 후, 0.2% Triton X-100이 포함된 PBS로 상온에서 15분 동안 처리하였다. 그런 후 hFLS를 0.1% BSA가 포함된 PBS로 세척 후 FACS tube로 옮긴 후, 1차 항체와 4℃에서 15분 동안 반응시켰다. 1차 항체로는 TSPAN2에 대한 mAb 하이브리도마 (RA1Ab, RA2Ab, RA3Ab) 배양액을 이용하였다. 이때 정상 섬유아세포(fibroblast)를 negative control로 사용하였다. FACS buffer (0.1% BSA와 0.05% 아지드화 나트륨(sodium azide)이 포함된 PBS)로 세척 후 FITC(Fluorescein isothiocyanate)로 표지된 goat anti-mouse IgG (H+L) F(ab’)2 fragment (Jackson ImmunoResearch)와 4℃에서 15분 동안 반응시켰다. FACS buffer로 세척 후 BD FACS Calibur로 hFLS를 분석하였다.In order to analyze the expression of TSPAN2 from human fibroblast-like synoviocytes (hFLS), three types of cells obtained from rheumatoid arthritis patients stored in the laboratory of Kangwon National University Medical School were used. The hFLS was fixed with paraformaldehyde for 15 minutes at room temperature, washed with PBS containing 0.1% BSA (bovine serum albumin), and then 15 minutes at room temperature with PBS containing 0.2% Triton X-100. For a while. Then, hFLS was washed with PBS containing 0.1% BSA, transferred to a FACS tube, and reacted with the primary antibody at 4°C for 15 minutes. As a primary antibody, a mAb hybridoma (RA1Ab, RA2Ab, RA3Ab) culture medium for TSPAN2 was used. At this time, normal fibroblasts were used as negative control. Goat anti-mouse IgG (H+L) F(ab')2 fragment labeled with FITC (Fluorescein isothiocyanate) after washing with FACS buffer (PBS containing 0.1% BSA and 0.05% sodium azide) (Jackson ImmunoResearch) and reacted at 4°C for 15 minutes. After washing with FACS buffer, hFLS was analyzed by BD FACS Calibur.
그 결과 도 2a 내지 도 2c와 같이, 인간 류마티스 관절염 hFLS에서 TSPAN2가 발현된 것을 확인하였다. TSPAN2에 대한 RA1Ab, RA2Ab, RA3Ab 항체 가운데 RA1Ab가 가장 강력하게 결합하는 것으로 나타났으며, RA2Ab와 RA3Ab의 결합은 상대적으로 약하였다.As a result, it was confirmed that TSPAN2 was expressed in human rheumatoid arthritis hFLS as shown in FIGS. 2A to 2C. Of the RA1Ab, RA2Ab, and RA3Ab antibodies to TSPAN2, it was found that RA1Ab binds most strongly, and the binding between RA2Ab and RA3Ab is relatively weak.
특히, 류마티스 관절염 환자 3명에서 얻은 hFLS가 이들 3가지 항체에 대한 결합 반응에서 유사한 경향을 보인 것은 TSPAN2가 환자에 따라 편차를 보이지 않고 류마티스 관절염 hFLS에 일반적으로 발현되어 있을 가능성을 시사하고 있다. In particular, the hFLS obtained in three patients with rheumatoid arthritis showed a similar trend in the binding response to these three antibodies, suggesting the possibility that TSPAN2 does not show a variation among patients and is generally expressed in rheumatoid arthritis hFLS.
실시예 4 : 류마티스 관절염 활막 세포에서 TSPAN2 발현 확인 및 억제 시 영향 확인Example 4: Confirmation of TSPAN2 expression in rheumatoid arthritis synovial cells and its effect upon inhibition
류마티스 관절염 활막 세포에서 TSPAN2의 발현을 확인하고, 더불어 항체를 이용하여 TSPAN2 발현 억제 시의 영향을 확인하였다. 이를 위한 인간 단일 항체(Human monoclonal) TSPAN2 (앱클론제작), 염소 다클론 액틴 항체(goat polyclonal actin antibodies)와 2차 항체(secondary antibodies, Santa Cruz, CA, USA)를 준비하였다. 더불어 ELISA에 사용된 인터루킨-1β(Interleukin-1beta, IL-1β), 종양 괴사 인자 알파(tumor necrosis factor-alpha, TNF-α), 인터페론-감마(interferon-gamma, IFN-γ)는 peprotech에서 구입했다.TSPAN2 expression was confirmed in rheumatoid arthritis synovial cells, and an effect of suppressing TSPAN2 expression was also confirmed using an antibody. Human monoclonal TSPAN2 (manufactured by Applon), goat polyclonal actin antibodies and secondary antibodies (Secondary antibodies, Santa Cruz, CA, USA) were prepared for this purpose. In addition, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) used in ELISA are purchased from peprotech. did.
더불어 Amaxa Human Dermal Fibroblast Nucleofector 키트 (NHDF-성인)를 사용하여 1 내지 5μg의 항-TSPAN2(anti-TSPAN2) 항체를 2~5×10 5(계대 4~6) 섬유아세포 유사 활막 세포에 형질감염시켰다. In addition, 1-5 μg of anti-TSPAN2 (anti-TSPAN2) antibody was transfected into 2-5×10 5 (passage 4-6) fibroblast-like synovial cells using the Amaxa Human Dermal Fibroblast Nucleofector Kit (NHDF-adult). .
그런 후, hFLSs는 6-웰 플레이트에서 10 % FCS를 갖는 DMEM에서 적절한 시간 동안 배양되었고, 0.1 % FCS를 갖는 DMEM에서 동기화되었다. hFLSs는 15분 동안 배양배지로 처리하였고 세포 용해물은 이전에 기술된 바와 같이 수득하였다. 전체 세포 용해물(50μg)을 Tris-글리신(glycine)으로 완충된 10% SDS-PAGE에서 분획하고 니트로셀룰로오스 멤브레인 (nitrocellulose membrane, Biorad, Hercules, CA, USA)으로 옮겼다. 막을 실온에서 1시간 동안 0.05 % Tween 20/Tris-완충 식염수(TBS) 중의 5% 탈지 우유(skim milk)로 차단시킨 후 1차 항체(1 : 1000)로 항온 배양하였다. 그다음 블롯을 2차 항체에서 실온에서 2시간 동안 배양하였다. 면역반응성의 단백질(Immunoreactive protein)은 Enhanced chemiluminescence (Perkin Elmer, Waltham, MA, USA)와 자동 방사선 촬영으로 검출되었으며, NIH Image (버전 1.63)를 사용하여 분석되었고 액틴 발현에 대해 정상화되었다.The hFLSs were then incubated in DMEM with 10% FCS in 6-well plates for a suitable time and synchronized in DMEM with 0.1% FCS. hFLSs were treated with culture medium for 15 minutes and cell lysates were obtained as previously described. Total cell lysate (50 μg) was fractionated on 10% SDS-PAGE buffered with Tris-glycine and transferred to a nitrocellulose membrane (Biorad, Hercules, CA, USA). The membrane was blocked with 5% skim milk in 0.05% Tween 20/Tris-buffered saline (TBS) for 1 hour at room temperature and then incubated with primary antibody (1:1000). The blot was then incubated for 2 hours at room temperature in the secondary antibody. Immunoreactive proteins were detected by enhanced chemiluminescence (Perkin Elmer, Waltham, MA, USA) and autoradiography, analyzed using NIH Image (version 1.63) and normalized to actin expression.
그 결과 도 3에 나타낸 바와 같이, 류마티스 관절염 활막 세포에서의 TSPAN2 발현이 항체 처리에 의하여 농도의존적으로 억제됨을 확인하였다. 또한 도 4a와 같이 항-TSPAN2 항체를 처리한 실험군에서의 TNF-α의 억제가 정상군과 비슷한 수준으로 이루어짐을 알 수 있었다. As a result, as shown in FIG. 3, it was confirmed that TSPAN2 expression in rheumatoid arthritis synovial cells was suppressed in a concentration-dependent manner by antibody treatment. In addition, it can be seen that the inhibition of TNF-α in the experimental group treated with the anti-TSPAN2 antibody was achieved at a level similar to that of the normal group as shown in FIG. 4A.
특히 도 4b와 같이, 항-TSPAN2 항체를 처리한 실험군에서의 IFN-γ의 증가가 정상군과 비슷한 수준으로 이루어짐을 확인하였다. In particular, as shown in Figure 4b, it was confirmed that the increase in IFN-γ in the experimental group treated with the anti-TSPAN2 antibody was made to a level similar to the normal group.
더불어 도 4c와 같이, 류마티스 관절염의 연골과 뼈 침식의 원인 물질인 IL-1β가 항-TSPAN2 항체를 처리한 실험군에서 정상군과 비슷한 수준으로 감소됨을 확인하였다. 따라서 항-TSPAN2 항체의 처리가 류마티스 관절염에 대하여 뚜렷한 치료 효과를 보임을 확인하였다. In addition, as shown in Fig. 4c, it was confirmed that IL-1β, a causative agent of cartilage and bone erosion of rheumatoid arthritis, was reduced to a level similar to that of the normal group in the experimental group treated with anti-TSPAN2 antibody. Therefore, it was confirmed that treatment with anti-TSPAN2 antibody showed a clear therapeutic effect against rheumatoid arthritis.
실시예 5 : 류마티스 관절염 동물모델에서 활액 조직의 변화 확인Example 5: Confirmation of changes in synovial tissue in rheumatoid arthritis animal model
실시예 1에서의 류마티스 관절염 동물 모델에 대해서 항-TSPAN2 항체를 처리한 경우 활액 조직이 어떻게 변화하는 지를 확인하였다. When the anti-TSPAN2 antibody was treated with respect to the rheumatoid arthritis animal model in Example 1, it was confirmed how synovial tissue changes.
항체는 일주에 한번 피하주사 하였으며 weight bearing 결과와 행동관찰 실험이 끝난 4주 후에 CO 2를 이용하여 랫을 희생시키고 관절을 포함한 하측 다리를 잘라 탈회 후 고정하였다. 그 후 H&E(헤마톡실린&에오신) 염색으로 활액 조직을 염색하여 비교 분석하였다.Antibodies were injected subcutaneously once a week, and rats were sacrificed using CO 2 after weight bearing results and behavior observation experiments, and the lower leg including the joint was cut and fixed after deliming. After that, the synovial tissue was stained with H&E (hematoxylin & eosin) staining for comparative analysis.
그 결과, 도 5a 내지 도 5c에서 보듯이, 정상 활액 조직(도 5a)에서는 활막 내막 세포층이 잘 보존되어 있고, 염증성 침윤이 그다지 관찰되지 않은 상태를 나타냈고, 류마티스 관절염을 유도하는 경우(도 5b) 염증에 의해서 조직이 상당히 손상되었고, 염증성 침윤이 상당히 진행되는 것을 확인할 수 있었다. 이에 비해서 류마티스 관절염 모델에 대해서 항-TSPAN2 항체를 처리하는 경우(도 5c) 정상 활액 조직과 유사하게 활막 내막 세포층이 잘 보존되어 있고, 염증성 침윤은 그다지 관찰되지 않음을 확인하여 류마티스에 대한 치료 효과가 우수한 것을 확인할 수 있었다.As a result, as shown in FIGS. 5A to 5C, in the normal synovial tissue (FIG. 5A), the synovial membrane layer was well preserved, and inflammatory infiltration was not observed much, and when inducing rheumatoid arthritis (FIG. 5B) ) It was confirmed that the tissue was significantly damaged by inflammation, and the inflammatory infiltration proceeded significantly. In contrast, when the anti-TSPAN2 antibody was treated against the rheumatoid arthritis model (FIG. 5C), it was confirmed that the synovial membrane layer was well preserved, and inflammatory infiltration was not observed much, similar to normal synovial tissue. It was confirmed that it was excellent.
실시예Example 6 : 6: 랫트Rat 류마티스Rheumatism 관절염 모델에서 In the arthritis model TSPAN2TSPAN2 억제에 따른 Restraint 류마티스Rheumatism 관절염의 치료 효과 Treatment effect of arthritis
실시예 1에서의 류마티스 관절염 동물 모델에 대해서 항-TSPAN2 항체를 처리한 경우 류마티스 관절염에 대한 치료 효과를 확인하였다.When the anti-TSPAN2 antibody was treated with respect to the rheumatoid arthritis animal model in Example 1, the therapeutic effect on rheumatoid arthritis was confirmed.
일단, 통증 행동에 대한 테스트로 동적 무게 베어링(weight bearing) 시험을 수행하였다. 랫트의 이동 경로의 아래에 장착된 로드 셀 센서 (CB1-K2, DACELL, Korea)에 의해서 행동을 측정하였고, 각 테스트는 3회 반복되었다.First, a dynamic weight bearing test was performed as a test for pain behavior. Behavior was measured by a load cell sensor (CB1-K2, DACELL, Korea) mounted under the rat's travel path, and each test was repeated three times.
weight bearing 시험 결과, 도 6a에서 보듯이, 대조군에 비해 처리군이 투여한 발의 통증이 줄어들어 절룩거리거나 다리를 끌지않고 균형에 맞게 걷는 것을 확인할 수 있었다. As a result of the weight bearing test, as shown in FIG. 6A, it was confirmed that the pain of the foot administered by the treatment group was reduced compared to the control group, so that walking in a balanced manner without limping or dragging the leg was observed.
또한, 류마티스가 유발된 쥐의 발바닥 두께도 도 1에서와 같이 시간이 지남에 따라 대조군에 비해 처리군이 부종이 줄어 발바닥 두께가 줄어드는 것을 알 수 있었다. 부종의 경우 1주차에는 약간 감소한 결과를 나타났으나, 2주 이후부터는 크게 감소하여 류마티스에 의한 부종이 크게 감소함을 알 수 있었다.In addition, it was found that the thickness of the soles of the rats caused by rheumatism also decreased in swelling in the treatment group compared to the control group as time passed, as shown in FIG. 1. In the case of edema, there was a slight decrease in the 1st week, but it was found that the edema caused by rheumatism decreased significantly after 2 weeks.
이상 살펴본 바와 같이, 본 발명에 따르면 TSPAN-2의 발현 또는 활성을 억제할 시 투여 1주 후부터 류마티스 관절염에 의한 붓기가 완화되며 2주 후에는 정상 대조군과 유사할 정도로 증상이 치료된 것을 확인할 수 있으므로, TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 또는 활성 억제제를 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아 류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 예방, 개선 또는 치료용 제제로 활용할 수 있으며, 후보물질을 투여하여 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정함으로써 류마티스 관절염에 치료 효과가 있는 물질을 선별할 수 있다.As described above, according to the present invention, when suppressing the expression or activity of TSPAN-2, swelling due to rheumatoid arthritis is relieved from 1 week after administration, and after 2 weeks, it can be confirmed that symptoms have been treated to a degree similar to that of the normal control group. Prevention of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia, a TSPAN-2 protein or an mRNA expression or activity inhibitor encoding the same , It can be used as a preparation for improvement or treatment, and by measuring the expression level of TSPAN-2 protein or mRNA encoding it by administering a candidate substance, a substance having a therapeutic effect on rheumatoid arthritis can be selected.

Claims (11)

  1. 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 약학적 조성물.Type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis, containing as an active ingredient an inhibitor of the expression or activity of a tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it And Rheumatic multiple muscle pain is selected from the group consisting of a pharmaceutical composition for preventing or treating any one or more diseases.
  2. 제1항에 있어서, 상기 TSPAN-2의 발현 또는 활성 억제제는 TSPAN-2 단백질 또는 이를 암호화하는 mRNA에 특이적으로 결합하는 항체, 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 천연추출물 및 합성화합물로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 약학적 조성물. The method of claim 1, wherein the expression or activity inhibitor of TSPAN-2 is an antibody, compound, peptide, peptide mimetics, aptamer, natural extract and synthetic specifically binding to TSPAN-2 protein or mRNA encoding the same. Pharmaceutical composition, characterized in that any one selected from the group consisting of compounds.
  3. TSPAN-2의 발현 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 치료 방법.Type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis, and the step of administering a pharmaceutical composition comprising an inhibitor of expression or activity of TSPAN-2 as an active ingredient to a subject other than humans. A method of treating any one or more diseases selected from the group consisting of rheumatoid multiple muscle pain.
  4. 테트라스파닌-2(Tetraspanin-2, TSPAN-2)의 발현 또는 활성 억제제를 유효성분으로 포함하는, 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 개선용 식품 조성물.Type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple myopathy, comprising an inhibitor of the expression or activity of tetraspanin-2 (TSPraspanin-2, TSPAN-2) as an active ingredient Food composition for preventing or improving any one or more diseases selected from the group consisting of.
  5. (a) 대조군인 류마티스 관절염이 발병된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계;(a) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal with rheumatoid arthritis as a control;
    (b) 상기 실험동물에서 류마티스 관절염을 치료할 수 있을 것으로 예상되는 후보물질을 투여하는 단계;(b) administering a candidate substance expected to be able to treat rheumatoid arthritis in the experimental animal;
    (c) 실험군인 상기 후보물질이 투여된 실험동물의 시료로부터 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및(c) measuring the expression level of TSPAN-2 protein or mRNA encoding it from a sample of an experimental animal to which the candidate substance, an experimental group, is administered; And
    (d) 대조군에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 실험군에서 측정된 것과 비교하여, 대조군에서 측정된 것보다도 실험군에서 측정된 것이 낮은 수준을 나타내는 경우, 상기 후보물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는, 류마티스 관절염의 예방 또는 치료용 제제의 스크리닝 방법.(d) When the expression level of the TSPAN-2 protein or mRNA encoding it measured in the control group is compared with that measured in the experimental group, the candidate substance is rheumatoid when the level measured in the experimental group is lower than that measured in the control group. A method for screening a preparation for preventing or treating rheumatoid arthritis, comprising determining that it can be used as a preventive agent for preventing the development of arthritis or a therapeutic agent for treating rheumatoid arthritis.
  6. (a) TSPAN-2 단백질 또는 이를 암호화하는 mRNA를 발현하는 분리된 활막 세포에 류마티스 관절염 치료 후보 물질을 처리하는 단계;(A) treating a candidate substance for treating rheumatoid arthritis in isolated synovial cells expressing TSPAN-2 protein or mRNA encoding the same;
    (b) 상기 후보 물질이 처리된 분리된 활막 세포에서 TSPAN-2 단백질 또는 이를 암호화하는 mRNA의 발현 수준을 측정하는 단계; 및(b) measuring the expression level of TSPAN-2 protein or mRNA encoding it in isolated synovial cells treated with the candidate substance; And
    (c) 상기 (b) 단계에서 측정된 TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 수준이 후보 물질이 처리되지 않은 분리된 활막 세포에 비해 낮은 수준을 나타내는 경우, 상기 후보 물질을 류마티스 관절염의 발병을 방지하는 예방용 제제 또는 류마티스 관절염을 치료하는 치료용 제제로서 사용할 수 있을 것으로 판정하는 단계를 포함하는, 류마티스 관절염의 예방 또는 치료용 제제의 스크리닝 방법.(c) When the level of TSPAN-2 protein or mRNA expression encoding it measured in step (b) is lower than that of isolated synovial cells in which the candidate substance is not treated, the candidate substance is prevented from developing rheumatoid arthritis. A method of screening for an agent for preventing or treating rheumatoid arthritis, comprising the step of determining that it can be used as a preventive agent for preventing or a therapeutic agent for treating rheumatoid arthritis.
  7. 제5항 또는 제6항에 있어서, TSPAN-2 단백질 또는 이를 암호화하는 mRNA 발현 수준의 측정은 역전사 중합효소 연쇄반응(Reverse Transcription-Polymerase chain Reaction, RT-PCR), 효소면역분석법(ELISA), 면역조직화학, 웨스턴 블랏(Western Blotting) 및 유세포 분석법(FACS)으로 구성된 군으로부터 선택된 어느 하나의 방법으로 측정하는 것을 특징으로 하는 스크리닝 방법.The method of claim 5 or 6, wherein the TSPAN-2 protein or mRNA expression level encoding the same is measured by Reverse Transcription-Polymerase chain Reaction (RT-PCR), Enzyme Immunoassay (ELISA), Immunity. Screening method characterized in that it is measured by any one method selected from the group consisting of histology, Western blotting and flow cytometry (FACS).
  8. 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환 예방 또는 치료용 제제를 제조하기 위한 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제의 용도.Tetraspanin-2 (Tetraspanin-) for the preparation of a preparation for the prevention or treatment of any one or more diseases selected from the group consisting of rheumatoid arthritis type 1, rheumatoid arthritis type 2, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid polymyalgia. 2, TSPAN-2) Use of inhibitors for the expression or activity of proteins or mRNAs encoding them.
  9. 제8항에 있어서, 상기 TSPAN-2의 발현 또는 활성 억제제는 TSPAN-2 단백질 또는 이를 암호화하는 mRNA에 특이적으로 결합하는 항체, 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 천연추출물 및 합성화합물로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 용도. The method of claim 8, wherein the expression or activity inhibitor of TSPAN-2 is an antibody, compound, peptide, peptide analog (mimetics), aptamer, natural extract and synthetic specifically binding to TSPAN-2 protein or mRNA encoding the same. Use characterized in that it is any one selected from the group consisting of compounds.
  10. 테트라스파닌-2(Tetraspanin-2, TSPAN-2) 단백질 또는 이를 암호화하는 mRNA의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로하는 개체에 투여하는 단계를 포함하는 제1형 류마티스 관절염, 제2형 류마티스 관절염, 임신성 류마티스 관절염, 소아류마티스 관절염 및 류마티스다발근육통증으로 이루어진 군에서 선택된 어느 하나 이상의 질환의 치료 방법.Type 1 comprising administering to the individual in need thereof an effective amount of a composition comprising an inhibitor or an expression or activity inhibitor of tetraspanin-2 (Tetraspanin-2, TSPAN-2) protein or mRNA encoding it. A method for treating any one or more diseases selected from the group consisting of rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, pediatric rheumatoid arthritis and rheumatoid multiple myalgia.
  11. 제10항에 있어서, 상기 TSPAN-2의 발현 또는 활성 억제제는 TSPAN-2 단백질 또는 이를 암호화하는 mRNA에 특이적으로 결합하는 항체, 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 천연추출물 및 합성화합물로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 치료 방법.The method of claim 10, wherein the expression or activity inhibitor of TSPAN-2 is an antibody, compound, peptide, peptide mimetics, aptamer, natural extract and synthesis that specifically binds to TSPAN-2 protein or mRNA encoding the same. A treatment method characterized in that it is any one selected from the group consisting of compounds.
PCT/KR2020/000580 2019-01-14 2020-01-13 Composition for preventing, alleviating or treating rheumatoid arthritis, containing tetraspanin-2 inhibitor as active ingredient WO2020149593A2 (en)

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