WO2020171274A1 - Peptide ayant une activité prophylactique ou thérapeutique contre une maladie inflammatoire ou auto-immune et utilisation associée - Google Patents

Peptide ayant une activité prophylactique ou thérapeutique contre une maladie inflammatoire ou auto-immune et utilisation associée Download PDF

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WO2020171274A1
WO2020171274A1 PCT/KR2019/002415 KR2019002415W WO2020171274A1 WO 2020171274 A1 WO2020171274 A1 WO 2020171274A1 KR 2019002415 W KR2019002415 W KR 2019002415W WO 2020171274 A1 WO2020171274 A1 WO 2020171274A1
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seq
disease
autoimmune
amino acid
inflammatory
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Korean (ko)
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김용애
김지선
정지호
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Hankuk University of Foreign Studies HUFS
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Hankuk University of Foreign Studies HUFS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is an inflammatory disease or autoimmune disease comprising any one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4 It is for a peptide having a prophylactic or therapeutic activity of.
  • the present invention also relates to a pharmaceutical composition or an anti-inflammatory composition for preventing or treating inflammatory diseases or autoimmune diseases comprising any one or more peptides of SEQ ID NOs: 1 to 4.
  • the present invention also relates to a method for treating an inflammatory disease or an autoimmune disease comprising any one or more peptides of SEQ ID NOs: 1 to 4, or to use in the manufacture of a therapeutic drug.
  • Immune tolerance refers to unresponsiveness to a specific antigen and is distinguished from immunodeficiency in which the immune system is not entirely immune. This is important for maintaining immune homeostasis and suppressing inflammation in the body, so if it is deficient, it may cause diseases such as inflammatory diseases or autoimmune diseases.
  • arthritis is largely classified into two categories, and there are over 100 different types.
  • first category a disease in which inflammation and pain occur in bones or ligaments due to damage or degenerative changes in the cartilage that protects joints, and is commonly called'degenerative arthritis'.
  • the second category is ‘inflammatory arthritis,’ which includes all types caused by an excessive immune system attacking the lining of the joint.
  • Rheumatoid arthritis is a representative of the inflammatory arthritis, which is known to be caused by genetic or environmental factors, and is also a representative autoimmune disease. Rheumatoid arthritis suffers from about 1% of the population, and is known to be more common in women than in men due to the influence of female hormones.
  • rheumatoid arthritis is an imbalance between pro-inflammatory cytokines and anti-inflammatory cytokines.
  • cytokines such as TNF- ⁇ , IL-1, or IL-6
  • immune cells such as lymphocytes and macrophages are activated.
  • the inflammation-inducing cytokines collect inflammation-inducing cytokines into the joint and directly activate osteoclasts. In addition, it activates T cells and B cells, and promotes catabolism (decomposition) of cartilage and cartilage cells.
  • the pharmaceutical industry is largely divided into a low-molecular chemical pharmaceutical industry produced through chemical synthesis, and a high-molecular biopharmaceutical industry produced through biological methods, mainly for proteins and vaccines.
  • chemical drugs have a small molecular weight and are easily absorbed in the body, many drugs are developed for oral administration, such as pills, but biopharmaceuticals are difficult to absorb because of their high molecular weight, proteins, etc., may be decomposed in the stomach, or more stable in aqueous solutions. It is mainly developed in the form of injection.
  • biopharmaceutical market created by utilizing the functions and information of living organisms is growing and changing faster than the chemically synthesized drug market.
  • Biopharmaceuticals have fewer side effects due to their high specificity, and have a low probability of adverse immune reactions, as well as alternative drugs for proteins lacking in the body. It has the advantage of being able to use it.
  • since it uses biologically-derived substances it has low toxicity and high safety, and has excellent therapeutic effects in intractable and chronic diseases.
  • the manufacturing process is difficult because it has a large molecular weight and a complex molecular structure compared to synthetic drugs.
  • Peptides can be mass-produced by mechanical synthesis, have low antigenicity due to their short length, and have the advantages of low production cost due to a simple production process, and thus are attracting attention as a product that replaces existing protein therapeutics.
  • low-molecular-weight peptides act in vivo and are discharged to the outside of the body, so there is no risk of toxicity in the body.
  • the risk of side effects such as the occurrence of antibodies due to immune reactions is low, and since it specifically binds to a target substance, it can exhibit high activity.
  • the K562 protein which was first isolated from the leukemia cell line K562 cell culture, is known as a new anti-inflammatory candidate substance capable of balancing the imbalance between pro-inflammatory and anti-inflammatory factors as an inhibitory regulator of inflammatory cytokine expression.
  • the protein translated from methionine residue 316 of the full length of the K562 protein is called truncated K562 protein (tK562, 19 kDa), and it is confirmed that it exhibits an inflammatory cytokine inhibitory effect similar to that of the full length of the K562 protein. Became.
  • Non-Patent Document 1 discloses that the IL-10 receptor is expressed in a B-cell chronic lymphocytic leukemia cell line, and proliferation is inhibited when the cell line is treated with IL-10.
  • the mechanism of development of inflammatory diseases, in particular autoimmune arthritis is not yet clear, and treatment is still limited.
  • Non-Patent Document 1 Jesper Jurlander et al., ‘Characterization of Interleukin-10 Receptor Expression on B-cell Chronic Lymphocytic Leukemia cells’ (Blood, Vol 89, No 11 (June 1), 1997: pp 4146-4152)
  • the present inventors have made great efforts to provide a peptide having an anti-inflammatory effect or an autoimmune disease prevention or treatment effect, safety, and suitable size (molecular weight) for development as a pharmaceutical, as a biological agent.
  • a peptide (epitope) essential to substantially induce an anti-inflammatory effect was isolated, which was smaller than the full length of tK562 and confirmed that it more effectively inhibits inflammatory cytokine secretion, and the present invention was completed.
  • an object of the present invention is an inflammatory disease comprising any one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4 Or to provide a peptide having a prophylactic or therapeutic activity of an autoimmune disease.
  • Another object of the present invention is to provide a pharmaceutical composition or an anti-inflammatory composition for preventing or treating inflammatory diseases or autoimmune diseases comprising any one or more peptides of SEQ ID NOs: 1 to 4.
  • Another object of the present invention is to provide a method for treating an inflammatory disease or an autoimmune disease comprising any one or more of the peptides of SEQ ID NOs: 1 to 4, or for use in preparing a therapeutic drug.
  • the present invention comprises any one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4 It provides a peptide having a prophylactic or therapeutic activity of an inflammatory disease or an autoimmune disease.
  • the present invention also includes a peptide comprising the amino acid sequence of SEQ ID NO: 1;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 2;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 3;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 4;
  • a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases including any one or more peptides selected from the group consisting of; A method of treating an inflammatory disease or an autoimmune disease; To use in the manufacture of drugs for treating inflammatory diseases or autoimmune diseases; Provides.
  • the nucleotide sequence encoding SEQ ID NO: 1 is SEQ ID NO: 7
  • the nucleotide sequence encoding SEQ ID NO: 2 is SEQ ID NO: 8
  • the nucleotide sequence encoding SEQ ID NO: 3 is SEQ ID NO: 9
  • the nucleotide sequence encoding the SEQ ID NO: 4 may be SEQ ID NO: 10.
  • the inflammatory disease is pancreatitis, chronic hepatitis, esophagitis, gastritis, colitis, pneumonia, bronchitis, sore throat, myocardial infarction, heart failure, Alzheimer's, arthritis, kidney failure, psoriasis, anemia, diabetes and fibrosis. It may be any one or more selected from the group consisting of.
  • the arthritis is osteoarthritis, degenerative arthritis, inflammatory arthritis, rheumatoid arthritis, detachable osteochondritis, joint ligament damage, meniscus damage, joint misalignment, avascular necrosis, and juvenile idiopathic arthritis. It may be any one or more selected from the group consisting of.
  • the autoimmune disease is alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune Hepatitis, autoimmune ovariitis and orchitis, autoimmune thrombocytopenia, Behcet's disease, vesicular pemphigus, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunity syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scar pemphigus, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, abdominal complex cold globulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic lung Fibrosis, id
  • the present invention also includes a peptide comprising the amino acid sequence of SEQ ID NO: 1;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 2;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 3;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 4;
  • composition comprising any one or more peptides selected from the group consisting of.
  • K562 cytokine' to'tK562' of the present invention means a protein (cytokine) translated from methionine residue 316 of the entire sequence of the K562 protein (cytokine).
  • 'Epitope' of the present invention means a peptide containing a single or a plurality of peptides.
  • 'Anti-inflammatory' of the present invention means that factors or symptoms related to inflammation are suppressed or alleviated so that inflammation is not deepened.
  • peptides such as K562 cytokine which are conventionally used for the prevention or treatment of inflammatory diseases, have problems in development as pharmaceuticals, such as that their size or molecular weight is too large to be absorbed in the body.
  • problems in development as pharmaceuticals such as that their size or molecular weight is too large to be absorbed in the body.
  • research or development of a peptide having a small size or molecular weight and excellent effect is required.
  • the peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4 according to the present invention is an epitope substantially necessary for causing an anti-inflammatory effect in the K562 cytokine to the truncated K562 cytokine, and has a full length of the K562 cytokine to the truncated K562 cytokine. Compared to the case of use, it has an excellent inhibitory effect on the secretion of inflammatory cytokines, and its size or molecular weight is small, so it is suitable to be used as a composition having an effect of preventing or treating inflammatory or autoimmune diseases.
  • a three-dimensional structure of a truncated K562 cytokine (tK562) was first generated through homology modeling using an independent computational simulation technique (FIG. 1). Specifically, a protein having high homology to tK562 was found through PSI-BLAST search, and various template proteins were produced through multiple sequence alignment (MSA). After homology modeling of tK562 and the various template proteins was performed, the structure was optimized through an energy minimization process to determine the three-dimensional structure of tK562 (FIG. 3).
  • the PSI-BLAST generates a position-specific scoring matrix (PSSM) from multiple sequence alignments (MSA) of sequences having a score equal to or greater than a threshold value by a protein-protein sequence similarity search method (FIG. 2).
  • PSSM can find sequences with high scores (i.e., high similarity) through repeated searches and consequently detect distant relationships between proteins.
  • proteins having high homology with a portion of the sequence were selected because there is no protein having high homology to the entire sequence thereof.
  • Multiple sequence alignments align three or more sequences of similar length to produce a concise and informative summary of sequence data, infer homology from the results, and verify evolutionary relationships between sequences.
  • IL-10 inhibits IL-1, IL-6, TNF- ⁇ and IFN- ⁇ , which are cytokines that are frequently produced in autoimmune diseases such as rheumatoid arthritis.
  • the binding of IL-10 and IL-10 receptor (IL-10R- ⁇ ) modulates immune suppression signals to inhibit the synthesis of pro-inflammatory cytokines, and regulates the growth and differentiation of T cells and B cells to trigger an immune stimulating function.
  • IL-10 phosphorylates tyrosine kinase (TYK2) and Janus kinase (JAK1) step by step by binding to the IL-10 receptor in a dimer form, and by phosphorylating STAT 3, consequently transmitting a signal of anti-inflammatory action. do.
  • tyrosine 496 phosphorylated by tK562 is the site of Janus kinase (Example 2).
  • the tK562 structure produced through homology modeling independently constructed in the present invention was produced as a dimer using a docking protocol, and then compared with IL-10, it was confirmed that both sequence or structural similarity was found (Example 3). , In particular, it was confirmed that the portion (active site) in which IL-10 binds to the IL-10 receptor is significantly similar (Example 4).
  • the present inventors selected short epitopes that may have prophylactic or therapeutic activity against inflammatory diseases or autoimmune diseases among the tK562 moieties having high homology with the active site of IL-10, and the peptides of SEQ ID NOs 1 to 4 of the present invention It was proposed as (AIP-YK1 to 4) (Table 1).
  • SEQ ID NO: 5 represents the amino acid sequence of tK562
  • SEQ ID NO: 6 represents the nucleotide sequence of tK562.
  • Th17 cell differentiation experiment was performed to confirm whether the AIP-YK1 to AIP-YK4 epitopes of the present invention have substantially anti-inflammatory activity.
  • Th17 cells play a key role in immune defense of fungi and bacteria, but are known as cells that cause various autoimmune diseases by producing IL-17 or IL-22, which are inflammatory cytokines.
  • all of the epitopes of the present invention exhibited the same or superior level of anti-inflammatory activity as tK562 (SEQ ID NO: 5 or SEQ ID NO: 6), and among them, AIP-YK4 has anti-inflammatory activity similar to that of VIP (neuropeptide vasoactive intestinal peptide). It showed inflammatory activity (Example 5). That is, it was confirmed that the sites necessary to substantially exhibit anti-inflammatory activity among K562 were AIP-YK1 to AIP-YK4.
  • the VIP neuropeptide vasoactive intestinal peptide
  • the VIP is a neuro (nerve) peptide having a 28 amino acid sequence (His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn) As, it is known to have an effect of reducing inflammation or autoimmune components of rheumatoid arthritis.
  • the present invention is an inflammatory disease or autologous disease comprising any one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4 It is possible to provide a peptide having a prophylactic or therapeutic activity of an immune disease.
  • the peptide comprising the amino acid sequence of SEQ ID NO: 3 may have prophylactic or therapeutic activity against inflammatory diseases or autoimmune diseases even in the form of amino acids added to its N-terminus and C-terminus, which is in the form of the amino acid sequence of SEQ ID NO: 1 Can be
  • the present invention can provide a peptide having prophylactic or therapeutic activity for inflammatory diseases or autoimmune diseases comprising the amino acid sequence of SEQ ID NO: 4.
  • the peptide comprising the amino acid sequence of SEQ ID NO: 4 may have prophylactic or therapeutic activity against inflammatory diseases or autoimmune diseases even in the form of amino acids added to its N-terminus and C-terminus, which is in the form of the amino acid sequence of SEQ ID NO: 2 Can be
  • the present invention is a peptide comprising the amino acid sequence of SEQ ID NO: 1;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 2;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 3;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 4;
  • a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases including any one or more peptides selected from the group consisting of; A method of treating an inflammatory disease or an autoimmune disease; To use in the manufacture of drugs for treating inflammatory diseases or autoimmune diseases; Can provide.
  • the pharmaceutical composition of the present invention may contain, without limitation, any nucleotide sequence encoding each of the amino acid sequences of SEQ ID NOs: 1 to 4;
  • the nucleotide sequence encoding SEQ ID NO: 1 is SEQ ID NO: 7
  • the nucleotide sequence encoding SEQ ID NO: 2 is SEQ ID NO: 8
  • the nucleotide sequence encoding SEQ ID NO: 3 is SEQ ID NO: 9
  • SEQ ID NO: 4 The encoding nucleotide sequence is preferably SEQ ID NO: 10.
  • the inflammatory disease of the present invention may correspond without limitation if it is an inflammatory disease involving Th17 or IL-17, but pancreatitis, chronic hepatitis, esophagitis, gastritis, colitis, pneumonia, bronchitis, sore throat, myocardial infarction, heart failure, Alzheimer's, arthritis , Renal failure, psoriasis, anemia, diabetes and fibrosis is preferably any one or more selected from the group consisting of, more preferably arthritis.
  • the arthritis of the present invention is any selected from the group consisting of osteoarthritis, degenerative arthritis, inflammatory arthritis, rheumatoid arthritis, detachable osteochondritis, joint ligament injury, meniscus damage, joint misalignment, avascular necrosis, and juvenile idiopathic arthritis.
  • One or more is preferable, but is not limited thereto.
  • the autoimmune disease of the present invention may correspond without limitation if it is an autoimmune disease involving Th17 or IL-17, but alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison disease, adrenal gland Autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune ovariitis and orchitis, autoimmune thrombocytopenia, Behcet's disease, vesicular pemphigus, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue Immune Dysfunction Syndrome, Chronic Inflammatory Demyelinating Polyneuropathy, Churg-Strauss Syndrome, Scarring Pleuropathy, CREST Syndrome, Cold Aggregate Disease, Crohn's Disease, Disc Lupus, Abdominal Complex Cold Globulinemia, Fibromyalgia-Fibromyositis, Glomerulonephritis, Graves Disease, Guillain-Barre syndrome, Hashimoto's thyroid
  • composition of the present invention may be in various oral or parenteral dosage forms.
  • buffers e.g., saline or PBS
  • antioxidants e.g., bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
  • bacteriostatic agents e.g., EDTA or glutathione
  • chelating agents e.g., EDTA or glutathione
  • fillers e.g., bulking agents, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
  • adjuvants e.g., Aluminum hydroxide
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in one or more compounds, such as starch (corn starch, wheat starch, rice starch, potato Starch), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl -It is prepared by mixing cellulose or gelatin.
  • starch corn starch, wheat starch, rice starch, potato Starch
  • calcium carbonate sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl -It is prepared by mixing cellulose or gelatin.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, or syrups.
  • various excipients such as humectants, sweeteners, fragrances or preservatives are included. I can.
  • cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier and a preservative may be additionally included.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations or suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • injectable ester such as ethyl oleate
  • a base for suppositories witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like may be used.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, it is used externally to the skin; Intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection; Transdermal administration; Alternatively, it may be formulated in the form of a nasal inhalant according to a method known in the art.
  • suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil.
  • suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used.
  • the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
  • transdermal administration means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.
  • the compounds used according to the invention can be prepared using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, pressurized pack or It can be conveniently delivered from a nebulizer in the form of an aerosol spray.
  • a pressurized aerosol the dosage unit can be determined by providing a valve that delivers a metered amount.
  • gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a formula generally known for all pharmaceutical chemistry.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • a pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug sensitivity, administration time of the patient. , Route of administration and rate of excretion, duration of treatment, factors including concurrent 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 administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple.
  • the total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long period of time in multiple doses. . It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.
  • the dosage of the pharmaceutical composition of the present invention varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
  • a daily dosage based on the peptides of the present invention when administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day.
  • per 1 kg of body weight per day preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per day, may be administered in 1 to several times.
  • the dosage amount does not limit the scope of the present invention in any way.
  • composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
  • the pharmaceutical composition of the present invention may also be provided in the form of an external preparation containing peptides as active ingredients.
  • an external application for skin additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents (foaming agent), fragrance, surfactant, water, ionic emulsifier, nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocker, wetting agent, essential oil, dye, pigment, hydrophilic activator, It may contain adjuvants commonly used in the field of dermatology such as lipophilic active agents or any other ingredients commonly used in skin external preparations such as lipid vesicles. In addition, the above ingredients may be introduced in amounts generally used in the field of dermatology.
  • the pharmaceutical composition for preventing and treating inflammatory or autoimmune diseases of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, patch, gel, cream or spray, but is not limited thereto.
  • the present invention is a peptide comprising the amino acid sequence of SEQ ID NO: 1;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 2;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 3;
  • a peptide comprising the amino acid sequence of SEQ ID NO: 4;
  • an anti-inflammatory composition comprising any one or more peptides selected from the group consisting of.
  • Peptides comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 4 of the present invention can significantly inhibit the secretion or expression of inflammatory cytokines such as IL-17, thereby preventing or treating inflammatory or autoimmune diseases
  • a composition comprising any one or more of the above peptides is effective as a pharmaceutical composition for the prevention or treatment of inflammatory diseases or autoimmune diseases, a method of treatment, a use for use in the manufacture of a therapeutic drug, or an anti-inflammatory composition.
  • 1 shows a computer simulation step performed for homology modeling of truncated K562 cytokines.
  • FIG 3 shows the structure of a truncated K562 cytokine determined through homology modeling of the present invention.
  • Fig. 4 shows the results of comparison of amino acid sequences of IL-10 (top) and truncated K562 cytokine (bottom) and their structures. The higher the similarity of both sequences is, the darker blue is displayed, and the lower the similarity, the brighter the color, and eventually it turns to white.
  • FIG. 5 shows the structure of a truncated K562 cytokine dimer and amino acid sequences of SEQ ID NOs: 1 (AIP-YK1), 2 (AIP-YK2), 3 (AIP-YK3) and 4 (AIP-YK4).
  • Peptides of SEQ ID NOs: 1 (AIP-YK1, pep 1), 2 (AIP-YK2, pep 2), 3 (AIP-YK3, pep 3) and 4 (AIP-YK4, pep 4) of the present invention are IL-17 It was confirmed that pep 4 showed an anti-inflammatory effect similar to that of VIP (neuropeptide vasoactive intestinal peptide), a positive control.
  • VIP neuropeptide vasoactive intestinal peptide
  • the phosphorylation pattern was analyzed to confirm the mechanism of anti-inflammatory effect of the K562 cytokine to the truncated K562 cytokine (tK562).
  • transgenic mouse expressing the full-length K562 cytokine to tK562 was prepared, and the phosphorylation pattern of its cell signaling protein was analyzed.
  • IL-10 and truncated K562 cytokine similarly act on the IL-10 receptor to exhibit anti-inflammatory effects, and by comparing tK562 and IL-10, it is essential to show the actual anti-inflammatory effect in tK562.
  • sequence and structural similarities between tK562 and IL-10 were compared, and the docking simulation between tK562 and IL-10R- ⁇ was performed using the docking protocol of the ZDOCK algorithm.
  • a tK562 dimer was created to find all possible binding modes based on desolvation energy, static electricity, and structural complementarity, and a tK562 epitope having high homology with the active site of IL-10 was selected.
  • tK562 Epitope amino acid amount Amino acid sequence Sequence number AIP-YK1 31 TKTPRDKERERYRERERDRERDRERDRER One AIP-YK2 31 GMSNSYAECYPATMDDMAVDSDEEVDYSKMD 2 AIP-YK3 26 PRDKERERYRERERDRERDRDRERDR 3 AIP-YK4 18 YPATMDDMAVDSDEEVDY 4
  • the sequences of the two proteins have a high homology of about 48%, and in particular, the sequence regions having the homology are IL-10 and IL-10 receptors (IL-10R- ⁇ ) Was confirmed to correspond to the binding portion, that is, the active site of IL-10.
  • IL-10R- ⁇ IL-10 receptors
  • tK562 epitopes AIP-YK1, AIP-YK2, AIP-YK3 and AIP-YK4 having a total of four short peptide sequences were finally selected. .
  • the molecular weight of each epitope was less than 2 kDa.
  • naive CD4 + T cells were isolated from the spleen of wild-type Balb/c mice using a magnetic activated cell sorting (MACS) CD4+ T cell separation kit (Miltenyi Biotec, Cologne, Germany) according to the manufacturer's instructions. I did. The isolated naive CD4+ T cells were treated with phosphate buffered saline (PBS), VIP (neuropeptide vasoactive intestinal peptide) or AIP-YK 1 to 4, and then cultured in RPMI medium for 3 days.
  • PBS phosphate buffered saline
  • VIP neuropeptide vasoactive intestinal peptide
  • AIP-YK 1 AIP-YK 1
  • the RPMI medium is 1 g/ml anti-CD3 (eBioscience, San Diego, CA, USA), 1 g/ml anti-CD28 (eBioscience), 2 ng/ml TGF- ⁇ (R&D system) to stimulate Th17 cell differentiation. , Minneapolis, MN, USA), 20 ng/ml IL-6 (BioLegend, San Diego, CA, USA), 5 ⁇ g/ml anti-IFN- ⁇ (BioLegend) and 5 g/ml anti-IL-4 (BioLegend ). After incubation, the levels of IL-17 secreted from Th17 cells were measured by qPCR by stimulation with anti-CD3 and anti-CD28 antibodies for 24 hours.
  • the concentration of IL-17 cytokine was 935 pg/ml when inflammation was induced (TH17[W1]), 547 pg/ml when treated with a positive control VIP, AIP-YK1 (pep 1), 2 (pep 2). , 3 (pep 3) or 4 (pep 4) treatment was 748, 666, 750 or 569 pg/ml, respectively. That is, it was confirmed that the epitopes selected in the present invention can effectively inhibit IL-17, and in particular, AIP-YK4 exhibits the best IL-17 inhibitory effect at a level similar to that of VIP.
  • the peptides provided in the present invention are effective in preventing or treating anti-inflammatory effects or autoimmune diseases by separating peptides (epitopes) essential to substantially produce anti-inflammatory effects from truncated K562 cytokines (tK562) As it is a peptide of a suitable size (molecular weight) to be developed as a product, it has high industrial applicability.
  • SEQ ID NO: 1 is a sequence of AIP-YK1 peptide (epitope) having an anti-inflammatory effect or a prophylactic or therapeutic effect of an autoimmune disease provided by the present invention, and is a 31 amino acid sequence.
  • SEQ ID NO: 2 is a sequence of an AIP-YK2 peptide (epitope) having an anti-inflammatory effect or a prophylactic or therapeutic effect of an autoimmune disease provided by the present invention, and is a 31 amino acid sequence.
  • SEQ ID NO: 3 is a sequence of an AIP-YK3 peptide (epitope) having an anti-inflammatory effect or an effect of preventing or treating autoimmune diseases provided by the present invention, and is a 26 amino acid sequence.
  • SEQ ID NO: 4 is a sequence of an AIP-YK4 peptide (epitope) having an anti-inflammatory effect or an effect of preventing or treating autoimmune diseases provided by the present invention, and is an 18 amino acid sequence.
  • SEQ ID NO: 5 is the amino acid sequence of the truncated K562 cytokine (tK562).
  • SEQ ID NO: 6 is the nucleotide sequence of the truncated K562 cytokine (tK562).
  • SEQ ID NO: 7 is the nucleotide sequence of AIP-YK1 (SEQ ID NO: 1).
  • SEQ ID NO: 8 is the nucleotide sequence of AIP-YK2 (SEQ ID NO: 2).
  • SEQ ID NO: 9 is the nucleotide sequence of AIP-YK3 (SEQ ID NO: 3).
  • SEQ ID NO: 10 is the nucleotide sequence of AIP-YK4 (SEQ ID NO: 4).

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Abstract

La présente invention concerne un peptide comprenant au moins une séquence d'acides aminés choisie dans le groupe constitué par la séquence d'acides aminés SEQ ID NO : 1, la séquence d'acides aminés SEQ ID NO : 2, la séquence d'acides aminés SEQ ID NO : 3, et la séquence d'acides aminés SEQ ID NO : 4, et ayant une activité prophylactique ou thérapeutique contre une maladie inflammatoire ou auto-immune. La présente invention concerne également une composition pharmaceutique ou anti-inflammatoire comprenant au moins l'un des peptides de SEQ ID NO : 1 à 4 destinée à la prophylaxie ou la thérapie d'une maladie inflammatoire ou auto-immune. La présente invention concerne en outre une utilisation d'au moins l'un des peptides de SEQ ID NO : 1 à 4 dans une méthode thérapeutique pour une maladie inflammatoire ou auto-immune ou dans la préparation d'un médicament thérapeutique pour une maladie inflammatoire ou auto-immune. Le peptide comprenant l'une quelconque des séquences d'acides aminés de SEQ ID NO : 1 à 4 peut inhiber de manière significative la sécrétion ou l'expression de cytokines inflammatoires telles que IL-17, etc. et a ainsi, une activité prophylactique ou thérapeutique dirigée contre une maladie inflammatoire ou auto-immune. Par conséquent, une composition comprenant au moins l'un des peptides est efficace pour une utilisation dans une composition pharmaceutique destinée à la prophylaxie ou la thérapie d'une maladie inflammatoire ou auto-immune, dans une méthode thérapeutique, et dans la préparation d'un médicament thérapeutique, ou en tant que composition anti-inflammatoire.
PCT/KR2019/002415 2019-02-20 2019-02-28 Peptide ayant une activité prophylactique ou thérapeutique contre une maladie inflammatoire ou auto-immune et utilisation associée Ceased WO2020171274A1 (fr)

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EP4321528A4 (fr) * 2021-04-09 2025-04-30 Ajou University Industry-Academic Cooperation Foundation Peptide pour prévenir ou traiter des maladies inflammatoires
JP7792727B2 (ja) * 2021-11-24 2025-12-26 ウィングスタビオ インク アミロイド前駆体タンパク質に対する阻害活性を有するペプチドおよびその用途

Citations (5)

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Publication number Priority date Publication date Assignee Title
US5612195A (en) * 1991-12-02 1997-03-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Recombinant production of a human interferon (IFN)-gamma antagonist cytokine
US6207812B1 (en) * 1998-03-13 2001-03-27 Rhode Island Hospital Chondrosarcoma associated genes
KR20090037487A (ko) * 2006-07-26 2009-04-15 바이오마크 파마슈티칼스 리미티드 염증 매개물질의 분비를 감쇄시키는 방법 및 그에 유용한 펩티드
KR20120089800A (ko) * 2009-06-29 2012-08-13 투 투 바이오테크 리미티드 신규 항체 및 치료 및 진단 방법에서 그것의 사용
US9737588B2 (en) * 2013-04-16 2017-08-22 Cellinbio Co., Ltd Method for treating arthritis using IK factor or nucleic acid encoding IK factor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5612195A (en) * 1991-12-02 1997-03-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Recombinant production of a human interferon (IFN)-gamma antagonist cytokine
US6207812B1 (en) * 1998-03-13 2001-03-27 Rhode Island Hospital Chondrosarcoma associated genes
KR20090037487A (ko) * 2006-07-26 2009-04-15 바이오마크 파마슈티칼스 리미티드 염증 매개물질의 분비를 감쇄시키는 방법 및 그에 유용한 펩티드
KR20120089800A (ko) * 2009-06-29 2012-08-13 투 투 바이오테크 리미티드 신규 항체 및 치료 및 진단 방법에서 그것의 사용
US9737588B2 (en) * 2013-04-16 2017-08-22 Cellinbio Co., Ltd Method for treating arthritis using IK factor or nucleic acid encoding IK factor

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