WO2022098051A1 - Novel peptide with anti-inflammatory and tissue regeneration actions - Google Patents

Novel peptide with anti-inflammatory and tissue regeneration actions Download PDF

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Publication number
WO2022098051A1
WO2022098051A1 PCT/KR2021/015692 KR2021015692W WO2022098051A1 WO 2022098051 A1 WO2022098051 A1 WO 2022098051A1 KR 2021015692 W KR2021015692 W KR 2021015692W WO 2022098051 A1 WO2022098051 A1 WO 2022098051A1
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Prior art keywords
peptide
present
absent
composition
group
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PCT/KR2021/015692
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French (fr)
Korean (ko)
Inventor
이철호
김용훈
김경심
노정란
이경륜
김병찬
최동희
김재훈
강은정
고준
최영근
이인복
서윤정
최정현
장동호
박혜연
박정호
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한국생명공학연구원
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    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides

Definitions

  • the present invention relates to novel peptides having anti-inflammatory and tissue regeneration action and uses thereof.
  • Inflammation is a complex biological response of the immune system to clear harmful extrinsic and endogenous signals or pathogens and initiate the healing process.
  • Various infectious factors such as bacteria and viruses, ultraviolet light, heavy metals, cholesterol, asbestos and many nanomaterials, cause inflammation.
  • Inflammatory response is essential for recovery from infection or wound healing process, but uncontrolled inflammatory response or chronic inflammation is not only caused by diseases such as arthritis, metabolic disease, hepatitis, enteritis, gastritis, gastric ulcer, esophagitis, dermatitis, encephalitis, depression, anxiety, etc. It can also cause disability, cognitive impairment, memory impairment, degenerative brain disease, and developmental disability.
  • the inflammatory response is often one of the causes of damage by causing a decrease in the function of cells, tissues, and organs.
  • diseases such as skin aging, loss of muscle strength, and hair loss are diseases caused by tissue damage, and recent attention has been focused on developing therapeutic agents that can effectively inhibit such tissue damage.
  • the present inventors developed the peptide of the present invention after intensive research efforts to develop a therapeutic agent capable of effectively inhibiting the onset and exacerbation of inflammatory diseases and tissue damage-related diseases, which are recently increasing in incidence in modern people, and developed anti-inflammatory and tissue regeneration
  • the present invention was completed by confirming that the effect was excellent.
  • X1 is Ser or absent
  • X2 is Ile or absent
  • X3 is Lys or absent
  • X4 is Gly or absent
  • X5 is Ala or absent
  • X6 is Tyr or absent
  • X2 is Val or absent
  • X3 is Ser or absent
  • X4 is Ser or absent
  • X5 is Ile or absent
  • X6 is Lys or absent.
  • Another object of the present invention is to provide a polynucleotide encoding the peptide and a vector comprising the polynucleotide.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including the peptide.
  • Another object of the present invention is to provide a composition for tissue regeneration comprising the peptide.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating tissue damage-related diseases, including the peptide.
  • Another object of the present invention is to provide a health functional food composition, feed composition, veterinary medicine, quasi-drug, and cosmetic composition comprising the peptide.
  • Another object of the present invention is to provide a method for preventing or treating an inflammatory disease, comprising administering the peptide to an individual.
  • Another object of the present invention is to provide a method for tissue regeneration, comprising administering the peptide to a subject.
  • the peptide of the present invention has anti-inflammatory and tissue regeneration effects, it can be used for tissue regeneration as well as prevention, improvement and treatment of inflammatory diseases.
  • PEP001 is SEQ ID NO: 1
  • PEP002 is SEQ ID NO: 2
  • PEP003 is SEQ ID NO: 3
  • PEP004 is SEQ ID NO: 4
  • PEP005 is SEQ ID NO: 5
  • Amuc_1409 is a functional fragment except for the leader sequence of SEQ ID NO: 6 refers to SEQ ID NO:7.
  • 1 is a comparison result of knee joint thickness and weight bearing measurement results by administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of degenerative arthritis (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 Amuc_1409 vs. vehicle group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.001 PEP001 vs. vehicle group, #p ⁇ 0.05, ##p ⁇ 0.01, ###p ⁇ 0.001 PEP002 vs. vehicle $p ⁇ 0.05, $$$p ⁇ 0.001 PEP003 vs. vehicle, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.001 PEP004 vs. vehicle, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.001 PEP005 vs. vehicle group).
  • Figure 4 is the result of confirming the change in the daily arthritis index (A), ankle joint thickness (B) and edema (C) by Amuc_1409 administration in an animal model of rheumatoid arthritis (p ⁇ 0.05, **p ⁇ 0.01 vs. vehicle group) ).
  • A daily arthritis index change
  • B ankle joint thickness
  • C edema change
  • PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of rheumatoid arthritis ( ⁇ p ⁇ 0.01) , ⁇ p ⁇ 0.001 PEP001 vs. vehicle group, ##p ⁇ 0.01, ###p ⁇ 0.001 PEP002 vs. vehicle group, $p ⁇ 0.05, $$p ⁇ 0.01, $$$p ⁇ 0.001 PEP003 vs. vehicle group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.001 PEP004 vs. vehicle group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.001 PEP005 vs. vehicle group).
  • 11 is an analysis result of liver damage change by administration of Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 in an acute hepatitis animal model (*p ⁇ 0.05 vs. vehicle group).
  • 13 is an analysis result of weight change by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model ( ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01 PEP004 vs. vehicle group, ⁇ p ⁇ 0.01 PEP005 vs. vehicle) army).
  • 16 is an analysis result of changes in the expression of inflammatory marker genes in the colon by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model (*p ⁇ 0.05, **p ⁇ 0.01 vs. vehicle group).
  • 17 is a result of analysis of gastric mucosal damage by Amuc_1409 administration in an animal model of gastritis/gastric ulcer (*p ⁇ 0.05 vs. vehicle group).
  • 19 is an analysis result of gastric mucosal damage by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of gastritis/gastric ulcer (*p ⁇ 0.05 vs. vehicle group).
  • 20 is an inflammatory marker gene analysis result by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an HCl/ethanol-induced gastritis/gastric ulcer model (*p ⁇ 0.05 vs. vehicle group).
  • Figure 21 is a comparison of ear thickness (Figure 21B) and weight change (Figure 21C) per area (Figure 21C) of mouse ear erythema and edema change by Amuc_1409 administration in an animal model of dermatitis ( Figure 21A) (**p ⁇ 0.01) vs. vehicle group).
  • FIG. 22 is a photograph taken of changes in mouse ear erythema and edema by administration of PEP001, PEP002, PEP003, PEP004, and PEP005 in an animal model of dermatitis (FIG. 22A), comparing ear thickness (FIG. 22B) and weight change per area (FIG. 22C) (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.05 vs. vehicle group).
  • 25 is a gene analysis result of brain inflammation index by administration of Amuc_1409 in a depression and anxiety model (*p ⁇ 0.05 vs. vehicle group).
  • 26 is an analysis result of FST, TST immobility posture, and EPM open arm time by Amuc_1409 administration in a depression and anxiety model (*p ⁇ 0.05 vs. vehicle group).
  • FIG. 29 is a graph showing the effect of improving cognitive function and memory in a novel object recognition test (NORT) according to administration of Amuc_1409 and PEP002, PEP003, PEP004 and PEP005 in a cognitive dysfunction model (A and C) , new object detection time; B and D, number of new object detections. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.01)
  • FIG. 30 is a graph showing the effect of improving cognitive function and memory in a novel object recognition test (NORT) for new objects by administration of Amuc_1409 and PEP002, PEP003, PEP004 and PEP005 in a natural aging animal model (A and C, new object detection time; B and D, number of new object detections) (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.01).
  • NERT novel object recognition test
  • Figure 31 shows the time of sniffing behavior to determine the degree of social interaction and social curiosity between two mice after administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 to developmentally disabled animal model mice. (A and C) and the number of times (B and D) analysis results (*p ⁇ 0.05, ***p ⁇ 0.01).
  • Figure 32 is a photograph and graph showing the comparison of the wound healing effect by day according to the application of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in the full-thickness skin defect wound-inducing animal model (*p ⁇ 0.05, **p ⁇ 0.01, * **p ⁇ 0.001 Amuc_1409 vs. vehicle group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01 PEP001 vs. vehicle group, #p ⁇ 0.05, ##p ⁇ 0.01, ###p ⁇ 0.001 PEP002 vs. vehicle group, $p ⁇ 0.05, $$p ⁇ 0.01 PEP003 vs. vehicle group, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.001 PEP004 vs. vehicle group, ⁇ p ⁇ 0.01, ⁇ p ⁇ 0.001 PEP005 vs. vehicle army).
  • Figure 33 confirms the effect of improving muscle strength and muscle mass by administration of Amuc_1409 in an aging animal model (*p ⁇ 0.05 vs. vehicle group).
  • 35 is a result of analysis of muscle strength, muscle mass, and protein synthesis-related gene expression in muscle cells according to PEP001 administration in an aging animal model (*p ⁇ 0.05 vs. vehicle group).
  • Figure 36 is a result of muscle atrophy-related gene analysis by treatment with PEP004 and PEP005 in C2C12 myoblasts (*p ⁇ 0.05 vs. vehicle group).
  • FIG. 37 is a macroscopic photograph (FIG. 37A) and a scoring graph (FIG. 37B) observing the progress of hair growth on the 21st day after application of Amuc_1409 (*p ⁇ 0.05 vs. vehicle group).
  • Fig. 38 shows the results of H&E staining by Amuc_1409 skin application (Fig. 38A), histological number of follicles (Fig. 38B), hair follicle depth (Fig. 38C), dermal thickness (Fig. 38D) and subcutaneous thickness (Fig. 38) after the end of the experimental period in an animal model. 38E) This is a change comparison result (**p ⁇ 0.01, ***p ⁇ 0.001 vs. vehicle group).
  • Fig. 40 shows the results of analysis of gene expression related to collagen degradation and synthesis according to Amuc_1409 treatment in human skin keratinocytes (p ⁇ 0.05).
  • One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 4 or 5.
  • the peptide of the present invention may include a sequence represented by the following general formula 1:
  • X1 is Ser or absent
  • X2 is Ile or absent
  • X3 is Lys or absent
  • X4 is Gly or absent
  • X5 is Ala or absent
  • X6 is Tyr or absent.
  • X1 may be Ser and X2 may be Ile.
  • X1 to X6 may be absent.
  • X1 may be Ser
  • X2 may be Ile
  • X3 may be Lys
  • X4 may be Gly
  • X5 may be Ala
  • X6 may be Tyr.
  • the peptide of the present invention may include a sequence represented by the following general formula 2:
  • X2 is Val or absent
  • X3 is Ser or absent
  • X4 is Ser or absent
  • X5 is Ile or absent
  • X6 is Lys or absent.
  • X6 may be Lys.
  • X1 to X6 may be absent.
  • X1 may be Ala
  • X2 may be Val
  • X3 may be Ser
  • X4 may be Ser
  • X5 may be Ile
  • X6 may be Lys.
  • absence for example "X5 is Ile or absent" should be understood to mean that amino acid residues directly adjacent to the absent amino acid are directly linked to each other by conventional amide bonds.
  • the peptides provided in the present invention include salt forms thereof.
  • salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
  • metal salts include alkali metal salts such as sodium salts, potassium salts and the like; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts and the like; aluminum salts and so on
  • the peptide of the present invention comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-7.
  • the present invention is not limited thereto, and peptides exhibiting the same activity as the sequence are included without limitation.
  • amino acid sequence of SEQ ID NO: 7 of the present invention corresponds to a sequence except for the leader sequence in the amino acid sequence of SEQ ID NO: 6, it contains the amino acid sequence of SEQ ID NO: 7, or consists essentially of SEQ ID NO: 7 (consisting of the amino acid sequence of SEQ ID NO: 7) Peptides essentially of, or consisting of, are also included within the scope of the peptides of the present invention.
  • the peptide of the present invention may consist of 3 to 150 consecutive amino acids among the amino acid sequence of SEQ ID NO: 6 including the amino acid sequence of SEQ ID NO: 4 or 5.
  • the peptide of the present invention may be a peptide derived from Akkermansia muciniphila strain.
  • the peptide of the present invention can be produced according to a method for synthesizing a peptide known in the art. For example, it may be converted into the peptide molecule of the present invention under physiological conditions.
  • the peptide of the present invention may be synthesized according to a solid-phase synthesis process and a liquid-phase synthesis process. That is, the peptide provided in the present invention can be produced by repeatedly condensing a partial peptide or amino acid constituting the peptide molecule, a peptide to be synthesized, and the remaining portion in a desired order.
  • the target peptide can be produced by removing the protecting group.
  • Another aspect of the present invention provides a polynucleotide encoding the peptide.
  • Another aspect of the present invention provides a vector comprising the polynucleotide.
  • the polynucleotide of the present invention is a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds, and is a DNA or RNA strand of a certain length or longer, which means a polynucleotide encoding the peptide according to the present invention. do.
  • a known vector such as a plasmid vector, a cosmid vector, or a bacteriophage vector
  • the vector is any known vector using DNA recombination technology. It can be easily prepared by those skilled in the art according to the method
  • peptide analog having a function corresponding to the peptide provided by the present invention is also included in the scope of the present invention.
  • peptide analog refers to a non-naturally occurring amino acid sequence, or a modified naturally occurring amino acid sequence.
  • the peptide of the present invention may be in a form in which a carrier material is linked.
  • Examples of the carrier material usable in the present invention may be selected from the group consisting of immunoglobulin Fc region, albumin, transferrin, and polyethylene glycol (PEG), but is not limited thereto.
  • PEG non-specifically binds to a specific site or various sites of the target peptide to increase the molecular weight of the peptide, thereby inhibiting loss by kidney and preventing hydrolysis, and does not cause any special side effects.
  • binding of PEG to a foreign peptide can sometimes inhibit recognition of antigenic sites present in the foreign peptide by immune cells. Specifically, by inhibiting phagocytosis and intracellular proteolysis of the peptide by antigen-presenting cells, the possibility of the peptide acting as an antigen can be reduced.
  • the peptide of the present invention may be linked to the carrier material through a linker.
  • the linker may be a peptidic or non-peptidyl polymer.
  • non-peptidyl polymers usable in the present invention include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl It may be selected from the group consisting of ethers, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof.
  • PLA polylactic acid
  • PLGA polylactic-glycolic acid
  • lipid polymers chitins, hyaluronic acid, and combinations thereof.
  • the peptide of the present invention is in addition to a moiety that can be selected from the group consisting of PEG, monosaccharides, fluorophores, chromophores, radioactive compounds and cell-penetrating peptides. can be joined.
  • the peptides of the present invention are phosphorylation, sulfation, acrylation, and glycosylation in some cases to the extent that the activity of the molecule is not entirely altered. ), methylation, farnesylation, acetylation, and amidation may be modified.
  • the peptide of the present invention may be present in glycosylated, PEGylated, amidated, esterified, acylated, acetylated and/or alkylated form.
  • the peptides provided in the present invention may have anti-inflammatory and/or regenerative properties.
  • one aspect of the present invention provides a use for preventing or treating inflammatory diseases of the peptide of the present invention.
  • Another aspect of the present invention provides the use of the peptide of the present invention for tissue regeneration.
  • anti inflammation refers to inhibiting inflammation
  • the inflammation is one of the biological tissue's defense responses to certain stimuli, and is a complex complex that can cause tissue deterioration, circulatory disturbance and exudation, and tissue proliferation. say lesion.
  • Inflammation is part of innate immunity, and as in other animals, human innate immunity recognizes patterns on the surface of cells that are specific to pathogens. Phagocytes recognize cells with such a surface as non-self and attack pathogens. When pathogens break through the body's physical barriers, an inflammatory response can occur. The inflammatory response is a part of the immune defense mechanism, but if it occurs excessively or continuously, acute or chronic inflammatory diseases are induced.
  • Inflammation occurs in various mechanisms in the body, such as hormone secretion, cytokines, and C-reactive protein (CRP), and there are many related substances. Among them, various cytokines secreted from immune cells regulate the immune system, and some promote inflammation. Therefore, the expression level of intracellular cytokines can be an indicator of inflammatory response activation.
  • CRP C-reactive protein
  • inflammatory disease means a disease caused by an inflammatory reaction
  • the inflammatory disease is, for example, increased expression of one or more inflammatory markers selected from the group consisting of Tnf ⁇ , Il-1 ⁇ , Il-6, Cox2, iNos, CCl2, Cd11b, F4/80, Ifn ⁇ ; And/or it may be caused by a decrease in the expression of an anti-inflammatory marker consisting of Il-10 and Il-1rn.
  • one or more inflammatory markers selected from the group consisting of Tnf ⁇ , Il-1 ⁇ , Il-6, Cox2, iNos, CCl2, Cd11b, F4/80, Ifn ⁇
  • an anti-inflammatory marker consisting of Il-10 and Il-1rn.
  • it is not limited thereto.
  • the inflammatory disease of the present invention is a disease selected from the group consisting of arthritis, metabolic disease, hepatitis, enteritis, gastritis, gastric ulcer, esophagitis, dermatitis, encephalitis, depression, anxiety disorder, cognitive impairment, memory disorder, degenerative brain disease and developmental disorder. can but not limited to this
  • Inflammatory diseases of the present invention include both acute and chronic diseases.
  • arthritis of the present invention means an inflammatory disease occurring in the joints.
  • the arthritis includes osteoarthritis, rheumatoid arthritis, chronic rheumatoid arthritis, degenerative arthritis, bruised arthritis, gouty arthritis, atrophic arthritis, chronic inflammatory arthritis, deformable arthritis, infectious arthritis, menopausal arthritis, monoarthritis, hypertrophic arthritis, suppurative arthritis do.
  • the "metabolic disease” of the present invention is a generic term for diseases caused by metabolic disorders in a living body.
  • the metabolic disease is characterized by a change in the function of the intestinal barrier, a change in the concentration of LPS in the blood, an occurrence of intestinal inflammation, a change in intestinal mucus, an increase in insulin resistance, a decrease in insulin sensitivity, an increase in fasting blood sugar, an increase in insulin resistance, an increase in glucose tolerance, and creatinine in the blood.
  • urea nitrogen (BUN), uric acid may indicate one or more of an increase in the concentration of creatine kinase, or may be a disease caused by the above-mentioned phenomenon or a disease having the above-mentioned phenomenon as a prognostic symptom, but is not limited thereto.
  • the metabolic disease may be selected from the group consisting of insulin resistance disease, obesity, diabetes, fatty liver, nonalcoholic fatty liver, hyperlipidemia, kidney damage, arteriosclerosis, dyslipidemia and hypertension, for example, obesity, diabetes, insulin resistance and dyslipidemia, but is not limited thereto.
  • Hepatitis in the present invention means an inflammatory disease occurring in the liver.
  • the hepatitis is acute or chronic hepatitis, cirrhosis, fatty liver, liver failure, liver cancer, alcoholic fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis ( Liver fibrosis) and Liver cirrhosis.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • liver fibrosis Liver fibrosis
  • Liver cirrhosis Liver cirrhosis
  • the hepatitis may be acute hepatitis or non-alcoholic hepatitis, but is not limited thereto.
  • the non-alcoholic hepatitis includes, without limitation, those that occur due to causes other than alcohol, for example, metabolic diseases such as obesity, diabetes, dyslipidemia, metabolic syndrome, or a high-fat diet that is the cause of the metabolic diseases. It may be caused by, but not limited to.
  • enterritis refers to an inflammatory disease occurring in the intestine.
  • the “enteritis” is used in the same sense as “inflammatory bowel disease", and includes ulcerative colitis, Crohn's disease, intestinal Bechet's disease, hemorrhagic rectal ulcer, and ileal capsuleitis. do.
  • stomach in the present invention means an inflammatory disease occurring in the stomach.
  • the gastritis is used in the sense of including stomach diseases such as stomach cramps, gastritis, gastric ulcer, duodenitis, duodenal ulcer.
  • Esophagitis of the present invention means an inflammatory disease occurring in the esophagus.
  • the esophagitis includes all those caused by various causes such as bacterial infection, gastric contents or acid reflux, drugs, and may be, for example, gastroesophageal reflux disease, but is not limited thereto.
  • Dermatitis of the present invention means an inflammatory disease occurring on the skin.
  • the dermatitis is allergic contact dermatitis, urticaria, alopecia dermatitis (dry skin on the lower leg), atopic dermatitis, contact dermatitis such as irritant contact dermatitis and poison ivy-induced contact dermatitis, eczema, gravitational dermatitis, monetary dermatitis , otitis externa, perioral dermatitis and seborrheic dermatitis.
  • Encephalitis in the present invention means an inflammatory disease occurring in the brain.
  • inflammatory cells When inflammatory cells are excessively activated in the brain, the secretion of inflammatory cytokines increases, and brain cell damage can be induced by this overactivation of the brain inflammatory response. Accordingly, the encephalitis is closely related to cognitive impairment, memory impairment, and degenerative brain disease. The encephalitis includes neuroinflammation.
  • “Depression” in the present invention refers to a disease that causes a decrease in daily functioning by causing various cognitive and psychosomatic symptoms with a decrease in motivation and depression as the main symptoms.
  • the "anxiety disorder” of the present invention is a kind of mental disorder, and it is a widespread, very unpleasant, and vaguely anxious feeling, accompanied by related physical symptoms (chest palpitation, sweating, etc.) and behavioral symptoms (irritability, pacing, etc.). When anxious, the entire brain enters an arousal state, and therefore, an anxiety disorder causes disturbances in peripheral behavior, autonomic nervous system, sensation, and perception. Anxiety disorders can also be associated with various combinations of psychological and physical signs of anxiety that are not attributable to real danger but appear as an aggression (panic disorder) or a persistent state (generalized anxiety disorder).
  • Cognitive impairment refers to a disease caused by a decrease in cognitive function (cognitive ability) such as memory ability, temporal and spatial grasping ability, judgment, language ability or calculation ability due to brain damage.
  • the "memory disorder” of the present invention is a pathological condition in which it is difficult or impossible to remember things or recall past experiences, and diseases caused by memory loss, such as forgetfulness, instantaneous memory loss, short-term memory loss, Includes long-term memory loss and transient amnesia. However, it is not limited thereto.
  • the peptide of the present invention may be used to improve memory and/or cognitive ability, but is not limited thereto.
  • degenerative brain disease refers to a disease occurring in the brain among degenerative diseases.
  • the degenerative brain disease includes Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, and Huntington's disease. However, it is not limited thereto.
  • Developmental disability of the present invention refers to a state in which mental and physical development is not developed as much as the age, and is 25% behind the normal expectation of the age in the developmental screening test.
  • Common symptoms of developmental disorders are difficulties in understanding and using language, difficulties in overall understanding of social situations and formation of human relationships, and a pattern of clinging to specific objects and repeating certain behavioral procedures.
  • Developmental disorders in the present invention include intellectual disabilities, cerebral palsy, pervasive developmental disorders, developmental speech disorders, functional disorders of special senses including sight or hearing, learning disorders, attention deficit hyperactivity disorder, epilepsy, and combinations thereof. It may be selected from the group consisting of, but is not limited thereto.
  • the pervasive developmental disorder may be selected from the group consisting of autism spectrum disorder, Asperger's syndrome, Childhood Disintegrative Disorder, Rett Syndrome, atypical autism spectrum disorder, and combinations thereof,
  • developmental disorders can include all disorders occurring in developmental areas such as perception, cognition, movement, and language, and include learning disabilities, communication disorders, motor dysfunction, cerebral palsy, genetic disorders, and chromosome disorders.
  • Down Syndrome; Fragile X Syndrome may include all developmental disorders with delay in development or function. However, it is not limited thereto.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, including the peptide provided by the present invention.
  • the peptides of the present invention and inflammatory diseases are the same as described above.
  • prevention means any action that inhibits or delays the onset of a disease by administering the peptide according to the present invention.
  • treatment refers to any action in which the symptoms of a suspected disease and onset individual are improved or beneficially changed by administering the peptide according to the present invention.
  • the pharmaceutical composition of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition.
  • the pharmaceutical composition is each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods to be used.
  • carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used for suspensions, solutions, emulsions, and syrups. there is.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
  • Another aspect of the present invention provides a health functional food composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
  • the food composition of the present invention can be ingested on a daily basis, it is very useful because it can be expected to prevent or improve the effect of diseases.
  • the food composition of the present invention includes the form of pills, powder, granules, needles, tablets, capsules or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example, There are beverages, gum, tea, vitamin complexes, and health supplements.
  • the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated by administration of the peptide of the present invention, for example, the severity of symptoms.
  • the food composition of the present invention includes the form of pills, powder, granules, needles, tablets, capsules or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example, There are beverages, gum, tea, vitamin complexes, and health supplements.
  • the food supplement additives include food supplement additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.
  • Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents eg, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like.
  • it may contain the pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination.
  • the health supplement includes health functional food and health food.
  • the functional food is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects.
  • function (sex) means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.
  • the food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food.
  • the food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of the drug using food as a raw material, and has excellent portability, Food can be ingested as an adjuvant for enhancing the effect of preventing or improving cognitive dysfunction and neuroinflammation.
  • Another aspect of the present invention provides a feed composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
  • the composition for feed may include a feed additive.
  • the feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.
  • feed refers to any natural or artificial diet, one-meal, etc., or a component of the one-meal, which is suitable for or suitable for the animal to eat, ingest, and digest.
  • the type of feed is not particularly limited, and feed commonly used in the art may be used.
  • Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in mixture of two or more.
  • Another aspect of the present invention provides a method for preventing or treating an inflammatory disease comprising administering to an individual the peptide provided in the present invention.
  • the "individual" of the present invention means any animal, such as a rat, livestock, or human, that is likely to or has developed a disease.
  • humans may be excluded from the subject of the present invention, but is not limited thereto.
  • the peptide may be administered through any general route as long as it can reach the target tissue.
  • the peptide may be administered in the form of a pharmaceutical composition.
  • the treatment method of the present invention not only a method of administering the peptide provided by the present invention alone, but also a combination therapy may be considered.
  • Other agents used in the combination therapy may be included without limitation as long as they are known to be effective in preventing or treating inflammatory diseases, and may be provided in an amount effective to produce a desired result in the prevention or treatment of inflammatory diseases.
  • the treatment method of the present invention can be achieved by administering a therapeutically effective amount of the first therapeutic agent comprising the peptide provided in the present invention and, optionally, the second therapeutic agent or factor.
  • the pharmaceutical composition of the present invention is not particularly limited thereto, but routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered through
  • the oral composition may be coated with the active agent or formulated to be protected from degradation in the stomach.
  • the pharmaceutical composition may be an enteric coating to be protected from decomposition from the stomach, but is not limited thereto.
  • the composition may be administered by any device capable of transporting the active agent to a target cell.
  • the peptide of the present invention is useful for preventing or treating inflammatory diseases can be used
  • Another aspect of the present invention provides an animal pharmaceutical composition for preventing or treating inflammatory diseases comprising the peptide provided in the present invention.
  • the peptides and inflammatory diseases are the same as described above.
  • the animal means all animals, such as mice, livestock, and humans, that are likely to develop or have a disease, and may be animals other than humans in one embodiment, but is not limited thereto.
  • composition of the present invention when used as an veterinary pharmaceutical composition, the composition may be used as it is or may be used together with other pharmaceuticals or quasi-drug ingredients, and may be appropriately used according to a conventional method, but is not limited thereto.
  • the mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, improvement or therapeutic treatment).
  • Another aspect of the present invention provides a quasi-drug for preventing or improving inflammatory diseases comprising the peptide provided by the present invention.
  • quasi-drug refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals.
  • quasi-drugs exclude products used for medicinal purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body.
  • the quasi-drug composition of the present invention may be prepared from one or more selected from the group consisting of body cleanser, foam, soap, mask, ointment, cream, lotion, essence and spray, but is not limited thereto.
  • Another aspect of the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
  • the cosmetic composition may be prepared in various forms according to a conventional cosmetic preparation method.
  • the cosmetic composition may be prepared in the form of a cosmetic product, lotion, cream, lotion, etc. containing the peptide of the present invention, which may be used by diluting it with a conventional cleansing solution, astringent solution and moisturizing solution.
  • the cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions.
  • the peptide content of the present invention may be included in an effective amount to achieve the effect of preventing or improving inflammatory diseases. For example, it may be contained in an amount of 0.001 to 10% by weight based on the total weight of the composition, specifically, it may be contained in an amount of about 0.01 to 1% by weight, but is not limited thereto.
  • the formulation of the cosmetic composition is a solution, an external ointment, a cream, a foam, a nutritional lotion, a flexible lotion, a perfume, a pack, a soft water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath agent, a sunscreen cream, a sun oil, a suspension , emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch or spray.
  • the cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent , inorganic salts, colorants, antioxidants, disinfectants, preservatives, fragrances, etc. may be appropriately mixed, but the present invention is not limited thereto.
  • one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent , inorganic salts, colorants, antioxidants, disinfectants, preservatives, fragrances, etc. may be appropriately mixed, but the present invention is not limited thereto.
  • the peptide provided in the present invention is characterized in that it has a regeneration effect.
  • one aspect of the present invention provides the use of the above-described peptide of the present invention for tissue regeneration.
  • the tissue may be selected from skin, muscle, hair, hair follicles and hair roots.
  • the tissue regeneration may be selected from skin regeneration, muscle tissue regeneration, wound healing, muscle strength enhancement, hair loss prevention, wool promotion, hair growth promotion, damaged hair improvement, and hair regeneration.
  • regeneration means suppressing the deterioration of the function of cells, tissues, or organs or restoring the reduced function.
  • Decreased function of cells, tissues, or organs may be due to damage to cells, tissues, or organs. Such damage may be caused by various factors such as radiation therapy, drug treatment, surgery, infection, inflammation, degenerative diseases, autoimmune diseases, and aging. Meanwhile, the “regeneration” may be achieved by promoting cell differentiation, but is not limited thereto.
  • the peptide provided by the present invention may have the ability to regenerate muscle tissue.
  • the regeneration of the muscle tissue may be achieved by myoblast differentiation, but is not limited thereto.
  • myocytes are muscle cells in an undifferentiated state, and when myocytes are differentiated into skeletal muscle cells, muscle tissue is formed, so differentiation of myocytes is also called myogenesis.
  • Factors involved in the differentiation of these myoblasts include Mef2, Serum response factor (SRF), MyoD, Myf5, Myf6, myogenin and myosin heavy chain, and the expression level of these factors By measuring, it is possible to determine whether myoblasts are differentiated.
  • the regeneration of the muscle tissue may be achieved by inhibiting myoblast atrophy.
  • myoblast atrophy For example, by measuring the expression levels of atrogin1 and MuRF1, which are factors involved in muscle atrophy, it can be determined whether or not atrophy of myoblasts is suppressed.
  • composition for tissue regeneration comprising the peptide provided in the present invention.
  • composition for tissue regeneration may be used for the prevention or treatment of tissue damage-related diseases. Therefore, another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of tissue damage-related diseases comprising the peptide provided in the present invention as an active ingredient.
  • the peptide and pharmaceutical composition are the same as described above.
  • the tissue of the present invention may be a muscle tissue, and the tissue damage-related disease may be a muscle-related disease.
  • the muscle-related disease is muscle dystrophy (muscular dystrophy), muscular atrophy (muscular atropy), sarcopenia (muscular sarcopenia), myositis (Myositis), polymyositis, peripheral vascular disease (peripheral vascular disease) and fibrosis ( fibrosis) may be selected from the group consisting of, but is not limited thereto.
  • the tissue in the present invention may be selected from hair, hair follicles, and hair roots.
  • the composition for tissue regeneration may exhibit effects such as preventing hair loss, promoting hair growth, promoting hair growth, improving damaged hair, and regenerating hair. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for preventing, treating or improving hair loss. However, it is not limited thereto.
  • tissue regeneration in the present invention may be wound healing.
  • the wound healing means treating a wound caused by damage to cells, and the wound is a meaning encompassing all damage to the living body, and is also referred to as a wound.
  • the wound healing may refer to any action of administering the composition of the present invention to a wounded individual to inhibit or delay the deterioration of the wound, or to improve or benefit the symptoms of the wound. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for preventing or treating wounds or wounds.
  • the wound may be, for example, a skin tissue, but is not limited thereto.
  • the peptides provided by the present invention may exhibit an effect of inhibiting and/or improving skin aging. Inhibition and/or improvement of skin aging may also be referred to as “skin regeneration”. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for skin regeneration.
  • Skin aging of the present invention refers to the appearance of symptoms such as reduced elasticity, reduced gloss, wrinkles, weakened regeneration, or severe dryness in the skin, and may be caused by the passage of time or external environment.
  • the skin aging includes both intrinsic aging that appears naturally with the passage of time and photoaging that occurs in the skin due to ultraviolet rays.
  • the synthesis amount of collagen, hyaluronic acid, elastin, proteoglycan, fibronectin and/or its precursors decreases, and the expression of degrading enzymes of the components increases Or a phenomenon such as a decrease in the expression of the synthetase of the component may appear.
  • Skin aging inhibition of the present invention may also be referred to as “skin regeneration”, and may include an increase in the amount of synthesis of collagen or a precursor thereof in skin cells.
  • the skin cells include skin keratinocytes and skin fibroblasts.
  • the inhibition of skin aging of the present invention may include an increase in collagen synthetase activity and/or inhibition of collagenase activity.
  • the collagen synthetase may be Col1a1 and/or Col3a1.
  • the collagen-degrading enzyme may be MMP-1 and/or MMP-3. However, it is not limited thereto.
  • the inhibition of skin aging of the present invention is to increase skin moisture, increase skin elasticity, increase skin thickness, improve skin wrinkles, alleviate skin irritation, recover skin damage and/or improve skin tone may include However, it is not limited thereto.
  • Another aspect of the present invention provides a health functional food composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
  • the peptide, tissue regeneration and health functional food are as described above.
  • Another aspect of the present invention provides a feed composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
  • Another aspect of the present invention provides an veterinary pharmaceutical composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
  • the peptide, tissue regeneration, feed, and veterinary medicine are the same as described above.
  • Another aspect of the present invention provides a method for preventing or treating tissue damage-related diseases comprising administering to an individual the peptide provided by the present invention.
  • Another aspect of the present invention provides a method for tissue regeneration comprising administering to an individual the peptide provided by the present invention.
  • a method of administering the peptide provided by the present invention alone not only a method of administering the peptide provided by the present invention alone, but also a combination therapy may be considered.
  • Other agents used in the combination therapy may be included without limitation as long as they are known to have a tissue regeneration effect, and may be provided in an amount effective to produce a desired result in tissue regeneration.
  • the peptide, subject, administration, tissue damage-related disease and prevention or treatment method are the same as described above.
  • tissue regeneration and differentiation-related genes increased according to the promotion of cell differentiation by administration of the peptide of the present invention, and accordingly, it was confirmed that muscle tissue, skin tissue, hair, and hair root tissue were regenerated,
  • the peptide of the present invention can be usefully used for the prevention and treatment of tissue-related diseases.
  • Another aspect of the present invention provides a quasi-drug composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
  • Another aspect of the present invention provides a cosmetic composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
  • the peptide, tissue regeneration, quasi-drug composition and cosmetic composition are the same as described above.
  • PEP001 is SEQ ID NO: 1
  • PEP002 is SEQ ID NO: 2
  • PEP003 is SEQ ID NO: 3
  • PEP004 is SEQ ID NO: 4
  • PEP005 is SEQ ID NO: 5
  • Amuc_1409 is functional except for the leader sequence of SEQ ID NO: 6 It refers to the fragment SEQ ID NO:7.
  • Example 1 Efficacy verification of novel peptides in arthritis animal models
  • Example 1-1 Efficacy verification of novel peptides in degenerative arthritis animal model
  • the diameter of the knee joint was measured using a caliper to evaluate edema caused by inflammatory changes in the joint.
  • the knee joint tissue of the mouse was fixed with 10% neutral buffered formalin, and the process of removing calcification from the bones with 0.5 M EDTA solution was performed. Then, the joint tissue was embedded in paraffin to prepare a 5 ⁇ m joint section, and the degree of damage to the cartilage tissue was analyzed histopathologically by staining with Safranin O. At this time, the degree of damage to the cartilage tissue was evaluated by checking the degree of safranin O staining of the section, and scoring was performed according to OARSI (Osteoarthritis Research Society International grade) guidelines. The scoring criteria according to the OARSI guidelines are as follows (Table 1).
  • the group administered with Amuc_1409, PEP002, PEP003 and PEP004 compared to the PBS buffer group on the 1st day after MIA injection.
  • the group administered with Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 had knee joint compared to the group administered with PBS buffer. was significantly decreased (p ⁇ 0.05, Student's t-test) (Fig. 1).
  • the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 significantly increased the decrease in weight bearing rate by MIA injection compared to the PBS buffer administration group ( p ⁇ 0.05, Student's t-test) (Fig. 1).
  • the PBS buffer administration group showed a marked decrease in safranin O staining and cartilage thickness, but compared with Amuc_1409, PEP001, PEP002, PEP003,
  • the group administered with PEP004 and PEP005 showed only a slight loss of safranin O in the cartilage area, and it was observed that the overall cartilage morphology was maintained.
  • Example 1-2 Efficacy verification of novel peptides in rheumatoid arthritis animal model
  • bovine type 2 collagen (CII) was dissolved in 0.1 M acetic acid solution to 2 mg/ml and then dialyzed with phosphate buffered saline to M. tuberculosis (tuberculosis). ) containing Complete Fred's Adjuvant (CFA, Chondrex) in the same amount and emulsified, and then 100 ⁇ l of the emulsified collagen solution was added to the base of the tail of 8-week-old male DBA/1J mice (i.e., 100 ⁇ g/100 ⁇ l) intradermal injection to induce primary immunity (primary immunity).
  • CFA Complete Fred's Adjuvant
  • the same CII was mixed with an equal amount of CFA and then injected intradermally into the base of the tail of the mouse by 100 ⁇ l (ie, 100 ⁇ g/100 ⁇ l) to induce a secondary immune response (secondary immunization).
  • a boosting reaction was induced by intraperitoneal injection of 40 ⁇ g of lipopolysaccharide (LPS) per animal, and the experiment was terminated on the 35th day after the primary immunization.
  • LPS lipopolysaccharide
  • the severity of joint inflammation was evaluated at 2-day intervals.
  • the arthritis evaluation was used by adding up the scores from the four legs according to the scale below.
  • the score criteria according to the arthritis evaluation are as follows (Table 2).
  • the thickness of the ankle joint was measured with a caliper, pictures for each test group were taken, and the test animals were killed. proceeded. Then, a 5 ⁇ m joint section was prepared by embedding the mouse hind paw tissue in paraffin, stained with hematoxylin and eosin (H&E), and histopathologically, the severity of joint inflammation was evaluated. At this time, the evaluation of arthritis was used by averaging the scores based on the following scale. The score criteria according to the arthritis evaluation are as follows.
  • the group administered with Amuc_1409 compared to the group administered with the PBS buffer decreased close to a significant level.
  • the arthritis index significantly decreased in the group administered with Amuc_1409 (p ⁇ 0.01, Student's t-test).
  • the thickness of the ankle joint was significantly reduced in the group administered with Amuc_1409 compared to the group administered with PBS buffer. It was confirmed that the swelling of the hind paws was overall reduced (p ⁇ 0.05, Student's t-test) (FIG. 4).
  • the arthritis index significantly decreased in the PEP001-administered group from the 30th day after the first CII injection to the end of the experiment compared to the PBS buffer-administered group, and PEP002 And the PEP003 administration group had a significant decrease in the arthritis index from the 26th day after the first CII injection to the end of the experiment, the PEP004 administration group significantly decreased the arthritis index from the 32nd day after the first CII injection to the end of the experiment, and the PEP005 administration group showed a significant decrease in the CII 1 From the 28th day after the primary injection to the end of the experiment, the arthritis index decreased significantly.
  • the thickness of the ankle joint was significantly reduced in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 compared to the group administered with the PBS buffer.
  • the edema of the hind paws was overall reduced in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 (p ⁇ 0.05, Student's t-test) (FIG. 5).
  • the peptide of the present invention is effective in preventing and treating arthritis.
  • Obesity/diabetes and metabolic disease animal models accompanied by chronic inflammation were induced by free feeding on a high-fat diet for 45 weeks or 8 weeks using 8-week-old SPF male C57BL/6J mice.
  • Amuc_1409 was fed to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 45 weeks, or PEP001, PEP002, and PEP001, PEP002, to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 8 weeks.
  • PEP003, PEP004 and PEP005 in PBS buffer were orally administered to mice once a day at the same time until the end of the test.
  • PBS buffer was orally administered in the same way.
  • fasting blood glucose was measured using ACCU-CHECK in a small amount of blood obtained through a wound at the tip of the tail of a mouse fasted for 16 hours, and blood was collected from the orbital venous plexus using a heparin-treated capillary tube. The collected blood was immediately centrifuged at 10,000 rpm (4°C) for 10 minutes to separate plasma. analyzed.
  • the blood glucose of the group administered with Amuc_1409 decreased compared to the group administered with PBS buffer from 30 minutes to 120 minutes of insulin administration. It showed resistance improvement efficacy, and as a result of converting the graph of the insulin resistance experiment into AUC (area under the curve), it was confirmed that the group administered with Amuc_1409 was reduced close to a significant level compared to the group administered with PBS buffer. In addition, when fasting blood glucose was measured after fasting for 16 hours at the end of the test, it was confirmed that the group administered with Amuc_1409 significantly decreased (p ⁇ 0.05, Student's t-test) ( FIG. 8 ).
  • the initial body weight of the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 decreased compared to the group administered with PBS buffer, and the group administered with PEP003, PEP004 and PEP005 decreased significantly.
  • the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 showed an anti-obesity effect as all decreased compared to the group administered with PBS buffer (p ⁇ 0.05, Student's t-test) (FIG. 9) .
  • the blood glucose of the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 decreased compared to the group administered with PBS buffer at 30 minutes of insulin administration.
  • the effect of improving insulin resistance by a high-fat diet was reduced compared to the group administered with PBS buffer in all groups administered with PEP001, PEP002, PEP003, PEP004 and PEP005 as a result of converting the graph of the insulin resistance experiment into AUC.
  • TC and LDL-C concentrations which are blood lipid indicators, did not show a significant difference between the Amuc_1409 administration group and the PBS buffer administration group, but decreased in the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups compared to the PBS buffer administration group.
  • TC significantly decreased in the PEP003-administered group, and showed a tendency to decrease in the PEP004 and PEP005-administered groups.
  • the peptide of the present invention has a preventive and therapeutic effect on various metabolic diseases.
  • Example 3 Efficacy verification of novel peptides in hepatitis animal model
  • Example 3-1 Efficacy verification of novel peptides in acute hepatitis animal model
  • ConA Concanavalin A
  • SPF specific pathogen free mice
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • Sections with a thickness of 5 ⁇ m were prepared by embedding in paraffin and stained with hematoxylin and eosin (H&E).
  • ALT was significantly decreased in the group administered with Amuc_1409, PEP002, PEP003, and PEP004 compared to the group administered with the PBS buffer, and was close to a significant level in the group administered with PEP001 and PEP005. was confirmed to be lowered.
  • AST decreased significantly in the group administered with Amuc_1409 compared to the group administered with PBS buffer, and decreased close to a significant level in the group administered with PEP001, and showed a tendency to decrease in the groups administered with PEP002, PEP003, PEP004 and PEP005 (p ⁇ 0.05, Student's t). -test) (Fig. 11).
  • Example 3-2 Efficacy verification of novel peptides in nonalcoholic steatohepatitis animal model
  • the nonalcoholic hepatitis model was induced by free feeding on a high-fat diet for 45 weeks or 8 weeks using 8-week-old SPF male C57BL/6J mice.
  • Amuc_1409 was fed to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 45 weeks, or PEP001, PEP002, and PEP001, PEP002, to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 8 weeks
  • PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 2.4 ⁇ M
  • 150 ⁇ l per animal was orally administered to mice once a day at the same time until the end of the test.
  • PBS buffer was orally administered in the same way.
  • RNA was added to Trizol and homogenized to extract RNA.
  • Reverse transcriptase was added to the obtained RNA and reacted to synthesize cDNA, followed by quantitative real-time PCR (RT-qPCR) by adding SYBR Green and primers, followed by Tnf ⁇ , Il-1 ⁇ , Il-10 and Il-
  • RT-qPCR quantitative real-time PCR
  • Tnf ⁇ was significantly reduced in the PEP003, PEP004 and PEP005 administration groups compared to the PBS buffer administration group, and Il-1 ⁇ was It decreased close to a significant level in the PEP001 and PEP004 administration groups, and significantly decreased in the Amuc_1409, PEP002, PEP003 and PEP005 administration groups.
  • Il-10 an anti-inflammatory index, significantly increased in the groups administered with Amuc_1409 and PEP001, PEP003, PEP004 and PEP005 compared to the group administered with PBS buffer, and showed a tendency to increase close to a significant level in the group administered with PEP002.
  • An additional anti-inflammatory index, Il-1rn was significantly increased in the Amuc_1409 administration group compared to the PBS buffer administration group (p ⁇ 0.05, Student's t-test) (Fig. 12).
  • the peptide of the present invention is effective in preventing and treating hepatitis.
  • PEP002, PEP003, PEP004 and PEP005 were dissolved in PBS buffer at a concentration of 4.3 ⁇ M, and 150 ⁇ l per animal was orally administered at the same time once a day, and the same amount of PBS buffer was orally administered to the control group.
  • the rate of increase in body weight due to the recovery of enteritis was confirmed through oral administration 6 times for 6 days.
  • the test was terminated after oral administration three times for 3 days.
  • the length of the colon is measured, and a part of the distal colon is rapidly cooled in liquid nitrogen, RNA is extracted using Trizol, reverse transcriptase is added and reacted to synthesize cDNA, and SYBR Green and primer are added.
  • RT-qPCR quantitative real-time PCR
  • the Il-6 gene expression level was quantified.
  • a part of the distal colon was fixed in 10% NBF at room temperature for more than 24 hours, then embedded in paraffin to prepare a 5 ⁇ m-thick section and stained with hematoxylin and eosin (H&E). H&E-stained tissue sections were observed under a light microscope to evaluate the degree of histopathological enteritis using a histologic colitis scoring system (Table 4).
  • the group administered with PEP002, PEP004 and PEP005 significantly increased compared to the group administered with the PBS buffer, and showed a tendency to increase in the group administered with PEP003 (p ⁇ 0.05, Student's t-test) (Fig. 14). ).
  • the peptide of the present invention has a preventive and therapeutic effect on inflammatory bowel disease.
  • Example 5 Efficacy verification of novel peptides in gastritis/gastric ulcer animal models
  • Gastritis/gastric ulcer model was performed using 7-week-old male C57BL/6J mice from 3 days before induction so that the concentrations of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 were 6.7 ⁇ M in PBS buffer and then 150 ⁇ l per animal once a day. The mice were orally administered at the same time.
  • 0.2 ml of 0.3 M HCl/60% ethanol was orally administered, and the tissue was excised after 1 hour to take pictures of the inner side of the stomach and compare the degree of gastric damage.
  • the measurement of gastritis/gastric ulcer index was converted by scoring according to the degree of mucosal damage. If there is no damage, there is at least one site of bleeding corrosion corresponding to 0 points, minor circular bleeding erosion less than 5 mm, 1 point for one site, 2 points for a site with a length greater than 5 mm, and 2 points for bleeding corrosion. 3 points, 4 points for bleeding corrosion with a length of 5 mm or more and 2 mm or less, 5 points for 2-3 bleeding corrosion corresponding to 4 points, 6 points for 4-5 pieces, 6 points for 6 points or more A score of 7 and 8 points were given if there was hemorrhagic erosion throughout the gastric mucosa.
  • the peptide of the present invention is effective in preventing and treating gastritis and gastric ulcer.
  • Example 6 Efficacy verification of novel peptides in dermatitis animal model
  • the dermatitis model was performed by applying 2.5 ⁇ g of TPA (12-otetrade-canoyl-phorbol-13-acetate) dissolved in 25 ⁇ l acetone to the right ear of an 8-week-old male C57BL/6J mouse one day before sample application. Skin edema and inflammation were induced, and 25 ⁇ l of acetone, a TPA solvent, was applied to the left ear and used as a negative control.
  • TPA (12-otetrade-canoyl-phorbol-13-acetate) dissolved in 25 ⁇ l acetone
  • the thickness of the ears was measured to check the degree of edema and inflammation relief of the ears, and both ears were photographed for each test group.
  • the weight per area of the ear was measured, and the mouse ear was fixed in 10% neutral buffered formalin at room temperature for at least 24 hours, then embedded in paraffin to prepare a 5 ⁇ m-thick section, and hematoxylin and (H&E) staining with eosin (Eosin).
  • H&E hematoxylin and
  • Eosin hematoxylin and eosin
  • the group administered with PEP001, PEP002, PEP003, PEP004, and PEP005 markedly reduced erythema and edema of the ear compared to the group administered with PBS buffer.
  • the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 markedly reduced erythema and edema of the ear compared to the group administered with PBS buffer.
  • the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 significantly decreased compared to the group administered with the PBS buffer (p ⁇ 0.05, Student's t-test) ( FIG. 22 ).
  • the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004, and PEP005 showed improvement in ear tissue edema and inflammatory cells by TPA application compared to the PBS buffer administration group. Infiltration was markedly reduced ( FIG. 23 ).
  • the peptide of the present invention is effective in preventing and treating skin inflammatory diseases.
  • Example 7 Efficacy verification of novel peptides in animal models of depression and anxiety disorders
  • LPS lipopolysaccharide
  • TST tail suspension test
  • FST forced swim test
  • Elevated plus-maze was performed to analyze the effect on anxiety level.
  • the EPM device consists of two open arms (50 ⁇ 10 cm) intersected with two closed arms (50 ⁇ 10 cm) at a height of 50 cm from the floor, connected by an area of 10 ⁇ 10 cm in the center.
  • the mice were placed in the center of the maze and using a video camera, the time spent on the open arm was measured for a total of 5 min, and the ratio of time spent on the open arm was calculated as [(time spent in open arm/time spent in open and closed arms) Time spent in) ⁇ 100] was calculated and recorded.
  • the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups significantly decreased the retention time of immobility due to tail suspension and forced swimming compared to the PBS buffer administration group (p ⁇ 0.05, Student's). t-test)
  • the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups showed anxiolytic efficacy by increasing the time spent in the open arm compared to the PBS buffer administration group (p ⁇ 0.05, Student's t-test) ) (Fig. 26).
  • Example 8 Efficacy verification of novel peptides in animal models of cognitive/memory disorders
  • Example 8-1 Efficacy verification of novel peptides in Alzheimer's disease animal model
  • Amuc_1409 was orally administered to 5-month-old male APP/PS1 Alzheimer's disease mice with amyloid deposition and cognitive and memory impairments observed in the brain, 5 ⁇ g daily for 5 days a week, and administered for 8 weeks. After dissolving PEP001 in PBS buffer to a concentration of 2.4 ⁇ M, 150 ⁇ l per animal was orally administered 5 days a week for 8 weeks. The control group (vehicle) was orally administered with PBS in the same amount (volume). In addition, normal mice (Non-Tg) not administered with the novel protein fragment were used as a control group.
  • a novel object recognition test was performed to test the cognitive function improvement effect in APP/PS1 Alzheimer's disease mice administered with Amuc_1409 and PEP001. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
  • Example 8-2 Efficacy verification of novel peptides in animal models of cognitive/memory impairment
  • Cognitive and memory impairments were induced by intraperitoneal injection of LPS at 250 ⁇ g/kg/day for 7 days in male C57BL/6J mice.
  • Akkermansia muciniphila-derived protein Amuc_1409 was orally administered 5 ⁇ g daily from one week before LPS administration until the cognitive and memory test was performed.
  • PEP002, PEP003, PEP004 and PEP005 were tested for cognition and memory from three days before LPS administration. After dissolving in PBS buffer so that the concentration became 2.4 ⁇ M until completion, 150 ⁇ l per animal was orally administered to the mouse every day at the same time once a day.
  • the control group (vehicle) was orally administered with PBS in the same amount (volume).
  • a novel object recognition test was performed to test the effect of improving cognitive function in cognitive and memory impaired mice administered with Amuc_1409 or PEP002, PEP003, PEP004 and PEP005. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
  • Example 8-3 Verification of cognitive/memory efficacy of novel peptides in naturally aging mice
  • a novel object recognition test was performed to test the cognitive function improvement effect in naturally aging mice administered with Amuc_1409 and PEP002, PEP003, PEP004 and PEP005. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
  • the peptide of the present invention has the effect of preventing and treating cognition and memory disorders as well as preventing and treating degenerative brain diseases, and improving cognition and memory.
  • BTBR mice (BTBR T + Itpr3 tf /J (BTBR)), a developmental disability model, were used as experimental animals.
  • mice To 3-week-old male BTBR mice, Amuc_1409 was orally administered 5 ⁇ g daily for 5 days a week, and PBS (Phosphate-buffered saline) was administered to the control group. After dissolving PEP001, PEP002, PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 2.4 ⁇ M, 150 ⁇ l per animal was orally administered to mice once a day at the same time for 5 days a week. Behavioral evaluation was performed by observing the social behavior of the mice 4 weeks after administration. The mice were allowed to freely ingest sterilized feed and water in a breeding facility in a specific pathogen-free environment in which the temperature was maintained at 22-24° C., and were bred while maintaining a 12-hour day and night cycle.
  • PBS Phosphate-buffered saline
  • the sniffing time (A) and the number of times (B) were measured by observing the sniffing behavior with other mice for 3 minutes. It was confirmed that the social behavior of the mice orally administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 was significantly increased compared to the group administered with the vehicle to the BTBR mice (p ⁇ 0.05, Student's t-test) (FIG. 31) ).
  • the full-thickness skin defect wound induction model was designed to show a full-thickness wounding after anesthetizing 9-week-old specific pathogen free (SPF) male C57BL/6J mice, about 1.0 cm from the base of the tail of the mouse.
  • SPF pathogen free
  • a full-thickness wound was made on the back of the tail of the mouse that had fallen off using a 10 ⁇ 3mm sterile dissection mass No. 10.
  • the wound was compressed to stop bleeding, and the wound was covered with a film spray dressing (Cavilon, 3M).
  • the wound area of the animals of each test group was individually photographed using a digital camera at a constant height from the wound for 28 days at intervals of 7 days from the day of wound induction, and the wound area was used using the Image J Soft program. was measured.
  • the peptide of the present invention has an effect on damage and regeneration of body tissues including skin tissues.
  • Example 11 Verification of tissue regeneration promoting efficacy of novel peptides
  • novel peptide of the present invention has the effect of promoting tissue regeneration, it was confirmed whether the regeneration of muscle tissue and muscle strength were improved.
  • Example 11-1 Validation of muscle strength improvement and muscle regeneration efficacy of new peptides in aging animal models
  • the aged mouse was asked to hold the wire mesh connected to the strength probe of the grip strength meter with all four legs, and then carefully pulled the tail backward to measure the maximum grip strength at the moment of holding the wire mesh and holding it. did In the case of muscle weight, after administration, the gastrocnemius (GC), soleus, and tibialis anterior (TA) muscles from both hind paws of each group of aged mice were carefully separated not to be damaged, and then each weight was measured and the weight of the mouse was used. After correction, the muscle weight compared to the body weight was calculated.
  • GC gastrocnemius
  • TA tibialis anterior
  • the TA muscle was fixed in 10% formalin, and a frozen section sample (8 ⁇ m) was prepared to prepare a slide.
  • the prepared slides were washed with PBS buffer, then permeated with 0.5% Triton X-100 and blocked, and treated with a primary antibody (anti-laminin).
  • a fluorescent secondary antibody Alexa Fluor 488 Goat anti-rabbit IgG was applied, and finally, DAPI was treated for nuclear staining.
  • the muscle tissue was photographed using a confocal microscope, and the cross-sectional area of the muscle fibers was measured from at least 500 to 3500 ⁇ m 2 or more using image analysis software (ImageInside). The distribution was calculated for each size.
  • RNA-like growth factor involved in protein synthesis in muscle cells
  • RT-qPCR quantitative real-time PCR
  • Example 11-2 Verification of muscle regeneration efficacy of novel peptides in myoblast model
  • myoblast C2C12 myoblast
  • DMEM medium containing 10% FBS and 1% antibotics
  • C2C12 cells were replaced with a differentiation medium containing 2% horse serum and treated with PEP004 and PEP005 at a concentration of 64 nM for a time to induce differentiation to form myotubes. was observed.
  • Reverse transcriptase was added to the obtained RNA and reacted to synthesize cDNA, and quantitative real-time PCR (RT-qPCR) was performed by adding SYBR Green and primers, and then atrogin-1 and MuRF1, factors involved in muscular atrophy, By measuring the expression level, it was confirmed whether the atrophy of the differentiated myoblasts was inhibited.
  • the peptide of the present invention has the effect of promoting muscle regeneration, strengthening of muscle strength, and improving muscle strength.
  • Example 12 Verification of hair regeneration and hair growth promoting efficacy of novel peptides
  • Criteria for scoring evaluation of gross hair growth Criteria score not growing at all 0 Less than 30% of the hair removal area darkens the skin color Hair does not grow in the epilation area One About 30-70% of the hair removal area darkens the skin color Hair does not grow in the epilation area 2 More than 70% of the hair removal area darkens the skin color Less than 30% growth in the hair removal site 3 More than 70% of the hair removal area darkens the skin color 30-70% growth at the hair removal site 4 More than 70% of the hair removal area darkens the skin color More than 70% growth in the hair removal area 5 More than 90% of the hair removal site grows 6
  • the mouse skin tissue was fixed in 10% NBF at room temperature for more than 24 hours, then embedded in paraffin to prepare a 5 ⁇ m thick section, and then treated with hematoxylin and eosin. (H&E) staining.
  • H&E staining The histological changes of the hair follicle tissue were observed using the H&E-stained tissue sections under an optical microscope.
  • the quantity and depth of hair follicles and the thickness of the dermis and subcutaneous tissue were measured under an optical microscope, and the hair growth cycle was evaluated.
  • the number of hair follicles the number of hair follicles present in the subcutaneous tissue was counted using an optical microscope at 100 magnification.
  • the depth of hair follicles and changes in the dermis and subcutaneous thickness were measured through the Image J program using a scale bar.
  • the group applied with Amuc_1409 significantly increased the number of hair follicles in the subcutaneous fat layer compared to the group applied with Vehicle, and the depth of hair follicles and the thickness of the dermis and subcutaneous tissue were significantly thicker than the group applied with Vehicle. lost (p ⁇ 0.05, Student's t-test) ( FIG. 38 ).
  • the peptide of the present invention has the effect of promoting hair regeneration and hair growth.
  • human skin keratinocytes were used.
  • HaCaT Human dermal keratinocytes
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin under conditions of 37°C and 5% CO2.
  • the cell density reached 80%, the number of 4x105 cells per well was aliquoted in a 6-well plate and cultured for 24 hours.
  • Amuc_1409 was pretreated at a concentration of 30 nM for 1 hour and washed twice with PBS (phosphate buffered saline) after 1 hour, using an ultraviolet (UVB) irradiation device (CL-1000 UV crosslinker; UVP, USA).
  • UVB ultraviolet irradiation device
  • Trizol (Invitrogen, USA) was added to cells in each well to confirm the expression of collagen degrading enzymes MMP-1 and MMP-3 genes and collagen synthesizing enzymes Col1a1 and Col3a1 genes in relation to skin wrinkles caused by skin aging.
  • cDNA was synthesized using 1 ug of each RNA.
  • the peptide of the present invention has the effect of suppressing symptoms related to skin aging and promoting skin regeneration.

Abstract

The present invention relates to a novel peptide with anti-inflammatory and tissue regeneration actions, and a use thereof.

Description

항염 및 조직 재생작용을 갖는 신규 펩타이드Novel peptides with anti-inflammatory and tissue regeneration action
본 발명은 항염 및 조직 재생작용을 갖는 신규 펩타이드 및 이의 용도에 관한 것이다.The present invention relates to novel peptides having anti-inflammatory and tissue regeneration action and uses thereof.
염증은 유해한 외인성 및 내인성 신호 또는 병원균을 제거하고 치유 과정을 시작하기 위한 면역 체계의 복잡한 생물학적 반응이다. 박테리아 및 바이러스, 자외선, 중금속, 콜레스테롤, 석면 및 많은 나노 물질과 같은 다양한 감염 요인은 염증을 유발한다. 염증 반응은 감염이나 상처 치유 과정에서 회복되기 위해서는 필수적이지만, 조절되지 않는 염증 반응이나 만성 염증은 관절염, 대사성 질환, 간염, 장염, 위염, 위궤양, 식도염, 피부염 등의 질환뿐 아니라 뇌염, 우울증, 불안장애, 인지장애, 기억장애, 퇴행성 뇌질환 및 발달장애를 유발하기도 한다.Inflammation is a complex biological response of the immune system to clear harmful extrinsic and endogenous signals or pathogens and initiate the healing process. Various infectious factors, such as bacteria and viruses, ultraviolet light, heavy metals, cholesterol, asbestos and many nanomaterials, cause inflammation. Inflammatory response is essential for recovery from infection or wound healing process, but uncontrolled inflammatory response or chronic inflammation is not only caused by diseases such as arthritis, metabolic disease, hepatitis, enteritis, gastritis, gastric ulcer, esophagitis, dermatitis, encephalitis, depression, anxiety, etc. It can also cause disability, cognitive impairment, memory impairment, degenerative brain disease, and developmental disability.
한편 염증반응은 종종 세포, 조직, 기관의 기능 저하를 유발하여 손상을 유발하는 하나의 원인이 되기도 한다.On the other hand, the inflammatory response is often one of the causes of damage by causing a decrease in the function of cells, tissues, and organs.
예를 들면 피부 노화, 근력 손실, 탈모 등의 질환은 조직이 손상되어 발생하는 질환으로, 최근 이러한 조직의 손상을 효과적으로 억제할 수 있는 치료제 개발에 관심이 집중되고 있다. For example, diseases such as skin aging, loss of muscle strength, and hair loss are diseases caused by tissue damage, and recent attention has been focused on developing therapeutic agents that can effectively inhibit such tissue damage.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
WO2014-200651WO2014-200651
WO2020-127904WO2020-127904
본 발명자들은 최근 현대인들에게서 발병이 높아지고 있는 염증 질환, 조직 손상 관련 질환의 발병 및 악화를 효과적으로 억제할 수 있는 치료제를 개발하기 위해 예의 연구 노력한 끝에, 본 발명의 펩타이드를 개발하고, 항염 및 조직 재생 효과가 우수한 것을 확인하여 본 발명을 완성하였다.The present inventors developed the peptide of the present invention after intensive research efforts to develop a therapeutic agent capable of effectively inhibiting the onset and exacerbation of inflammatory diseases and tissue damage-related diseases, which are recently increasing in incidence in modern people, and developed anti-inflammatory and tissue regeneration The present invention was completed by confirming that the effect was excellent.
본 발명의 목적은 하기 일반식 1 또는 2의 서열을 포함하는 펩타이드를 제공하는 것이다:It is an object of the present invention to provide a peptide comprising the sequence of Formula 1 or 2:
[일반식 1][General formula 1]
Ala-Val-Ser-X1-X2-X3-X4-X5-X6Ala-Val-Ser-X1-X2-X3-X4-X5-X6
여기서, X1은 Ser 또는 부존재;Here, X1 is Ser or absent;
X2는 Ile 또는 부존재;X2 is Ile or absent;
X3은 Lys 또는 부존재;X3 is Lys or absent;
X4는 Gly 또는 부존재;X4 is Gly or absent;
X5는 Ala 또는 부존재; 및X5 is Ala or absent; and
X6은 Tyr 또는 부존재함,X6 is Tyr or absent;
[일반식 2][General Formula 2]
X1-X2-X3-X4-X5-X6-Gly-Ala-TyrX1-X2-X3-X4-X5-X6-Gly-Ala-Tyr
여기서, X1은 Ala 또는 부존재;where X1 is Ala or absent;
X2는 Val 또는 부존재;X2 is Val or absent;
X3은 Ser 또는 부존재;X3 is Ser or absent;
X4는 Ser 또는 부존재;X4 is Ser or absent;
X5는 Ile 또는 부존재; 및X5 is Ile or absent; and
X6은 Lys 또는 부존재함.X6 is Lys or absent.
본 발명의 다른 목적은 상기 펩타이드를 코딩하는 폴리뉴클레오티드 및 상기 폴리뉴클레오티드를 포함하는 벡터를 제공하는 것이다.Another object of the present invention is to provide a polynucleotide encoding the peptide and a vector comprising the polynucleotide.
본 발명의 다른 목적은 상기 펩타이드를 포함하는, 염증질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including the peptide.
본 발명의 다른 목적은 상기 펩타이드를 포함하는, 조직 재생용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for tissue regeneration comprising the peptide.
본 발명의 다른 목적은 상기 펩타이드를 포함하는, 조직 손상 관련 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating tissue damage-related diseases, including the peptide.
본 발명의 다른 목적은 상기 펩타이드를 포함하는 건강기능식품 조성물, 사료 조성물, 동물 의약품, 의약외품 및 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition, feed composition, veterinary medicine, quasi-drug, and cosmetic composition comprising the peptide.
본 발명의 다른 목적은 상기 펩타이드를 개체에 투여하는 단계를 포함하는, 염증질환 예방 또는 치료방법을 제공하는 것이다. Another object of the present invention is to provide a method for preventing or treating an inflammatory disease, comprising administering the peptide to an individual.
본 발명의 다른 목적은 상기 펩타이드를 개체에 투여하는 단계를 포함하는, 조직 재생 방법을 제공하는 것이다.Another object of the present invention is to provide a method for tissue regeneration, comprising administering the peptide to a subject.
본 발명의 펩타이드는 항염 및 조직 재생 효과를 가지는바, 염증질환의 예방, 개선 및 치료뿐 아니라 조직 재생 용도로도 사용될 수 있다.Since the peptide of the present invention has anti-inflammatory and tissue regeneration effects, it can be used for tissue regeneration as well as prevention, improvement and treatment of inflammatory diseases.
본 발명의 도면에서 PEP001은 서열번호 1, PEP002는 서열번호 2, PEP003은 서열번호 3, PEP004는 서열번호 4, PEP005는 서열번호 5를, Amuc_1409은 서열번호 6의 단백질의 leader sequence를 제외한 기능적 단편인 서열번호 7을 지칭한다.In the drawings of the present invention, PEP001 is SEQ ID NO: 1, PEP002 is SEQ ID NO: 2, PEP003 is SEQ ID NO: 3, PEP004 is SEQ ID NO: 4, PEP005 is SEQ ID NO: 5, Amuc_1409 is a functional fragment except for the leader sequence of SEQ ID NO: 6 refers to SEQ ID NO:7.
도 1은 퇴행성 관절염 동물모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 무릎 관절 두께 및 체중 부하 측정 결과 비교 결과이다(*p<0.05, **p<0.01, ***p<0.001 Amuc_1409 vs. vehicle군, ∫p<0.05, ∫∫p<0.01, ∫∫∫p<0.001 PEP001 vs. vehicle군, #p<0.05, ##p<0.01, ###p<0.001 PEP002 vs. vehicle군 $p<0.05, $$$p<0.001 PEP003 vs. vehicle군, †p<0.05, ††p<0.01, †††p<0.001 PEP004 vs. vehicle군, ‡p<0.05, ‡‡‡p<0.001 PEP005 vs. vehicle군).1 is a comparison result of knee joint thickness and weight bearing measurement results by administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of degenerative arthritis (*p<0.05, **p<0.01, ***p< 0.001 Amuc_1409 vs. vehicle group, ∫p<0.05, ∫∫p<0.01, ∫∫∫p<0.001 PEP001 vs. vehicle group, #p<0.05, ##p<0.01, ###p<0.001 PEP002 vs. vehicle $p<0.05, $$$p<0.001 PEP003 vs. vehicle, †p<0.05, ††p<0.01, †††p<0.001 PEP004 vs. vehicle, ‡p<0.05, ‡‡‡ p<0.001 PEP005 vs. vehicle group).
도 2는 퇴행성 관절염 동물모델에서 Amuc_1409 투여에 의한 Safranin O 염색을 통한 무릎 관절 연골 손상 변화 비교 결과이다(**p<0.01 vs. vehicle군).2 is a comparison result of knee articular cartilage damage through Safranin O staining by Amuc_1409 administration in an animal model of degenerative arthritis (**p<0.01 vs. vehicle group).
도 3은 퇴행성 관절염 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 Safranin O 염색을 통한 무릎 관절 연골 손상 변화 비교 결과이다(*p<0.05, **p<0.01, ***p<0.001 vs. vehicle군).3 is a comparison result of knee articular cartilage damage through Safranin O staining by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of degenerative arthritis (*p<0.05, **p<0.01, ***p< 0.001 vs. vehicle group).
도 4는 류마티스 관절염 동물모델에서 Amuc_1409 투여에 의한 일자별 관절염 지수 변화 (A) 발목 관절 두께 (B) 및 부종의 변화 (C)를 확인한 결과이다(p<0.05, **p<0.01 vs. vehicle군).Figure 4 is the result of confirming the change in the daily arthritis index (A), ankle joint thickness (B) and edema (C) by Amuc_1409 administration in an animal model of rheumatoid arthritis (p<0.05, **p<0.01 vs. vehicle group) ).
도 5는 류마티스 관절염 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 일자별 관절염 지수 변화 (A) 발목 관절 두께 (B) 및 부종의 변화 (C) 를 비교한 것이다(∫∫p<0.01, ∫∫∫p<0.001 PEP001 vs. vehicle군, ##p<0.01, ###p<0.001 PEP002 vs. vehicle군, $p<0.05, $$p<0.01, $$$p<0.001 PEP003 vs. vehicle군, †p<0.05, ††p<0.01, †††p<0.001 PEP004 vs. vehicle군, ‡p<0.05, ‡‡‡p<0.001 PEP005 vs. vehicle군).5 is a comparison of the daily arthritis index change (A) ankle joint thickness (B) and edema change (C) by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of rheumatoid arthritis (∫∫p<0.01) , ∫∫∫p<0.001 PEP001 vs. vehicle group, ##p<0.01, ###p<0.001 PEP002 vs. vehicle group, $p<0.05, $$p<0.01, $$$p<0.001 PEP003 vs. vehicle group, †p<0.05, ††p<0.01, †††p<0.001 PEP004 vs. vehicle group, ‡p<0.05, ‡‡‡p<0.001 PEP005 vs. vehicle group).
도 6은 류마티스 관절염 동물모델에서 실험기간 종료 후 Amuc_1409 투여에 의한 H&E 염색결과 (A) 및 조직병리학적 관절염 지수 (B) 변화를 비교한 것이다 (**p<0.01 vs. vehicle군).6 is a comparison of H&E staining results (A) and histopathological arthritis index (B) changes by Amuc_1409 administration after the end of the experimental period in an animal model of rheumatoid arthritis (**p<0.01 vs. vehicle group).
도 7은 류마티스 관절염 동물모델에서 실험기간 종료 후 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 H&E 염색결과 (A) 및 조직병리학적 관절염 지수 (B) 변화를 비교한 것이다(*p<0.05, ***p<0.001).7 is a comparison of changes in H&E staining results (A) and histopathological arthritis index (B) by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 after the end of the experimental period in an animal model of rheumatoid arthritis (*p<0.05, ***p<0.001).
도 8은 비만/당뇨 대사질환 동물모델에서 Amuc_1409 투여에 의한 체중, 복부지방 무게, 인슐린 저항성 관련 지표 및 공복혈당 분석 결과이다 (p<0.05).8 shows the results of analysis of body weight, abdominal fat weight, insulin resistance-related indicators and fasting blood glucose by Amuc_1409 administration in an animal model of obesity/diabetic metabolic disease (p<0.05).
도 9는 비만/당뇨 대사질환 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 따른 체중, 복부지방 무게, 인슐린 저항성 관련 지표 및 공복혈당 분석 결과이다(#p<0.05 PEP002, $p<0.05 PEP003, †p<0.05 PEP004, ‡p<0.05 PEP005 vs. vehicle군).9 shows the results of analysis of body weight, abdominal fat weight, insulin resistance-related indicators and fasting blood glucose according to administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of obesity/diabetic metabolic disease (#p<0.05 PEP002, $p<0.05) PEP003, †p<0.05 PEP004, ‡p<0.05 PEP005 vs. vehicle group).
도 10은 비만/당뇨 대사질환 동물모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 따른 혈중 지질 관련 지표의 변화 분석 결과이다(*p<0.05 vs. vehicle군).10 is an analysis result of changes in blood lipid-related indicators according to administration of Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of obesity/diabetic metabolic disease (*p<0.05 vs. vehicle group).
도 11은 급성 간염 동물모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 간손상 변화 분석 결과이다(*p<0.05 vs. vehicle군).11 is an analysis result of liver damage change by administration of Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 in an acute hepatitis animal model (*p<0.05 vs. vehicle group).
도 12는 고지방 식이 유도 비알코올성 지방간염 동물모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 따른 간내 염증 관련 유전자 발현 변화 분석 결과이다(*p<0.05, **p<0.05 vs. vehicle군).12 is an analysis result of intrahepatic inflammation-related gene expression change according to administration of Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 in a high-fat diet-induced nonalcoholic steatohepatitis animal model (*p<0.05, **p<0.05 vs. vehicle) army).
도 13은 염증성 장질환 마우스 모델에서 PEP002, PEP003, PEP004 및 PEP005 투여에 의한 체중 변화 분석 결과이다(†p<0.05, ††p<0.01 PEP004 vs. vehicle군, ‡‡p<0.01 PEP005 vs. vehicle군).13 is an analysis result of weight change by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model (†p<0.05, ††p<0.01 PEP004 vs. vehicle group, ‡‡p<0.01 PEP005 vs. vehicle) army).
도 14는 염증성 장질환 마우스 모델에서 PEP002, PEP003, PEP004 및 PEP005 투여에 의한 결장 길이 변화 분석 결과이다(*p<0.05, **p<0.01 vs. vehicle군).14 is an analysis result of colon length change by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model (*p<0.05, **p<0.01 vs. vehicle group).
도 15는 염증성 장질환 마우스 모델에서 PEP002, PEP003, PEP004 및 PEP005 투여에 의한 결장의 조직 병리학적 변화 분석 결과이다 (*p<0.05, **p<0.01, ***p<0.001 vs. vehicle군).15 is a result of analysis of histopathological changes in the colon by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model (*p<0.05, **p<0.01, ***p<0.001 vs. vehicle group. ).
도 16은 염증성 장질환 마우스 모델에서 PEP002, PEP003, PEP004 및 PEP005 투여에 의한 결장의 염증 지표 유전자 발현 변화 분석 결과이다(*p<0.05, **p<0.01 vs. vehicle군).16 is an analysis result of changes in the expression of inflammatory marker genes in the colon by administration of PEP002, PEP003, PEP004 and PEP005 in an inflammatory bowel disease mouse model (*p<0.05, **p<0.01 vs. vehicle group).
도 17은 위염/위궤양 동물모델에서 Amuc_1409 투여에 의한 위 점막 손상 분석 결과이다(*p<0.05 vs. vehicle군).17 is a result of analysis of gastric mucosal damage by Amuc_1409 administration in an animal model of gastritis/gastric ulcer (*p<0.05 vs. vehicle group).
도 18은 위염/위궤양 모델에서 Amuc_1409 투여에 의한 염증 지표 유전자 분석 결과이다(*p<0.05 vs. vehicle군).18 is an inflammatory index gene analysis result by Amuc_1409 administration in a gastritis/gastric ulcer model (*p<0.05 vs. vehicle group).
도 19는 위염/위궤양 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 위 점막 손상 분석 결과이다(*p<0.05 vs. vehicle군).19 is an analysis result of gastric mucosal damage by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of gastritis/gastric ulcer (*p<0.05 vs. vehicle group).
도 20은 HCl/ethanol 유도 위염/위궤양 모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 염증 지표 유전자 분석 결과이다(*p<0.05 vs. vehicle군).20 is an inflammatory marker gene analysis result by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in an HCl/ethanol-induced gastritis/gastric ulcer model (*p<0.05 vs. vehicle group).
도 21은 피부염 동물모델에서 Amuc_1409 투여에 의한 마우스 귀 홍반 및 부종의 변화 촬영한 사진 (도 21A) 귀 두께 (도 21B) 및 일정면적당 무게 변화 (도 21C) 를 비교한 것이다(**p<0.01 vs. vehicle군).Figure 21 is a comparison of ear thickness (Figure 21B) and weight change (Figure 21C) per area (Figure 21C) of mouse ear erythema and edema change by Amuc_1409 administration in an animal model of dermatitis (Figure 21A) (**p<0.01) vs. vehicle group).
도 22는 피부염 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 마우스 귀 홍반 및 부종의 변화 촬영한 사진 (도 22A) 귀 두께 (도 22B) 및 일정면적당 무게 변화 (도 22C)를 비교한 것이다(*p<0.05, **p<0.01, ***p<0.05 vs. vehicle군).22 is a photograph taken of changes in mouse ear erythema and edema by administration of PEP001, PEP002, PEP003, PEP004, and PEP005 in an animal model of dermatitis (FIG. 22A), comparing ear thickness (FIG. 22B) and weight change per area (FIG. 22C) (*p<0.05, **p<0.01, ***p<0.05 vs. vehicle group).
도 23은 피부염 동물모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 조직병리학적 마우스 귀 조직의 H&E 염색 결과이다.23 is a result of H&E staining of histopathological mouse ear tissue by administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of dermatitis.
도 24는 우울증 및 불안 모델에서 Amuc_1409 투여에 의한 FST, TST 부동자세 및 EPM 열린 arm에 머문 시간 분석 결과이다(*p<0.05 vs. vehicle군).24 is an analysis result of FST, TST immobility posture, and EPM stay time in an open arm by Amuc_1409 administration in a depression and anxiety model (*p<0.05 vs. vehicle group).
도 25는 우울증 및 불안 모델에서 Amuc_1409 투여에 의한 뇌염증 지표 유전자 분석 결과이다(*p<0.05 vs. vehicle군).25 is a gene analysis result of brain inflammation index by administration of Amuc_1409 in a depression and anxiety model (*p<0.05 vs. vehicle group).
도 26은 우울증 및 불안 모델에서 Amuc_1409 투여에 의한 FST, TST 부동자세 및 EPM 열린 arm 시간 분석 결과이다(*p<0.05 vs. vehicle군).26 is an analysis result of FST, TST immobility posture, and EPM open arm time by Amuc_1409 administration in a depression and anxiety model (*p<0.05 vs. vehicle group).
도 27은 우울증 및 불안 모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여에 의한 뇌염증 지표 유전자 분석 결과이다(*p<0.05 vs. vehicle군).27 is a result of analysis of brain inflammation index genes by administration of PEP001, PEP002, PEP003, PEP004 and PEP005 in a depression and anxiety model (*p<0.05 vs. vehicle group).
도 28은 알츠하이머병 마우스 동물모델에서 Amuc_1409와 PEP001 투여에 의한 새로운 사물에 대한 인지기능 및 기억력 개선 효과를 나타낸 그래프 (A 및 C, 새로운 물체 탐지 시간; B 및 D, 새로운 물체 탐지 횟수)이다(*p<0.05, **p<0.01, ***p<0.01).28 is a graph (A and C, new object detection time; B and D, number of new object detection) showing the effect of improving cognitive function and memory for new objects by administration of Amuc_1409 and PEP001 in an Alzheimer's disease mouse animal model (* p<0.05, **p<0.01, ***p<0.01).
도 29는 인지기능 장애 모델에서 Amuc_1409와 PEP002, PEP003, PEP004 및 PEP005 투여에 따른 새로운 사물에 대한 인지테스트(novel object recognition test; NORT)에서의 인지 기능 및 기억력 개선 효과를 나타낸 그래프이다 (A 및 C, 새로운 물체 탐지 시간; B 및 D, 새로운 물체 탐지 횟수. *p<0.05, **p<0.01, ***p<0.01)29 is a graph showing the effect of improving cognitive function and memory in a novel object recognition test (NORT) according to administration of Amuc_1409 and PEP002, PEP003, PEP004 and PEP005 in a cognitive dysfunction model (A and C) , new object detection time; B and D, number of new object detections. *p<0.05, **p<0.01, ***p<0.01)
도 30은 자연노화 동물모델에서 Amuc_1409와 PEP002, PEP003, PEP004 및 PEP005 투여에 의한 새로운 사물에 대한 인지테스트(novel object recognition test; NORT)에서의 인지 기능 및 기억력 개선 효과를 나타낸 그래프 (A 및 C, 새로운 물체 탐지 시간; B 및 D, 새로운 물체 탐지 횟수)이다(*p<0.05, **p<0.01, ***p<0.01).30 is a graph showing the effect of improving cognitive function and memory in a novel object recognition test (NORT) for new objects by administration of Amuc_1409 and PEP002, PEP003, PEP004 and PEP005 in a natural aging animal model (A and C, new object detection time; B and D, number of new object detections) (*p<0.05, **p<0.01, ***p<0.01).
도 31은 발달 장애 동물모델 마우스에 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005의 투여 후, 두 마리의 마우스 간의 사회적 상호작용 및 사회적 호기심의 정도를 확인하기 위한 상호작용 행동(sniffing behavior)의 시간(A와 C) 및 횟수(B와 D) 분석 결과이다(*p<0.05, ***p<0.01).Figure 31 shows the time of sniffing behavior to determine the degree of social interaction and social curiosity between two mice after administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 to developmentally disabled animal model mice. (A and C) and the number of times (B and D) analysis results (*p<0.05, ***p<0.01).
도 32는 전층 피부결손 창상 유발 동물 모델에서 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 도포에 따른 일자별 창상 치유 효과를 비교하여 나타낸 사진과 그래프이다(*p<0.05, **p<0.01, ***p<0.001 Amuc_1409 vs. vehicle군, ∫p<0.05, ∫∫p<0.01 PEP001 vs. vehicle군, #p<0.05, ##p<0.01, ###p<0.001 PEP002 vs. vehicle군, $p<0.05, $$p<0.01 PEP003 vs. vehicle군, ††p<0.01, †††p<0.001 PEP004 vs. vehicle군, ‡‡p<0.01, ‡‡‡p<0.001 PEP005 vs. vehicle군).Figure 32 is a photograph and graph showing the comparison of the wound healing effect by day according to the application of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in the full-thickness skin defect wound-inducing animal model (*p<0.05, **p<0.01, * **p<0.001 Amuc_1409 vs. vehicle group, ∫p<0.05, ∫∫p<0.01 PEP001 vs. vehicle group, #p<0.05, ##p<0.01, ###p<0.001 PEP002 vs. vehicle group, $p<0.05, $$p<0.01 PEP003 vs. vehicle group, ††p<0.01, †††p<0.001 PEP004 vs. vehicle group, ‡‡p<0.01, ‡‡‡p<0.001 PEP005 vs. vehicle army).
도 33은 노화동물모델에서 Amuc_1409 투여에 의한 근력 및 근육량 개선 효과를 확인한 것이다(*p<0.05 vs. vehicle군).Figure 33 confirms the effect of improving muscle strength and muscle mass by administration of Amuc_1409 in an aging animal model (*p<0.05 vs. vehicle group).
도 34는 노화동물모델에서 Amuc_1409 투여에 의한 근위축 개선 효과를 확인한 것이다(*p<0.05 vs. vehicle군).34 shows the improvement of muscle atrophy by administration of Amuc_1409 in an aging animal model (*p<0.05 vs. vehicle group).
도 35는 노화동물모델에서 PEP001 투여에 따른 근력, 근육양 및 근육세포 내 단백질 합성 관련 유전자 발현 분석 결과이다(*p<0.05 vs. vehicle군).35 is a result of analysis of muscle strength, muscle mass, and protein synthesis-related gene expression in muscle cells according to PEP001 administration in an aging animal model (*p<0.05 vs. vehicle group).
도 36은 C2C12 근원세포에서 PEP004 및 PEP005 처리에 의한 근 위축 관련 유전자 분석 결과이다(*p<0.05 vs. vehicle군).Figure 36 is a result of muscle atrophy-related gene analysis by treatment with PEP004 and PEP005 in C2C12 myoblasts (*p<0.05 vs. vehicle group).
도 37은 Amuc_1409 도포 후 21일 째 모발성장 경과를 관찰한 육안 사진 (도 37A)과 스코어링 그래프 (도 37B)이다(*p<0.05 vs. vehicle군).37 is a macroscopic photograph (FIG. 37A) and a scoring graph (FIG. 37B) observing the progress of hair growth on the 21st day after application of Amuc_1409 (*p<0.05 vs. vehicle group).
도 38은 동물모델에서 실험기간 종료 후 Amuc_1409 피부 도포에 의한 H&E 염색결과 (도 38A), 조직학적 모낭 수 (도 38B), 모낭 깊이 (도 38C), 진피 두께 (도 38D) 및 피하 두께 (도 38E) 변화 비교 결과이다(**p<0.01, ***p<0.001 vs. vehicle군).Fig. 38 shows the results of H&E staining by Amuc_1409 skin application (Fig. 38A), histological number of follicles (Fig. 38B), hair follicle depth (Fig. 38C), dermal thickness (Fig. 38D) and subcutaneous thickness (Fig. 38) after the end of the experimental period in an animal model. 38E) This is a change comparison result (**p<0.01, ***p<0.001 vs. vehicle group).
도 39는 동물모델에서 실험기간 종료 후 Amuc_1409 피부 도포에 의한 조직학적 발모주기 비율 변화 비교 결과이다(***p<0.001 vs. vehicle군).39 is a comparison result of histological hair growth cycle rate change by applying Amuc_1409 to the skin after the end of the experimental period in an animal model (***p<0.001 vs. vehicle group).
도 40은 사람의 피부 각질세포에서 Amuc_1409 처리에 따른 콜라겐 분해 및 합성 관련 유전자 발현 분석 결과이다(p<0.05).Fig. 40 shows the results of analysis of gene expression related to collagen degradation and synthesis according to Amuc_1409 treatment in human skin keratinocytes (p<0.05).
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 발명에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 의도된다.In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.
본 발명의 하나의 양태는 서열번호 4 또는 5의 아미노산 서열을 포함하는 펩타이드를 제공한다.One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 4 or 5.
일 구현예로, 본 발명의 펩타이드는 다음 일반식 1로 표시되는 서열을 포함할 수 있다:In one embodiment, the peptide of the present invention may include a sequence represented by the following general formula 1:
[일반식 1][General formula 1]
Ala-Val-Ser-X1-X2-X3-X4-X5-X6Ala-Val-Ser-X1-X2-X3-X4-X5-X6
여기서, X1은 Ser 또는 부존재;Here, X1 is Ser or absent;
X2는 Ile 또는 부존재;X2 is Ile or absent;
X3은 Lys 또는 부존재;X3 is Lys or absent;
X4는 Gly 또는 부존재;X4 is Gly or absent;
X5는 Ala 또는 부존재; 및X5 is Ala or absent; and
X6은 Tyr 또는 부존재함.X6 is Tyr or absent.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 1에서, X1은 Ser이고 X2는 Ile일 수 있다.In any one of the above-described embodiments, in Formula 1, X1 may be Ser and X2 may be Ile.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 1에서, X1 내지 X6은 부존재하는 것일 수 있다. In any one of the above-described embodiments, in Formula 1, X1 to X6 may be absent.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 1에서, X1은 Ser이고, X2는 Ile이고, X3은 Lys이고, X4는 Gly이고, X5는 Ala이고, X6은 Tyr일 수 있다.In any one of the above embodiments, in Formula 1, X1 may be Ser, X2 may be Ile, X3 may be Lys, X4 may be Gly, X5 may be Ala, and X6 may be Tyr.
일 구현예로, 본 발명의 펩타이드는 다음 일반식 2로 표시되는 서열을 포함할 수 있다:In one embodiment, the peptide of the present invention may include a sequence represented by the following general formula 2:
[일반식 2][General Formula 2]
X1-X2-X3-X4-X5-X6-Gly-Ala-TyrX1-X2-X3-X4-X5-X6-Gly-Ala-Tyr
여기서, X1은 Ala 또는 부존재;where X1 is Ala or absent;
X2는 Val 또는 부존재;X2 is Val or absent;
X3은 Ser 또는 부존재;X3 is Ser or absent;
X4는 Ser 또는 부존재;X4 is Ser or absent;
X5는 Ile 또는 부존재; 및X5 is Ile or absent; and
X6은 Lys 또는 부존재함.X6 is Lys or absent.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 2에서, X6은 Lys 일 수 있다.In any one of the above-described embodiments, in Formula 2, X6 may be Lys.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 2에서, X1 내지 X6은 부존재하는 것일 수 있다. In any one of the above-described embodiments, in Formula 2, X1 to X6 may be absent.
전술한 구현예 중 어느 하나의 구현예로, 상기 일반식 2에서, X1은 Ala이고, X2는 Val이고, X3은 Ser이고, X4는 Ser이고, X5는 Ile이고, X6은 Lys일 수 있다.In any one of the above-described embodiments, in Formula 2, X1 may be Ala, X2 may be Val, X3 may be Ser, X4 may be Ser, X5 may be Ile, and X6 may be Lys.
상기 용어 "부존재" 예를 들어 "X5는 Ile 또는 부존재한다" 는 것은, 부존재하는 아미노산에 직접 인접한 아미노산 잔기가 통상적인 아미드 결합에 의해 서로 직접 연결된 것으로 이해되어야 한다.The term "absence", for example "X5 is Ile or absent", should be understood to mean that amino acid residues directly adjacent to the absent amino acid are directly linked to each other by conventional amide bonds.
본 발명에서 제공하는 펩타이드는 그의 염 형태를 포함한다. 이와 같은 염의 예는 금속 염, 암모늄 염, 유기 염기와의 염, 무기 산과의 염, 유기 산과의 염, 염기성 또는 산성 아미노산과의 염, 및 기타 등등을 포함한다. 금속 염의 바람직한 예는 알칼리 금속 염 예컨대 나트륨 염, 칼륨 염 및 기타 등등; 알칼리성 토류 금속 염 예컨대 칼슘 염, 마그네슘 염, 바륨 염 및 기타 등등; 알루미늄 염 및 기타 등등을 포함한다The peptides provided in the present invention include salt forms thereof. Examples of such salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferred examples of metal salts include alkali metal salts such as sodium salts, potassium salts and the like; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts and the like; aluminum salts and so on
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 서열번호 1 내지 7 중 어느 하나의 아미노산 서열을 포함하거나, 상기 아미노산 서열로 필수적으로 구성되거나(consisting essentially of), 또는 상기 아미노산 서열로 구성될 수 있다. 그러나 이에 제한되지 않고, 상기 서열과 동일한 활성을 나타내는 펩타이드는 제한 없이 포함된다. 예를 들어, 본 발명의 서열번호 7의 아미노산 서열은 서열번호 6의 아미노산 서열에서 leader sequence만을 제외한 서열에 상응하므로, 서열번호 7의 아미노산 서열을 포함하거나, 서열번호 7로 필수적으로 구성되거나(consisting essentially of), 또는 구성되는 펩타이드도 본 발명의 펩타이드의 범위에 포함된다.In any one of the preceding embodiments, the peptide of the present invention comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-7. can be composed of However, the present invention is not limited thereto, and peptides exhibiting the same activity as the sequence are included without limitation. For example, since the amino acid sequence of SEQ ID NO: 7 of the present invention corresponds to a sequence except for the leader sequence in the amino acid sequence of SEQ ID NO: 6, it contains the amino acid sequence of SEQ ID NO: 7, or consists essentially of SEQ ID NO: 7 (consisting of the amino acid sequence of SEQ ID NO: 7) Peptides essentially of, or consisting of, are also included within the scope of the peptides of the present invention.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 서열번호 4 또는 5의 아미노산 서열을 포함하는 서열번호 6의 아미노산 서열 중에서 연속하는 3개 내지 150개의 아미노산으로 이루어진 것일 수 있다.In any one of the above embodiments, the peptide of the present invention may consist of 3 to 150 consecutive amino acids among the amino acid sequence of SEQ ID NO: 6 including the amino acid sequence of SEQ ID NO: 4 or 5.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는, 아커만시아 뮤시니필라(Akkermansia muciniphila) 균주 유래의 펩타이드일 수 있다.In any one of the above-described embodiments, the peptide of the present invention may be a peptide derived from Akkermansia muciniphila strain.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 당업계에 공지된 펩타이드 합성 방법에 따라 생산될 수 있다. 일 예로, 생리적 조건하에 본 발명의 펩타이드 분자로 전환되는 것일 수 있다. 일 예로, 고상 합성 과정 및 액상 합성 과정에 따라 본 발명의 펩타이드를 합성할 수 있다. 즉, 본 발명에서 제공하는 펩타이드는 상기 펩타이드 분자를 구성할 수 있는 부분적 펩타이드 또는 아미노산, 합성되어야 하는 펩타이드 그리고 나머지 부분을 원하는 순서에 따라 반복 축합시킴으로써 생산될 수 있다. 바람직한 순서를 갖는 생산물이 보호기를 갖는 경우, 목적 펩타이드는 보호기를 제거함으로써 생산될 수 있다. In any one of the above-described embodiments, the peptide of the present invention can be produced according to a method for synthesizing a peptide known in the art. For example, it may be converted into the peptide molecule of the present invention under physiological conditions. For example, the peptide of the present invention may be synthesized according to a solid-phase synthesis process and a liquid-phase synthesis process. That is, the peptide provided in the present invention can be produced by repeatedly condensing a partial peptide or amino acid constituting the peptide molecule, a peptide to be synthesized, and the remaining portion in a desired order. When the product having the desired order has a protecting group, the target peptide can be produced by removing the protecting group.
본 발명의 다른 하나의 양태는 상기 펩타이드를 코딩하는 폴리뉴클레오티드를 제공한다.Another aspect of the present invention provides a polynucleotide encoding the peptide.
본 발명의 다른 하나의 양태는 상기 폴리뉴클레오티드를 포함하는 벡터를 제공한다.Another aspect of the present invention provides a vector comprising the polynucleotide.
본 발명의 폴리뉴클레오티드는 뉴클레오티드 단위체(monomer)가 공유결합에 의해 사슬모양으로 길게 이어진 뉴클레오티드의 중합체(polymer)이며, 일정한 길이 이상의 DNA 또는 RNA 가닥으로서, 본 발명에 따른 펩타이드를 코딩하는 폴리뉴클레오티드를 의미한다.The polynucleotide of the present invention is a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds, and is a DNA or RNA strand of a certain length or longer, which means a polynucleotide encoding the peptide according to the present invention. do.
또한, 본 발명의 폴리뉴클레오티드는 상기 펩타이드를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 펩타이드의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있다. 즉, 본 발명의 폴리뉴클레오티드는 이와 동등한 활성을 갖는 펩타이드를 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다.In addition, in the polynucleotide of the present invention, various modifications can be made in the coding region within a range that does not change the amino acid sequence of the peptide expressed from the coding region in consideration of codons preferred by the organism in which the peptide is to be expressed, Various modifications or modifications can be made within the range that does not affect the expression of the gene even in parts other than the coding region. That is, as long as the polynucleotide of the present invention encodes a peptide having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
본 발명의 재조합 벡터는 세포 내에 도입하여 본 발명의 펩타이드를 발현시키기 위한 수단으로서, 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 등 공지의 벡터를 사용할 수 있으며, 벡터는 DNA 재조합 기술을 이용한 임의의 공지된 방법에 따라 당업자가 용이하게 제조할 수 있다As a means for introducing the recombinant vector of the present invention into a cell to express the peptide of the present invention, a known vector such as a plasmid vector, a cosmid vector, or a bacteriophage vector may be used, and the vector is any known vector using DNA recombination technology. It can be easily prepared by those skilled in the art according to the method
본 발명에서 제공하는 펩타이드에 상응하는 기능을 가지는 펩타이드 유사체 또한 본 발명의 범위에 포함된다. 상기 용어 "펩타이드 유사체"는 비-천연 발생 아미노산 서열, 또는 변형된 천연 발생 아미노산 서열을 지칭한다.A peptide analog having a function corresponding to the peptide provided by the present invention is also included in the scope of the present invention. The term “peptide analog” refers to a non-naturally occurring amino acid sequence, or a modified naturally occurring amino acid sequence.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 캐리어 물질이 연결된 형태일 수 있다. In any one of the above-described embodiments, the peptide of the present invention may be in a form in which a carrier material is linked.
본 발명에 사용가능한 캐리어 물질의 예를 일부만 들자면 면역글로불린 Fc 영역, 알부민, 트랜스페린 및 폴리에틸렌글리콜(PEG)로 구성된 군에서 선택될 수 있으나, 이에 제한되지는 않는다. Examples of the carrier material usable in the present invention may be selected from the group consisting of immunoglobulin Fc region, albumin, transferrin, and polyethylene glycol (PEG), but is not limited thereto.
PEG는 목적 펩타이드의 특정 부위 또는 다양한 부위에 비특이적으로 결합하여 펩타이드의 분자량을 증가시켜 신장에 의한 소실을 억제하고 가수분해를 방지하는데 효과가 있으며 특별한 부작용도 일으키지 않는다. 이에 더하여 외래 펩타이드에 PEG를 결합시키면 때로는 외래 펩타이드에 존재하는 항원 부위가 면역세포에 의해 인식되는 것을 저해할 수 있다. 구체적으로는 펩타이드의 항원제시세포(antigen-presenting cell)에 의한 대식작용 및 세포 내 분해작용(proteolysis)을 저해하여 펩타이드가 항원으로 작용할 가능성을 낮출 수 있다.PEG non-specifically binds to a specific site or various sites of the target peptide to increase the molecular weight of the peptide, thereby inhibiting loss by kidney and preventing hydrolysis, and does not cause any special side effects. In addition, binding of PEG to a foreign peptide can sometimes inhibit recognition of antigenic sites present in the foreign peptide by immune cells. Specifically, by inhibiting phagocytosis and intracellular proteolysis of the peptide by antigen-presenting cells, the possibility of the peptide acting as an antigen can be reduced.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 상기 캐리어 물질과 링커를 통해 연결될 수 있다. 상기 링커는 펩타이드성 또는 비펩타이드성 중합체일 수 있다.In any one of the above-described embodiments, the peptide of the present invention may be linked to the carrier material through a linker. The linker may be a peptidic or non-peptidyl polymer.
본 발명에 사용 가능한 비펩타이드성 중합체의 예를 일부만 들자면 폴리에틸렌 글리콜, 폴리프로필렌 글리콜, 에틸렌 글리콜과 프로필렌 글리콜의 공중합체, 폴리옥시 에틸화 폴리올, 폴리비닐 알콜, 폴리사카라이드, 덱스트란, 폴리비닐 에틸 에테르, PLA(폴리락트산, polylactic acid) 및 PLGA(폴리락틱-글리콜산, polylactic-glycolic acid)와 같은 생분해성 고분자, 지질 중합체, 키틴류, 히아루론산 및 이들의 조합으로 구성된 군으로부터 선택될 수 있다. 이 기술 분야에 이미 알려진 전술한 예시 비펩타이드 중합체의 유도체 및 당해 분야의 기술 수준에서 용이하게 제조할 수 있는 유도체들도 본 발명의 범위에 포함된다.Examples of non-peptidyl polymers usable in the present invention include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl It may be selected from the group consisting of ethers, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Derivatives of the above-described exemplary non-peptide polymers already known in the art and derivatives that can be easily prepared at the level of skill in the art are also included in the scope of the present invention.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 PEG, 단당류, 형광단, 발색단, 방사성 화합물 및 세포 투과 펩타이드(cell-penetrating peptide)로 이루어진 군으로부터 선택될 수 있는 모이어티에 추가로 접합될 수 있다.In any one of the preceding embodiments, the peptide of the present invention is in addition to a moiety that can be selected from the group consisting of PEG, monosaccharides, fluorophores, chromophores, radioactive compounds and cell-penetrating peptides. can be joined.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 분자의 활성을 전체적으로 변경시키지 않는 범위에서, 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation), 아세틸화(acetylation) 및 아미드화(amidation) 등으로 수식(modification)될 수 있다. In any one of the above-described embodiments, the peptides of the present invention are phosphorylation, sulfation, acrylation, and glycosylation in some cases to the extent that the activity of the molecule is not entirely altered. ), methylation, farnesylation, acetylation, and amidation may be modified.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 펩타이드는 글리코실화, 페길화(PEGylation), 아미드화, 에스테르화, 아실화, 아세틸화 및/또는 알킬화 된 형태로 존재하는 것일 수 있다. In any one of the above embodiments, the peptide of the present invention may be present in glycosylated, PEGylated, amidated, esterified, acylated, acetylated and/or alkylated form.
그러나 전술한 예시에 제한되지 않는다.However, it is not limited to the above-described example.
본 발명에서 제공하는 펩타이드는 항염 및/또는 재생 작용을 가지는 것일 수 있다.The peptides provided in the present invention may have anti-inflammatory and/or regenerative properties.
따라서, 본 발명의 하나의 양태는 본 발명의 펩타이드의 염증질환 예방 또는 치료 용도를 제공한다. Accordingly, one aspect of the present invention provides a use for preventing or treating inflammatory diseases of the peptide of the present invention.
본 발명의 다른 하나의 양태는, 본 발명의 펩타이드의 조직 재생 용도를 제공한다.Another aspect of the present invention provides the use of the peptide of the present invention for tissue regeneration.
본 발명에서 "항염(anti inflammation)" 은 염증을 억제하는 것을 말하며, 상기 염증은 어떤 자극에 대한 생체 조직의 방어반응의 하나로서, 조직 변질, 순환 장애와 삼출, 조직 증식을 병발할 수 있는 복잡한 병변을 말한다. In the present invention, "anti inflammation" refers to inhibiting inflammation, and the inflammation is one of the biological tissue's defense responses to certain stimuli, and is a complex complex that can cause tissue deterioration, circulatory disturbance and exudation, and tissue proliferation. say lesion.
염증은 선천성 면역의 일부이며 다른 동물에서처럼 인간의 선천성 면역은 병원체에 특이적으로 존재하는 세포 표면의 패턴을 인식한다. 식세포는 그런 표면을 가진 세포를 비자기로 인식하고 병원체를 공격한다. 병원균이 신체의 물리적 장벽을 깨고 들어온다면 염증 반응이 일어날 수 있다. 염증반응은 면역학적 방어기전의 일부이지만, 과도하거나 지속적으로 발생되면 급성 또는 만성 염증질환이 유발된다. Inflammation is part of innate immunity, and as in other animals, human innate immunity recognizes patterns on the surface of cells that are specific to pathogens. Phagocytes recognize cells with such a surface as non-self and attack pathogens. When pathogens break through the body's physical barriers, an inflammatory response can occur. The inflammatory response is a part of the immune defense mechanism, but if it occurs excessively or continuously, acute or chronic inflammatory diseases are induced.
염증 발생은 호르몬 분비, 사이토카인, C-반응성 단백질(C-reactive protein, CRP) 등 체내 여러 기전에서 이루어지며, 관련된 물질도 매우 많다. 이 중 면역세포에서 분비되는 다양한 사이토카인은 면역계를 조절하며, 일부는 염증을 촉진 시키기도 한다. 따라서, 세포 내 사이토카인의 발현량이 염증 반응 활성화의 지표가 될 수 있다.Inflammation occurs in various mechanisms in the body, such as hormone secretion, cytokines, and C-reactive protein (CRP), and there are many related substances. Among them, various cytokines secreted from immune cells regulate the immune system, and some promote inflammation. Therefore, the expression level of intracellular cytokines can be an indicator of inflammatory response activation.
본 발명에서 "염증질환"은 염증반응에 의해 발생하는 질환을 의미한다,In the present invention, "inflammatory disease" means a disease caused by an inflammatory reaction,
상기 염증질환은 예를 들어 Tnfα, Il-1β, Il-6, Cox2, iNos, CCl2, Cd11b, F4/80, Ifnγ 로 이루어지는 군에서 선택되는 1 이상의 염증성 마커 발현 증가; 및/또는 Il-10 및 Il-1rn으로 이루어지는 항염 마커의 발현 감소에 의해 발생하는 것일 수 있다. 그러나 이에 제한되지 않는다.The inflammatory disease is, for example, increased expression of one or more inflammatory markers selected from the group consisting of Tnfα, Il-1β, Il-6, Cox2, iNos, CCl2, Cd11b, F4/80, Ifnγ; And/or it may be caused by a decrease in the expression of an anti-inflammatory marker consisting of Il-10 and Il-1rn. However, it is not limited thereto.
본 발명의 염증질환은 관절염, 대사성 질환, 간염, 장염, 위염, 위궤양, 식도염, 피부염, 뇌염, 우울증, 불안장애, 인지장애, 기억장애, 퇴행성 뇌질환 및 발달장애로 이루어진 군에서 선택되는 질환일 수 있다. 그러나 이에 제한되지 않는다The inflammatory disease of the present invention is a disease selected from the group consisting of arthritis, metabolic disease, hepatitis, enteritis, gastritis, gastric ulcer, esophagitis, dermatitis, encephalitis, depression, anxiety disorder, cognitive impairment, memory disorder, degenerative brain disease and developmental disorder. can but not limited to this
본 발명의 염증질환은 급성 또는 만성 질환을 모두 포함한다.Inflammatory diseases of the present invention include both acute and chronic diseases.
본 발명의 "관절염"은 관절에 발생하는 염증 질환을 의미한다. 상기 관절염은 골관절염, 류마티스성 관절염, 만성 류마티스성 관절염, 퇴행성 관절염, 타박성 관절염, 통풍성 관절염, 위축성 관절염, 만성 염증성 관절염, 변형성 관절염, 감염성 관절염, 폐경기 관절염, 단절성 관절염, 비대증 관절염, 화농성 관절염을 포함한다. "Arthritis" of the present invention means an inflammatory disease occurring in the joints. The arthritis includes osteoarthritis, rheumatoid arthritis, chronic rheumatoid arthritis, degenerative arthritis, bruised arthritis, gouty arthritis, atrophic arthritis, chronic inflammatory arthritis, deformable arthritis, infectious arthritis, menopausal arthritis, monoarthritis, hypertrophic arthritis, suppurative arthritis do.
본 발명의 "대사성 질환" 은 생체 내 물질대사 장애에 의해서 발생하는 질환을 질환을 총칭하는 것이다. 상기 대사성 질환은 장내 방어막(intestinal barrier)의 기능 변화, 혈중 LPS의 농도 변화, 장내 염증 발생, 장내 점액질 변화, 인슐린 저항성 증가, 인슐린 민감도 감소, 공복혈당 증가, 인슐린 내성 증가, 글루코스 내성 증가, 혈중 크레아티닌, 요소 질소(BUN), 요산, 크레아틴 키나제 농도 증가 중 1 이상의 현상을 나타내는 것이거나, 상기 현상으로 인한 것 내지는 상기 현상을 전조 증상으로 하는 질환일 수 있으나, 이에 제한되지 않는다.The "metabolic disease" of the present invention is a generic term for diseases caused by metabolic disorders in a living body. The metabolic disease is characterized by a change in the function of the intestinal barrier, a change in the concentration of LPS in the blood, an occurrence of intestinal inflammation, a change in intestinal mucus, an increase in insulin resistance, a decrease in insulin sensitivity, an increase in fasting blood sugar, an increase in insulin resistance, an increase in glucose tolerance, and creatinine in the blood. , urea nitrogen (BUN), uric acid, and may indicate one or more of an increase in the concentration of creatine kinase, or may be a disease caused by the above-mentioned phenomenon or a disease having the above-mentioned phenomenon as a prognostic symptom, but is not limited thereto.
상기 대사성 질환은 인슐린 저항성 질환, 비만, 당뇨병, 지방간, 비알콜성 지방간, 고지혈증, 신장 손상, 동맥경화증, 이상지질혈증 및 고혈압으로 이루어진 군으로부터 선택된 것일 수 있고, 예를 들어 비만, 당뇨, 인슐린 저항성 및 이상지질혈증 중에서 선택될 수 있으나, 이에 제한되지 않는다.The metabolic disease may be selected from the group consisting of insulin resistance disease, obesity, diabetes, fatty liver, nonalcoholic fatty liver, hyperlipidemia, kidney damage, arteriosclerosis, dyslipidemia and hypertension, for example, obesity, diabetes, insulin resistance and dyslipidemia, but is not limited thereto.
본 발명의 "간염"은 간에서 발생하는 염증 질환을 의미한다. 상기 간염은 급성 또는 만성 간염, 간경변, 지방간, 간부전, 간암, 알코올성 지방간, 비알코올 지방간질환(non-alcoholic fatty liver disease, NAFLD), 비알코올성 지방간염(non-alcoholic steatohepatitis, NASH), 간섬유화 (Liver fibrosis) 및 간경화 (Liver cirrhosis)를 포함한다. 간세포 및 간 조직 염증이 지속되면 장기간에 걸쳐 간세포가 파괴되고 재생하는 과정이 반복되어 간에 섬유조직과 재생 결절이 증가하고, 간경변 또는 간경화가 진행될 수 있다. 예를 들어 상기 간염은 급성 간염 또는 비알코올성 간염일 수 있으나 이에 제한되는 것은 아니다. 상기 비알코올성 간염은 알코올 이외의 원인으로 발병하는 것은 제한없이 포함하며, 예를 들어 비만, 당뇨, 이상지질혈증, 대사증후군 등의 대사질환을 원인으로 하거나, 상기 대사질환의 원인인 고지방식이에 의해 발생하는 것일 수 있으나 이에 제한되지는 않는다."Hepatitis" in the present invention means an inflammatory disease occurring in the liver. The hepatitis is acute or chronic hepatitis, cirrhosis, fatty liver, liver failure, liver cancer, alcoholic fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis ( Liver fibrosis) and Liver cirrhosis. If hepatocytes and liver tissue inflammation continues, the process of destruction and regeneration of hepatocytes is repeated over a long period of time, leading to an increase in fibrous tissue and regenerating nodules in the liver, and cirrhosis or cirrhosis of the liver. For example, the hepatitis may be acute hepatitis or non-alcoholic hepatitis, but is not limited thereto. The non-alcoholic hepatitis includes, without limitation, those that occur due to causes other than alcohol, for example, metabolic diseases such as obesity, diabetes, dyslipidemia, metabolic syndrome, or a high-fat diet that is the cause of the metabolic diseases. It may be caused by, but not limited to.
본 발명의 "장염"은 장에서 발생하는 염증 질환을 의미한다. 상기 "장염"은 "염증성 장질환"과 동일한 의미로 사용되며, 궤양성 대장염(ulcerative colitis), 크론병(Chrohn's disease), 장형 베체트병(intestinal Bechet's disease), 출혈성 직장 궤양, 및 회장낭염을 포함한다.In the present invention, "enteritis" refers to an inflammatory disease occurring in the intestine. The "enteritis" is used in the same sense as "inflammatory bowel disease", and includes ulcerative colitis, Crohn's disease, intestinal Bechet's disease, hemorrhagic rectal ulcer, and ileal capsuleitis. do.
본 발명의 "위염"은 위에 발생하는 염증 질환을 의미한다. 상기 위염은 위경련, 위염, 위궤양, 십이지장염, 십이지장궤양과 같은 위질환을 포함하는 의미로 사용된다."Gastritis" in the present invention means an inflammatory disease occurring in the stomach. The gastritis is used in the sense of including stomach diseases such as stomach cramps, gastritis, gastric ulcer, duodenitis, duodenal ulcer.
본 발명의 "식도염" 은 식도에 발생하는 염증 질환을 의미한다. 상기 식도염은 세균 감염이나, 위의 내용물 또는 위산 역류, 약물 등 다양한 원인에 의해 발생하는 것을 모두 포함하며, 일 예로 역류성 식도염 일 수 있으나 이에 제한되지는 않는다."Esophagitis" of the present invention means an inflammatory disease occurring in the esophagus. The esophagitis includes all those caused by various causes such as bacterial infection, gastric contents or acid reflux, drugs, and may be, for example, gastroesophageal reflux disease, but is not limited thereto.
본 발명의 "피부염"은 피부에 발생하는 염증 질환을 의미한다. 상기 피부염은 알러지성 접촉 피부염, 두드러기, 무피지성 피부염 (하퇴상의 건조한 피부), 아토피 피부염, 접촉 피부염, 이를 테면 자극 접촉 피부염 및 옻-유도 접촉 피부염, 습진, 인력(gravitational) 피부염, 화폐상 피부염, 외이도염, 구강 주위 피부염 및 지루성 피부염을 포함한다."Dermatitis" of the present invention means an inflammatory disease occurring on the skin. The dermatitis is allergic contact dermatitis, urticaria, alopecia dermatitis (dry skin on the lower leg), atopic dermatitis, contact dermatitis such as irritant contact dermatitis and poison ivy-induced contact dermatitis, eczema, gravitational dermatitis, monetary dermatitis , otitis externa, perioral dermatitis and seborrheic dermatitis.
본 발명의 "뇌염" 은 뇌에서 발생하는 염증 질환을 의미한다. 뇌에서 염증세포가 과하게 활성화 되면 염증성 싸이토카인의 분비가 많아지고 이러한 뇌염증 반응의 과활성화에 의해 뇌세포 손상이 유발될 수 있다. 따라서 상기 뇌염은, 인지장애, 기억장애, 퇴행성 뇌질환과 밀접한 관련을 가진다. 상기 뇌염은 신경 염증(neuroinflammation)을 포함한다."Encephalitis" in the present invention means an inflammatory disease occurring in the brain. When inflammatory cells are excessively activated in the brain, the secretion of inflammatory cytokines increases, and brain cell damage can be induced by this overactivation of the brain inflammatory response. Accordingly, the encephalitis is closely related to cognitive impairment, memory impairment, and degenerative brain disease. The encephalitis includes neuroinflammation.
본 발명의 "우울증"은 의욕 저하와 우울감을 주요 증상으로 하여 다양한 인지 및 정신 신체적 증상을 일으켜 일상 기능의 저하를 가져오는 질환을 의미한다. "Depression" in the present invention refers to a disease that causes a decrease in daily functioning by causing various cognitive and psychosomatic symptoms with a decrease in motivation and depression as the main symptoms.
본 발명의 "불안 장애"는 정신 질환의 일종으로서 광범위하게 매우 불쾌한, 그리고 막연히 불안한 느낌으로, 관련된 신체 증상 (가슴 두근거림, 진땀 등)과 행동 증상 (과민성, 서성댐 등)을 동반한다. 불안할 때 뇌 전체는 각성(arousal) 상태에 들어가며, 따라서 불안 장애로 인해 말초 행동, 자율신경계, 감각, 지각 등에 지장이 생긴다. 또한 불안 장애는 실제적인 위험에 기인하지 않고 공격(공황 장애)이나 지속적인 상태(일반화된 불안 장애)로서 나타나는 심리적이고 육체적인 불안 징후들의 다양한 조합과 관련될 수 있다. The "anxiety disorder" of the present invention is a kind of mental disorder, and it is a widespread, very unpleasant, and vaguely anxious feeling, accompanied by related physical symptoms (chest palpitation, sweating, etc.) and behavioral symptoms (irritability, pacing, etc.). When anxious, the entire brain enters an arousal state, and therefore, an anxiety disorder causes disturbances in peripheral behavior, autonomic nervous system, sensation, and perception. Anxiety disorders can also be associated with various combinations of psychological and physical signs of anxiety that are not attributable to real danger but appear as an aggression (panic disorder) or a persistent state (generalized anxiety disorder).
본 발명의 "인지장애"는 뇌 손상에 의해 기억능력, 시공간 파악능력, 판단력, 언어능력 또는 계산능력 등의 인지기능(인지능력)이 저하되어 유발된 질환을 의미한다. 본 발명의 "기억장애"는 사물을 기억하거나 과거의 경험을 생각해내는 일이 어렵거나 아주 불가능한 병적인 상태로서, 기억력 저하에 의해 발생하는 질환, 예를 들어 건망증, 순간기억상실, 단기기억상실, 장기기억상실, 일과성 기억장애를 포함한다. 그러나 이에 제한되지 않는다. 일 예로, 본 발명의 펩타이드는 기억력 및/또는 인지능력의 개선에도 사용될 수 있으나 이에 제한되지 않는다. "Cognitive impairment" of the present invention refers to a disease caused by a decrease in cognitive function (cognitive ability) such as memory ability, temporal and spatial grasping ability, judgment, language ability or calculation ability due to brain damage. The "memory disorder" of the present invention is a pathological condition in which it is difficult or impossible to remember things or recall past experiences, and diseases caused by memory loss, such as forgetfulness, instantaneous memory loss, short-term memory loss, Includes long-term memory loss and transient amnesia. However, it is not limited thereto. For example, the peptide of the present invention may be used to improve memory and/or cognitive ability, but is not limited thereto.
본 발명의 "퇴행성 뇌질환(degenerative brain disease)" 은 퇴행성 질환 중 뇌에서 발생하는 질환을 의미한다. 일 예로, 상기 퇴행성 뇌질환은 알츠하이머, 파킨슨병, 루게릭병, 경도 인지장애, 뇌졸중, 헌팅턴 병을 포함한다. 그러나 이에 제한되지 않는다.As used herein, "degenerative brain disease" refers to a disease occurring in the brain among degenerative diseases. For example, the degenerative brain disease includes Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, mild cognitive impairment, stroke, and Huntington's disease. However, it is not limited thereto.
본 발명의 "발달장애 (Developmental Disability)"는 정신, 신체적인 발달에서 나이만큼 발달하지 않은 상태로서 발달 선별 검사에서 해당 연령의 정상 기대치보다 25% 정도 뒤쳐져 있는 경우를 의미한다. 발달장애의 공통적인 증상은 언어를 이해하고 사용하는데 어려움을 겪으며, 사회적 상황의 전반적인 이해와 인간 관계 형성에서 어려움을 보이며 특정 사물에 특이하게 집착하고, 일정한 행동 절차를 반복하는 패턴을 보인다. 본 발명에서의 발달장애는 지적 장애, 뇌성마비, 전반적 발달장애, 발달성 언어장애, 시각 또는 청각을 포함하는 특수 감각의 기능장애, 학습장애, 주의력결핍 과잉행동장애, 뇌전증 및 이들의 조합으로 구성된 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 전반적 발달장애는 자폐범주성장애, 아스퍼거 증후군, 아동기 붕괴성 장애(Childhood Disintegrative Disorder), 레트 증후군(Rett Syndrome), 비정형 자폐범주성장애 및 이들의 조합으로 구성된 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다. 이 밖에도 발달장애는 지각, 인지, 운동, 언어 등의 발달영역에서 발생하는 모든 장애를 포함할 수 있으며, 학습장애, 의사소통장애, 운동기능장애, 뇌성마비(Cerebral Palsy), 유전장애, 염색체장애(Down Syndrome; 다운 증후군, Fragile X Syndrome) 등 발달이나 기능에서 지체가 있는 모든 발달장애를 포함할 수 있다. 그러나 이에 제한되지 않는다. "Developmental disability" of the present invention refers to a state in which mental and physical development is not developed as much as the age, and is 25% behind the normal expectation of the age in the developmental screening test. Common symptoms of developmental disorders are difficulties in understanding and using language, difficulties in overall understanding of social situations and formation of human relationships, and a pattern of clinging to specific objects and repeating certain behavioral procedures. Developmental disorders in the present invention include intellectual disabilities, cerebral palsy, pervasive developmental disorders, developmental speech disorders, functional disorders of special senses including sight or hearing, learning disorders, attention deficit hyperactivity disorder, epilepsy, and combinations thereof. It may be selected from the group consisting of, but is not limited thereto. In addition, the pervasive developmental disorder may be selected from the group consisting of autism spectrum disorder, Asperger's syndrome, Childhood Disintegrative Disorder, Rett Syndrome, atypical autism spectrum disorder, and combinations thereof, However, the present invention is not limited thereto. In addition, developmental disorders can include all disorders occurring in developmental areas such as perception, cognition, movement, and language, and include learning disabilities, communication disorders, motor dysfunction, cerebral palsy, genetic disorders, and chromosome disorders. (Down Syndrome; Fragile X Syndrome) may include all developmental disorders with delay in development or function. However, it is not limited thereto.
본 발명의 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는, 염증질환 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, including the peptide provided by the present invention.
본 발명의 펩타이드, 염증질환에 대해서는 전술한 바와 같다.The peptides of the present invention and inflammatory diseases are the same as described above.
본 발명에서 "예방" 이란 본 발명에 따른 펩타이드 투여에 의해 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미한다. 본 발명에서 "치료"는 본 발명에 따른 펩타이드 투여에 의해 질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, "prevention" means any action that inhibits or delays the onset of a disease by administering the peptide according to the present invention. In the present invention, "treatment" refers to any action in which the symptoms of a suspected disease and onset individual are improved or beneficially changed by administering the peptide according to the present invention.
본 발명의 약학 조성물은, 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 구체적으로, 상기 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명에서, 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물과 이의 분획물들에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. Specifically, the pharmaceutical composition is each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods to be used. can In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used for suspensions, solutions, emulsions, and syrups. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 염증질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a health functional food composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
본 발명의 식품 조성물은 일상적으로 섭취하는 것이 가능하기 때문에, 질환의 예방 또는 개선 효과를 기대할 수 있어 매우 유용하다.Since the food composition of the present invention can be ingested on a daily basis, it is very useful because it can be expected to prevent or improve the effect of diseases.
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함하며, 본 발명의 조성물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있다.The food composition of the present invention includes the form of pills, powder, granules, needles, tablets, capsules or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example, There are beverages, gum, tea, vitamin complexes, and health supplements.
본 발명의 용어, "개선"이란, 본 발명의 펩타이드 투여로 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated by administration of the peptide of the present invention, for example, the severity of symptoms.
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함하며, 본 발명의 조성물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있다.The food composition of the present invention includes the form of pills, powder, granules, needles, tablets, capsules or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example, There are beverages, gum, tea, vitamin complexes, and health supplements.
본 발명의 식품 조성물에서 포함할 수 있는 필수 성분으로 본 발명에서 제공하는 펩타이드 외에 다른 성분에는 특별히 제한이 없으며 통상의 식품과 같이 여러가지 생약추출물, 식품 보조 첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 또한, 상기 식품 보조 첨가제는 당업계에 통상적인 식품 보조 첨가제, 예를 들어 향미제, 풍미제, 착색제, 충진제, 안정화제 등을 포함한다.As an essential ingredient that can be included in the food composition of the present invention, there is no particular limitation on other ingredients other than the peptide provided in the present invention. there is. In addition, the food supplement additives include food supplement additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.
상기 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외에 향미제로서 천연 향미제(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제(치즈, 초콜릿 등), 펙트산 및 이의 염, 알긴산 및 이의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination.
본 발명에서 상기 건강보조식품은 건강기능식품 및 건강식품 등을 포함한다.In the present invention, the health supplement includes health functional food and health food.
상기 건강 기능(성) 식품(functional food)이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 "기능(성)"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 식품의 제형 또한 식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 식품은 인지기능 장애, 신경 염증의 예방 또는 개선의 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of the drug using food as a raw material, and has excellent portability, Food can be ingested as an adjuvant for enhancing the effect of preventing or improving cognitive dysfunction and neuroinflammation.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 염증질환 예방 또는 개선용 사료 조성물을 제공한다.Another aspect of the present invention provides a feed composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
상기 사료용 조성물은 사료 첨가제를 포함할 수 있다. 본 발명의 사료첨가제는 사료관리법상의 보조사료에 해당한다.The composition for feed may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.
본 발명에서 용어, "사료"는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미한다.As used herein, the term “feed” refers to any natural or artificial diet, one-meal, etc., or a component of the one-meal, which is suitable for or suitable for the animal to eat, ingest, and digest.
상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박 류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in mixture of two or more.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 개체에 투여하는 단계를 포함하는 염증질환의 예방 또는 치료방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating an inflammatory disease comprising administering to an individual the peptide provided in the present invention.
본 발명의 "개체"는 질환이 발병될 가능성이 있거나 또는 발병된 쥐, 가축, 인간 등의 모든 동물을 의미한다. 또한 본 발명의 개체에서 인간은 제외될 수 있으나 이에 제한되지 않는다.The "individual" of the present invention means any animal, such as a rat, livestock, or human, that is likely to or has developed a disease. In addition, humans may be excluded from the subject of the present invention, but is not limited thereto.
본 발명의 질환을 치료하는 방법에 있어서 상기 펩타이드의 투여는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 일 예로 상기 펩타이드는 약학 조성물의 형태로 투여될 수 있다. In the method of treating a disease of the present invention, the peptide may be administered through any general route as long as it can reach the target tissue. For example, the peptide may be administered in the form of a pharmaceutical composition.
본 발명의 치료방법에 있어서 본 발명에서 제공하는 펩타이드를 단독으로 투여하는 방법뿐만 아니라 병용 요법 또한 고려될 수 있다. 상기 병용 요법에 사용되는 다른 제제는 염증질환의 예방 또는 치료 효과가 있는 것으로 알려져 있는 것이면 제한없이 포함 될 수 있으며, 염증질환의 예방 또는 치료에서 원하는 결과를 생산하는 데에 효과적인 양으로 제공될 수 있다. 일 예로 본 발명의 치료방법은 본 발명에서 제공하는 펩타이드를 포함하는 제1 치료제제와, 선택적으로, 제2 치료제제 또는 인자의 치료적 유효량을 투여함으로써 달성될 수 있다. 이는 양쪽 치료제제를 모두 포함하는 단일 조성물 또는 약제학적 제형을 투여하거나, 하나의 조성물이 본 발명에서 제공하는 펩타이드를 포함하고 다른 것이 제2 치료제제를 포함하는 2개의 별개 조성물 또는 제형을 동시 혹은 순차적으로 투여함으로써 달성될 수 있으나, 이에 제한되지는 않는다. In the treatment method of the present invention, not only a method of administering the peptide provided by the present invention alone, but also a combination therapy may be considered. Other agents used in the combination therapy may be included without limitation as long as they are known to be effective in preventing or treating inflammatory diseases, and may be provided in an amount effective to produce a desired result in the prevention or treatment of inflammatory diseases. . For example, the treatment method of the present invention can be achieved by administering a therapeutically effective amount of the first therapeutic agent comprising the peptide provided in the present invention and, optionally, the second therapeutic agent or factor. This includes administering a single composition or pharmaceutical formulation containing both therapeutic agents, or simultaneously or sequentially administering two separate compositions or formulations, wherein one composition contains the peptide provided in the present invention and the other contains a second therapeutic agent. It can be achieved by administering to, but is not limited thereto.
본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여 등의 경로를 통해 투여 될 수 있다. 다만, 경구 투여 시에는 위산에 의해 유효성분이 변성될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화될 수 있다. 일 예로 상기 약학 조성물은 위에서부터의 분해로 보호되도록 장용 코팅(enteric coating)된 것일 수 있으나 이에 제한되지 않는다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. The pharmaceutical composition of the present invention is not particularly limited thereto, but routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered through However, since the active ingredient may be denatured by gastric acid during oral administration, the oral composition may be coated with the active agent or formulated to be protected from degradation in the stomach. For example, the pharmaceutical composition may be an enteric coating to be protected from decomposition from the stomach, but is not limited thereto. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.
본 발명의 구체예에서는 본 발명의 펩타이드 투여에 의해 염증성 사이토카인 발현 억제, 항염 사이토카인 발현의 증가 및 염증에 조직 손상이 억제되는 것을 확인하였는 바, 본 발명의 펩타이드를 염증질환 예방 또는 치료에 유용하게 사용할 수 있다.In an embodiment of the present invention, it was confirmed that inhibition of inflammatory cytokine expression, increase of anti-inflammatory cytokine expression, and tissue damage due to inflammation by administration of the peptide of the present invention was confirmed, the peptide of the present invention is useful for preventing or treating inflammatory diseases can be used
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 염증질환 예방 또는 치료용 동물 의약품 조성물을 제공한다.Another aspect of the present invention provides an animal pharmaceutical composition for preventing or treating inflammatory diseases comprising the peptide provided in the present invention.
상기 펩타이드, 염증질환에 대해서는 전술한 바와 같다.The peptides and inflammatory diseases are the same as described above.
상기 동물은 질환이 발병될 가능성이 있거나 또는 발병된 쥐, 가축, 인간 등의 모든 동물을 의미하며, 일 구현예로 인간을 제외한 동물 일 수 있으나, 이에 제한되지는 않는다.The animal means all animals, such as mice, livestock, and humans, that are likely to develop or have a disease, and may be animals other than humans in one embodiment, but is not limited thereto.
본 발명의 조성물을 동물의약품 조성물로 사용할 경우, 상기 조성물을 그대로 사용하거나 다른 의약품 또는 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있으며 이에 제한되는 것은 아니다. 유효성분의 혼합양은 사용 목적(예방, 건강, 개선 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the composition of the present invention is used as an veterinary pharmaceutical composition, the composition may be used as it is or may be used together with other pharmaceuticals or quasi-drug ingredients, and may be appropriately used according to a conventional method, but is not limited thereto. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, improvement or therapeutic treatment).
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 염증질환 예방 또는 개선용 의약외품을 제공한다.Another aspect of the present invention provides a quasi-drug for preventing or improving inflammatory diseases comprising the peptide provided by the present invention.
본 발명에서 "의약외품"은 사람이나 동물의 질병을 진단, 치료, 개선, 경감, 처치 또는 예방할 목적으로 사용되는 물품들 중 의약품보다 작용이 경미한 물품들을 의미한다. 예를 들어 약사법에 따르면 의약외품이란 의약품의 용도로 사용되는 물품을 제외한 것으로, 사람ㆍ동물의 질병 치료나 예방에 쓰이는 제품, 인체에 대한 작용이 경미하거나 직접 작용하지 않는 제품 등이 포함된다.In the present invention, "quasi-drug" refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs exclude products used for medicinal purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body.
본 발명의 상기 의약외품 조성물은 바디 클렌저, 폼, 비누, 마스크, 연고제, 크림, 로션, 에센스 및 스프레이로 이루어진 군에서 선택되는 하나 이상으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The quasi-drug composition of the present invention may be prepared from one or more selected from the group consisting of body cleanser, foam, soap, mask, ointment, cream, lotion, essence and spray, but is not limited thereto.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 염증질환 예방 또는 개선용 화장료 조성물을 제공한다.Another aspect of the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising the peptide provided in the present invention.
상기 화장료 조성물은 통상의 화장료 제조방법에 따라, 다양한 형태로 제조될 수 있다. 예를 들어, 상기 화장료 조성물은 본 발명의 펩타이드를 함유하는 향장 제품, 화장수, 크림, 로션 등의 형태로 제조될 수 있으며, 이는 통상의 클렌징액, 수렴액 및 보습액으로 희석하여 사용될 수 있다. The cosmetic composition may be prepared in various forms according to a conventional cosmetic preparation method. For example, the cosmetic composition may be prepared in the form of a cosmetic product, lotion, cream, lotion, etc. containing the peptide of the present invention, which may be used by diluting it with a conventional cleansing solution, astringent solution and moisturizing solution.
또한, 상기 화장료 조성물은 화장료 조성물 분야에서 통상적으로 사용되는 안정화제, 용해화제, 비타민, 안료, 및 향료와 같은 통상적인 보조제를 포함할 수 있다. 상기 화장료 조성물에 있어서, 본 발명의 펩타이드 함량은 염증질환의 예방 또는 개선이라는 효과를 달성하기에 유효한 양으로 포함될 수 있다. 예를 들면 조성물 총 중량에 대하여 0.001 ∼ 10 중량%의 함량으로 함유될 수 있고, 구체적으로는 약 0.01 ∼ 1 중량%의 함량으로 함유될 수 있으나 이에 제한되지 않는다.In addition, the cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition, the peptide content of the present invention may be included in an effective amount to achieve the effect of preventing or improving inflammatory diseases. For example, it may be contained in an amount of 0.001 to 10% by weight based on the total weight of the composition, specifically, it may be contained in an amount of about 0.01 to 1% by weight, but is not limited thereto.
상기 화장료 조성물의 제형은 용액, 외용 연고, 크림, 폼, 영양 화장수, 유연 화장수, 향수, 팩, 유연수, 유액, 메이크업 베이스, 에센스, 비누, 액체 세정료, 입욕제, 선 스크린 크림, 선 오일, 현탁액, 유탁액, 페이스트, 겔, 로션, 파우더, 비누, 계면 활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패치 또는 스프레이일 수 있으나, 이에 제한되는 것은 아니다.The formulation of the cosmetic composition is a solution, an external ointment, a cream, a foam, a nutritional lotion, a flexible lotion, a perfume, a pack, a soft water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath agent, a sunscreen cream, a sun oil, a suspension , emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch or spray.
상기 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한 담체를 1 종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면 활성제, 보습제, 저급 알코올, 증점제, 킬레이트제, 무기염류, 색소, 산화방지제, 살균제, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다The cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent , inorganic salts, colorants, antioxidants, disinfectants, preservatives, fragrances, etc. may be appropriately mixed, but the present invention is not limited thereto.
본 발명에서 제공하는 펩타이드는 재생(regeneration) 효과를 갖는 것을 특징으로 한다. The peptide provided in the present invention is characterized in that it has a regeneration effect.
따라서, 본 발명의 하나의 양태는 전술한 본 발명의 펩타이드의 조직 재생 용도를 제공한다. Accordingly, one aspect of the present invention provides the use of the above-described peptide of the present invention for tissue regeneration.
일 구현예로, 상기 조직은 피부, 근육, 모발, 모낭 및 모근 중에서 선택되는 것일 수 있다. In one embodiment, the tissue may be selected from skin, muscle, hair, hair follicles and hair roots.
일 구현예로, 상기 조직 재생은 피부 재생, 근육조직 재생, 상처 치유, 근력 증진, 탈모 방지, 양모 촉진, 발모 촉진, 손상 모발 개선 및 모발 재생 중에서 선택되는 것일 수 있다. In one embodiment, the tissue regeneration may be selected from skin regeneration, muscle tissue regeneration, wound healing, muscle strength enhancement, hair loss prevention, wool promotion, hair growth promotion, damaged hair improvement, and hair regeneration.
본 발명에서 "재생" 이란 세포, 조직, 기관의 기능 저하를 억제하거나 저하된 기능을 회복하는 것을 의미한다. 세포, 조직, 기관의 기능 저하는 세포, 조직, 기관의 손상으로 인한 것일 수 있다. 이러한 손상은 방사선치료, 약물치료, 수술, 감염, 염증, 퇴행성 질환, 자가면역질환, 노화 등 다양한 요인에 의해 발생할 수 있다. 한편 상기 "재생"은 세포 분화 촉진에 의해 달성될 수 있으나 이에 제한되지 않는다.In the present invention, "regeneration" means suppressing the deterioration of the function of cells, tissues, or organs or restoring the reduced function. Decreased function of cells, tissues, or organs may be due to damage to cells, tissues, or organs. Such damage may be caused by various factors such as radiation therapy, drug treatment, surgery, infection, inflammation, degenerative diseases, autoimmune diseases, and aging. Meanwhile, the "regeneration" may be achieved by promoting cell differentiation, but is not limited thereto.
일 구현예로 본 발명에서 제공하는 펩타이드는 근육 조직 재생능을 갖는 것일 수 있다. In one embodiment, the peptide provided by the present invention may have the ability to regenerate muscle tissue.
상기 근육 조직의 재생은 근원세포 분화에 의해 달성될 수 있으나 이에 제한되지 않는다.The regeneration of the muscle tissue may be achieved by myoblast differentiation, but is not limited thereto.
본 발명에서 "근원세포"는 분화되지 않은 상태에 있는 근육세포로, 근원세포가 골격근세포로 분화되면 근육조직이 형성되므로 근원세포의 분화는 근발생(myogenesis)이라고도 칭한다. 이러한 근원세포의 분화에 관여하는 인자는 Mef2, SRF(Serum response factor), MyoD, Myf5, Myf6, 마이오제닌(myogenin) 및 미오신헤비체인(myosin heavy chain) 등이 있으며, 이들 인자의 발현수준을 측정하여 근원세포 분화 여부를 판단할 수 있다. In the present invention, "myocytes" are muscle cells in an undifferentiated state, and when myocytes are differentiated into skeletal muscle cells, muscle tissue is formed, so differentiation of myocytes is also called myogenesis. Factors involved in the differentiation of these myoblasts include Mef2, Serum response factor (SRF), MyoD, Myf5, Myf6, myogenin and myosin heavy chain, and the expression level of these factors By measuring, it is possible to determine whether myoblasts are differentiated.
다른 예로, 상기 근육 조직의 재생은 근원세포 위축 억제에 의해 달성될 수 있다. 일 예로 근위축 관여 인자인 atrogin1 및 MuRF1의 발현 수준을 측정하여 근원세포의 위축 억제 여부를 판단할 수 있다.As another example, the regeneration of the muscle tissue may be achieved by inhibiting myoblast atrophy. For example, by measuring the expression levels of atrogin1 and MuRF1, which are factors involved in muscle atrophy, it can be determined whether or not atrophy of myoblasts is suppressed.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 포함하는 조직 재생용 조성물을 제공한다.Another aspect of the present invention provides a composition for tissue regeneration comprising the peptide provided in the present invention.
상기 조직 재생용 조성물은 조직 손상 관련 질환의 예방 또는 치료 용도로 사용될 수 있다. 따라서 본 발명의 다른 하나의 양태는 전술한 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는 조직 손상 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The composition for tissue regeneration may be used for the prevention or treatment of tissue damage-related diseases. Therefore, another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of tissue damage-related diseases comprising the peptide provided in the present invention as an active ingredient.
상기 펩타이드, 약학적 조성물에 대해서는 전술한 바와 같다.The peptide and pharmaceutical composition are the same as described above.
일 구현예로 본 발명의 조직은 근육조직일 수 있으며, 조직 손상 관련 질환은 근육 관련 질환일 수 있다.In one embodiment, the tissue of the present invention may be a muscle tissue, and the tissue damage-related disease may be a muscle-related disease.
일 예로, 상기 근육 관련 질환은 근디스트로피 (muscular dystrophy), 근위축증(muscular atropy), 근감소증 (muscular sarcopenia), 근육염 (Myositis), 다발성 근육염 (polymyositis), 말초혈관 질환 (peripheral vascular disease) 및 섬유증 (fibrosis)으로 구성된 군에서 선택되는 것일 수 있으나, 이에 제한되지 않는다.For example, the muscle-related disease is muscle dystrophy (muscular dystrophy), muscular atrophy (muscular atropy), sarcopenia (muscular sarcopenia), myositis (Myositis), polymyositis, peripheral vascular disease (peripheral vascular disease) and fibrosis ( fibrosis) may be selected from the group consisting of, but is not limited thereto.
일 구현예로 본 발명에서의 조직은 모발, 모낭, 모근에서 선택되는 것일 수 있다. In one embodiment, the tissue in the present invention may be selected from hair, hair follicles, and hair roots.
상기 조직 재생용 조성물은 탈모 방지, 양모 촉진, 발모 촉진, 손상 모발 개선, 모발 재생 등의 효과를 나타내는 것일 수 있다. 따라서, 본 발명의 조직 재생용 조성물은 탈모 예방, 치료 또는 개선용 조성물로 사용될 수 있다. 그러나 이에 제한되지 않는다.The composition for tissue regeneration may exhibit effects such as preventing hair loss, promoting hair growth, promoting hair growth, improving damaged hair, and regenerating hair. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for preventing, treating or improving hair loss. However, it is not limited thereto.
일 구현예로, 본 발명에서의 조직 재생은 상처 치유 일 수 있다. 상기 상처 치유는 세포의 손상으로 인한 상처를 치료하는 것을 의미하며, 상기 상처는 생체가 손상된 것을 모두 아우르는 의미로서, 창상이라고도 한다. 상기 상처 치유는 본 발명의 조성물을 상처가 난 개체에 투여하여, 상처가 악화되는 것을 억제 또는 지연시키거나, 상처의 증세가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미할 수 있다. 따라서, 본 발명의 조직 재생용 조성물은 상처 또는 창상의 예방 또는 치료용 조성물로 사용될 수 있다. In one embodiment, tissue regeneration in the present invention may be wound healing. The wound healing means treating a wound caused by damage to cells, and the wound is a meaning encompassing all damage to the living body, and is also referred to as a wound. The wound healing may refer to any action of administering the composition of the present invention to a wounded individual to inhibit or delay the deterioration of the wound, or to improve or benefit the symptoms of the wound. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for preventing or treating wounds or wounds.
상기 상처는 예를 들어 피부 조직에 발생한 것일 수 있으나 이에 제한되지 않는다.The wound may be, for example, a skin tissue, but is not limited thereto.
일 구현예로, 본 발명에서 제공하는 펩타이드는 피부 노화 억제 및/또는 개선 효과를 나타낼 수 있다. 피부 노화의 억제 및/또는 개선은 "피부 재생" 으로도 지칭할 수 있다. 따라서, 본 발명의 조직 재생용 조성물은 피부 재생용 조성물로 사용될 수 있다.In one embodiment, the peptides provided by the present invention may exhibit an effect of inhibiting and/or improving skin aging. Inhibition and/or improvement of skin aging may also be referred to as “skin regeneration”. Therefore, the composition for tissue regeneration of the present invention can be used as a composition for skin regeneration.
본 발명의 "피부 노화"는 피부에 탄력 감소, 윤기 감소, 주름 생성, 재생력 약화 또는 심한 건조 등의 증상이 나타나는 것으로, 시간의 흐름 또는 외부 환경 등에 의해 유발될 수 있다. 상기 피부 노화는 세월의 흐름에 따라 자연적으로 나타나는 내인성 노화 및 자외선으로 인해 피부에 생기는 광노화를 모두 포함한다. 본 발명의 피부 노화에 의해 피부 세포에서는 콜라겐(collagen), 히알루론산(hyaluronic acid), 엘라스틴(elastin), 프로테오글리칸(proteoglycan) 피브로넥틴 및/또는 그 전구체의 합성량 감소, 상기 성분의 분해효소의 발현 증가 또는 상기 성분의 합성효소의 발현 감소 등의 현상이 나타날 수 있다."Skin aging" of the present invention refers to the appearance of symptoms such as reduced elasticity, reduced gloss, wrinkles, weakened regeneration, or severe dryness in the skin, and may be caused by the passage of time or external environment. The skin aging includes both intrinsic aging that appears naturally with the passage of time and photoaging that occurs in the skin due to ultraviolet rays. In skin cells due to skin aging of the present invention, the synthesis amount of collagen, hyaluronic acid, elastin, proteoglycan, fibronectin and/or its precursors decreases, and the expression of degrading enzymes of the components increases Or a phenomenon such as a decrease in the expression of the synthetase of the component may appear.
본 발명의 "피부노화 억제"는, "피부 재생"으로도 지칭할 수 있으며, 피부 세포의 콜라겐 또는 그 전구체의 합성량 증가를 포함할 수 있다. 상기 피부 세포는 피부 각질 세포 및 피부 섬유아세포를 포함한다. "Skin aging inhibition" of the present invention may also be referred to as "skin regeneration", and may include an increase in the amount of synthesis of collagen or a precursor thereof in skin cells. The skin cells include skin keratinocytes and skin fibroblasts.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 피부노화의 억제는 콜라겐 합성효소 활성의 증가 및/또는 콜라겐 분해효소 활성의 억제를 포함할 수 있다. 일 예로, 상기 콜라겐 합성효소는 Col1a1 및/또는 Col3a1 일 수 있다. 일 예로, 상기 콜라겐 분해효소는 MMP-1 및/또는 MMP-3 일 수 있다. 그러나, 이에 제한되지 않는다.In any one of the above embodiments, the inhibition of skin aging of the present invention may include an increase in collagen synthetase activity and/or inhibition of collagenase activity. For example, the collagen synthetase may be Col1a1 and/or Col3a1. For example, the collagen-degrading enzyme may be MMP-1 and/or MMP-3. However, it is not limited thereto.
전술한 구현예 중 어느 하나의 구현예로, 본 발명의 피부노화의 억제는 피부 보습 증가, 피부 탄력 증진, 피부 두께 증가, 피부 주름 개선, 피부 자극 완화, 피부 손상 회복 및/또는 피부 색조 개선을 포함할 수 있다. 그러나, 이에 제한되지 않는다. In any one of the above-described embodiments, the inhibition of skin aging of the present invention is to increase skin moisture, increase skin elasticity, increase skin thickness, improve skin wrinkles, alleviate skin irritation, recover skin damage and/or improve skin tone may include However, it is not limited thereto.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는 조직 재생용 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a health functional food composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
상기 펩타이드, 조직 재생 및 건강기능식품에 대해서는 전술한 바와 같다.The peptide, tissue regeneration and health functional food are as described above.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는 조직 재생용 사료 조성물을 제공한다.Another aspect of the present invention provides a feed composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는 조직 재생용 동물 의약품 조성물을 제공한다. Another aspect of the present invention provides an veterinary pharmaceutical composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
상기 펩타이드, 조직 재생, 사료 및 동물 의약품에 대해서는 전술한 바와 같다.The peptide, tissue regeneration, feed, and veterinary medicine are the same as described above.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 개체에 투여하는 단계를 포함하는 조직 손상 관련 질환의 예방 또는 치료방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating tissue damage-related diseases comprising administering to an individual the peptide provided by the present invention.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 개체에 투여하는 단계를 포함하는 조직 재생 방법을 제공한다. Another aspect of the present invention provides a method for tissue regeneration comprising administering to an individual the peptide provided by the present invention.
본 발명의 치료방법에 있어서 본 발명에서 제공하는 펩타이드를 단독으로 투여하는 방법뿐만 아니라 병용 요법 또한 고려될 수 있다. 상기 병용 요법에 사용되는 다른 제제는 조직 재생 효과가 있는 것으로 알려져 있는 것이면 제한없이 포함 될 수 있으며, 조직 재생에서 원하는 결과를 생산하는 데에 효과적인 양으로 제공될 수 있다.In the treatment method of the present invention, not only a method of administering the peptide provided by the present invention alone, but also a combination therapy may be considered. Other agents used in the combination therapy may be included without limitation as long as they are known to have a tissue regeneration effect, and may be provided in an amount effective to produce a desired result in tissue regeneration.
상기 펩타이드, 개체, 투여, 조직 손상 관련 질환 및 예방 또는 치료방법에 대해서는 전술한 바와 같다.The peptide, subject, administration, tissue damage-related disease and prevention or treatment method are the same as described above.
본 발명의 구체예에서는 본 발명의 펩타이드 투여에 의해 세포 분화 촉진에 따른 조직 재생 및 분화 관련 유전자의 발현증가를 확인하였고, 이에 따라 근조직, 피부조직 및 모발, 모근 조직이 재생되는 것을 확인하였는 바, 본 발명의 펩타이드를 조직 관련 질환 예방 및 치료에 유용하게 사용할 수 있다.In an embodiment of the present invention, it was confirmed that the expression of tissue regeneration and differentiation-related genes increased according to the promotion of cell differentiation by administration of the peptide of the present invention, and accordingly, it was confirmed that muscle tissue, skin tissue, hair, and hair root tissue were regenerated, The peptide of the present invention can be usefully used for the prevention and treatment of tissue-related diseases.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는, 조직 재생용 의약외품 조성물을 제공한다. Another aspect of the present invention provides a quasi-drug composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
본 발명의 다른 하나의 양태는 본 발명에서 제공하는 펩타이드를 유효성분으로 포함하는, 조직 재생용 화장료 조성물을 제공한다.Another aspect of the present invention provides a cosmetic composition for tissue regeneration comprising the peptide provided in the present invention as an active ingredient.
상기 펩타이드, 조직 재생, 의약외품 조성물 및 화장료 조성물에 대해서는 전술한 바와 같다.The peptide, tissue regeneration, quasi-drug composition and cosmetic composition are the same as described above.
이하 본 발명을 실시예 및 실험예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예 및 실험예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예 및 실험예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.
한편 본원발명의 실시예에서, PEP001은 서열번호 1, PEP002는 서열번호 2, PEP003은 서열번호 3, PEP004는 서열번호 4, PEP005는 서열번호 5를, Amuc_1409는 서열번호 6의 leader sequence를 제외한 기능적 단편인 서열번호 7을 지칭한다.Meanwhile, in an embodiment of the present invention, PEP001 is SEQ ID NO: 1, PEP002 is SEQ ID NO: 2, PEP003 is SEQ ID NO: 3, PEP004 is SEQ ID NO: 4, PEP005 is SEQ ID NO: 5, Amuc_1409 is functional except for the leader sequence of SEQ ID NO: 6 It refers to the fragment SEQ ID NO:7.
실시예 1: 관절염 동물모델에서 신규 펩타이드의 효능 검증Example 1: Efficacy verification of novel peptides in arthritis animal models
실시예 1-1: 퇴행성 관절염 동물모델에서 신규 펩타이드의 효능 검증Example 1-1: Efficacy verification of novel peptides in degenerative arthritis animal model
실시예 1-1-1: 실험 방법Example 1-1-1: Experimental method
퇴행성 관절염 모델은 9주령의 특정 병원체 부재 (specific pathogen free, SPF) 수컷 C57BL/6J 마우스를 이용하여 마취하고 오른쪽 무릎관절 주변을 깨끗이 제모한 후, MIA 용액 (Monosodium iodoacetate 0.5 mg in 10μl 0.9% sterile saline)을 10μl씩 무릎관절 강 내에 주입하여 관절염을 유도하였다. In the degenerative arthritis model, 9-week-old specific pathogen free (SPF) male C57BL/6J mice were anesthetized, the area around the right knee joint was removed and the MIA solution (Monosodium iodoacetate 0.5 mg in 10 μl 0.9% sterile saline) was anesthetized. ) was injected into the knee joint cavity by 10 μl to induce arthritis.
MIA (Monosodium iodoacetate) 용액 주입 3일 전부터 Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005를 6.7 μM 농도로 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 복강으로 주사하였다. Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 후 3일째 (0 day)에 MIA 주입 1시간 전 추가 1회 복강 주사 하였으며, MIA 주입 후 1일 1회 동일 시간에 약물을 복강 주사하고, MIA 주입 30일째 실험을 종료하였다. 대조군에는 PBS 완충액 동일량을 복강으로 주사하였다. From 3 days before MIA (Monosodium iodoacetate) solution injection, Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 were dissolved in PBS buffer at a concentration of 6.7 μM, and 150 μl per animal was injected intraperitoneally once a day at the same time. After administration of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005, on the 3rd day (0 day), an additional intraperitoneal injection was administered 1 hour before MIA injection, and the drug was intraperitoneally injected once a day after MIA injection at the same time, The experiment was terminated on day 30 of MIA injection. In the control group, the same amount of PBS buffer was injected intraperitoneally.
MIA 주입 후 2일 간격으로 7일 동안 (0, 1, 3, 5, 7 days) 캘리퍼스를 이용하여 무릎관절의 직경을 측정하여 관절의 염증성 변화로 일어나는 부종을 평가하였다.After MIA injection, for 7 days (0, 1, 3, 5, 7 days) at intervals of 2 days, the diameter of the knee joint was measured using a caliper to evaluate edema caused by inflammatory changes in the joint.
MIA 주입 후 2주에 인카파시턴스 시험기 (incapacitance tester)를 사용하여 뒷다리 체중 부하 (weight bearing)를 측정함으로서 관절염의 유발 및 진행정도를 간접적으로 평가하였다. 이때 관절염이 심할수록 해당 다리의 체중 부하가 감소한다. 양쪽의 발의 체중 부하 무게 (g)를 각각 측정하고, 본 실험에서 그 값은 전체 뒷다리에 대한 관절염이 유발된 오른쪽 뒷다리 비율 (%)로 나타내었다.Two weeks after the MIA injection, the induction and progression of arthritis were indirectly evaluated by measuring the hind limb weight bearing using an incapacitance tester. At this time, the more severe the arthritis, the lower the weight bearing of the corresponding leg. The weight-bearing weight (g) of both feet was measured, and in this experiment, the value was expressed as the ratio (%) of the right hind limb induced with arthritis to the entire hind limb.
Figure PCTKR2021015692-appb-img-000001
Figure PCTKR2021015692-appb-img-000001
실험 종료 후 시험동물을 희생시킨 후, 마우스의 무릎 관절 조직을 10% 중성완충 포르말린으로 고정하고 0.5 M EDTA 용액으로 뼈에서 석회질을 제거하는 과정을 진행하였다. 그 다음 관절 조직을 파라핀에 포매하여 5 μm의 관절 절편을 준비하고, 사프라닌 O (Safranin O) 염색을 진행하여 조직병리학적으로 연골 조직의 손상 정도를 분석하였다. 이 때 연골 조직의 손상 정도는 절편의 사프라닌 O 염색 정도를 확인하여 평가하였고, OARSI (Osteoarthritis Research Society International grade) 가이드 라인에 따라 점수화하였다. OARSI 가이드 라인에 따른 점수 기준은 다음과 같다 (표 1).After sacrificing the test animals after the end of the experiment, the knee joint tissue of the mouse was fixed with 10% neutral buffered formalin, and the process of removing calcification from the bones with 0.5 M EDTA solution was performed. Then, the joint tissue was embedded in paraffin to prepare a 5 μm joint section, and the degree of damage to the cartilage tissue was analyzed histopathologically by staining with Safranin O. At this time, the degree of damage to the cartilage tissue was evaluated by checking the degree of safranin O staining of the section, and scoring was performed according to OARSI (Osteoarthritis Research Society International grade) guidelines. The scoring criteria according to the OARSI guidelines are as follows (Table 1).
Figure PCTKR2021015692-appb-img-000002
Figure PCTKR2021015692-appb-img-000002
실시예 1-1-2: 실험 결과Example 1-1-2: Experimental results
MIA 유도 퇴행성 관절염 모델에서 MIA 주입 후 2일 간격으로 7일 동안 캘리퍼스를 이용한 무릎 관절의 직경을 측정한 결과, MIA 주입 후 1일째에는 PBS 완충액 투여군에 비해 Amuc_1409와 PEP002, PEP003 및 PEP004를 투여한 군에서 유의하게 감소하였고 PEP001 및 PEP005 투여군은 감소하는 경향을 보였으며, MIA 주입 후 3, 5, 7일째에는 PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 무릎 관절의 두께가 유의하게 감소하였다 (p<0.05, Student's t-test) (도 1).As a result of measuring the diameter of the knee joint using calipers for 7 days at 2 day intervals after MIA injection in the MIA-induced degenerative arthritis model, the group administered with Amuc_1409, PEP002, PEP003 and PEP004 compared to the PBS buffer group on the 1st day after MIA injection. In the group administered with PEP001 and PEP005, there was a tendency to decrease, and on days 3, 5, and 7 after MIA injection, the group administered with Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 had knee joint compared to the group administered with PBS buffer. was significantly decreased (p<0.05, Student's t-test) (Fig. 1).
MIA 주입 2주와 4주후 incapacitance tester를 이용한 체중 부하 측정 결과 PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 MIA 주입에 의한 체중 부하율의 감소가 유의하게 증가하였다 (p<0.05, Student's t-test) (도 1).As a result of weight bearing measurement using an incapacitance tester 2 and 4 weeks after MIA injection, the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 significantly increased the decrease in weight bearing rate by MIA injection compared to the PBS buffer administration group ( p<0.05, Student's t-test) (Fig. 1).
또한, 마우스 무릎 관절 조직에서 사프라닌 O 염색을 통한 조직병리학적 소견을 확인한 결과 PBS 완충액 투여군에서는 사프라닌 O 염색 및 연골 두께의 뚜렷한 감소를 보였으나, 그에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 연골 부위에 사프라닌 O의 약간의 소실만 보였으며 전체 연골 형태는 유지됨을 관찰할 수 있었다. 연골파괴 정도를 OARSI score에 의해 정량적으로 수치화하였을 때 PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 유의하게 감소하였다 (p<0.05, Student's t-test) (도 2 및 도 3).In addition, as a result of confirming histopathological findings through safranin O staining in mouse knee joint tissue, the PBS buffer administration group showed a marked decrease in safranin O staining and cartilage thickness, but compared with Amuc_1409, PEP001, PEP002, PEP003, The group administered with PEP004 and PEP005 showed only a slight loss of safranin O in the cartilage area, and it was observed that the overall cartilage morphology was maintained. When the degree of cartilage destruction was quantitatively quantified by OARSI score, the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 significantly decreased compared to the group administered with PBS buffer (p<0.05, Student's t-test) (Fig. 2 and 3).
실시예 1-2: 류마티스 관절염 동물모델에서 신규 펩타이드의 효능 검증Example 1-2: Efficacy verification of novel peptides in rheumatoid arthritis animal model
실시예 1-2-1: 실험 방법Example 1-2-1: Experimental method
류마티스 관절염 (collagen induced arthritis, CIA)모델은 소의 2형 콜라겐 (CII)을 2 mg/ml이 되도록 0.1 M 아세트산 용액에 녹인 후 식염수 완충액 (phosphate buffered saline)으로 투석하여 M. 투베르쿨로시스 (tuberculosis)를 함유하는 Complete Fred's Adjuvant (CFA, Chondrex)와 동량으로 혼합하여 에멀젼화 (emulsification)한 후, 에멀젼화된 콜라겐 용액을 8주령의 수컷 DBA/1J 마우스의 꼬리 기저부에 마리당 100 μl씩 (즉 100 μg/100 μl) 피내 주사하여 1차 면역을 유도하였다 (1차 면역). 1차 면역 후 21일째에 동일한 CII를 동량의 CFA와 섞은 후 100 μl씩 (즉 100 μg/100 μl) 마우스의 꼬리 기저부에 피내 주사하여 2차 면역 반응을 유도하였다 (2차 면역). 1차 면역 후 28일째에 리포폴리사카라이드 (lipopolysaccharide, LPS)를 마리당 40 μg씩 복강 주사하여 boosting 반응을 유도하였으며, 1차 면역 후 35일째에 실험을 종료하였다.In a rheumatoid arthritis (collagen induced arthritis, CIA) model, bovine type 2 collagen (CII) was dissolved in 0.1 M acetic acid solution to 2 mg/ml and then dialyzed with phosphate buffered saline to M. tuberculosis (tuberculosis). ) containing Complete Fred's Adjuvant (CFA, Chondrex) in the same amount and emulsified, and then 100 μl of the emulsified collagen solution was added to the base of the tail of 8-week-old male DBA/1J mice (i.e., 100 μg/100 μl) intradermal injection to induce primary immunity (primary immunity). On the 21st day after the primary immunization, the same CII was mixed with an equal amount of CFA and then injected intradermally into the base of the tail of the mouse by 100 µl (ie, 100 µg/100 µl) to induce a secondary immune response (secondary immunization). On the 28th day after the primary immunization, a boosting reaction was induced by intraperitoneal injection of 40 μg of lipopolysaccharide (LPS) per animal, and the experiment was terminated on the 35th day after the primary immunization.
1차 면역 후 18일째부터 실험종료일까지 Amuc_1409는 13.4 μM 농도로 그리고 PEP001, PEP002, PEP003, PEP004 및 PEP005는 6.7 μM 농도로 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 복강으로 주사하였다. 대조군에는 PBS 완충액 동일량을 복강으로 주사하였다.From the 18th day after the first immunization to the end of the experiment, Amuc_1409 was dissolved in PBS buffer at a concentration of 13.4 μM and PEP001, PEP002, PEP003, PEP004 and PEP005 at a concentration of 6.7 μM. injected. In the control group, the same amount of PBS buffer was injected intraperitoneally.
1차 면역 후 18일째부터 실험종료일까지 2일 간격으로 관절 염증의 위중도를 평가하였다. 이때 관절염 평가는 네 다리에서 아래의 척도에 따라 매긴 점수를 합산하여 사용하였다. 관절염 평가에 따른 점수 기준은 다음과 같다 (표 2).From the 18th day after the primary immunization to the end of the experiment, the severity of joint inflammation was evaluated at 2-day intervals. At this time, the arthritis evaluation was used by adding up the scores from the four legs according to the scale below. The score criteria according to the arthritis evaluation are as follows (Table 2).
Figure PCTKR2021015692-appb-img-000003
Figure PCTKR2021015692-appb-img-000003
실험 종료일에 발목 관절의 두께를 캘리퍼스로 측정하고, 시험군별 사진을 촬영하였으며 시험동물을 치사시킨 후, 마우스의 뒷발을 10% 중성완충 포르말린으로 고정하고 0.5 M EDTA 용액으로 뼈에서 석회질을 제거하는 과정을 진행하였다. 그 다음 마우스 뒷발조직을 파라핀에 포매하여 5 μm의 관절 절편을 준비하고, 헤마톡실린 (Hematoxylin)과 에오신 (Eosin)으로 (H&E) 염색하고, 조직병리학적으로 관절 염증의 위중도를 평가하였다. 이때 관절염 평가는 아래의 척도에 따라 매긴 점수를 평균하여 사용하였다. 관절염 평가에 따른 점수 기준은 다음과 같다.At the end of the experiment, the thickness of the ankle joint was measured with a caliper, pictures for each test group were taken, and the test animals were killed. proceeded. Then, a 5 μm joint section was prepared by embedding the mouse hind paw tissue in paraffin, stained with hematoxylin and eosin (H&E), and histopathologically, the severity of joint inflammation was evaluated. At this time, the evaluation of arthritis was used by averaging the scores based on the following scale. The score criteria according to the arthritis evaluation are as follows.
Figure PCTKR2021015692-appb-img-000004
Figure PCTKR2021015692-appb-img-000004
실시예 1-2-2: 실험 결과Example 1-2-2: Experimental results
류마티스 관절염 모델에서 관절 염증의 위중도를 평가한 결과 CII 1차 주사 후 30일째에는 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 유의한 수준에 근접하게 저하되었으며, 그 이후부터 실험 종료일까지는 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 관절염 지수가 유의하게 감소하였다 (p<0.01, Student's t-test). 또한 실험 종료시점의 발목 관절의 두께를 측정한 결과 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 발목 관절의 두께가 유의하게 감소하였고, 육안사진 촬영 결과에서도 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 뒷발의 부종이 전체적으로 감소함을 확인하였다 (p<0.05, Student's t-test) (도 4).As a result of evaluating the severity of joint inflammation in the rheumatoid arthritis model, on the 30th day after the first CII injection, the group administered with Amuc_1409 compared to the group administered with the PBS buffer decreased close to a significant level. In comparison, the arthritis index significantly decreased in the group administered with Amuc_1409 (p<0.01, Student's t-test). Also, as a result of measuring the thickness of the ankle joint at the end of the experiment, the thickness of the ankle joint was significantly reduced in the group administered with Amuc_1409 compared to the group administered with PBS buffer. It was confirmed that the swelling of the hind paws was overall reduced (p<0.05, Student's t-test) (FIG. 4).
PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 관절 염증의 위중도를 평가한 결과 PBS 완충액 투여군에 비해 PEP001 투여군은 CII 1차 주사 후 30일째부터 실험 종료일까지 관절염 지수가 유의하게 감소하였고, PEP002 및 PEP003 투여군은 CII 1차 주사 후 26일째부터 실험 종료일까지 관절염 지수가 유의하게 감소하였고, PEP004투여군은 CII 1차 주사 후 32일째부터 실험 종료일까지 관절염 지수가 유의하게 감소하였고, PEP005 투여군은 CII 1차 주사 후 28일째부터 실험 종료일까지 관절염 지수가 유의하게 감소하였다. 또한 실험 종료시점의 발목 관절의 두께를 측정한 결과 PBS 완충액 투여군에 비해 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 발목 관절의 두께가 유의하게 감소하였고, 육안사진 촬영 결과에서도 PBS 완충액 투여군에 비해 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 뒷발의 부종이 전체적으로 감소함을 확인하였다 (p<0.05, Student's t-test) (도 5). As a result of evaluating the severity of joint inflammation in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005, the arthritis index significantly decreased in the PEP001-administered group from the 30th day after the first CII injection to the end of the experiment compared to the PBS buffer-administered group, and PEP002 And the PEP003 administration group had a significant decrease in the arthritis index from the 26th day after the first CII injection to the end of the experiment, the PEP004 administration group significantly decreased the arthritis index from the 32nd day after the first CII injection to the end of the experiment, and the PEP005 administration group showed a significant decrease in the CII 1 From the 28th day after the primary injection to the end of the experiment, the arthritis index decreased significantly. Also, as a result of measuring the thickness of the ankle joint at the end of the experiment, the thickness of the ankle joint was significantly reduced in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 compared to the group administered with the PBS buffer. In comparison, it was confirmed that the edema of the hind paws was overall reduced in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 (p<0.05, Student's t-test) (FIG. 5).
또한, 마우스 뒷발 조직에서 H&E 염색을 통해 조직병리학적 소견을 확인한 결과 PBS 완충액 투여군은 중등도의 염증세포 침윤, 활막의 명확한 증식 및 관절강 내 pannus 형성이 관찰된 반면 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 PBS 완충액 투여군에서 관찰된 조직병리학적인 변화들이 뚜렷이 감소하였으며, 정량적인 조직병리학적인 관절염 지수 역시 PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 유의하게 감소하였다 (p<0.05, Student's t-test) (도 6 및 도 7).In addition, as a result of confirming histopathological findings through H&E staining in mouse hindpaw tissue, moderate inflammatory cell infiltration, clear proliferation of synovial membrane, and pannus formation in the joint cavity were observed in the PBS buffer-administered group, whereas Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and In the group administered with PEP005, the histopathological changes observed in the group administered with the PBS buffer were remarkably reduced, and the quantitative histopathological arthritis index was also compared to the group administered with the PBS buffer. significantly decreased (p<0.05, Student's t-test) ( FIGS. 6 and 7 ).
이를 통해 본 발명의 펩타이드가 관절염 예방 및 치료 효과가 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention is effective in preventing and treating arthritis.
실시예 2: 대사질환 동물모델에서 신규 펩타이드의 효능 검증Example 2: Efficacy verification of novel peptides in animal models of metabolic diseases
실시예 2-1: 실험 방법Example 2-1: Experimental method
비만/당뇨 및 만성 염증이 동반되는 대사질환 동물모델은 8주령의 SPF 수컷 C57BL/6J 마우스를 이용하여 고지방 식이를 45주 또는 8주간 자유 급이함으로써 유도하였다. 45주간 고지방 식이를 급이한 마우스에 추가 3주간 고지방 식이를 급이함과 함께 Amuc_1409를, 또는 8주간 고지방 식이를 급이한 마우스에 추가 3주간 고지방 식이를 급이함과 함께 PEP001, PEP002, PEP003, PEP004 및 PEP005를 2.4 μM 농도가 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 시험 종료 시까지 마우스 경구로 투여하였다. 대조군에는 PBS 완충액을 동일한 방법으로 경구투여 하였다.Obesity/diabetes and metabolic disease animal models accompanied by chronic inflammation were induced by free feeding on a high-fat diet for 45 weeks or 8 weeks using 8-week-old SPF male C57BL/6J mice. Amuc_1409 was fed to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 45 weeks, or PEP001, PEP002, and PEP001, PEP002, to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 8 weeks. After dissolving PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 2.4 μM, 150 μl per animal was orally administered to mice once a day at the same time until the end of the test. To the control group, PBS buffer was orally administered in the same way.
모든 동물에 대하여 투여 개시시 투여 직전 일반증상을 관찰하고 군 분리 후 시험 종료시까지 체중을 측정하였다.For all animals, general symptoms were observed immediately before administration at the start of administration, and body weights were measured after group separation until the end of the test.
시험물질 투여 개시 후 2주차에 4시간 절식시킨 마우스에 인슐린 (휴마로그, 1 unit/kg)을 복강주사 후 30분, 60분, 90분 및 120분의 혈당을 꼬리 끝부분의 상처를 통해 확보한 소량의 혈액에서 ACCU-CHECK (Roche)을 이용하여 측정함으로써 인슐린 부하 실험 (insulin tolerance test, ITT)을 실시하였다.After intraperitoneal injection of insulin (Humalog, 1 unit/kg) into mice fasted for 4 hours in the 2nd week after the start of test substance administration, blood glucose at 30 minutes, 60 minutes, 90 minutes and 120 minutes is secured through the wound at the tip of the tail An insulin tolerance test (ITT) was performed by measuring a small amount of blood using ACCU-CHECK (Roche).
시험 종료시 16시간 절식시킨 마우스의 꼬리 끝부분의 상처를 통해 확보한 소량의 혈액에서 ACCU-CHECK을 이용하여 공복혈당을 측정하고, 안와정맥총에서 heparin 처리된 capillary tube를 이용하여 채혈하였다. 채취한 혈액은 즉시 10,000 rpm (4℃)에서 10분간 원심분리하여 혈장을 분리한 후 자동혈액생화학분석기를 통해 혈액 이상지질 지표인 total cholesterol (TC) 과 low density lipoprotein-cholesterol (LDL-C)을 분석하였다.At the end of the test, fasting blood glucose was measured using ACCU-CHECK in a small amount of blood obtained through a wound at the tip of the tail of a mouse fasted for 16 hours, and blood was collected from the orbital venous plexus using a heparin-treated capillary tube. The collected blood was immediately centrifuged at 10,000 rpm (4℃) for 10 minutes to separate plasma. analyzed.
실시예 2-2: 실험 결과Example 2-2: Experimental results
비만/당뇨와 만성 염증이 동반되는 대사질환 동물모델에서 Amuc_1409를 투여한 군의 초기 대비 체중이 PBS 완충액 투여군에 비해 감소하였으며, 복부지방의 경우 Amuc_1409를 투여한 군에서 유의하게 감소하여 항비만 효과를 나타내었다 (p<0.05, Student's t-test) (도 8).In an animal model of metabolic disease accompanied by obesity/diabetes and chronic inflammation, the initial body weight of the group administered with Amuc_1409 was reduced compared to the group administered with PBS buffer, and in the case of abdominal fat, the group administered with Amuc_1409 significantly decreased, indicating an anti-obesity effect. (p<0.05, Student's t-test) (Fig. 8).
인슐린 저항성 실험에서 Amuc_1409를 투여한 군의 혈당이 인슐린 투여 30분부터 120분까지 PBS 완충액 투여군에 비해 감소하였으며, Amuc_1409를 투여한 군은 120분까지 감소된 혈당이 유의하게 유지되어 고지방 식이에 의한 인슐린 저항성 개선 효능을 보였고, 인슐린 저항성 실험의 그래프를 AUC (area under the curve)로 환산한 결과 Amuc_1409를 투여한 군에서 PBS 완충액을 투여한 군에 비해 유의한 수준에 근접하게 감소된 것을 확인하였다. 또한, 시험 종료시 16시간 절식시킨 후 공복혈당을 측정하였을 때 Amuc_1409를 투여한 군에서 유의하게 감소된 것을 확인하였다 (p<0.05, Student's t-test) (도 8).In the insulin resistance experiment, the blood glucose of the group administered with Amuc_1409 decreased compared to the group administered with PBS buffer from 30 minutes to 120 minutes of insulin administration. It showed resistance improvement efficacy, and as a result of converting the graph of the insulin resistance experiment into AUC (area under the curve), it was confirmed that the group administered with Amuc_1409 was reduced close to a significant level compared to the group administered with PBS buffer. In addition, when fasting blood glucose was measured after fasting for 16 hours at the end of the test, it was confirmed that the group administered with Amuc_1409 significantly decreased (p<0.05, Student's t-test) ( FIG. 8 ).
비만/당뇨와 만성 염증이 동반되는 대사질환 동물모델에서 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군의 초기 대비 체중이 PBS 완충액 투여군에 비해 모두 감소하였으며, PEP003, PEP004 및 PEP005를 투여한 군에서 유의하게 감소하였다. 복부지방의 경우, PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 PBS 완충액을 투여한 군에 비해 모두 감소하여 항비만 효과를 나타내었다 (p<0.05, Student's t-test) (도 9).In an animal model of metabolic disease accompanied by obesity/diabetes and chronic inflammation, the initial body weight of the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 decreased compared to the group administered with PBS buffer, and the group administered with PEP003, PEP004 and PEP005 decreased significantly. In the case of abdominal fat, the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 showed an anti-obesity effect as all decreased compared to the group administered with PBS buffer (p<0.05, Student's t-test) (FIG. 9) .
인슐린 저항성 실험에서 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군의 혈당이 인슐린 투여 30분에 모두 PBS 완충액 투여군에 비해 감소하였으며, PEP003과 PEP004 투여군의 경우 120분까지 감소된 혈당이 유의하게 유지되어 고지방 식이에 의한 인슐린 저항성 개선 효능을 보였고, 인슐린 저항성 실험의 그래프를 AUC로 환산한 결과 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 모든 군에서 PBS 완충액을 투여한 군에 비해 감소된 것을 확인하였다. 또한, 시험 종료시 16시간 절식시킨 후 공복혈당을 측정하였을 때 PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 감소된 것을 확인하였다 (p<0.05, Student's t-test) (도 9).In the insulin resistance experiment, the blood glucose of the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 decreased compared to the group administered with PBS buffer at 30 minutes of insulin administration. As a result, it was confirmed that the effect of improving insulin resistance by a high-fat diet was reduced compared to the group administered with PBS buffer in all groups administered with PEP001, PEP002, PEP003, PEP004 and PEP005 as a result of converting the graph of the insulin resistance experiment into AUC. did In addition, when fasting blood glucose was measured after fasting for 16 hours at the end of the test, it was confirmed that PEP002, PEP003, PEP004 and PEP005 were decreased in the group administered (p<0.05, Student's t-test) (FIG. 9).
혈중 지질 지표인 TC 및 LDL-C 농도가 Amuc_1409 투여군과 PBS 완충액 투여군사이에 큰 차이를 보이지 않았으나, PEP001, PEP002, PEP003, PEP004 및 PEP005 투여군에서 PBS 완충액 투여군에 비해 감소하였다. TC의 경우 PEP003 투여군에서 유의하게 감소하였으며, PEP004와 PEP005를 투여한 군에서 감소하는 경향을 보였다. 또한 LDL-C의 경우 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 모든 군에서 PBS 완충액을 투여한 군보다 유의하게 감소하여 이상지질혈증 개선 효능을 보였다 (p<0.05, Student's t-test) (도 10).TC and LDL-C concentrations, which are blood lipid indicators, did not show a significant difference between the Amuc_1409 administration group and the PBS buffer administration group, but decreased in the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups compared to the PBS buffer administration group. TC significantly decreased in the PEP003-administered group, and showed a tendency to decrease in the PEP004 and PEP005-administered groups. In addition, in the case of LDL-C, all groups administered with PEP001, PEP002, PEP003, PEP004, and PEP005 showed a significantly reduced effect on dyslipidemia than the group administered with PBS buffer (p<0.05, Student's t-test) ( Fig. 10).
이를 통해, 본 발명의 펩타이드가 다양한 대사성 질환의 예방 및 치료 효과를 가지는 것을 확인하였다.Through this, it was confirmed that the peptide of the present invention has a preventive and therapeutic effect on various metabolic diseases.
실시예 3: 간염 동물모델에서 신규 펩타이드의 효능 검증Example 3: Efficacy verification of novel peptides in hepatitis animal model
실시예 3-1: 급성 간염 동물모델에서 신규 펩타이드의 효능 검증Example 3-1: Efficacy verification of novel peptides in acute hepatitis animal model
실시예 3-1-1: 실험 방법Example 3-1-1: Experimental method
급성 간염 모델은 8주령의 특정 병원체 부재 (specific pathogen free, SPF) 수컷 C57BL/6J 마우스를 이용하여 ConA (Concanavalin A)를 1.5 mg/ml이 되도록 PBS 완충액 (phosphate buffered saline)에 녹인 후 100 μl/10 g 기준으로 마우스 체중에 대비하여 꼬리 정맥을 통해 주사함으로써 유도하였다. In the acute hepatitis model, ConA (Concanavalin A) was dissolved in phosphate buffered saline to 1.5 mg/ml using 8-week-old specific pathogen free (SPF) male C57BL/6J mice, and then 100 μl/ It was induced by injection through the tail vein against the mouse body weight on a 10 g basis.
Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005는 ConA 투여 3일 전부터 6.7 μM 농도가 되도록 PBS 완충액에 녹인 후 Amuc_1409는 마리당 210 μl씩 그리고 PEP001, PEP002, PEP003, PEP004 및 PEP005는 마리당 150 μl씩 1일 1회 동일 시간에 마우스 복강으로 주사하였고, ConA 주사 30분 전에 추가 1회 복강주사 하였으며, ConA 정맥주사 9시간 후에 실험을 종료하였다. 대조군에는 PBS 완충액을 동일 방법으로 정맥주사 하였다.After dissolving Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 6.7 μM from 3 days before ConA administration, 210 μl of Amuc_1409 per animal and 150 μl of PEP001, PEP002, PEP003, PEP004 and PEP005 per animal for 1 day The mouse was intraperitoneally injected once at the same time, and another intraperitoneal injection was administered 30 minutes before ConA injection, and the experiment was terminated 9 hours after ConA intravenous injection. In the control group, PBS buffer was intravenously injected in the same way.
ConA 정맥주사 9시간 후 실험종료 시점에 안와정맥총에서 heparin 처리된 capillary tube를 이용하여 채혈 하고, 채취한 혈액은 즉시 10,000 rpm (4℃)에서 10분간 원심분리하여 혈장을 분리한 후, 자동혈액생화학분석기 (Beckman Coulter AU480 automatic analyzer, Beckman Coulter)를 통해 간손상 지표인 alanine aminotransferase (ALT) 및 aspartate aminotransferase (AST)를 분석하였고, 간을 적출하여 그 일부를 10% NBF에 24시간 이상 상온에서 고정한 후 파라핀으로 포매하여 5 μm 두께의 절편을 준비하고 헤마톡실린 (Hematoxylin)과 에오신 (Eosin)으로 (H&E) 염색하였다.After 9 hours of ConA intravenous injection, at the end of the experiment, blood was collected from the orbital venous plexus using a heparin-treated capillary tube, and the collected blood was immediately centrifuged at 10,000 rpm (4℃) for 10 minutes to separate plasma, followed by automatic blood biochemistry. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicators of liver damage, were analyzed through an analyzer (Beckman Coulter AU480 automatic analyzer, Beckman Coulter). After the liver was excised and part of it was fixed in 10% NBF at room temperature for more than 24 hours, Sections with a thickness of 5 μm were prepared by embedding in paraffin and stained with hematoxylin and eosin (H&E).
실시예 3-1-2: 실험 결과Example 3-1-2: Experimental results
급성 간염 모델에서 간손상 지표인 ALT와 AST를 분석한 결과, ALT는 PBS 완충액 투여군에 비해 Amuc_1409와 PEP002, PEP003 및 PEP004를 투여한 군에서 유의성 있게 감소하였고 PEP001 및 PEP005 투여군에서 유의한 수준에 근접하게 저하된 것을 확인하였다. 한편 AST는 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 유의성 있게 감소하였고 PEP001 투여군에서 유의한 수준에 근접하게 저하되었으며 PEP002, PEP003, PEP004 및 PEP005 투여군에서 감소하는 경향을 보였다 (p<0.05, Student's t-test) (도 11).As a result of analyzing ALT and AST, which are indicators of liver damage in the acute hepatitis model, ALT was significantly decreased in the group administered with Amuc_1409, PEP002, PEP003, and PEP004 compared to the group administered with the PBS buffer, and was close to a significant level in the group administered with PEP001 and PEP005. was confirmed to be lowered. On the other hand, AST decreased significantly in the group administered with Amuc_1409 compared to the group administered with PBS buffer, and decreased close to a significant level in the group administered with PEP001, and showed a tendency to decrease in the groups administered with PEP002, PEP003, PEP004 and PEP005 (p<0.05, Student's t). -test) (Fig. 11).
또한, 간조직에서 H&E 염색을 통해 조직병리학적 소견을 확인한 결과, PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 ConA 유도 급성 간염 발생에 따른 간내 염증세포 침윤 및 간 실질의 괴사소가 뚜렷하게 감소하였다 (도 11).In addition, as a result of confirming histopathological findings through H&E staining in liver tissue, the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004, and PEP005 compared to the group administered with the PBS buffer, the infiltration of inflammatory cells in the liver due to the occurrence of ConA-induced acute hepatitis and Necrosis of the liver parenchyma was significantly reduced ( FIG. 11 ).
실시예 3-2: 비알코올성 간염 (nonalcoholic steatohepatitis) 동물모델에서 신규 펩타이드의 효능 검증Example 3-2: Efficacy verification of novel peptides in nonalcoholic steatohepatitis animal model
실시예 3-2-1: 실험 방법Example 3-2-1: Experimental method
비알코올성 간염 모델은 8주령의 SPF 수컷 C57BL/6J 마우스를 이용하여 고지방 식이를 45주간 또는 8주간 자유 급이함으로써 유도하였다. 45주간 고지방 식이를 급이한 마우스에 추가 3주간 고지방 식이를 급이함과 함께 Amuc_1409를, 또는 8주간 고지방 식이를 급이한 마우스에 추가 3주간 고지방 식이를 급이함과 함께 PEP001, PEP002, PEP003, PEP004 및 PEP005를 2.4 μM 농도가 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 시험 종료 시까지 마우스 경구로 투여하였다. 대조군에는 PBS 완충액을 동일 방법으로 경구투여 하였다.The nonalcoholic hepatitis model was induced by free feeding on a high-fat diet for 45 weeks or 8 weeks using 8-week-old SPF male C57BL/6J mice. Amuc_1409 was fed to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 45 weeks, or PEP001, PEP002, and PEP001, PEP002, to mice fed a high-fat diet for an additional 3 weeks to mice fed a high-fat diet for 8 weeks After dissolving PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 2.4 μM, 150 μl per animal was orally administered to mice once a day at the same time until the end of the test. To the control group, PBS buffer was orally administered in the same way.
실험 종료시 적출한 간의 일부를 Trizol을 넣고 균질화하여 RNA를 추출하였다. 확보한 RNA에 역전사효소 (reverse transcriptase)를 첨가하고 반응시켜 cDNA를 합성하고 SYBR Green과 프라이머를 첨가하여 정량적 실시간 PCR (RT-qPCR)을 진행한 뒤 Tnfα, Il-1β, Il-10 및 Il-1rn 등의 유전자 발현 정도를 정량화하였다.At the end of the experiment, a part of the liver that was extracted was added to Trizol and homogenized to extract RNA. Reverse transcriptase was added to the obtained RNA and reacted to synthesize cDNA, followed by quantitative real-time PCR (RT-qPCR) by adding SYBR Green and primers, followed by Tnfα, Il-1β, Il-10 and Il- The expression level of genes such as 1rn was quantified.
실시예 3-2-2: 실험 결과Example 3-2-2: Experimental results
비알코올성 간염 모델의 간조직에서 유전자 발현을 확인한 결과, 고지방 식이 유도 비알코올성 간염에서 증가하는 염증성 지표들 중 Tnfα는 PEP003, PEP004 및 PEP005 투여군에서 PBS 완충액 투여군에 비해 유의하게 감소하였고, Il-1β는 PEP001 및 PEP004 투여군에서 유의한 수준에 근접하게 저하되었으며 Amuc_1409와 PEP002, PEP003 및 PEP005 투여군에서 유의성 있게 감소하였다. 반면 항염 지표인 Il-10은 Amuc_1409와 PEP001, PEP003, PEP004 및 PEP005 투여군에서 PBS 완충액 투여군에 비해 유의하게 증가하였고, PEP002 투여군에서 유의한 수준에 근접하게 증가하는 경향을 보였다. 추가적인 항염 지표인 Il-1rn은 PBS 완충액 투여군에 비해 Amuc_1409 투여군에서 유의성 있게 증가하였다 (p<0.05, Student’s t-test) (도 12).As a result of confirming gene expression in the liver tissue of the nonalcoholic hepatitis model, among the inflammatory markers increasing in high-fat diet-induced nonalcoholic hepatitis, Tnfα was significantly reduced in the PEP003, PEP004 and PEP005 administration groups compared to the PBS buffer administration group, and Il-1β was It decreased close to a significant level in the PEP001 and PEP004 administration groups, and significantly decreased in the Amuc_1409, PEP002, PEP003 and PEP005 administration groups. On the other hand, Il-10, an anti-inflammatory index, significantly increased in the groups administered with Amuc_1409 and PEP001, PEP003, PEP004 and PEP005 compared to the group administered with PBS buffer, and showed a tendency to increase close to a significant level in the group administered with PEP002. An additional anti-inflammatory index, Il-1rn, was significantly increased in the Amuc_1409 administration group compared to the PBS buffer administration group (p<0.05, Student's t-test) (Fig. 12).
이를 통해, 본 발명의 펩타이드가 간염 예방 및 치료 효과가 있음을 확인하였다. Through this, it was confirmed that the peptide of the present invention is effective in preventing and treating hepatitis.
실시예 4: 염증성 장질환 동물모델에서 신규 펩타이드의 효능 검증Example 4: Efficacy verification of novel peptides in inflammatory bowel disease animal model
실시예 4-1: 실험 방법Example 4-1: Experimental method
본 발명의 펩타이드의 염증성 장질환 예방 및 치료 효능을 확인하기 위하여, 장염 동물 모델을 이용하여 실험을 수행하였다.In order to confirm the efficacy of the peptide of the present invention for preventing and treating inflammatory bowel disease, an experiment was performed using an animal model of enteritis.
장염 모델은 10주령의 특정 병원체 부재 (specific pathogen free, SPF) 수컷 C57BL/6J 마우스를 이용하여 DSS를 1.2%가 되도록 음수에 녹인 후 6일 동안 음수를 통해 투여하여 장염을 유도하고, 이후 물로 교체하여 회복 기간을 주었다. In the enteritis model, using 10-week-old specific pathogen free (SPF) male C57BL/6J mice, DSS was dissolved in negative water to 1.2%, and then administered through negative water for 6 days to induce enteritis, and then replaced with water. This gave him a period of recovery.
회복 기간 동안 PEP002, PEP003, PEP004 및 PEP005는 4.3 μM 농도로 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 경구 투여하였고, 대조군에는 PBS 완충액 동일량을 경구로 투여하였다. During the recovery period, PEP002, PEP003, PEP004 and PEP005 were dissolved in PBS buffer at a concentration of 4.3 μM, and 150 μl per animal was orally administered at the same time once a day, and the same amount of PBS buffer was orally administered to the control group.
회복기간 동안 6일간 6회 경구 투여를 통해 장염의 회복으로 인한 체중의 증가율을 확인하였다. 또한 결장의 길이와 결장 내 염증 정도 및 결장 조직의 회복 정도를 확인하기 위하여, 3일간 3회 경구 투여 후 시험을 종료하였다. During the recovery period, the rate of increase in body weight due to the recovery of enteritis was confirmed through oral administration 6 times for 6 days. In addition, in order to confirm the length of the colon, the degree of inflammation in the colon, and the degree of recovery of the colon tissue, the test was terminated after oral administration three times for 3 days.
실험 종료 시 결장의 길이를 측정하고, 원위결장의 일부는 액체질소에 급속 냉각하여 Trizol을 이용하여 RNA를 추출하여 역전사효소 (reverse transcriptase)를 첨가하고 반응시켜 cDNA를 합성하고 SYBR Green과 프라이머를 첨가하여 정량적 실시간 PCR (RT-qPCR)을 진행한 뒤 Il-6 유전자 발현 정도를 정량화하였다. 또한 원위결장 일부는 10% NBF에 24시간 이상 상온에서 고정한 후 파라핀으로 포매하여 5 μm 두께의 절편을 준비하고 헤마톡실린 (Hematoxylin)과 에오신 (Eosin)으로 (H&E) 염색하였다. H&E 염색된 조직 절편을 광학현미경으로 관찰하여 조직병리학적인 장염의 정도를 조직학적 장염 점수 체계 (histologic colitis scoring system)을 이용하여 평가하였다 (표 4).At the end of the experiment, the length of the colon is measured, and a part of the distal colon is rapidly cooled in liquid nitrogen, RNA is extracted using Trizol, reverse transcriptase is added and reacted to synthesize cDNA, and SYBR Green and primer are added. After performing quantitative real-time PCR (RT-qPCR), the Il-6 gene expression level was quantified. In addition, a part of the distal colon was fixed in 10% NBF at room temperature for more than 24 hours, then embedded in paraffin to prepare a 5 μm-thick section and stained with hematoxylin and eosin (H&E). H&E-stained tissue sections were observed under a light microscope to evaluate the degree of histopathological enteritis using a histologic colitis scoring system (Table 4).
Figure PCTKR2021015692-appb-img-000005
Figure PCTKR2021015692-appb-img-000005
실시예 4-2: 실험 결과Example 4-2: Experimental results
분석결과, 장염 모델에서 체중 증가율 분석 결과 PBS 완충액 투여군에 비해 PEP004 및 PEP005를 투여한 군에서 유의하게 증가하였고, PEP002 및 PEP003를 투여한 군에서는 증가하는 경향을 보였다 (p<0.05, Student's t-test) (도 13).As a result of the analysis, as a result of analyzing the weight gain in the enteritis model, the group administered with PEP004 and PEP005 significantly increased compared to the group administered with PBS buffer, and the group administered with PEP002 and PEP003 showed a tendency to increase (p<0.05, Student's t-test). ) (Fig. 13).
또한 결장의 길이 측정 결과 PBS 완충액 투여군에 비해 PEP002, PEP004 및 PEP005를 투여한 군에서 유의하게 증가하였고, PEP003를 투여한 군에서 증가하는 경향을 보였다 (p<0.05, Student's t-test) (도 14).In addition, as a result of measuring the length of the colon, the group administered with PEP002, PEP004 and PEP005 significantly increased compared to the group administered with the PBS buffer, and showed a tendency to increase in the group administered with PEP003 (p<0.05, Student's t-test) (Fig. 14). ).
H&E 염색을 통한 조직학적 검사 결과 PBS 완충액 투여군에 비해 PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 장내 염증의 정도나 범위가 감소하였고, crypt의 손상 정도가 유의하게 감소하는 것을 관찰하였다 (p<0.05, Student's t-test) (도 15).As a result of histological examination through H&E staining, it was observed that the degree or range of intestinal inflammation was reduced in the group administered with PEP002, PEP003, PEP004 and PEP005 compared to the group administered with PBS buffer, and the degree of crypt damage was significantly reduced (p< 0.05, Student's t-test) ( FIG. 15 ).
또한 PBS 완충액 투여군에 비해 PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 Il-6의 유전자 발현이 유의하게 감소하였다 (p<0.05, Student's t-test) (도 16).In addition, the gene expression of Il-6 was significantly reduced in the group administered with PEP002, PEP003, PEP004 and PEP005 compared to the PBS buffer administration group (p<0.05, Student's t-test) ( FIG. 16 ).
이를 통해 본 발명의 펩타이드가 염증성 장질환의 예방 및 치료 효과를 가지는 것을 확인하였다.Through this, it was confirmed that the peptide of the present invention has a preventive and therapeutic effect on inflammatory bowel disease.
실시예 5: 위염/위궤양 동물모델에서 신규 펩타이드의 효능 검증Example 5: Efficacy verification of novel peptides in gastritis/gastric ulcer animal models
실시예 5-1: 실험 방법Example 5-1: Experimental method
위염/위궤양 모델은 7주령 수컷 C57BL/6J 마우스를 사용하여 유도 3일 전부터 Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005의 농도가 6.7 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 경구로 투여하였다.Gastritis/gastric ulcer model was performed using 7-week-old male C57BL/6J mice from 3 days before induction so that the concentrations of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 were 6.7 μM in PBS buffer and then 150 μl per animal once a day. The mice were orally administered at the same time.
급성 위염/위궤양 유발을 위해 0.3 M HCl/60% ethanol을 각 0.2 ml씩 경구로 투여하고 1시간 후 조직을 적출하여 위장 내면의 사진을 찍고 위의 손상 정도를 비교하였다.For acute gastritis/gastric ulcer induction, 0.2 ml of 0.3 M HCl/60% ethanol was orally administered, and the tissue was excised after 1 hour to take pictures of the inner side of the stomach and compare the degree of gastric damage.
위염/위궤양 지표의 측정은 점막 손상 정도에 따라 점수를 매겨 환산하였다. 손상이 없을 경우는 0점, 5 mm 미만 정도의 미미한 원모양의 출혈 부식 한 부위에는 1점, 5 mm 보다 큰 길이의 출혈 부식 한 부위에는 2점, 2점에 해당하는 출혈 부식 부위가 하나 이상일 때는 3점, 5 mm 이상의 길이 및 2 mm 이하 넓이의 출혈 부식에는 4점, 4 점에 해당하는 출혈 부식이 2-3개인 경우 5점, 4-5개인 경우는 6점, 6개 이상인 경우에는 7점 그리고 위 점막 전체적으로 출혈 부식이 있는 경우 8점을 매겼다.The measurement of gastritis/gastric ulcer index was converted by scoring according to the degree of mucosal damage. If there is no damage, there is at least one site of bleeding corrosion corresponding to 0 points, minor circular bleeding erosion less than 5 mm, 1 point for one site, 2 points for a site with a length greater than 5 mm, and 2 points for bleeding corrosion. 3 points, 4 points for bleeding corrosion with a length of 5 mm or more and 2 mm or less, 5 points for 2-3 bleeding corrosion corresponding to 4 points, 6 points for 4-5 pieces, 6 points for 6 points or more A score of 7 and 8 points were given if there was hemorrhagic erosion throughout the gastric mucosa.
투여한 Amuc_1409 및 PEP001, PEP002, PEP003, PEP004 및 PEP005의 추가적인 항염 활성을 확인하기 위하여 위 점막 조직에서 염증 관련 유전자인 Tnfα, Il-1β, iNos, Cox2 및 Il-10의 mRNA 발현을 확인하였다.In order to confirm the additional anti-inflammatory activity of the administered Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005, the mRNA expression of inflammation-related genes Tnfα, Il-1β, iNos, Cox2 and Il-10 was confirmed in gastric mucosal tissues.
실시예 5-2: 실험 결과Example 5-2: Experimental results
실시예 5-1에서 제조한 위염/위궤양 모델에서 위 점막 손상 정도를 점수화하였을 때, Amuc_1409를 투여한 군의 위 점막 손상 정도가 PBS 완충액을 투여한 군에 비해 유의하게 감소하였다 (p<0.05, Student's t-test) (도 17).When the degree of gastric mucosal damage was scored in the gastritis/gastric ulcer model prepared in Example 5-1, the degree of gastric mucosal damage in the group administered with Amuc_1409 was significantly reduced compared to the group administered with PBS buffer (p<0.05, Student's t-test) (FIG. 17).
또한 RT-qPCR을 통한 위 점막 염증 관련 유전자들의 발현을 분석한 결과 PBS 완충액을 투여한 군보다 Amuc_1409를 투여한 군에서 Tnfα 및 iNos의 mRNA발현이 유의하게 감소하였고, 항염 사이토카인인 Il-10의 mRNA 발현은 증가하였다 (p<0.05, Student's t-test) (도 18).In addition, as a result of analyzing the expression of gastric mucosal inflammation-related genes through RT-qPCR, mRNA expression of Tnfα and iNos was significantly reduced in the group administered with Amuc_1409 than in the group administered with PBS buffer, and the anti-inflammatory cytokine Il-10 mRNA expression increased (p<0.05, Student's t-test) ( FIG. 18 ).
위염/위궤양 모델에서 위 점막 손상 정도를 점수화하였을 때, PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군의 위 점막 손상 정도가 PBS 완충액을 투여한 군에 비해 유의하게 감소하였다 (p<0.05, Student's t-test) (도 19).When the degree of gastric mucosal damage was scored in the gastritis/gastric ulcer model, the degree of gastric mucosal damage in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 was significantly reduced compared to the group administered with PBS buffer (p<0.05, Student's t-test) ( FIG. 19 ).
또한 RT-qPCR을 통한 위 점막 염증 관련 유전자들의 발현을 분석한 결과 PBS 완충액을 투여한 군보다 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 Tnfα, Il-1β, iNos 및 Cox2의 발현이 유의하게 감소하였다 (p<0.05, Student’s t-test) (도 20).In addition, as a result of analyzing the expression of gastric mucosal inflammation-related genes through RT-qPCR, the expression of Tnfα, Il-1β, iNos and Cox2 was higher in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 than in the group administered with PBS buffer. decreased significantly (p<0.05, Student's t-test) (FIG. 20).
이를 통해, 본 발명의 펩타이드가 위염 및 위궤양의 예방 및 치료에 효과가 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention is effective in preventing and treating gastritis and gastric ulcer.
실시예 6: 피부염 동물모델에서 신규 펩타이드의 효능 검증Example 6: Efficacy verification of novel peptides in dermatitis animal model
실시예 6-1: 실험 방법Example 6-1: Experimental method
피부염 모델은 시료 적용 하루 전 8주령의 수컷 C57BL/6J 마우스의 양쪽 귀를 제모 한 후 마우스의 오른쪽 귀에 25μl 아세톤에 용해된 TPA(12-otetrade-canoyl-phorbol-13-acetate) 2.5μg를 도포하여 피부 부종 및 염증을 유도하였고, 왼쪽 귀는 TPA 용매인 아세톤 25μl을 도포하여 음성 대조군으로 이용하였다.The dermatitis model was performed by applying 2.5 μg of TPA (12-otetrade-canoyl-phorbol-13-acetate) dissolved in 25 μl acetone to the right ear of an 8-week-old male C57BL/6J mouse one day before sample application. Skin edema and inflammation were induced, and 25 μl of acetone, a TPA solvent, was applied to the left ear and used as a negative control.
TPA 도포 3일 전부터 Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005를 6.7 μM 농도로 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 복강으로 주사하였다. Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 후 3일째 (0 day)에 TPA 도포 30분 전 1회 복강 주사 하였으며, TPA 주입 후 3시간째에 약물을 추가 1회 복강 주사하였다. 대조군에는 PBS 완충액 동일량을 복강으로 주사하였고 TPA 도포 26시간 후 실험을 종료하였다From 3 days before TPA application, Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 were dissolved in PBS buffer at a concentration of 6.7 μM, and 150 μl per animal was injected intraperitoneally into mice once a day at the same time. After administration of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005, on the 3rd day (0 day), one intraperitoneal injection was performed 30 minutes before TPA application, and 3 hours after TPA injection, the drug was additionally injected intraperitoneally. In the control group, the same amount of PBS buffer was injected intraperitoneally, and the experiment was terminated 26 hours after TPA application.
TPA 도포 26시간 후 귀의 부종 및 염증 완화 정도를 확인하고자 귀의 두께를 측정하고, 시험군 별 양쪽 귀를 사진 촬영하였다. 시험동물을 희생시킨 후 귀의 일정 면적당 무게를 측정 하였고, 마우스의 귀를 10% 중성완충 포르말린에 24시간 이상 상온에서 고정한 후 파라핀으로 포매하여 5 μm 두께의 절편을 준비하고 헤마톡실린 (Hematoxylin)과 에오신 (Eosin)으로 (H&E) 염색하였다. H&E 염색된 조직 절편을 광학현미경으로 귀의 부종 및 염증세포 침윤 정도를 확인하였다.26 hours after TPA application, the thickness of the ears was measured to check the degree of edema and inflammation relief of the ears, and both ears were photographed for each test group. After sacrificing the test animal, the weight per area of the ear was measured, and the mouse ear was fixed in 10% neutral buffered formalin at room temperature for at least 24 hours, then embedded in paraffin to prepare a 5 μm-thick section, and hematoxylin and (H&E) staining with eosin (Eosin). The degree of ear edema and inflammatory cell infiltration was checked for H&E-stained tissue sections under a light microscope.
실시예 6-2: 실험 결과Example 6-2: Experimental results
피부염 모델에서 TPA 도포 26시간 후 귀 육안사진 촬영 결과 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 귀의 홍반 및 부종이 전체적으로 감소됨을 확인하였고, 귀 두께를 측정한 결과 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 감소하는 경향을 보였다. 또한 귀 무게를 측정한 결과 PBS 완충액 투여군에 비해 Amuc_1409를 투여한 군에서 유의하게 감소하였다 (p<0.05, Student's t-test) (도 21).In the dermatitis model, it was confirmed that erythema and edema of the ear were overall reduced in the group administered with Amuc_1409 compared to the group administered with PBS buffer as a result of taking a visual photograph of the ear 26 hours after application of TPA in the dermatitis model. showed a decreasing trend in the group. In addition, as a result of measuring the ear weight, it was significantly decreased in the group administered with Amuc_1409 compared to the group administered with the PBS buffer (p<0.05, Student's t-test) ( FIG. 21 ).
PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 TPA 도포 26시간 후 귀 육안사진 촬영 결과 PBS 완충액 투여군에 비해 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 귀의 홍반 및 부종이 뚜렷하게 감소됨을 확인하였고, 귀 두께를 측정한 결과 PBS 완충액 투여군에 비해 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 유의하게 감소하였다. 또한 귀 무게를 측정한 결과 PBS 완충액 투여군에 비해 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 유의하게 감소하였다 (p<0.05, Student's t-test) (도 22).In the group administered with PEP001, PEP002, PEP003, PEP004, and PEP005, as a result of taking a visual photograph of the ear 26 hours after TPA application, the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 markedly reduced erythema and edema of the ear compared to the group administered with PBS buffer. As a result of measuring the ear thickness, it was significantly decreased in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 compared to the group administered with PBS buffer. In addition, as a result of measuring the ear weight, the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 significantly decreased compared to the group administered with the PBS buffer (p<0.05, Student's t-test) ( FIG. 22 ).
또한, 마우스 귀 조직에서 H&E 염색을 통해 조직병리학적 소견을 확인한 결과 PBS 완충액 투여군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군은 TPA 도포에 의한 귀 조직의 부종 및 염증 세포의 침윤이 뚜렷하게 감소하였다 (도 23).In addition, as a result of confirming histopathological findings through H&E staining in mouse ear tissues, the group administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004, and PEP005 showed improvement in ear tissue edema and inflammatory cells by TPA application compared to the PBS buffer administration group. Infiltration was markedly reduced ( FIG. 23 ).
이를 통해, 본 발명의 펩타이드가 피부 염증 질환의 예방 및 치료 효과가 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention is effective in preventing and treating skin inflammatory diseases.
실시예 7: 우울증 및 불안 장애 동물모델에서 신규 펩타이드의 효능 검증Example 7: Efficacy verification of novel peptides in animal models of depression and anxiety disorders
실시예 7-1: 실험 방법Example 7-1: Experimental method
무게가 20~22 g인 수컷 7주령의 C57BL/6J 마우스를 대한바이오링크로부터 공급받았으며, 동물은 실험 당일까지 고형 사료와 물을 충분히 공급하고 온도 22±2℃, 습도 55±15%, 12시간 명/암 주기의 환경을 유지하며 1주간 적응시킨 후 실험에 사용하였다.7-week-old male C57BL/6J mice weighing 20-22 g were supplied from Daehan Biolink, and the animals were supplied with solid food and water sufficiently until the day of the experiment, temperature 22±2℃, humidity 55±15%, 12 hours. The light/dark cycle was maintained and the environment was acclimatized for 1 week before being used in the experiment.
우울증 및 불안을 유도하기 3일 전부터 Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005의 농도가 6.7 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 경구로 투여하였다. From 3 days before the induction of depression and anxiety, the concentration of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 was dissolved in PBS buffer to 6.7 μM, and 150 μl per animal was orally administered to mice once a day at the same time.
Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여 3일 째 되는 날 우울증 및 불안을 유도하기 위하여 PBS 완충액에 용해된 lipopolysaccharide (LPS)를 0.8 mg/kg 농도로 복강 내 주사하였다.On the third day of administration of Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005, lipopolysaccharide (LPS) dissolved in PBS buffer was intraperitoneally injected at a concentration of 0.8 mg/kg to induce depression and anxiety.
우울증에 미치는 영향을 분석하기 위하여 꼬리 현수 (Tail suspension test, TST) 및 강제 수영 시험법 (Forced swim test, FST)을 실시하였다. TST는 하얀색 아크릴로 제작된 오픈된 상자에 꼬리 현수 장치를 설치하고 마우스의 꼬리가 바닥에 닿지 않을 높이로 거꾸로 매달아 놓은 뒤 6분 동안 같은 환경에서 관찰하면서 부동자세 (immobility)를 유지하는 시간을 측정하였다. FST는 발과 꼬리가 닿지 않는 깊이의 실린더에서 마우스의 부동성을 측정하여 우울 증상인 절망의 정도를 확인하는 것으로, 25℃ 내외의 물을 아크릴로 된 투명 원통 (45 cm 깊이x18 cm 직경)의 바닥으로부터 30 cm까지 채우고 마우스를 물에 노출시켜 6분 동안 수영하면서 부동자세 (immobility)를 유지하는 시간을 측정하였다. FST와 TST 분석은 총 6분 촬영 중 시작 후 2분은 적응시간으로 간주하고, 종료 전 4분 동안 움직이지 않은 총 시간을 측정하여 기록하였다.To analyze the effect on depression, tail suspension test (TST) and forced swim test (FST) were performed. TST measures the length of time the mouse maintains immobility while observing in the same environment for 6 minutes after installing a tail suspension device in an open box made of white acrylic and hanging it upside down at a height so that the tail does not touch the floor. did FST is to measure the immobility of mice in a cylinder at a depth where the feet and tail do not touch to determine the degree of despair, which is a depressive symptom. It was filled up to 30 cm from and the mice were exposed to water, and the time to maintain the immobility posture (immobility) was measured while swimming for 6 minutes. For FST and TST analysis, 2 minutes after the start of a total of 6 minutes of shooting were considered as adaptation time, and the total time of no movement for 4 minutes before ending was measured and recorded.
불안 정도에 미치는 영향을 분석하기 위하여 고가식 십자미로 검사 (Elevated plus-maze, EPM)를 실시하였다. EPM 장치는 바닥으로부터 50 cm의 높이에 2개의 열린 arms (50Х10 cm)과 교차되어 있는 2 개의 닫힌 arms (50Х10 cm)으로 구성되어져 있고, 중앙에 10Х10 cm의 면적에 의해 연결되어 있다. 실험 시작 시, 생쥐를 maze의 중앙에 놓고 비디오 카메라를 사용하여 열린 arm에 머문 시간을 총 5분 동안 측정하고, 열린 arm에서 보낸 시간의 비율을 [(열린 arm에서 보낸 시간/열린 arm 및 닫힌 arm에서 보낸 시간)Х100] 계산하여 기록하였다.Elevated plus-maze (EPM) was performed to analyze the effect on anxiety level. The EPM device consists of two open arms (50Х10 cm) intersected with two closed arms (50Х10 cm) at a height of 50 cm from the floor, connected by an area of 10Х10 cm in the center. At the beginning of the experiment, the mice were placed in the center of the maze and using a video camera, the time spent on the open arm was measured for a total of 5 min, and the ratio of time spent on the open arm was calculated as [(time spent in open arm/time spent in open and closed arms) Time spent in) Х100] was calculated and recorded.
아울러, 우울증 및 불안 동물모델에서 Amuc_1409 및 PEP001, PEP002, PEP003, PEP004 및 PEP005의 투여에 따른 항염 활성을 확인하기 위하여 뇌조직 (hippocampus)에서 우울증 관련 주요 염증 유전자인 Ifnγ와 Il-10 mRNA 발현을 확인하였다.In addition, in order to confirm the anti-inflammatory activity according to the administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 in an animal model of depression and anxiety, the expression of Ifnγ and Il-10 mRNA, which are major depression-related inflammatory genes, was confirmed in the brain tissue (hippocampus). did
실시예 7-2: 실험 결과Example 7-2: Experimental results
우울증 및 불안 모델의 꼬리 현수 및 강제 수영 시험에서 Amuc_1409 투여군이 꼬리 현수 및 강제 수영에 의한 부동자세 유지시간이 PBS 완충액 투여군보다 유의적으로 감소하였다. 또한, 고가식 십자미로 시험에서 Amuc_1409 투여군이 PBS 완충액 투여군보다 열린 arm에서 보낸 시간이 증가하여 불안 완화 효능을 나타내었다 (p<0.05, Student's t-test) (도 24).In the tail suspension and forced swimming tests of the depression and anxiety models, the retention time of immobility by tail suspension and forced swimming in the Amuc_1409 administration group was significantly reduced than in the PBS buffer administration group. In addition, in the high-weight cross maze test, the time spent in the open arm of the Amuc_1409 administration group increased compared to the PBS buffer administration group, indicating anxiolytic efficacy (p<0.05, Student's t-test) ( FIG. 24 ).
RT-qPCR을 통한 뇌조직 (hippocampus) 염증 관련 유전자들의 발현을 분석한 결과 PBS 완충액을 투여한 군보다 Amuc_1409를 투여한 군에서 염증성 사이토카인인 Ifnγ mRNA 발현은 감소한 반면 항염증 사이토카인인 Il-10의 mRNA 발현이 증가하였다 (도 25).As a result of analyzing the expression of brain tissue (hippocampus) inflammation-related genes through RT-qPCR, the expression of Ifnγ mRNA, an inflammatory cytokine, was decreased in the group administered with Amuc_1409 than in the group administered with PBS buffer, whereas the anti-inflammatory cytokine Il-10 of mRNA expression was increased ( FIG. 25 ).
우울증 및 불안 모델의 꼬리 현수 및 강제 수영 시험에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여군이 꼬리 현수 및 강제 수영에 의한 부동자세 유지시간이 PBS 완충액 투여군보다 유의적으로 감소하였다 (p<0.05, Student's t-test) 또한, 고가식 십자미로 시험에서 PEP001, PEP002, PEP003, PEP004 및 PEP005 투여군이 PBS 완충액 투여군보다 열린 arm에서 보낸 시간이 증가하여 불안 완화 효능을 나타내었다 (p<0.05, Student's t-test) (도 26).In the tail suspension and forced swimming tests of the depression and anxiety models, the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups significantly decreased the retention time of immobility due to tail suspension and forced swimming compared to the PBS buffer administration group (p<0.05, Student's). t-test) In addition, in the high-pitch cross maze test, the PEP001, PEP002, PEP003, PEP004 and PEP005 administration groups showed anxiolytic efficacy by increasing the time spent in the open arm compared to the PBS buffer administration group (p<0.05, Student's t-test) ) (Fig. 26).
RT-qPCR을 통한 뇌조직 (hippocampus) 염증 관련 유전자들의 발현을 분석한 결과 PBS 완충액을 투여한 군보다 PEP001, PEP002, PEP003, PEP004 및 PEP005를 투여한 군에서 염증성 사이토카인인 Ifnγ mRNA 발현은 유의하게 감소한 반면 항염증 사이토카인인 Il-10의 mRNA 발현이 유의하게 증가하였다 (p<0.05, Student's t-test) (도 27).As a result of analyzing the expression of brain tissue (hippocampus) inflammation-related genes through RT-qPCR, the expression of Ifnγ mRNA, an inflammatory cytokine, was significantly higher in the group administered with PEP001, PEP002, PEP003, PEP004 and PEP005 than in the group administered with the PBS buffer. On the other hand, the mRNA expression of Il-10, an anti-inflammatory cytokine, was significantly increased (p<0.05, Student's t-test) (FIG. 27).
실시예 8: 인지/기억력 장애 동물모델에서 신규 펩타이드의 효능 검증Example 8: Efficacy verification of novel peptides in animal models of cognitive/memory disorders
실시예 8-1: 알츠하이머병 동물모델에서 신규 펩타이드의 효능 검증Example 8-1: Efficacy verification of novel peptides in Alzheimer's disease animal model
뇌에서 아밀로이드 침착과 인지 및 기억력 장애가 관찰되는 5개월령의 APP/PS1 알츠하이머병 수컷 마우스에 Amuc_1409를 주5일 동안 매일 5 ㎍씩 경구 투여하여 8주간 투여하였다. PEP001은 농도가 2.4 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1주일에 5일, 8주간 경구 투여하였다. 대조군(vehicle)에는 PBS를 동일한 양(volume)으로 경구 투여하였다. 또한 신규 단백질 단편을 투여하지 않은 정상 마우스(Non-Tg)를 대조군으로 사용하였다.Amuc_1409 was orally administered to 5-month-old male APP/PS1 Alzheimer's disease mice with amyloid deposition and cognitive and memory impairments observed in the brain, 5 μg daily for 5 days a week, and administered for 8 weeks. After dissolving PEP001 in PBS buffer to a concentration of 2.4 μM, 150 μl per animal was orally administered 5 days a week for 8 weeks. The control group (vehicle) was orally administered with PBS in the same amount (volume). In addition, normal mice (Non-Tg) not administered with the novel protein fragment were used as a control group.
Amuc_1409와 PEP001이 투여된 APP/PS1 알츠하이머병 마우스에서 인지기능 개선 효과를 테스트하기 위해 새로운 물체 인지 및 기억력 테스트(novel object recognition test)를 수행하였다. 24시간 전에 본 물체와 새로운 물체를 제공하였을 때 새로운 물체에 대한 탐색 시간 및 탐색 횟수를 측정하여 인지 및 기억력을 테스트하였다.A novel object recognition test was performed to test the cognitive function improvement effect in APP/PS1 Alzheimer's disease mice administered with Amuc_1409 and PEP001. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
분석결과, APP/PS1 알츠하이머병 마우스는 정상 마우스에 비해 새로운 물체 인지 및 기억력이 현저히 감소하여 새로운 물체(Novel object)와 익숙한 물체(Familiar object)를 잘 구분하지 못하는 것을 확인 하였으나, 8주간 Amuc_1409와 PEP001 투여 시 인지 및 기억력이 유의미하게 개선되는 것을 확인하였다 (p<0.05, Student's t-test) (도 28).As a result of the analysis, it was confirmed that APP/PS1 Alzheimer's disease mice had significantly reduced new object recognition and memory compared to normal mice, making it difficult to distinguish between novel objects and familiar objects. However, Amuc_1409 and PEP001 for 8 weeks It was confirmed that cognition and memory were significantly improved upon administration (p<0.05, Student's t-test) (FIG. 28).
실시예 8-2: 인지/기억장애 동물모델에서 신규 펩타이드의 효능 검증Example 8-2: Efficacy verification of novel peptides in animal models of cognitive/memory impairment
C57BL/6J 수컷 마우스에 LPS를 250 ㎍/kg/day로 복강에 7일 동안 매일 주사 투여하여 인지 및 기억력 장애를 유발하였다. 아커만시아 뮤시니필라 (Akkermansia muciniphila) 유래의 단백질 Amuc_1409은 LPS 투여 일주일 전부터 인지 및 기억력 테스트 수행 시까지 매일 5 μg씩 경구 투여하였으며 PEP002, PEP003, PEP004 및 PEP005는 LPS 투여 3일 전부터 인지 및 기억력 테스트 수행 시까지 농도가 2.4 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 매일 해당 마우스에 경구로 투여하였다. 대조군(vehicle)에는 PBS를 동일한 양(volume)으로 경구 투여하였다.Cognitive and memory impairments were induced by intraperitoneal injection of LPS at 250 μg/kg/day for 7 days in male C57BL/6J mice. Akkermansia muciniphila-derived protein Amuc_1409 was orally administered 5 μg daily from one week before LPS administration until the cognitive and memory test was performed. PEP002, PEP003, PEP004 and PEP005 were tested for cognition and memory from three days before LPS administration. After dissolving in PBS buffer so that the concentration became 2.4 μM until completion, 150 μl per animal was orally administered to the mouse every day at the same time once a day. The control group (vehicle) was orally administered with PBS in the same amount (volume).
Amuc_1409 또는 PEP002, PEP003, PEP004 및 PEP005를 투여한 인지 및 기억력 장애 마우스에서 인지기능 개선 효과를 테스트하기 위해 새로운 물체 인지 및 기억력 테스트(novel object recognition test)를 수행하였다. 24시간 전에 본 물체와 새로운 물체를 제공하였을 때 새로운 물체에 대한 탐색 시간 및 탐색 횟수를 측정하여 인지 및 기억력을 테스트하였다.A novel object recognition test was performed to test the effect of improving cognitive function in cognitive and memory impaired mice administered with Amuc_1409 or PEP002, PEP003, PEP004 and PEP005. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
분석결과, 마우스에 LPS 투여 시 새로운 물체에 대한 인지 및 기억력이 현저히 감소하여 새로운 물체(Novel object)와 익숙한 물체(Familiar object)를 잘 구분하지 못하는 것을 확인 하였으나, 마우스에 Amuc_1409 또는 PEP002, PEP003, PEP004 및 PEP005 투여 시, LPS 투여에 따른 염증반응에 의한 인지 및 기억력 손상이 억제되며, 대조군보다도 기억력 및 인지기능이 개선되는 것을 확인하였다 (p<0.05, Student's t-test) (도 29).As a result of the analysis, it was confirmed that when LPS was administered to mice, recognition and memory for new objects were significantly reduced, and it was confirmed that it was difficult to distinguish new objects (Novel objects) from familiar objects (Familiar objects). And it was confirmed that when PEP005 was administered, cognitive and memory impairment due to the inflammatory reaction according to LPS administration was suppressed, and memory and cognitive function were improved compared to the control group (p<0.05, Student's t-test) ( FIG. 29 ).
실시예 8-3: 자연 노화 마우스에서 신규 펩타이드의 인지/기억력 효능 검증Example 8-3: Verification of cognitive/memory efficacy of novel peptides in naturally aging mice
16.5개월령 수컷 마우스에 Amuc_1409를 매일 4.5 ㎍을 주 5일 동안 경구 투여하여 8주간 투여하였으며 16개월령 수컷 마우스에 PEP002, PEP003, PEP004 및 PEP005의 농도가 2.4 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 경구로 3주간 투여하였다. 대조군(vehicle)에는 PBS를 동일한 양(volume)으로 경구 투여하였다. 4.5 μg of Amuc_1409 was orally administered to 16.5-month-old male mice daily for 5 days a week for 8 weeks. After dissolving in PBS buffer so that the concentrations of PEP002, PEP003, PEP004 and PEP005 became 2.4 μM to 16-month-old male mice, 150 μl each It was orally administered to mice once a day at the same time for 3 weeks. The control group (vehicle) was orally administered with PBS in the same amount (volume).
Amuc_1409와 PEP002, PEP003, PEP004 및 PEP005가 투여된 자연 노화 마우스에서 인지기능 개선 효과를 테스트하기 위해 새로운 물체 인지 및 기억력 테스트(novel object recognition test)를 수행하였다. 24시간 전에 본 물체와 새로운 물체를 제공하였을 때 새로운 물체에 대한 탐색 시간 및 탐색 횟수를 측정하여 인지 및 기억력을 테스트하였다.A novel object recognition test was performed to test the cognitive function improvement effect in naturally aging mice administered with Amuc_1409 and PEP002, PEP003, PEP004 and PEP005. Recognition and memory were tested by measuring the search time and number of searches for the new object when the original object and the new object were provided 24 hours ago.
분석결과, 자연 노화 마우스는 새로운 물체에 대한 인지 및 기억력이 현저히 감소하여 새로운 물체(Novel object)와 익숙한 물체(Familiar object)를 잘 구분하지 못하는 것을 확인 하였으나, Amuc_1409와 PEP002, PEP003, PEP004 및 PEP005 투여 시 인지 및 기억력이 유의미하게 개선되는 것을 확인하였다 (p<0.05, Student's t-test) (도 30).As a result of the analysis, it was confirmed that naturally aging mice could not distinguish between novel objects and familiar objects well due to significantly reduced cognition and memory for new objects. It was confirmed that visual recognition and memory were significantly improved (p<0.05, Student's t-test) (FIG. 30).
이를 통해, 본 발명의 펩타이드가 퇴행성 뇌질환의 예방, 치료뿐만 아니라 인지, 기억장애의 예방, 치료 효과를 가지며, 인지 및 기억력의 개선효능이 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention has the effect of preventing and treating cognition and memory disorders as well as preventing and treating degenerative brain diseases, and improving cognition and memory.
실시예 9: 발달 장애 동물모델에서 신규 펩타이드의 효능 검증Example 9: Efficacy verification of novel peptides in developmental disorder animal models
발달 장애에 대한 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005 의 효능을 검증하기 위하여, 실험동물로서 발달 장애 모델인 BTBR 마우스(BTBR T+Itpr3tf/J(BTBR))를 사용하였다. In order to verify the efficacy of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 on developmental disorders, BTBR mice (BTBR T + Itpr3 tf /J (BTBR)), a developmental disability model, were used as experimental animals.
3주령의 수컷 BTBR 마우스에 대해 Amuc_1409는 매일 5 ㎍을 주 5일 동안 경구 투여하였으며 대조군은 PBS(Phosphate-buffered saline)을 투여하였다. PEP001, PEP002, PEP003, PEP004 및 PEP005는 농도가 2.4 μM이 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 주 5일동안 마우스 경구로 투여하였다. 투여 4주 후 마우스의 사회성 행동을 관찰하여 행동학적 평가를 수행하였다. 상기 마우스는 온도가 22~24℃로 유지되는 특정병원균이 없는 환경 (Specific pathogen free)의 사육시설에서 멸균된 사료와 물을 자유롭게 섭취하게 했으며, 12시간 주야 사이클을 유지하면서 사육하였다.To 3-week-old male BTBR mice, Amuc_1409 was orally administered 5 μg daily for 5 days a week, and PBS (Phosphate-buffered saline) was administered to the control group. After dissolving PEP001, PEP002, PEP003, PEP004 and PEP005 in PBS buffer to a concentration of 2.4 μM, 150 μl per animal was orally administered to mice once a day at the same time for 5 days a week. Behavioral evaluation was performed by observing the social behavior of the mice 4 weeks after administration. The mice were allowed to freely ingest sterilized feed and water in a breeding facility in a specific pathogen-free environment in which the temperature was maintained at 22-24° C., and were bred while maintaining a 12-hour day and night cycle.
발달장애 마우스의 사회성 행동을 분석하기 위해 다른 마우스와의 상호작용 행동(sniffing behavior)을 3분 동안 관찰하여 sniffing 하는 시간(A)와 횟수(B)를 측정하였다. BTBR 마우스에 비히클을 투여한 그룹에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 경구 투여한 마우스의 사회성 행동이 유의미하게 증가하는 것을 확인하였다 (p<0.05, Student's t-test) (도 31).To analyze the social behavior of the developmentally disabled mouse, the sniffing time (A) and the number of times (B) were measured by observing the sniffing behavior with other mice for 3 minutes. It was confirmed that the social behavior of the mice orally administered with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 was significantly increased compared to the group administered with the vehicle to the BTBR mice (p<0.05, Student's t-test) (FIG. 31) ).
상기 결과를 통해, Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005의 투여가 발달 장애에서 나타내는 사회성 행동 장애에 대해 예방 및 치료 효과가 있음을 확인하였는바, 본 발명에서 제공하는 펩타이드가 자폐 범주성 장애를 포함한 발달장애의 예방 및 치료, 개선에 효과적임을 알 수 있다.Through the above results, it was confirmed that the administration of Amuc_1409 and PEP001, PEP002, PEP003, PEP004 and PEP005 has a preventive and therapeutic effect on social behavioral disorders shown in developmental disorders. It can be seen that it is effective in the prevention, treatment, and improvement of developmental disorders, including
실시예 10: 신규 펩타이드의 조직 재생 효능 검증Example 10: Verification of tissue regeneration efficacy of novel peptides
신규 펩타이드의 조직 재생 효능을 확인하기 위하여 마우스 꼬리의 전층 피부결손으로 유도되는 창상 유발 모델을 이용하여 실험을 수행하였다.In order to confirm the tissue regeneration efficacy of the novel peptide, an experiment was performed using a wound induction model induced by a full-thickness skin defect of the tail of a mouse.
전층 피부결손 창상 유발 모델은 9주령의 특정 병원체 부재 (specific pathogen free, SPF) 수컷 C57BL/6J 마우스를 마취한 후 전층 상처 (full-thickness wounding)을 나타내기 위하여, 마우스의 꼬리 기저부에서 약 1.0cm 떨어진 마우스 꼬리의 등 부분에 10Х3mm 크기로 무균상태의 해부용 매스 10호를 사용하여 전층 상처를 만들었다. 상처를 압박하여 출혈을 멈추게 하였고, 상처를 필름 분무 드레싱 (film spray dressing; Cavilon, 3M)으로 덮었다.The full-thickness skin defect wound induction model was designed to show a full-thickness wounding after anesthetizing 9-week-old specific pathogen free (SPF) male C57BL/6J mice, about 1.0 cm from the base of the tail of the mouse. A full-thickness wound was made on the back of the tail of the mouse that had fallen off using a 10Х3mm sterile dissection mass No. 10. The wound was compressed to stop bleeding, and the wound was covered with a film spray dressing (Cavilon, 3M).
창상 유발 당일부터 28일 동안 Amuc_1409 또는 PEP001, PEP002, PEP003, PEP004 및 PEP005를 120 μM 농도로 vehicle (50% 에탄올, 20% 프로필렌글라이콜, 30% 물)에 녹인 후 마리당 25 μl씩 1일 1회 창상 부위에 도포하였다. 대조군에는 vehicle (50% 에탄올, 20% 프로필렌글라이콜, 30% 물) 동일량을 창상 부위에 도포하였다. 창상 유발 29일째에 시험을 종료하였다.After dissolving Amuc_1409 or PEP001, PEP002, PEP003, PEP004 and PEP005 in a vehicle (50% ethanol, 20% propylene glycol, 30% water) at a concentration of 120 μM for 28 days from the day of induction of the wound, 25 μl per animal per day 1 day It was applied to the ash wound site. In the control group, the same amount of vehicle (50% ethanol, 20% propylene glycol, 30% water) was applied to the wound site. The trial was terminated on the 29th day of wound induction.
창상 치유 효과를 관찰하기 위해서 창상 유발 당일부터 7일 간격으로 28일 동안 상처로부터 일정한 높이에서 디지털카메라를 이용해 각 시험군의 동물들의 창상 부위를 개별적으로 촬영하였고, Image J Soft 프로그램을 이용하여 창상 면적을 측정하였다. In order to observe the wound healing effect, the wound area of the animals of each test group was individually photographed using a digital camera at a constant height from the wound for 28 days at intervals of 7 days from the day of wound induction, and the wound area was used using the Image J Soft program. was measured.
분석결과, 전층 피부결손 창상 유발 동물모델에서 육안상으로 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 도포한 군에서는 Vehicle 도포군에 비해 상처 크기가 빠르게 감소하는 것을 확인하였다. 또한 창상 면적 측정 결과 vehicle 도포군에 비해 Amuc_1409와 PEP001, PEP002, PEP003, PEP004 및 PEP005를 도포한 군에서는 유의하게 창상 면적이 감소하였다 (p<0.05, Student's t-test) (도 32).As a result of the analysis, it was confirmed that the wound size was rapidly reduced in the group applied with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 compared to the Vehicle-applied group visually in the full-thickness skin defect wound-inducing animal model. Also, as a result of measuring the wound area, the wound area was significantly decreased in the group coated with Amuc_1409, PEP001, PEP002, PEP003, PEP004 and PEP005 compared to the vehicle-applied group (p<0.05, Student's t-test) (Fig. 32).
이를 통해, 본 발명의 펩타이드는 피부 조직을 포함하는 신체 조직의 손상 및 재생 효능이 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention has an effect on damage and regeneration of body tissues including skin tissues.
실시예 11: 신규 펩타이드의 조직 재생 촉진 효능 검증Example 11: Verification of tissue regeneration promoting efficacy of novel peptides
본 발명의 신규 펩타이드가 조직 재생 촉진 효능을 가지는 것을 확인하기 위하여, 근조직의 재생 촉진 및 근력 개선 여부를 확인하였다.In order to confirm that the novel peptide of the present invention has the effect of promoting tissue regeneration, it was confirmed whether the regeneration of muscle tissue and muscle strength were improved.
실시예 11-1. 노화동물모델에서 신규 펩타이드의 근력개선 및 근재생 효능 검증Example 11-1. Validation of muscle strength improvement and muscle regeneration efficacy of new peptides in aging animal models
자연 노화로 인한 근위축/근력감소 동물모델로는 80-83주령의 C57BL/6 수컷마우스를 이용하였다. 해당 노화마우스에 Amuc_1409이 2.4 μM 농도가 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 경구로 32주 동안 투여하였다. 대조군에는 PBS 완충액을 동일한 방법으로 경구투여 하였다.As an animal model for muscle atrophy/reduction of muscle strength due to natural aging, C57BL/6 male mice aged 80-83 weeks were used. After dissolving Amuc_1409 in PBS buffer to a concentration of 2.4 μM to the aged mice, 150 μl per mouse was orally administered once a day at the same time for 32 weeks. To the control group, PBS buffer was orally administered in the same way.
Amuc_1409 투여 16주차에 노화마우스가 grip strength meter의 근력 probe에 연결된 와이어망을 네 발로 붙잡도록 한 다음, 꼬리를 조심스럽게 뒤쪽 방향으로 잡아당겨 와이어망을 잡고 버티는 순간의 최대 악력 (grip strength)을 측정하였다. 근육 무게의 경우, 투여가 끝난 후 각 군의 노화마우스 양쪽 뒷발에서 gastrocnemius (GC), soleus 및 tibialis anterior (TA) 근육을 손상이 되지 않도록 주의하여 분리한 후 각각의 무게를 측정하고 마우스의 체중으로 보정하여 체중대비 근육무게를 산출하였다. At the 16th week of Amuc_1409 administration, the aged mouse was asked to hold the wire mesh connected to the strength probe of the grip strength meter with all four legs, and then carefully pulled the tail backward to measure the maximum grip strength at the moment of holding the wire mesh and holding it. did In the case of muscle weight, after administration, the gastrocnemius (GC), soleus, and tibialis anterior (TA) muscles from both hind paws of each group of aged mice were carefully separated not to be damaged, and then each weight was measured and the weight of the mouse was used. After correction, the muscle weight compared to the body weight was calculated.
실험 종료 후 각 군의 근섬유 크기를 분석하기 위하여 TA 근육을 10% 포르말린에 고정하고, 동결 섹션 샘플 (8 ㎛)을 준비하여 슬라이드를 제작하였다. 준비된 슬라이드를 PBS 완충 용액으로 세척한 후 0.5% 트리톤X-100으로 투과하고 블라킹한 후, 1차 항체 (anti-laminin)를 처리하였다. 24시간 후 형광을 띄는 2차 항체 (Alexa Fluor 488 Goat anti-rabbit IgG)를 적용하고 마지막으로 핵 염색을 위하여 DAPI를 처리하였다. 면역염색이 완료된 슬라이드는 공초점 (Confocal) 현미경을 사용하여 근육 조직을 촬영한 후 이미지 분석 소프트웨어 (ImageInside)를 활용하여 근섬유의 횡단면 크기 (cross-sectional area)를 최소 500 이하부터 3500 ㎛2 이상까지 크기별로 분포를 산출하였다.After the end of the experiment, in order to analyze the muscle fiber size of each group, the TA muscle was fixed in 10% formalin, and a frozen section sample (8 μm) was prepared to prepare a slide. The prepared slides were washed with PBS buffer, then permeated with 0.5% Triton X-100 and blocked, and treated with a primary antibody (anti-laminin). After 24 hours, a fluorescent secondary antibody (Alexa Fluor 488 Goat anti-rabbit IgG) was applied, and finally, DAPI was treated for nuclear staining. After immunostaining was completed, the muscle tissue was photographed using a confocal microscope, and the cross-sectional area of the muscle fibers was measured from at least 500 to 3500 μm 2 or more using image analysis software (ImageInside). The distribution was calculated for each size.
분석결과, 근력 노화도 실험에서 Amuc_1409 투여 16주차에 PBS 완충액 투여군의 악력과 비교하여 Amuc_1409를 투여한 군에서 유의하게 근력이 증가하였음을 확인하였다 (p<0.05, Student's t-test) (도 33A). 또한 실험 종료 후 측정한 GC, soleus 및 TA 근육무게 모두 Amuc_1409를 투여한 군에서 PBS 완충액 투여군 대비 증가하였으며, soleus 근육의 경우 유의하게 증가하여 노화에 의한 근육량 감소가 억제되는 것을 확인하였다 (p<0.05, Student's t-test) (도 33B-D).As a result of the analysis, it was confirmed that the muscle strength significantly increased in the group administered with Amuc_1409 compared to the grip strength of the group administered with PBS buffer at the 16th week of administration of Amuc_1409 in the muscle aging test (p<0.05, Student's t-test) (FIG. 33A). In addition, the GC, soleus and TA muscle weights measured after the end of the experiment were all increased in the group administered with Amuc_1409 compared to the group administered with PBS buffer, and the soleus muscle increased significantly, confirming that the decrease in muscle mass due to aging was suppressed (p<0.05). , Student's t-test) ( FIGS. 33B-D ).
또한, Amuc_1409 투여가 노화에 따른 근육 위축증 (atrophy)에 미치는 영향을 분석하기 위하여, 근육세포 기저막의 주요 성분 중 하나인 라미닌(laminin)을 면역염색하였다. 그 결과, PBS 완충액 투여군에 비해, Amuc_1409를 투여한 마우스의 TA 근육 내 근섬유 횡단면의 사이즈가 500 ㎛2 이하를 가지는 근섬유 수는 감소하였으며, 2,000 ㎛2 이상 크기의 근섬유는 유의하게 증가되어 있는 것을 관찰하였다 (p<0.05, Student's t-test) (도 34A-B). 또한 작은 사이즈부터 큰 사이즈를 모두 포함하는 전체 근섬유의 평균사이즈를 산출한 결과, Amuc_1409를 투여한 노화마우스의 전체 근섬유의 평균 사이즈가 PBS 완충액 투여군에 비해 유의하게 증가되어 근섬유 위축을 억제 또는 개선하는 효과가 있음을 확인하였다 (p<0.05, Student's t-test) (도 34C).In addition, in order to analyze the effect of Amuc_1409 administration on aging-related muscle atrophy, laminin, one of the main components of the basement membrane of muscle cells, was immunostained. As a result, compared to the PBS buffer administration group, it was observed that the number of muscle fibers having a cross-sectional size of 500 μm2 or less in the TA muscle of the mice administered with Amuc_1409 decreased, and the number of muscle fibers having a size of 2,000 μm2 or more increased significantly. (p<0.05, Student's t-test) (FIGS. 34A-B). In addition, as a result of calculating the average size of all muscle fibers including both small and large sizes, the average size of total muscle fibers in aged mice administered with Amuc_1409 was significantly increased compared to the PBS buffer administration group, thereby suppressing or improving muscle fiber atrophy. It was confirmed that there is (p<0.05, Student's t-test) (FIG. 34C).
한편 상기 Amuc_1409뿐 아니라 이의 단편이 가지는 효능도 확인하고자 82-83주령의 C57BL/6 수컷마우스를 이용하여 PEP001이 2.4 μM 농도가 되도록 PBS 완충액에 녹인 후 마리당 150 μl씩 1일 1회 동일 시간에 마우스 경구로 10주 동안 투여하였다. 대조군에는 PBS 완충액을 동일한 방법으로 경구투여 하였다. PEP001 투여 10주차에, Amuc_1409에서 측정한 것과 동일한 방식으로 최대 악력, 근육 무게를 산출하였다. Meanwhile, in order to check the efficacy of Amuc_1409 as well as fragments thereof, 82-83 week old C57BL/6 male mice were dissolved in PBS buffer so that PEP001 had a concentration of 2.4 μM, and then 150 μl of each mouse once a day at the same time. It was administered orally for 10 weeks. To the control group, PBS buffer was orally administered in the same way. At the 10th week of administration of PEP001, the maximum grip strength and muscle weight were calculated in the same manner as measured by Amuc_1409.
실험 종료시 적출한 GC 근육의 일부를 Trizol을 넣고 균질화하여 RNA를 추출하였다. 확보한 RNA에 역전사효소 (reverse transcriptase)를 첨가하고 반응시켜 cDNA를 합성하고 SYBR Green과 프라이머를 첨가하여 정량적 실시간 PCR (RT-qPCR)을 진행한 뒤 근육세포 내 단백질 합성에 관여하는 인슐린 유사성장인자 (insulin-like growth factor, IGF-1)의 유전자 발현 정도를 정량화하였다.At the end of the experiment, a part of the extracted GC muscle was added to Trizol and homogenized to extract RNA. Reverse transcriptase is added to the obtained RNA and reacted to synthesize cDNA. After quantitative real-time PCR (RT-qPCR) is performed by adding SYBR Green and primer, insulin-like growth factor involved in protein synthesis in muscle cells The gene expression level of (insulin-like growth factor, IGF-1) was quantified.
분석결과, 근력 노화도 실험에서 PEP001 투여 10주차에 PBS 완충액 투여군의 악력과 비교하여 PEP001을 투여한 군에서 유의하게 근력이 증가하였음을 확인하였다 (p<0.05, Student's t-test) (도 35A). 또한 실험 종료 후 측정한 GC, soleus 및 TA 근육무게 모두 PEP001을 투여한 군에서 PBS 완충액 투여군 대비 증가하였으며, GC 및 soleus 근육의 경우 유의하게 증가하여 노화에 의한 근육양 감소가 억제되는 것을 확인하였다 (p<0.05, Student's t-test) (도 35B-D).As a result of the analysis, it was confirmed that the muscle strength was significantly increased in the group administered with PEP001 compared to the grip strength of the group administered with PBS buffer at the 10th week of PEP001 administration in the muscle aging test (p<0.05, Student's t-test) (FIG. 35A). In addition, the GC, soleus, and TA muscle weights measured after the end of the experiment were all increased in the group administered with PEP001 compared to the group administered with PBS buffer, and the GC and soleus muscles significantly increased, confirming that the decrease in muscle mass due to aging was suppressed ( p<0.05, Student's t-test) (FIGS. 35B-D).
PEP001 투여 10주차에 적출한 GC 근육에서 근육세포 내 단백질 합성을 촉진하여 IGF-1의 유전자 발현이 PBS 완충액을 투여한 군과 비교하여 증가하는 경향이 관찰되었다 (도 35E). In the GC muscle harvested at the 10th week of administration of PEP001, it was observed that protein synthesis in muscle cells was promoted, thereby increasing the gene expression of IGF-1 compared to the group administered with the PBS buffer (FIG. 35E).
실시예 11-2. 근원세포 모델에서 신규 펩타이드의 근재생 효능 검증Example 11-2. Verification of muscle regeneration efficacy of novel peptides in myoblast model
근원세포 C2C12 (myoblast)를 6 well plate에 분주한 후, 10% FBS와 1% antibotics를 포함하는 DMEM 배지를 사용하여 37℃, 10% CO2 배양기에서 배양하였다. 그 후, 분화를 유도하기 위하여 C2C12 세포를 2% 말혈청 (Horse serum)을 포함하는 분화 배지로 교체하고 분화를 유도하는 시간 동안 PEP004 및 PEP005를 64 nM의 농도로 처리하여 근관세포 (myotube) 형성을 관찰하였다. After dispensing myoblast C2C12 (myoblast) in a 6 well plate, using DMEM medium containing 10% FBS and 1% antibotics, it was cultured in a 10% CO2 incubator at 37°C. Thereafter, in order to induce differentiation, C2C12 cells were replaced with a differentiation medium containing 2% horse serum and treated with PEP004 and PEP005 at a concentration of 64 nM for a time to induce differentiation to form myotubes. was observed.
근원세포를 분화시킨 후 근위축을 일으키는 덱사메타손 (dexamethason, 25 uM)을 PEP004 및 PEP005와 동시에 처리한 후, 세포에서 Trizol을 이용하여 RNA를 추출하였다. 확보한 RNA에 역전사효소 (reverse transcriptase)를 첨가하고 반응시켜 cDNA를 합성하고 SYBR Green과 프라이머를 첨가하여 정량적 실시간 PCR (RT-qPCR)을 진행한 뒤, 근 위축 관여 인자인 atrogin-1 및 MuRF1의 발현 수준을 측정하여 분화된 근원세포의 위축 억제 여부를 확인하였다. After myoblast differentiation, dexamethason (25 uM) causing muscle atrophy was simultaneously treated with PEP004 and PEP005, and RNA was extracted from the cells using Trizol. Reverse transcriptase was added to the obtained RNA and reacted to synthesize cDNA, and quantitative real-time PCR (RT-qPCR) was performed by adding SYBR Green and primers, and then atrogin-1 and MuRF1, factors involved in muscular atrophy, By measuring the expression level, it was confirmed whether the atrophy of the differentiated myoblasts was inhibited.
분석결과, C2C12 근원세포의 분화를 유도한 후 덱사메타손을 처리한 세포에서 근단백질 분해 유전자인 Atrogin-1과 MuRF1의 발현이 유의하게 증가한 반면, PEP004 및 PEP005를 덱사메타손과 함께 처리한 세포에서는 해당 유전자의 발현이 유의하게 감소하였다 (p<0.05, Student's t-test) (도 36).As a result of the analysis, the expression of myoprotein-degrading genes, Atrogin-1 and MuRF1, was significantly increased in cells treated with dexamethasone after inducing differentiation of C2C12 myoblasts, whereas in cells treated with PEP004 and PEP005 together with dexamethasone, the expression of the corresponding genes was increased. Expression was significantly decreased (p<0.05, Student's t-test) (FIG. 36).
이를 통해 본 발명의 펩타이드가 근재생 촉진 효과 및 근력 강화, 근력 개선 효과가 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention has the effect of promoting muscle regeneration, strengthening of muscle strength, and improving muscle strength.
실시예 12: 신규 펩타이드의 모발 재생 및 발모 촉진 효능 검증Example 12: Verification of hair regeneration and hair growth promoting efficacy of novel peptides
실시예 12-1: 실험 방법Example 12-1: Experimental method
발모촉진 효능을 확인하기 위하여, 등쪽 피부의 색이 핑크색을 보이는 휴지기 체모의 8주령 C57BL/6 마우스를 사용하였다. 마우스를 마취한 후 동물용 전기제모기를 이용하여 마우스 등판의 털을 제거하고 털이 제거된 부위에 Amuc_1409를 3.2 μM 농도로 vehicle (50% 에탄올, 20% 프로필렌글라이콜, 30% 물)에 녹인 후 마리당 200 μl씩 피펫(pipette)을 이용하여 23일 간 매일 1회 피부 도포하였다. 대조군에는 vehicle (50% 에탄올, 20% 프로필렌글라이콜, 30% 물) 동일량을 피부 도포하였다.In order to confirm the hair growth promoting effect, 8-week-old C57BL/6 mice with resting body hair showing a pink color of the skin on the back were used. After anesthetizing the mouse, remove the hair on the back of the mouse using an animal electric epilator, and dissolve Amuc_1409 in the vehicle (50% ethanol, 20% propylene glycol, 30% water) at a concentration of 3.2 μM in the hair-removed area. 200 μl per animal was applied to the skin once daily for 23 days using a pipette. In the control group, the same amount of vehicle (50% ethanol, 20% propylene glycol, 30% water) was applied to the skin.
각 군의 발모촉진 효과를 육안적으로 평가하기 위해 실험 시작 후 21일 째 마취한 후 모발성장을 관찰하였고 디지털 카메라를 이용하여 발모부위를 촬영하였다. 육안적으로 피부색 및 모발의 밀도를 관찰하여 다음 표 5의 스코어링 기준에 따라 점수화하여 그래프로 나타내었다 (표 5).In order to visually evaluate the effect of promoting hair growth in each group, hair growth was observed after anesthesia on the 21st day after the start of the experiment, and the hair growth area was photographed using a digital camera. The skin color and hair density were visually observed and scored according to the scoring criteria in Table 5 below, and presented as a graph (Table 5).
육안적 발모정도 평가 점수화 기준Criteria for scoring evaluation of gross hair growth
판정 기준Criteria 점수score
전혀 자라지 않음not growing at all 00
제모부위의 30% 미만으로 피부색이 어두워짐
제모부위에서 모발은 자라지 않음Less than 30% of the hair removal area darkens the skin color
Hair does not grow in the epilation area 		1One
제모부위의 30-70% 정도 피부색이 어두워짐
제모부위에서 모발은 자라지 않음About 30-70% of the hair removal area darkens the skin color
Hair does not grow in the epilation area 		22
제모부위의 70% 이상 피부색이 어두워짐
제모부위에서 30% 미만으로 자라남More than 70% of the hair removal area darkens the skin color
Less than 30% growth in the hair removal site 		33
제모부위의 70% 이상 피부색이 어두워짐
제모부위에서 30-70% 정도 자라남More than 70% of the hair removal area darkens the skin color
30-70% growth at the hair removal site 		44
제모부위의 70% 이상 피부색이 어두워짐
제모부위에서 70% 이상 자라남More than 70% of the hair removal area darkens the skin color
More than 70% growth in the hair removal area 		55
제모부위에서 90% 이상 자라남More than 90% of the hair removal site grows 66
실험 종료 후 시험동물을 치사시킨 후, 마우스의 피부조직을 10% NBF에 24시간 이상 상온에서 고정한 후 파라핀으로 포매하여 5 μm 두께의 절편을 준비하고 헤마톡실린 (Hematoxylin)과 에오신 (Eosin)으로 (H&E) 염색하였다. H&E 염색된 조직 절편을 광학현미경으로 모낭조직의 조직학적 변화를 관찰하였다. 광학현미경상에서 모낭의 수량 및 깊이와 진피 및 피하의 두께를 측정하였고 발모 주기를 평가하였다. 모낭의 수는 100배율 광학현미경으로 피하 조직에 존재하는 모낭 수를 계산하였다. 모낭의 깊이와 진피 및 피하의 두께 변화는 스케일 바를 이용하여 Image J 프로그램을 통해 측정하였다.After the test animal was killed, the mouse skin tissue was fixed in 10% NBF at room temperature for more than 24 hours, then embedded in paraffin to prepare a 5 μm thick section, and then treated with hematoxylin and eosin. (H&E) staining. The histological changes of the hair follicle tissue were observed using the H&E-stained tissue sections under an optical microscope. The quantity and depth of hair follicles and the thickness of the dermis and subcutaneous tissue were measured under an optical microscope, and the hair growth cycle was evaluated. For the number of hair follicles, the number of hair follicles present in the subcutaneous tissue was counted using an optical microscope at 100 magnification. The depth of hair follicles and changes in the dermis and subcutaneous thickness were measured through the Image J program using a scale bar.
실시예 12-2: 실험 결과Example 12-2: Experimental results
도포 후 21일 째 육안적으로 발모현상을 관찰하여 스코어링 한 결과 Amuc_1409를 도포한 군은 Vehicle 도포군에 비해 전체 면적 중에 모발이 자란 부위의 비율이 유의하게 증가하였다 (p<0.05, Student's t-test) (도 37).On the 21st day after application, hair growth was visually observed and scored. As a result, the proportion of hair growth in the total area in the group applied with Amuc_1409 was significantly increased in the group applied with Amuc_1409 compared to the group applied with Vehicle (p<0.05, Student's t-test). ) (FIG. 37).
H&E 염색을 통한 조직학적인 검사 결과 Amuc_1409를 도포한 군은 Vehicle 도포군에 비해 피하지방 층에 존재하는 모낭수가 유의하게 증가하였고, 모낭 깊이와 진피 및 피하 조직의 두께가 Vehicle 도포군에 비해 유의하게 두꺼워졌다 (p<0.05, Student's t-test) (도 38).As a result of histological examination through H&E staining, the group applied with Amuc_1409 significantly increased the number of hair follicles in the subcutaneous fat layer compared to the group applied with Vehicle, and the depth of hair follicles and the thickness of the dermis and subcutaneous tissue were significantly thicker than the group applied with Vehicle. lost (p<0.05, Student's t-test) ( FIG. 38 ).
또한 H&E 염색을 통한 조직학적으로 발모주기를 분석한 결과 Amuc_1409를 도포한 군은 Vehicle 도포군에 비해 성장기 단계의 모발 비율이 유의하게 증가하였다 (p<0.05, Student's t-test) (도 39).In addition, as a result of histological analysis of the hair growth cycle through H&E staining, the group applied with Amuc_1409 significantly increased the proportion of hair in the growth phase compared to the group applied with Vehicle (p<0.05, Student's t-test) (FIG. 39).
이를 통해 본 발명의 펩타이드가 모발 재생 및 발모 촉진 효능이 있음을 확인하였다.Through this, it was confirmed that the peptide of the present invention has the effect of promoting hair regeneration and hair growth.
실시예 13: 신규 펩타이드의 피부재생 효능 검증Example 13: Verification of skin regeneration efficacy of novel peptides
본 발명의 펩타이드의 피부 재생능을 확인하기 위하여 사람 피부 각질세포를 사용하였다.In order to confirm the skin regeneration ability of the peptide of the present invention, human skin keratinocytes were used.
사람의 피부 각질 세포주 (HaCaT)는 10% 소태아혈청 (Fetal Bovine Serum, FBS)과 1% 페니실린-스트렙토마이신을 포함하는 DMEM 배지에서 37℃, 5% CO2의 조건으로 배양하였다. 세포밀집도가 80%에 도달하면 6-웰 플레이트에 각 웰 당 4x105 세포의 수만큼 분주한 후 24시간 배양하였다. 24시간 후, Amuc_1409를 30 nM의 농도로 1시간 전처리하고 1시간 후 PBS (phosphate buffered saline)로 2회 세척한 후, 자외선 (UVB) 조사 장치 (CL-1000 UV crosslinker; UVP, USA)를 이용하여 10 mJ/cm2의 자외선을 조사하였다. 자외선 조사 후 혈청이 포함되지 않은 배지로 교환한 다음, 즉시 Amuc_1409를 8시간 처리하였다.Human dermal keratinocytes (HaCaT) were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin under conditions of 37°C and 5% CO2. When the cell density reached 80%, the number of 4x105 cells per well was aliquoted in a 6-well plate and cultured for 24 hours. After 24 hours, Amuc_1409 was pretreated at a concentration of 30 nM for 1 hour and washed twice with PBS (phosphate buffered saline) after 1 hour, using an ultraviolet (UVB) irradiation device (CL-1000 UV crosslinker; UVP, USA). Thus, 10 mJ/cm2 of ultraviolet light was irradiated. After UV irradiation, the medium was replaced with a serum-free medium, and then immediately treated with Amuc_1409 for 8 hours.
피부 노화에 따른 피부 주름과 관련하여, 콜라겐 분해 효소인 MMP-1 및 MMP-3 유전자와 콜라겐 합성 효소인 Col1a1 및 Col3a1 유전자의 발현을 확인하기 위하여 각 웰의 세포에서 트리졸 (Invitrogen, USA)을 이용하여 RNA를 분리한 뒤 nanodrop으로 정량한 후, 각 1 ug의 RNA를 사용하여 cDNA를 합성하였다. 합성된 cDNA에 MMP-1, MMP-3, Col1a1 및 Col3a1 주형 (primer)과 형광 염료인 AccuPower® 2X GreenStar™qPCR Master Mix (BIONEER, Korea)를 첨가한 혼합물을 real-time PCR 기계에서 실시간 중합효소 연쇄반응을 실시하였다.Trizol (Invitrogen, USA) was added to cells in each well to confirm the expression of collagen degrading enzymes MMP-1 and MMP-3 genes and collagen synthesizing enzymes Col1a1 and Col3a1 genes in relation to skin wrinkles caused by skin aging. After separation of RNA using nanodrop, cDNA was synthesized using 1 ug of each RNA. A mixture of MMP-1, MMP-3, Col1a1 and Col3a1 templates (primer) and a fluorescent dye, AccuPower® 2X GreenStar™ qPCR Master Mix (BIONEER, Korea), was added to the synthesized cDNA in a real-time PCR machine with real-time polymerase A chain reaction was carried out.
분석결과, 사람의 피부 각질세포에 UVB를 조사한 경우 콜라겐 분해 효소인 MMP-1과 MMP-3 유전자의 발현이 UVB를 조사하지 않은 군보다 유의하게 증가한 반면, 콜라겐 합성 효소인 Col1a1 및 Col3a1 유전자 발현은 유의하게 감소되어 피부세포의 광노화를 확인할 수 있었다. Amuc_1409를 처리한 세포에서 MMP-1과 MMP-3의 유전자 발현은 UVB를 조사한 대조군에 비하여 유의하게 감소하였으며, Col1a1 및 Col3a1 유전자 발현은 유의하게 증가하여 Amuc_1409이 UVB 조사에 의한 피부세포의 광노화를 억제하는 것을 확인할 수 있었다 (p<0.05, Student's t-test) (도 40).As a result of the analysis, when UVB was irradiated to human skin keratinocytes, the expression of MMP-1 and MMP-3 genes, which are collagen degrading enzymes, was significantly increased compared to the group not irradiated with UVB, whereas the expression of Col1a1 and Col3a1 genes, which are collagen synthetase, was decreased. It was significantly reduced, and photoaging of skin cells could be confirmed. In the cells treated with Amuc_1409, the gene expression of MMP-1 and MMP-3 was significantly decreased compared to the control group irradiated with UVB, and the expression of the Col1a1 and Col3a1 genes increased significantly, so that Amuc_1409 suppressed photoaging of skin cells by UVB irradiation. (p<0.05, Student's t-test) (FIG. 40).
이를 통해, 본 발명의 펩타이드가 피부 노화와 관련된 증상을 억제하고 피부 재생을 촉진하는 효과를 가지는 것을 확인하였다.Through this, it was confirmed that the peptide of the present invention has the effect of suppressing symptoms related to skin aging and promoting skin regeneration.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (15)

  1. 하기 일반식 1 또는 2의 서열을 포함하는 펩타이드:A peptide comprising the sequence of Formula 1 or 2:
    [일반식 1][General formula 1]
    Ala-Val-Ser-X1-X2-X3-X4-X5-X6Ala-Val-Ser-X1-X2-X3-X4-X5-X6
    여기서, X1은 Ser 또는 부존재;Here, X1 is Ser or absent;
    X2는 Ile 또는 부존재;X2 is Ile or absent;
    X3은 Lys 또는 부존재;X3 is Lys or absent;
    X4는 Gly 또는 부존재;X4 is Gly or absent;
    X5는 Ala 또는 부존재; 및X5 is Ala or absent; and
    X6은 Tyr 또는 부존재함,X6 is Tyr or absent;
    [일반식 2][General Formula 2]
    X1-X2-X3-X4-X5-X6-Gly-Ala-TyrX1-X2-X3-X4-X5-X6-Gly-Ala-Tyr
    여기서, X1은 Ala 또는 부존재;where X1 is Ala or absent;
    X2는 Val 또는 부존재;X2 is Val or absent;
    X3은 Ser 또는 부존재;X3 is Ser or absent;
    X4는 Ser 또는 부존재;X4 is Ser or absent;
    X5는 Ile 또는 부존재; 및X5 is Ile or absent; and
    X6은 Lys 또는 부존재함.X6 is Lys or absent.
  2. 제1항에 있어서, 상기 펩타이드는 서열번호 4 또는 5의 아미노산 서열을 포함하는 서열번호 6의 아미노산 서열 중에서 연속하는 3개 내지 150개의 아미노산으로 이루어진 것인, 펩타이드.The peptide of claim 1, wherein the peptide consists of 3 to 150 consecutive amino acids in the amino acid sequence of SEQ ID NO: 6 including the amino acid sequence of SEQ ID NO: 4 or 5.
  3. 제1항에 있어서, 상기 펩타이드는 서열번호 1 내지 7 중 어느 하나의 아미노산 서열로 이루어진 것인, 펩타이드.The peptide of claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 7.
  4. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 포함하는 염증질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating an inflammatory disease comprising the peptide of any one of claims 1 to 3.
  5. 제4항에 있어서, 상기 염증질환은 관절염, 대사성 질환, 간염, 장염, 위염, 위궤양, 식도염, 피부염, 뇌염, 우울증, 불안장애, 인지장애, 기억장애, 퇴행성 뇌질환 및 발달장애로 이루어진 군에서 선택되는 것인, 염증질환 예방 또는 치료용 약학적 조성물.According to claim 4, wherein the inflammatory disease is arthritis, metabolic disease, hepatitis, enteritis, gastritis, gastric ulcer, esophagitis, dermatitis, encephalitis, depression, anxiety disorder, cognitive impairment, memory impairment, degenerative brain disease and developmental disorders from the group consisting of A pharmaceutical composition for preventing or treating an inflammatory disease that is selected.
  6. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 포함하는 조직 재생용 조성물.A composition for tissue regeneration comprising the peptide of any one of claims 1 to 3.
  7. 제6항에 있어서, 상기 조직은 피부, 근육, 모발, 모낭 및 모근 중에서 선택되는 것인, 조직 재생용 조성물.The composition for tissue regeneration according to claim 6, wherein the tissue is selected from skin, muscle, hair, hair follicles and hair roots.
  8. 제6항에 있어서 상기 조직 재생은 피부 재생, 근육조직 재생, 상처 치유, 근력 증진, 탈모 방지, 양모 촉진, 발모 촉진, 손상 모발 개선 및 모발 재생 중에서 선택되는 것인, 조직 재생용 조성물.The composition for tissue regeneration according to claim 6, wherein the tissue regeneration is selected from skin regeneration, muscle tissue regeneration, wound healing, muscle strength enhancement, hair loss prevention, wool promotion, hair growth promotion, damaged hair improvement and hair regeneration.
  9. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 포함하는 염증질환 예방 또는 개선용 조성물로,A composition for preventing or improving inflammatory diseases comprising the peptide of any one of claims 1 to 3,
    상기 조성물은 건강기능식품, 사료, 동물 의약품, 의약외품 또는 화장료 조성물인 것인, 조성물.The composition is a health functional food, feed, veterinary medicine, quasi-drug or cosmetic composition, the composition.
  10. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 포함하는 조직 재생용 조성물로,A composition for tissue regeneration comprising the peptide of any one of claims 1 to 3,
    상기 조성물은 건강기능식품, 사료, 동물 의약품, 의약외품 또는 화장료 조성물인 것인, 조성물.The composition is a health functional food, feed, veterinary medicine, quasi-drug or cosmetic composition, the composition.
  11. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 포함하는, 조직 손상 관련 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of tissue damage-related diseases, comprising the peptide of any one of claims 1 to 3.
  12. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 개체에 투여하는 단계를 포함하는 염증질환 예방 또는 치료방법. A method for preventing or treating an inflammatory disease comprising administering the peptide of any one of claims 1 to 3 to an individual.
  13. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 개체에 투여하는 단계를 포함하는 조직 재생방법.A tissue regeneration method comprising the step of administering the peptide of any one of claims 1 to 3 to an individual.
  14. 제1항 내지 제3항 중 어느 한 항의 펩타이드를 코딩하는 폴리뉴클레오티드.A polynucleotide encoding the peptide of any one of claims 1 to 3.
  15. 제13항의 폴리뉴클레오티드를 포함하는 벡터.A vector comprising the polynucleotide of claim 13 .
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