WO2020046002A1 - Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers - Google Patents

Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers Download PDF

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WO2020046002A1
WO2020046002A1 PCT/KR2019/011037 KR2019011037W WO2020046002A1 WO 2020046002 A1 WO2020046002 A1 WO 2020046002A1 KR 2019011037 W KR2019011037 W KR 2019011037W WO 2020046002 A1 WO2020046002 A1 WO 2020046002A1
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seq
peptide
metabolic
disease
protein
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PCT/KR2019/011037
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French (fr)
Korean (ko)
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박윤정
정종평
이주연
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주식회사 나이벡
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Priority claimed from KR1020190105917A external-priority patent/KR102244161B1/en
Application filed by 주식회사 나이벡 filed Critical 주식회사 나이벡
Priority to EP23151983.6A priority Critical patent/EP4218788A1/en
Priority to DK19855142.6T priority patent/DK3862015T3/en
Priority to US17/272,010 priority patent/US20210340182A1/en
Priority to CN201980063987.2A priority patent/CN112912093A/en
Priority to EP19855142.6A priority patent/EP3862015B1/en
Publication of WO2020046002A1 publication Critical patent/WO2020046002A1/en

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    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to novel uses of peptides that inhibit the function and expression of multiple disease biomarkers, and more particularly, in relation to HDAC5, GDF15, ATF3, which are inflammatory and metabolic disease biomarkers, phosphorylation of HDAC5 protein, GDF15 Inflammatory, metabolic or fibrotic disease comprising as an active ingredient any one or more peptides represented by the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 8 having the function of inhibiting the expression of the protein, and / or ATF3 protein It relates to a pharmaceutical composition for treatment or prophylaxis.
  • Metabolic diseases including obesity and type 2 diabetes, are proliferating not only in Korea but also throughout the world. These metabolic diseases are known to be associated with chronic inflammatory responses (Nature, 2017, 542, 177). This phenomenon is observed systemically or tissue by tissue. Obesity in particular has been shown to chronically low levels of inflammation, which has been found to be initiated and controlled by the accumulation and change of inflammatory cells in adipose tissue. Chronic adipose tissue inflammation induces insulin resistance of adipocytes and inhibits the surplus energy accumulation function of adipose tissue.
  • Non-alcoholic fatty liver disease is a disease caused by accumulation of fat in the body, especially liver cells (more than 5%) due to obesity, diabetes, and lipid metabolism, regardless of drinking.
  • NASH non-alcoholic steatohepatitis
  • chromosomal DNA of eukaryotes is wound around a central protein called histone (Histone) and condensed to take the basic structure called nucleosomes.
  • Histone histone
  • the nucleosome structure is aggregated to form a chromatin structure.
  • the post-translational modification of histones is closely related to the structure of the chromatin structure.
  • acetylation, methylation, phosphorylation, ubiquitination and the like are known.
  • histone acetylation is related to histone acetylase (HAT) and histone deacetylation.
  • HDAC histone deacetylase
  • HDAC5 normally binds to nucleosomes of the inflammatory cytokine gene, and when exposed to proinflammatory factors such as lipopolysaccharide and TNF- ⁇ , HDAC5 phosphorylates and binds to protein chaperone 14-3-3. These conjugates exit the cytoplasm from the nucleus. After acetylation of histones begins, DNA structure is released, and mediators, such as NF-kB, bind to the genes of proinflammatory cytokines. Therefore, if inflammatory cytokines cannot be expressed if they are present in the nucleus by inhibiting phosphorylation of HDCA5, no inflammatory reaction occurs.
  • HDAC inhibitors have been developed, butyric acid (J. Biol. Chem., 1979, 254, 1716-1723) with cell cycle arrest, normalization and differentiation of transformed cells, cell cycle arrest, morphogenesis Trichostatin A, a metabolite of the microorganisms having it (Cancer Res., 1987, 47, 3688-3691, Exp. Cell Res., 1988, 177, 122-131, J. Biol. Chem., 1990, 265, 17174-17179 And Trapoxin (J. Antibiotics, 1990, 43, 1524-1534, J. Biol. Chem., 1993, 268, 22429-22435), which are metabolites of microorganisms having a cell proliferation inhibitory effect, and the like.
  • Activating transcription factor 3 (ATF3) protein is a component of the transcriptional factor mammalian activation transcription factor / cAMP responsive element-binding (CREB) protein. Induced by a variety of signals caused by a number of factors involved, it has been known to be involved in the complex process of intracellular stress response.
  • ATF3 protein acts as an activator or repressor of known target genes, with over 20 potential target genes of ATF3 known to date, including AdipoR1, AdipoR2, bNIP3, Cdc25A , CCL2, CCL4, Cyclin D1, FN-1, GLUT4, HIF-2 ⁇ , IFN- ⁇ IL-1 ⁇ , TWIST1, p53, p73, PDX-1, adiponectin and the like.
  • NF- ⁇ B in most cells including fibroblasts or epithelial cells, as well as in immune cells such as macrophage, mast cells, T cells, dendritic cells, etc.
  • Various pathways such as JNK, Erk, p38 and PKC induce ATF3, and induced ATF3 regulates transcription of various genes, resulting in apoptosis, cell proliferation, cell motility, DNA repair ( It is known to be involved in DNA repair and metabolism, among which NF- ⁇ B is also involved in inducing inflammatory responses.
  • ATF3 protein is also known as a biomarker of nonalcoholic liver disease caused by metabolic abnormalities such as diabetes and obesity (Kim et al., Journal of Hepatology 2017 vol. 67, 349-359).
  • the inflammatory response induced by ATF3 can be suppressed by a substance that modulates the expression or function of ATF3 protein, but until now, no inhibitor for ATF3 protein has been developed.
  • Growth differentiation factor 15 is a protein belonging to the transforming growth factor ⁇ (TGF- ⁇ ) superfamily, namely macrophage inhibitory cytokine 1, placental TGF- ⁇ prostate-derived factor (PDF), and nonsteroidal anti-inflammatory drug- Also called an activator gene.
  • GDF15 protein is known to be expressed in the liver of NAFLD patients, and as the progress of fibrosis increased, the expression level of GDF15 protein in liver tissue increased.
  • Expression of related proteins (SMAD2, SMAD3) was increased (Koo et al., Liver International. 2018; 38: 695-705).
  • GDF15 protein plays a role in developing cirrhosis in NAFLD and also acts as a biomarker. Although it is possible to reduce the progression of NAFLD by developing a substance that can inhibit the expression of GDF15, inhibitors to the GDF15 protein have not been developed until now.
  • the present inventors have an effect of inhibiting the phosphorylation of HDAC by the peptides previously developed by the present inventors, and also exhibit the effect of inhibiting the expression of liver disease markers ATF3 and GDF15, thereby generating various inflammatory cytokines. It was confirmed that it can be suppressed, and furthermore, the present invention was completed by confirming that the peptides were administered in arthritis animal models, colitis animal models, and fatty liver animal models, respectively, to show a therapeutic effect of the disease.
  • the present invention is a pharmaceutical for treating or preventing inflammation, metabolic or fibrotic disease comprising a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient To provide a composition.
  • the present invention also provides a use of the peptide for the treatment of inflammatory, metabolic or fibrotic disease.
  • the invention also provides the use of said composition for the manufacture of a medicament for the treatment of an inflammatory, metabolic or fibrotic disease.
  • the present invention also provides a method for preventing and treating an inflammatory, metabolic or fibrotic disease, comprising administering the peptide to a subject in need of treatment for an inflammatory, metabolic or fibrotic disease.
  • 1A shows the results of microarrays of phosphorylated proteins.
  • Figure 1b is the result of confirming the Western blot expression of the phosphorylated HDAC5 protein according to the change in the concentration of the peptide of SEQ ID NOS: 1 to 3.
  • Figure 1c is a Western blot results for comparing the phosphorylation inhibitory effect of the HDAC5 protein by the peptides of SEQ ID NOS: 1 to LMK, SAHA, a positive control.
  • Figure 2a is a result showing the hind paw swelling (%) according to the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • Figure 2b is a result showing the hind paw swelling inhibition (%) according to the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • Figure 3a is a three-dimensional image of microcities after administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • FIG. 3B shows bone volume (BV), bone volume / tissue volume (BV / TV), bone surface areas adjusted to BV (BS / BV), trabecular thickness (Tb. Th) is the result of analysis.
  • Figure 4a is a histogram of the joint observed under the microscope after administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • Figure 4b is a result showing the inflammation index after SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • Figure 5 shows the results of the concentration of TNF-a and IL-6 in the blood following the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
  • Figure 6a is a result of observation with a naked eye colon 8 days after administration of SEQ ID NO: 1 in colitis-derived animal model.
  • Figure 6b is the result of measuring the change in the length of the colon according to the administration of SEQ ID NO: 1 in colitis-derived animal model
  • Figure 6c is a result of measuring the expression of p40, TNF- ⁇ , IL-12 according to the administration of SEQ ID NO: 1 in colitis-derived animal model by ELISA.
  • Figure 7 shows the results confirmed by Western blot changes in the expression level of the liver disease markers GDF15 and ATF3 protein by the peptide treatment of SEQ ID NOS: 1-4.
  • FIG. 8 is a photograph observing the effect of reducing liver hypertrophy and subcutaneous fat according to the administration of SEQ ID NO: 1 in a fatty liver-derived animal model.
  • Figure 9a is a result of measuring the change in the length of the colon 10 days after the administration of SEQ ID NO: 1-4 in the DSS-induced inflammatory bowel disease animal model.
  • Figure 9b is the result of observation by H & E staining intestinal villus 10 days after the administration of SEQ ID NOS: 1-4 in DSS-induced inflammatory bowel disease animal model.
  • Figure 10a is a result of observing the inflammation and fat reduction effect of liver tissue in liver tissue according to SEQ ID NO: 1-6 in non-alcoholic steatohepatitis animal model through H & E staining.
  • Figure 10b is a result of observation through the Sirius red staining effect on the reduction of fibrosis in liver tissue following the administration of SEQ ID NO: 1-6 in non-alcoholic steatohepatitis animal model.
  • Figure 10c is a result confirmed by Masson trichrome staining the effect of reducing the fibrosis in liver tissue according to SEQ ID NO: 1-6 administration in the non-alcoholic steatohepatitis animal model.
  • 10d is a NAS score result for each group following the administration of SEQ ID NOs: 1-6 in a nonalcoholic steatohepatitis animal model.
  • the peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 8 inhibits the phosphorylation of HDAC5 against HDAC5, GDF15, ATF3 biomarkers of inflammation, metabolic or fibrotic disease, and inhibits the expression of GDF15 and ATF3 proteins. It has been shown to reduce the production of inflammatory cytokines and to reduce the associated nonalcoholic liver disease, fatty liver and subcutaneous fat caused by arthritis, periodontitis, atopy, inflammatory colitis, diabetes and obesity.
  • the present invention provides a pharmaceutical composition for treating or preventing inflammatory, metabolic or fibrotic diseases comprising a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient in one aspect. It is about.
  • the present invention relates to the use of the peptide for the treatment of inflammatory, metabolic or fibrotic disease.
  • the present invention relates to the use of said composition for the manufacture of a medicament for the treatment of an inflammatory, metabolic or fibrotic disease.
  • the present invention relates to a method for preventing and treating an inflammatory, metabolic or fibrotic disease comprising administering the peptide to a subject in need thereof.
  • the peptide is not only represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 8, but has at least 60% homology, at least 70% homology, or 80% It may have a homology or more than 90%, or may have a homology of 95% or more, for example, some amino acids (eg, 1 to 1) of the amino acid sequence of the peptide of SEQ ID NO: 1-8 6 amino acids) is replaced with another amino acid, or a portion (for example, 1 to 6 amino acids) of amino acids added or deleted, resulting in the peptide does not significantly affect the desired structure or function, or
  • the words selected from the group consisting of SEQ ID NOs: 1 to 8 It can be used to achieve the object of the appropriately substitute a single amino acid sequence invention will be apparent to those skilled in the art.
  • the inflammatory, metabolic or fibrotic disease may be a disease that is caused directly or indirectly by overexpression of the HDP5 protein superphosphorylated GDF15 protein and / or overexpression state of ATF3 protein in vivo.
  • the peptide may be characterized by inhibiting phosphorylation of HDAC5 protein, expression of GDF15 protein and / or expression of ATF3 protein.
  • the inflammatory, metabolic or fibrotic disease may be at least one disease selected from the group consisting of arthritis, inflammatory colitis, ulcerative enteritis, Crohn's disease, hepatitis, fatty liver, and obesity, but is not limited thereto.
  • Hyperphosphorylation of HDAC5 protein may be used for the treatment and prevention of any inflammatory, metabolic or fibrotic disease associated with overexpression of GDF15 protein and / or overexpression of ATF3 protein.
  • the hepatitis may be characterized by non-alcoholic steatohepatitis due to diabetes and / or obesity.
  • the fibrotic disease is characterized in that at least one selected from the group consisting of liver cirrhosis due to fibrosis, pulmonary fibrosis, obstructive pulmonary disease, heart failure, arteriosclerosis, chronic kidney failure, diabetes, keloids caused by postoperative sequelae, etc.
  • the hyperphosphorylation refers to a state in which phosphorylation of the protein is increased compared to an object that does not develop the disease, and the overexpression of the protein is compared to an object that does not develop the disease. Refers to an increased state of.
  • the pharmaceutical composition of the present invention may further include one or more pharmaceutically acceptable carriers in addition to the peptide.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention is formulated into any one formulation selected from the group consisting of injections, oral formulations, patches, solutions, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal administrations and suppositories. It may be, but is not limited thereto. These formulations may be prepared by conventional methods used in the art for formulation or by methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA and formulated into various formulations depending on the individual disease or component. Can be.
  • the therapeutic pharmaceutical composition may be formulated to further contain a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable adjuvant is an excipient, diluent, buffer, antimicrobial preservative, surfactant, antioxidant, thickener and viscosity modifier.
  • a pharmaceutically acceptable adjuvant is an excipient, diluent, buffer, antimicrobial preservative, surfactant, antioxidant, thickener and viscosity modifier.
  • the pharmaceutically acceptable adjuvant is an excipient, diluent, buffer, antimicrobial preservative, surfactant, antioxidant, thickener and viscosity modifier.
  • composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient.
  • the range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • the daily dosage of the peptides of SEQ ID NOS: 1 to 8 of the present invention is about 1 to 8 100 mg / kg, preferably 5 to 50 mg / kg, and may be administered once a day or divided into 2-3 times a week, but is not limited thereto.
  • the dosage regimen and dosage will vary depending on the age, weight and response of the individual patient. Appropriate dosage regimens and dosages can be readily selected by those of ordinary skill in the art that naturally consider such factors.
  • Peptides of SEQ ID NOS: 1 to 8 were sequentially synthesized from the N terminus by F-moc solid phase peptide synthesis.
  • the synthesized peptide sequences were cleaved from the resin, washed, lyophilized and separated and purified by liquid chromatography. Purified peptides were checked for molecular weight using MALDI-TOF analysis.
  • RAW 264.7 cells were starvated in 0.5% FBS for 2 hours, treated with 200 ⁇ M of SEQ ID NO: 1 peptide for 1 hour, followed by LPS (1 ⁇ g / mL) for 30 minutes at 37 ° C. I was.
  • Cell lysates of untreated cells (NT), LPS-treated cells, SEQ ID NO: 1 and LPS-treated cells were applied to a Phospho Explorer Antibody Microarray from Full Moon Biosystems.
  • the phosphor microarray can recognize 1318 antibodies related to 30 signaling systems. Each antibody was coated on standard-size coated glass microscope slides, and the slides were scanned with a GenePix 4100A scanner (Axon Instrument, Foster City, CA, USA). Data was analyzed with Genowiz 4.0TM (Ocimum Biosolutions, India). Phosphorylation ratio was calculated as follows.
  • phosphorylation ratio (phosphorylation level of the experimental group / non-phosphorylation level of the experimental group) / (phosphorylation level of the control group / non-phosphorylation level of the control group)
  • Proteins were lysed using RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibiotr and phosphatase inhibitor. Proteins were quantified by BCA protein assay and the expression levels of HDAC 5 and p65 protein were confirmed by western blot. In the case of Western blot, the sample was loaded in an 8% SDS PAGE gel with the size marker in the same amount and electrophoresed for about 2 hours, and then transferred to the nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and reacted with 1st antibody overnight at 1: 1000 ratio. Thereafter, the membrane was washed with TBST containing 0.1% tween-20, and reacted with HRP-attached 2nd antibody for 1 hour, and then chemiluminescence was confirmed by ECL substrate.
  • RIPA lysis buffer 25 mM Tris-HC
  • HDAC5 phosphorylation inhibition was better than LMK.
  • HDAC5 protein (Abcam, MA, USA) is dissolved in sodium acetate buffer (pH 4.5), and N-hydroxysuccinimide (NHS) and N-ethyl-N0- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) are crosslinkers.
  • NHS N-hydroxysuccinimide
  • EDAC N-ethyl-N0- (3-dimethylaminopropyl) -carbodiimide hydrochloride
  • Peptides of SEQ ID NO: 1 to 8 were reacted at various concentrations (15.625, 31.25, 62.5, 125, 250, 500 nM) at a rate of 30 ⁇ L / min, and the dissociation reaction was maintained for 5 minutes.
  • Rate constants for association (ka), dissociation (kd), and dissociation constant (KD) were calculated using the BIAevaluation software using a 1: 1 binding model.
  • Table 1 below shows the analysis results of Biacore showing the binding affinity between the peptides of SEQ ID NOs. 1 to 8 and the HDAC5 protein.
  • the peptides of SEQ ID NOs: 1 to 8 had KD values of about 10 ⁇ 7 , and the mismatch peptides showed about 10 ⁇ 4 . Since the smaller the KD value, the better the binding affinity, the peptides of SEQ ID NOs: 1 to 8 had excellent binding ability to the HDAC5 protein and could be predicted to reduce phosphorylation while binding to the HDAC5 protein.
  • bovine collagen type II was injected into a rat muscle mixture of 0.5 mL of 0.1 M acetic acid and 0.5 mL of Freund's incomplete adjuvant.
  • collagen induced arthritis (CIA) rats were prepared by further inoculating 250 ⁇ g of bovine collagen type II in 0.5 mL of 0.1 M acetic acid and 0.5 mL of Freund's incomplete adjuvant.
  • hind paw swelling In hind paw swelling (%), the CIA-induced group increased hind paw swelling in comparison with the normal group, and CIA-induced group injected with Etanercept (Enbrel®), a positive control, showed hind paw swelling between %) Decreased ( P ⁇ 0.05).
  • hind paw swelling In the CIA-induced group injected with SEQ ID NO: 1, hind paw swelling (%) decreased in a concentration-dependent manner.
  • hind paw swelling In the CIA-induced group injected with high concentration (30 mg / kg) of SEQ ID NO: 1 peptide, hind paw swelling (%) decreased between 38 and 49 days ( P ⁇ 0.05).
  • Microcomputed tomography was measured using SkyScan 1172 (SkyScan, Aartselaar, Belgium) to observe the degree of bone damage in the hind paw.
  • Total volume (TV; mm 3 ), bone volume (BV; mm 3 ), bone volume fraction (BV / TV;%) were analyzed using CTAn software. Rats were sacrificed on day 57 for histological analysis. Both hind paws and joints were harvested and fixed in 10% buffered formalin and demineralized in 30% citrate-buffered formic acid at 4 ° C for 3 days.
  • Paraffin blocks were made and then sectioned to a thickness of 4 ⁇ m, stained with hematoxylin and eosin (HE), and pathological scores were evaluated by light microscopy.
  • Joint inflammation index was measured based on the following criteria (Shiozawa et al., J Clin Invest. 1997; 99 (6): 1210-1216). 0, normal synovium; 1, synovial membrane hypertrophy and cell infiltrates; 2, pannus and cartilage erosion; 3, major erosion of cartilage and subchondral bone; and 4, loss of joint integrity and ankylosis.
  • BV bone volume
  • BV / TV bone volume / tissue volume
  • Tb.Th trabecular thickness
  • BV and BV / TV are parameters that can be compared to preserve the bone in samples of different sizes.
  • BS / BV is a parameter that indicates the bone surface is lost by erosion.
  • Tb.Th is a parameter that is contradictory to periarticular osteopenia by arthritis (Parfitt AM et al., J Bone Miner Res. 1987; 2 (6): 595-610).
  • the microscopic examination of the tissues revealed that the CIA-induced group showed chronic inflammation in synovial tissue and showed pannus formation, destruction of joints and bones, whereas the CIA-induced group injected with the peptide of SEQ ID NO: 1 concentration-dependent inflammation. The decrease of was confirmed.
  • the CIA-induced group injected with Etanercept showed similar results to the CIA-induced group injected with low concentration SEQID 1 (FIG. 4A).
  • the CIA-induced group (1.5 ⁇ 1.17) injected with the peptide of SEQ ID NO: 1 showed significantly lower inflammation index than the CIA-induced group (3.3 ⁇ 0.67; p ⁇ 0.01).
  • the CIA-induced group (2.3 ⁇ 1.16) injected with Etanercept was not significantly lower than the CIA-induced group. Therefore, the peptide of SEQ ID NO: 1 was found to have a higher effect of inhibiting the infiltration of inflammatory cells and reducing arthritis symptoms in CIA-induced rats than Etanercept (FIG. 4B).
  • the concentration of TNF- ⁇ was 128 pg / ml and IL-6 was 109 pg / ml.
  • the concentration of TNF- ⁇ was 40.3 pg / mL.
  • IL-6 concentration was 35.2 pg / mL, and when etanercept was injected, TNF- ⁇ concentration was 39 pg / mL and IL-6 concentration was 24 pg / mL (Fig. 5).
  • the peptide of SEQ ID NO: 1 was found to have an effect of reducing blood levels of inflammatory cytokines TNF- ⁇ and IL-6 to a similar degree to etanercept.
  • the peptides of SEQ ID NOs: 1 to 8 can be predicted to inhibit the phosphorylation of HDAC5, thereby inhibiting the production of inflammatory cytokines, thereby exhibiting anti-inflammatory activity and therapeutic effect of arthritis.
  • TNBS 2,4,6-trinitro benzene sulfonic acid
  • TNBS 50% (v / v) aqueous ethanol solution
  • Rat body weight was measured at 9:00 am daily, and colon length was measured using a vernier caliper (Mitutoyo, Japan) from the cecum to the anus, at the expense of the rat.
  • the colon of the group (TNBS) that induced colitis by TNBS injection was significantly reduced compared to the normal group, but the group injected with the peptide of SEQ ID NO: 1 Length increased similarly to normal group.
  • the mucosal tissue is collected from the muscle layer of the large intestine and the mucosal tissue is weighed, and 10 mg of the mucosal tissue is triple-washed with a buffer (50 mM Tris-HCl, pH 8.0). , Homogenized by dissolving in 150 mM NaCl, 0.1% sodium deoxycholate and 1 mM phenylmethysulfonyl fluoride). Then, p40, TNF- ⁇ , IL-12 were quantified using an ELISA kit (R & D, Minneapolis, MN, USA).
  • Proteins were lysed using RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Proteins were quantified by BCA protein assay and the expression levels of GDF15, ATF3, FAS and tubulin proteins were confirmed by western blot. In the case of Western blot, the sample was loaded in an 8% SDS PAGE gel with the size marker in the same amount and electrophoresed for about 2 hours, and then transferred to the nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and reacted with 1st antibody overnight at 1: 1000 ratio. After the membrane was washed with TBST containing 0.1% tween-20 and reacted with HRP-attached 2nd antibody for 1 hour, the chemiluminescence was confirmed by ECL substrate.
  • RIPA lysis buffer 25 mM Tris-HCl pH
  • a non-alcoholic hepatitis-induced animal model induced by a high fat diet was used.
  • Male C57BL / 6 mice (7 weeks old) were acclimated for 1 week, and 20 animals were controlled in normal diet, HFD induced obesity (DIO), and peptide low dose (LD SEQ ID NO: 1), respectively.
  • LD SEQ ID NO: 1 peptide low dose
  • 15mg / kg or high doses (HD SEQ ID NO: 1, 30mg / kg) were injected into each of the 5 groups per experimental group, such as the high-fat diet group was injected and fed to a high-fat diet or normal diet for 7 weeks.
  • the high-fat diet was fed a 60% high fat diet (Dyets, Inc). During the seven-week breeding, body weight and feed intake were measured twice a week. After breeding for seven weeks, animals were sacrificed on the 50th day, and liver and adipose tissues were collected and compared in size.
  • the SEQ ID NO: 1 peptide has an effect of treating liver hypertrophy and fatty liver, and also has an effect of suppressing obesity by reducing an increase in subcutaneous fat.
  • DSS was induced with inflammatory bowel disease and simultaneously injected with the synthetic peptide. Inflammation was induced in mice using 5% DSS as drinking water for 10 days, and at the same time, IP injection of synthetic peptides was performed. Normal, Defect, positive control (SAHA), immunoregulatory control (anti-TNF-a antibody), SEQ ID NO: 1-4 peptides were treated. After 10 days, after sacrifice by CO2 over-breathing, colon was secured and the length was compared (FIG. 9A). The tissue was fixed and treated to prepare a paraffin block, and H & E staining was performed to check whether the intestinal vill was damaged.
  • MCD Methionine / Choline Deficient diet mouse model
  • Male C57BL / 6J mice were used and non-alcoholic steatohepatitis was induced in a manner of self-leveling for 8 weeks with Methionine / Choline Deficient (MCD) diet.
  • MCD Methionine / Choline Deficient
  • the peptides of SEQ ID NOS: 1-6 were administered once daily by subcutaneous administration, and a total of 12 administrations were performed every three days. All animals were weighed twice weekly.
  • Animal necropsy and liver were secured and fixed in neutral buffered formalin solution. Paraffin blocks were prepared to confirm fat accumulation in liver tissues through H & E staining, and to determine the degree of fibrosis, and NAS score was evaluated.
  • NAS Score is a measure of ballooning degeneration, lobular inflammation, and steatosis. NAS score was also reduced by more than 50% in the peptide-treated group was confirmed that the non-alcoholic fatty liver is improved (Fig. 10d)
  • Peptides according to the present invention by inhibiting the phosphorylation of Histone deacetylase (HDAC), GDF15 protein expression, ATF3 protein expression inhibition, etc. to the biomarkers related to inflammatory and metabolic diseases, inflammatory cytokine (inflammatory cytokine) It can reduce the production of, it is effective in the prevention and treatment of metabolic diseases.
  • HDAC Histone deacetylase
  • GDF15 protein expression GDF15 protein expression
  • ATF3 protein expression inhibition etc.

Abstract

The present invention relates to a novel use of a peptide for inhibiting the functions and expressions of multiple disease biomarkers, and more specifically, to a pharmaceutical composition for treating or preventing inflammation, metabolic diseases or fibrotic diseases, the composition comprising, as an active ingredient, any one or more peptide among peptides which are represented by the amino acid sequences of SEQ ID NOs: 1 to 8, and which, for histone deacetylase 5 (HDAC5), GDF15 and ATF3 which are biomarkers associated with inflammation, metabolic diseases or fibrotic diseases, have the functions of inhibiting HDAC5 phosphorylation, inhibiting the expression of the GDF15 protein and inhibiting the expression of the ATF3 protein. The peptide, according to the present invention, enables the triple-blocking of the biomarkers associated with inflammation, metabolic diseases or fibrotic diseases by inhibiting HDAC phosphorylation, inhibiting the expression of the GDF15 protein and inhibiting the expression of the ATF3 protein, and thus the production of inflammatory cytokine may be reduced, and thus the peptide is effective in preventing and treating inflammatory, metabolic or fibrotic diseases.

Description

다중의 질환 바이오마커의 기능 및 발현을 억제하는 펩타이드의 신규한 용도Novel use of peptides to inhibit function and expression of multiple disease biomarkers
본 발명은 다중의 질환 바이오마커의 기능 및 발현을 억제하는 펩타이드의 신규한 용도에 관한 것으로, 더 상세하게는 염증 및 대사성 질환 바이오마커인 HDAC5, GDF15, ATF3와 관련하여, HDAC5 단백질의 인산화, GDF15 단백질의 발현, 및/또는 ATF3 단백질의 발현을 억제하는 기능을 가지고 있는 서열번호 1 내지 서열번호 8의 아미노산 서열로 표시되는 펩타이드 중 어느 하나 이상의 펩타이드를 유효성분으로 포함하는 염증, 대사성 또는 섬유화 질환의 치료 또는 예방용 약학적 조성물에 관한 것이다.The present invention relates to novel uses of peptides that inhibit the function and expression of multiple disease biomarkers, and more particularly, in relation to HDAC5, GDF15, ATF3, which are inflammatory and metabolic disease biomarkers, phosphorylation of HDAC5 protein, GDF15 Inflammatory, metabolic or fibrotic disease comprising as an active ingredient any one or more peptides represented by the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 8 having the function of inhibiting the expression of the protein, and / or ATF3 protein It relates to a pharmaceutical composition for treatment or prophylaxis.
비만, 제 2형 당뇨병을 포함한 대사 질환 (metabolic disease)은 우리나라 뿐만 아니라 세계적으로 급증하고 있는데, 이러한 대사 질환들은 만성적인 염증반응과 관계가 있다고 알려져 있다 (Nature, 2017, 542, 177). 이러한 현상은 전신적으로 또는 조직별로 관찰되고 있다. 특히 비만은 만성적으로 낮은 수준의 염증상태를 보이고 있으며, 이는 지방조직 내 염증세포의 축적 및 변화에 의해 시작되고 조절됨이 밝혀졌다. 만성적인 지방 조직 염증은 지방세포의 인슐린 저항성을 유도하고, 지방조직의 잉여 에너지 축적 기능을 저해한다. Metabolic diseases, including obesity and type 2 diabetes, are proliferating not only in Korea but also throughout the world. These metabolic diseases are known to be associated with chronic inflammatory responses (Nature, 2017, 542, 177). This phenomenon is observed systemically or tissue by tissue. Obesity in particular has been shown to chronically low levels of inflammation, which has been found to be initiated and controlled by the accumulation and change of inflammatory cells in adipose tissue. Chronic adipose tissue inflammation induces insulin resistance of adipocytes and inhibits the surplus energy accumulation function of adipose tissue.
또한, 알콜성 지방간은 우리나라에 흔하며, 알콜 섭취로 인해 간에서 지방합성이 촉진되고 정상적인 에너지 대사가 이루어지지 않음으로써 발병하며, 알콜 섭취 정도에 따라 간염이나 간경변으로 발전될 수 있다. 비알코올성 지방간질환 (Non-alcoholic fatty liver disease, NAFLD)은 음주와 관계없이 비만, 당뇨, 지질대사이상 등으로 인해 체내, 특히 간세포 내 지방이 축적(5% 이상)되어 생기는 질환으로 단순지방 축적에서부터 지방간(steatosis), 간세포의 염증반응(hepatocellular inflammation)을 나타내는 비알콜성 지방간염(non-alcoholic steatohepatitis, NASH), 진행 섬유화증(advanced fibrosis) 및 간경변(cirrhosis)까지 포함하는 넓은 범위의 질환을 의미한다. In addition, alcoholic fatty liver is common in Korea, it is caused by the ingestion of fatty liver in the liver due to alcohol intake and normal energy metabolism does not occur, depending on the degree of alcohol intake can develop hepatitis or cirrhosis. Non-alcoholic fatty liver disease (NAFLD) is a disease caused by accumulation of fat in the body, especially liver cells (more than 5%) due to obesity, diabetes, and lipid metabolism, regardless of drinking. Refers to a wide range of diseases including steatosis, non-alcoholic steatohepatitis (NASH), which represents hepatocellular inflammation, advanced fibrosis, and cirrhosis do.
현재 알콜성 또는 비알콜성 지방간 환자에게 사용되고 있는 치료제는 크게 두 가지로 분류되며 첫째로 위험인자의 교정을 통해 지방간을 치료 및 개선하는 약제인 비만치료제(orlistat), 인슐린저항치료제(metformin, pioglitazone, rosiglitazone), 고지혈증치료제(clofibrate, gemfibrozil, bezafibrate, atorvastatin,simvastatin) 등과, 두 번째로 지방간의 위험인자 교정과는 독립적으로, 이미 손상된 간세포 및 간기능 회복을 위한 약물로서 간세포 보호제(ursodeoxycholic acid 및 taurine), 항산화제 (vitamine E) 및 nutritional supporter(lectin, betaine, N-acetylcystein)등이 있으나, 그 치료 효과는 미미한 실정이다.Currently, there are two types of therapeutic agents used in alcoholic or non-alcoholic fatty liver patients. Firstly, orlistat, insulin resistance (metformin, pioglitazone, rosiglitazone), antihyperlipidemic drugs (clofibrate, gemfibrozil, bezafibrate, atorvastatin, simvastatin), and secondly, hepatoprotective agents (ursodeoxycholic acid and taurine) as agents for restoring hepatocellular and hepatic function that are already damaged, independent of fatty liver risk factor correction. , Antioxidants (vitamine E) and nutritional supporters (lectin, betaine, N-acetylcystein), but the therapeutic effect is insignificant.
한편, 진핵생물의 염색체 DNA는 히스톤 (Histone)이라는 중심 단백질에 감겨 응축되어 뉴클레오솜이라 불리는 기본 구조를 취한다. 또한, 그 뉴클레오솜 구조가 집합함으로써 크로마틴 구조를 형성하고 있다. 이 크로마틴 구조의 구성에는 히스톤의 번역 후 수식 (post translational modification)이 밀접하게 관계하고 있고, 그 번역 후 수식으로서, 아세틸화, 메틸화, 인산화, 유비퀴틴화 등이 알려져 있다. 그 중, 히스톤의 아세틸화 (acetylation)와 탈아세틸화 (deacetylation) 간의 균형은 유전자 발현에 매우 중요한 역할을 하고 있으며, 히스톤의 아세틸화는, 히스톤 아세틸화 효소(histone acetylase, HAT)와 히스톤 탈아세틸화 효소(histone deacetylase, HDAC)에 의해 가역적으로 제어된다. HDAC를 저해하면 HAT에 의한 히스톤의 아세틸화가 항진되고, 히스톤으로부터 유전자가 풀어지면서 유전자의 발현이 시작된다. HDAC은 면역반응, 세포주기, 분화, 사멸과 같은 다양한 생물학적 과정에 관여한다고 알려져 있다. 그 중 HDAC5는 class II HDAC (HDAC 4-7, 9, 10)에 속하고, class II HDAC은 조직 선택성이 있으며 세포내 신호 단백질인 p53, NF-κB, p65, c-jun transcription factors, JAK/STAT의 유전자를 탈아세틸화하여 전사를 조절하며, 이들 세포내 신호단백질은 다양한 염증 사이토카인의 생산에 관련된 중요한 매개체이다 (Gene. 2005;363:15-23). On the other hand, chromosomal DNA of eukaryotes is wound around a central protein called histone (Histone) and condensed to take the basic structure called nucleosomes. In addition, the nucleosome structure is aggregated to form a chromatin structure. The post-translational modification of histones is closely related to the structure of the chromatin structure. As the post-translational modifications, acetylation, methylation, phosphorylation, ubiquitination and the like are known. Among them, the balance between acetylation and deacetylation of histones plays a very important role in gene expression, and histone acetylation is related to histone acetylase (HAT) and histone deacetylation. Reversibly controlled by histone deacetylase (HDAC). Inhibition of HDAC promotes acetylation of histones by HAT and releases genes from histones to begin expression of genes. HDACs are known to be involved in a variety of biological processes such as immune response, cell cycle, differentiation, and death. Among them, HDAC5 belongs to class II HDAC (HDAC 4-7, 9, 10), class II HDAC is tissue selective and intracellular signal proteins p53, NF-κB, p65, c-jun transcription factors, JAK / Deacetylation of the genes of STATs regulates transcription, and these intracellular signaling proteins are important mediators involved in the production of various inflammatory cytokines (Gene. 2005; 363: 15-23).
HDAC5는 평상시에 염증성 사이토카인 유전자의 뉴클레오좀에 결합되어 있다가, 리포폴리사카라이드, TNF-α와 같은 염증유발 인자에 노출되게 되면, HDAC5가 인산화되면서 protein chaperone 14-3-3과 결합하고, 이들 결합체는 핵으로부터 세포질로 나가게 된다. 이후 히스톤에 아세틸레이션이 시작되면서, DNA구조가 풀리고, NF-kB와 같은 매개체가 염증유발 사이토카인의 유전자에 결합되면서, 유전자의 발현이 시작된다. 따라서, HDCA5의 인산화를 억제함으로써 핵내에 존재하게 해준다면 염증성 사이토카인이 발현될 수 없으므로 염증반응이 일어나지 않는다. HDAC5 normally binds to nucleosomes of the inflammatory cytokine gene, and when exposed to proinflammatory factors such as lipopolysaccharide and TNF-α, HDAC5 phosphorylates and binds to protein chaperone 14-3-3. These conjugates exit the cytoplasm from the nucleus. After acetylation of histones begins, DNA structure is released, and mediators, such as NF-kB, bind to the genes of proinflammatory cytokines. Therefore, if inflammatory cytokines cannot be expressed if they are present in the nucleus by inhibiting phosphorylation of HDCA5, no inflammatory reaction occurs.
HDAC 저해제들이 개발되었으며, 세포 주기 정지 작용, 형질 전환 세포의 정상화 및 분화 작용 등을 갖는 부티르산(J. Biol. Chem., 1979, 254, 1716-1723), 세포 주기 정지 작용, 형태 분화 작용 등을 갖는 미생물의 대사산물인 트리코스타틴 A(Cancer Res., 1987, 47, 3688-3691, Exp. Cell Res., 1988, 177, 122-131, J. Biol. Chem., 1990, 265, 17174-17179), 세포 증식 억제 작용을 갖는 미생물의 대사산물인 트라폭신(J. Antibiotics, 1990, 43, 1524-1534, J. Biol. Chem., 1993, 268, 22429-22435) 등을 들 수 있고, 항암제로서, vorinostat(SAHA), romidepsin(FK228), panobinostat(LBH-589) 등이 개발되었다. 그러나, 이들 저분자 합성물질 기반의 HDAC 저해제는 HDAC의 활성부위에 비가역적으로 결합하여 효소의 활성을 완전히 억제함으로써, 오히려 부작용을 일으킬 수 있다. 따라서, 기존의 저분자 합성물질 기반의 HDAC 저해제보다 부작용이 적으며 다른 기전을 가진 HDAC 저해제가 필요하다. HDAC inhibitors have been developed, butyric acid (J. Biol. Chem., 1979, 254, 1716-1723) with cell cycle arrest, normalization and differentiation of transformed cells, cell cycle arrest, morphogenesis Trichostatin A, a metabolite of the microorganisms having it (Cancer Res., 1987, 47, 3688-3691, Exp. Cell Res., 1988, 177, 122-131, J. Biol. Chem., 1990, 265, 17174-17179 And Trapoxin (J. Antibiotics, 1990, 43, 1524-1534, J. Biol. Chem., 1993, 268, 22429-22435), which are metabolites of microorganisms having a cell proliferation inhibitory effect, and the like. As the vorinostat (SAHA), romidepsin (FK228), panobinostat (LBH-589) and the like have been developed. However, these small molecule synthetic HDAC inhibitors may irreversibly bind to the active site of HDAC and completely inhibit the activity of the enzyme, thereby causing side effects. Therefore, HDAC inhibitors with different mechanisms are required and have fewer side effects than conventional low molecular synthetic HDAC inhibitors.
ATF3(Activating transcription factor 3) 단백질은 전사인자의 포유동물 활성전사조절인자/cAMP 반응요소-결합 단백질(mammalian activation transcription factor/cAMP responsive element-binding(CREB)protein)의 구성성분으로 ATF3 유전자는 암 발생에 관여하는 여러 요인들에 의해 나타나는 다양한 신호들에 의해 유도되며, 세포내 스트레스 반응의 복잡한 과정에 연관되어 있는 것으로 알려져 왔다. Activating transcription factor 3 (ATF3) protein is a component of the transcriptional factor mammalian activation transcription factor / cAMP responsive element-binding (CREB) protein. Induced by a variety of signals caused by a number of factors involved, it has been known to be involved in the complex process of intracellular stress response.
ATF3 단백질은 알려진 표적 유전자들의 활성인자(activator) 또는 억제제(repressor)로서 역할을 하고, 현재까지 문헌적으로 알려져 있는 ATF3의 잠재적 표적 유전자들은 20여 개가 넘으며, 여기에는 AdipoR1, AdipoR2, bNIP3, Cdc25A, CCL2, CCL4, Cyclin D1, FN-1, GLUT4, HIF-2α, IFN-γIL-1βα, TWIST1, p53, p73, PDX-1, 아디포넥틴(adiponectin) 등이 포함되는 것으로 알려져 있다. ATF3 protein acts as an activator or repressor of known target genes, with over 20 potential target genes of ATF3 known to date, including AdipoR1, AdipoR2, bNIP3, Cdc25A , CCL2, CCL4, Cyclin D1, FN-1, GLUT4, HIF-2α, IFN-γIL-1βα, TWIST1, p53, p73, PDX-1, adiponectin and the like.
또한, 대식세포(macrophage), 비만세포(mast cell), T 세포(T cell), 수지상 세포(dendritic cell) 등의 면역세포에서 뿐만 아니라 섬유아세포나 상피세포를 비롯한 대부분의 세포에서 NF-κB, JNK, Erk, p38, PKC 등 다양한 경로가 ATF3를 유도하고, 유도된 ATF3는 다양한 유전자들의 전사를 조절하여 세포사멸(apoptosis), 세포성장(cell proliferation), 세포이동(cell motility), DNA수선(DNA repair), 대사(metabolism)에 관여하며, 그 중에 NF-κB가 관계된 염증반응 유도에도 연관성이 있는 것으로 알려져 있다. ATF3 단백질의 발현은 당뇨, 비만과 같은 대사이상으로 발생한 비알코올성 간질환의 바이오마커로도 알려져 있다 (Kim et al., Journal of Hepatology 2017 vol. 67, 349-359). In addition, NF-κB in most cells including fibroblasts or epithelial cells, as well as in immune cells such as macrophage, mast cells, T cells, dendritic cells, etc. Various pathways such as JNK, Erk, p38 and PKC induce ATF3, and induced ATF3 regulates transcription of various genes, resulting in apoptosis, cell proliferation, cell motility, DNA repair ( It is known to be involved in DNA repair and metabolism, among which NF-κB is also involved in inducing inflammatory responses. Expression of ATF3 protein is also known as a biomarker of nonalcoholic liver disease caused by metabolic abnormalities such as diabetes and obesity (Kim et al., Journal of Hepatology 2017 vol. 67, 349-359).
따라서, ATF3 단백질의 발현 또는 기능을 조절하는 물질에 의해, ATF3에 의해 유도되는 염증반응을 억제할 수 있으나, 현재까지는 ATF3 단백질에 대한 저해제가 개발되지 않은 실정이다.Therefore, the inflammatory response induced by ATF3 can be suppressed by a substance that modulates the expression or function of ATF3 protein, but until now, no inhibitor for ATF3 protein has been developed.
GDF15(Growth differentiation factor 15)는 Transforming growth factor β (TGF- β) superfamily에 속하는 단백질로써, 다른 이름으로는 macrophage inhibitory cytokine 1, placental TGF-β prostate-derived factor (PDF), nonsteroidal anti-inflammatory drug-activator gene 등으로 불리기도 한다. GDF15 단백질은 NAFLD 환자들의 간에서 많이 발현되는 것으로 알려졌으며, 섬유화 진행 단계가 증가함에 따라, 간조직에서 GDF15 단백질의 발현량이 증가하였으며, 재조합 인간 GDF15 (rhGDF15)를 human hepatic stellate cells에 처리하면 간경화에 관련된 단백질 (SMAD2, SMAD3)의 발현이 증가하였다 (Koo et al., Liver International. 2018;38:695-705). 따라서, GDF15 단백질은 NAFLD에서 간경화로 발전시키는데 역할을 하며, 바이오마커로서의 작용도 한다. GDF15의 발현을 저해할 수 있는 물질을 개발하여 NAFLD의 진행을 감소시킬 수 있으나, 현재까지는 GDF15 단백질에 대한 저해제가 개발되지 않은 실정이다.Growth differentiation factor 15 (GDF15) is a protein belonging to the transforming growth factor β (TGF-β) superfamily, namely macrophage inhibitory cytokine 1, placental TGF-β prostate-derived factor (PDF), and nonsteroidal anti-inflammatory drug- Also called an activator gene. GDF15 protein is known to be expressed in the liver of NAFLD patients, and as the progress of fibrosis increased, the expression level of GDF15 protein in liver tissue increased. Treatment of recombinant hepatic GDF15 (rhGDF15) with human hepatic stellate cells prevented cirrhosis. Expression of related proteins (SMAD2, SMAD3) was increased (Koo et al., Liver International. 2018; 38: 695-705). Thus, GDF15 protein plays a role in developing cirrhosis in NAFLD and also acts as a biomarker. Although it is possible to reduce the progression of NAFLD by developing a substance that can inhibit the expression of GDF15, inhibitors to the GDF15 protein have not been developed until now.
이에, 본 발명자들은 본 발명자들에 의해 종래 개발된 펩타이드가 HDAC의 인산화를 억제하는 효과가 있고, 이와 더불어 간질환 마커인 ATF3 및 GDF15의 발현을 억제하는 효과를 나타내며, 이로써 다양한 염증성 사이토카인의 생성을 억제할 수 있음을 확인하였고, 나아가 관절염 동물 모델, 대장염 동물 모델 및 지방간 동물 모델에서 각각 상기 펩타이드를 투여함으로써 해당 질환의 치료 효과를 나타냄을 확인함으로써 본 발명을 완성하게 되었다. Accordingly, the present inventors have an effect of inhibiting the phosphorylation of HDAC by the peptides previously developed by the present inventors, and also exhibit the effect of inhibiting the expression of liver disease markers ATF3 and GDF15, thereby generating various inflammatory cytokines. It was confirmed that it can be suppressed, and furthermore, the present invention was completed by confirming that the peptides were administered in arthritis animal models, colitis animal models, and fatty liver animal models, respectively, to show a therapeutic effect of the disease.
발명의 요약Summary of the Invention
본 발명의 목적은 다중의 질환 바이오마커의 기능 및 발현을 억제하는 펩타이드의 신규한 용도를 제공하는 것이다. It is an object of the present invention to provide novel uses of peptides that inhibit the function and expression of multiple disease biomarkers.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 서열번호 8로 구성된 군 중 선택되는 어느 하나의 아미노산 서열로 표시되는 펩타이드를 유효성분으로 포함하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention is a pharmaceutical for treating or preventing inflammation, metabolic or fibrotic disease comprising a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient To provide a composition.
본 발명은 또한, 상기 펩타이드의 염증, 대사성 또는 섬유화 질환 치료 용도를 제공한다. The present invention also provides a use of the peptide for the treatment of inflammatory, metabolic or fibrotic disease.
본 발명은 또한, 염증, 대사성 또는 섬유화 질환 치료를 위한 약물의 제조를 위한 상기 조성물의 용도를 제공한다. The invention also provides the use of said composition for the manufacture of a medicament for the treatment of an inflammatory, metabolic or fibrotic disease.
본 발명은 또한, 상기 펩타이드를 염증, 대사성 또는 섬유화 질환 치료가 필요한 객체에 투여하는 단계를 포함하는 염증, 대사성 또는 섬유화 질환의 예방 및 치료방법을 제공한다.The present invention also provides a method for preventing and treating an inflammatory, metabolic or fibrotic disease, comprising administering the peptide to a subject in need of treatment for an inflammatory, metabolic or fibrotic disease.
도 1a는 인산화된 단백질의 마이크로어레이를 실시한 결과이다. 1A shows the results of microarrays of phosphorylated proteins.
도 1b는 서열번호 1 내지 3의 펩타이드의 농도 변화에 따른 인산화된 HDAC5 단백질의 발현량을 Western blot으로 확인한 결과이다.Figure 1b is the result of confirming the Western blot expression of the phosphorylated HDAC5 protein according to the change in the concentration of the peptide of SEQ ID NOS: 1 to 3.
도 1c는 서열번호 1 내지 3의 펩타이드에 의한 HDAC5 단백질의 인산화 억제 효과를 양성 대조군인 LMK, SAHA와 비교하기 위한 Western blot 결과이다.Figure 1c is a Western blot results for comparing the phosphorylation inhibitory effect of the HDAC5 protein by the peptides of SEQ ID NOS: 1 to LMK, SAHA, a positive control.
도 2a는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여에 따른 hind paw swelling (%)를 보여주는 결과이다. Figure 2a is a result showing the hind paw swelling (%) according to the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 2b는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여에 따른 hind paw swelling inhibition (%)을 보여주는 결과이다. Figure 2b is a result showing the hind paw swelling inhibition (%) according to the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 3a는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여 후 마이크로씨티의 3차원 이미지이다. Figure 3a is a three-dimensional image of microcities after administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 3b는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여에 따른 bone volume (BV), bone volume/tissue volume (BV/TV), bone surface areas adjusted to BV (BS/BV), trabecular thickness (Tb.Th)를 분석한 결과이다. FIG. 3B shows bone volume (BV), bone volume / tissue volume (BV / TV), bone surface areas adjusted to BV (BS / BV), trabecular thickness (Tb. Th) is the result of analysis.
도 4a는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여 후 현미경으로 관찰한 관절의 조직사진 결과이다. Figure 4a is a histogram of the joint observed under the microscope after administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 4b는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 후의 염증지수를 나타낸 결과이다. Figure 4b is a result showing the inflammation index after SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 5는 관절염 유도 동물모델에서 서열번호 1 및 etanercept 투여에 따른 혈중 내 TNF-a 와 IL-6의 농도 결과이다. Figure 5 shows the results of the concentration of TNF-a and IL-6 in the blood following the administration of SEQ ID NO: 1 and etanercept in arthritis induced animal model.
도 6a는 대장염 유도 동물모델에서 서열번호 1의 투여 후 8일 째 대장을 적출하여 육안으로 관찰한 결과이다.Figure 6a is a result of observation with a naked eye colon 8 days after administration of SEQ ID NO: 1 in colitis-derived animal model.
도 6b는 대장염 유도 동물모델에서 서열번호 1의 투여에 따른 대장의 길이 변화를 측정한 결과이다Figure 6b is the result of measuring the change in the length of the colon according to the administration of SEQ ID NO: 1 in colitis-derived animal model
도 6c는 대장염 유도 동물모델에서 서열번호 1의 투여에 따른 p40, TNF-α, IL-12의 발현을 ELISA로 측정할 결과이다. Figure 6c is a result of measuring the expression of p40, TNF-α, IL-12 according to the administration of SEQ ID NO: 1 in colitis-derived animal model by ELISA.
도 7은 서열번호 1 내지 4의 펩타이드 처리에 의한 간질환 마커인 GDF15와 ATF3 단백질의 발현량 변화를 Western blot으로 확인한 결과이다. Figure 7 shows the results confirmed by Western blot changes in the expression level of the liver disease markers GDF15 and ATF3 protein by the peptide treatment of SEQ ID NOS: 1-4.
도 8은 지방간 유도 동물모델에서 서열번호 1의 투여에 따른 간 비대증 및 피하지방 감소 효과를 관찰한 사진이다.8 is a photograph observing the effect of reducing liver hypertrophy and subcutaneous fat according to the administration of SEQ ID NO: 1 in a fatty liver-derived animal model.
도 9a는 DSS로 유도한 염증성 장질환 동물모델에서 서열번호 1-4의 투여 후 10일 째 대장의 길이 변화를 측정한 결과이다.Figure 9a is a result of measuring the change in the length of the colon 10 days after the administration of SEQ ID NO: 1-4 in the DSS-induced inflammatory bowel disease animal model.
도 9b는 DSS로 유도한 염증성 장질환 동물모델에서 서열번호 1-4의 투여 후 10일 째 대장내 융털을 H&E 염색하여 관찰한 결과이다.Figure 9b is the result of observation by H & E staining intestinal villus 10 days after the administration of SEQ ID NOS: 1-4 in DSS-induced inflammatory bowel disease animal model.
도 10a는 비알콜성 지방간염 동물모델에서 서열번호1-6 투여에 따른 간 조직내 간조직내 염증과 지방 감소효과를 H&E 염색을 통해 관찰한 결과이다.Figure 10a is a result of observing the inflammation and fat reduction effect of liver tissue in liver tissue according to SEQ ID NO: 1-6 in non-alcoholic steatohepatitis animal model through H & E staining.
도 10b는 비알콜성 지방간염 동물모델에서 서열번호1-6 투여에 따른 간 조직내 섬유화 감소 효과를 Sirius red 염색을 통해 관찰한 결과이다.Figure 10b is a result of observation through the Sirius red staining effect on the reduction of fibrosis in liver tissue following the administration of SEQ ID NO: 1-6 in non-alcoholic steatohepatitis animal model.
도 10c는 비알콜성 지방간염 동물모델에서 서열번호1-6 투여에 따른 간 조직내 섬유화 감소 효과를 Masson trichrome 염색을 통해 확인한 결과이다.Figure 10c is a result confirmed by Masson trichrome staining the effect of reducing the fibrosis in liver tissue according to SEQ ID NO: 1-6 administration in the non-alcoholic steatohepatitis animal model.
도 10d는 비알콜성 지방간염 동물모델에서 서열번호1-6 투여에 따른 군 별 NAS score 결과이다.10d is a NAS score result for each group following the administration of SEQ ID NOs: 1-6 in a nonalcoholic steatohepatitis animal model.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법 및 이하에 기술하는 실험 방법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.
본 발명에서는 서열번호 1 내지 8 중 어느 하나의 아미노산 서열로 표시되는 펩타이드가 염증, 대사성 또는 섬유화 질환 의 바이오마커인 HDAC5, GDF15, ATF3에 대해 HDAC5의 인산화를 억제하고, GDF15 및 ATF3 단백질의 발현을 감소시켜, 염증성 사이토카인의 생성을 감소시키고, 이와 연관된 관절염, 치주염, 아토피, 염증성 대장염, 당뇨 및 비만에 의해 발생하는 비알코올성 간질환, 지방간 및 피하지방 감소 효과를 나타냄을 확인하였다. In the present invention, the peptide represented by the amino acid sequence of any one of SEQ ID NOs: 1 to 8 inhibits the phosphorylation of HDAC5 against HDAC5, GDF15, ATF3 biomarkers of inflammation, metabolic or fibrotic disease, and inhibits the expression of GDF15 and ATF3 proteins. It has been shown to reduce the production of inflammatory cytokines and to reduce the associated nonalcoholic liver disease, fatty liver and subcutaneous fat caused by arthritis, periodontitis, atopy, inflammatory colitis, diabetes and obesity.
따라서, 본 발명은 일 관점에서 서열번호 1 내지 서열번호 8로 구성된 군 중 선택되는 어느 하나의 아미노산 서열로 표시되는 펩타이드를 유효성분으로 포함하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물에 관한 것이다.Therefore, the present invention provides a pharmaceutical composition for treating or preventing inflammatory, metabolic or fibrotic diseases comprising a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient in one aspect. It is about.
본 발명은 또 다른 관점에서, 상기 펩타이드의 염증, 대사성 또는 섬유화 질환 치료 용도에 관한 것이다. In another aspect, the present invention relates to the use of the peptide for the treatment of inflammatory, metabolic or fibrotic disease.
본 발명은 또 다른 관점에서, 염증, 대사성 또는 섬유화 질환 치료를 위한 약물의 제조를 위한 상기 조성물의 용도에 관한 것이다. In another aspect, the present invention relates to the use of said composition for the manufacture of a medicament for the treatment of an inflammatory, metabolic or fibrotic disease.
본 발명은 또 다른 관점에서, 상기 펩타이드를 염증 및 대사성 질환 치료가 필요한 객체에 투여하는 단계를 포함하는 염증, 대사성 또는 섬유화 질환의 예방 및 치료방법에 관한 것이다.In another aspect, the present invention relates to a method for preventing and treating an inflammatory, metabolic or fibrotic disease comprising administering the peptide to a subject in need thereof.
서열번호 1 GKCSTRGRKCCRRKK SEQ ID NO: 1 GKCSTRGRKCCRRKK
서열번호 2 (M1) GKCSTRGRKCMRRKKSEQ ID NO: 2 (M1) GKCSTRGRKCMRRKK
서열번호 3 (M2) GKCSTRGRKMCRRKKSEQ ID NO: 3 (M2) GKCSTRGRKMCRRKK
서열번호 4 (M3) GKCSTRGRKMMRRKKSEQ ID NO: 4 (M3) GKCSTRGRKMMRRKK
서열번호 5 (M4) GKMSTRGRKCCRRKK SEQ ID NO: 5 (M4) GKMSTRGRKCCRRKK
서열번호 6 (M5) GKMSTRGRKMCRRKK SEQ ID NO: 6 (M5) GKMSTRGRKMCRRKK
서열번호 7 (M6) GKMSTRGRKCMRRKK SEQ ID NO: 7 (M6) GKMSTRGRKCMRRKK
서열번호 8 (M7) GKMSTRGRKMMRRKKSEQ ID NO: 8 (M7) GKMSTRGRKMMRRKK
본 발명에서 상기 펩타이드는 서열번호 1 내지 8로 구성된 군 중 선택되는 어느 하나의 아미노산 서열로 표시되는 경우뿐만 아니라, 이와 60% 이상의 상동성을 가지고 있거나, 70% 이상의 상동성을 가지고 있거나, 80% 이상의 상동성을 가지고 있거나, 90% 이상의 상동성을 가지고 있거나, 95% 이상의 상동성을 가지고 있을 수 있으며, 예를 들어 서열번호 1 내지 8의 펩타이드의 아미노산 서열 중 일부 아미노산(예를 들어, 1 내지 6개의 아미노산)을 다른 아미노산으로 치환하거나, 아미노산을 일부(예를 들어, 1 내지 6개의 아미노산을) 추가 또는 결손시킨 경우로, 이로 인하여 상기 펩타이드가 목적하는 구조 또는 기능에 큰 영향이 없는 경우 내지는 상기 펩타이드의 안정성 등에 유리한 효과를 발휘하는 경우라면 서열번호 1 내지 8로 구성된 군 중 선택되는 어느 하나의 아미노산 서열을 적절히 대체하여 본 발명의 목적을 달성하기 위해 사용할 수 있음은 당업자에게 자명하다. In the present invention, the peptide is not only represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 8, but has at least 60% homology, at least 70% homology, or 80% It may have a homology or more than 90%, or may have a homology of 95% or more, for example, some amino acids (eg, 1 to 1) of the amino acid sequence of the peptide of SEQ ID NO: 1-8 6 amino acids) is replaced with another amino acid, or a portion (for example, 1 to 6 amino acids) of amino acids added or deleted, resulting in the peptide does not significantly affect the desired structure or function, or In the case of exerting an advantageous effect on the stability of the peptide or the like, the words selected from the group consisting of SEQ ID NOs: 1 to 8 It can be used to achieve the object of the appropriately substitute a single amino acid sequence invention will be apparent to those skilled in the art.
본 발명에서, 상기 염증, 대사성 또는 섬유화 질환 은 생체 내에서 HDAC5 단백질의 과인산화 GDF15 단백질의 과발현 및/또는 ATF3 단백질의 과발현 상태에 의해 직/간접적으로 발병하는 질환인 것을 특징으로 할 수 있으며, 상기 펩타이드는 HDAC5 단백질의 인산화, GDF15 단백질의 발현 및/또는 ATF3 단백질의 발현을 억제하는 것을 특징으로 할 수 있다.In the present invention, the inflammatory, metabolic or fibrotic disease may be a disease that is caused directly or indirectly by overexpression of the HDP5 protein superphosphorylated GDF15 protein and / or overexpression state of ATF3 protein in vivo. The peptide may be characterized by inhibiting phosphorylation of HDAC5 protein, expression of GDF15 protein and / or expression of ATF3 protein.
본 발명에서, 상기 염증, 대사성 또는 섬유화 질환은 관절염, 염증성 대장염, 궤양성 장염, 크론병, 간염, 지방간, 및 비만으로 구성된 군에서 선택되는 하나 이상의 질환인 것을 특징으로 할 수 있으나, 이에 한정되지는 않으며, HDAC5 단백질의 과인산화 GDF15 단백질의 과발현 및/또는 ATF3 단백질의 과발현과 관계된 모든 염증, 대사성 또는 섬유화 질환 의 치료 및 예방 용도로 활용될 수 있을 것이다. In the present invention, the inflammatory, metabolic or fibrotic disease may be at least one disease selected from the group consisting of arthritis, inflammatory colitis, ulcerative enteritis, Crohn's disease, hepatitis, fatty liver, and obesity, but is not limited thereto. Hyperphosphorylation of HDAC5 protein may be used for the treatment and prevention of any inflammatory, metabolic or fibrotic disease associated with overexpression of GDF15 protein and / or overexpression of ATF3 protein.
본 발명에서 상기 간염은 당뇨 및/또는 비만에 의한 비알코올성 지방간염인 것을 특징으로 할 수 있다.In the present invention, the hepatitis may be characterized by non-alcoholic steatohepatitis due to diabetes and / or obesity.
본 발명에서, 상기 섬유성 질환은 섬유화에 의한 간경화, 폐섬유증, 폐쇄성 폐질환, 심부전, 동맥경화, 만성신부전, 당뇨병, 수술후 후유증으로 발생하는 켈로이드 등으로 구성된 군에서 선택된 1종 이상인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.본 발명에서, 상기 과인산화란 해당 질환이 발병하지 않은 객체에 비해 해당 단백질의 인산화가 증가된 상태를 의미하고, 과발현이란 해당 질환이 발병하지 않은 객체에 비해 해당 단백질의 발현이 증가된 상태를 의미한다.In the present invention, the fibrotic disease is characterized in that at least one selected from the group consisting of liver cirrhosis due to fibrosis, pulmonary fibrosis, obstructive pulmonary disease, heart failure, arteriosclerosis, chronic kidney failure, diabetes, keloids caused by postoperative sequelae, etc. However, the present invention is not limited thereto. In the present invention, the hyperphosphorylation refers to a state in which phosphorylation of the protein is increased compared to an object that does not develop the disease, and the overexpression of the protein is compared to an object that does not develop the disease. Refers to an increased state of.
본 발명의 약학적 조성물은 펩타이드 이외에 약제학적으로 허용가능한 담체를 1 종 이상 더 포함할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical composition of the present invention may further include one or more pharmaceutically acceptable carriers in addition to the peptide. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
따라서, 본 발명의 조성물은 주사제, 경구투여용 제제, 패치제, 액제, 캡슐, 과립, 정제, 산제, 분무제, 연고제, 겔제, 점막투여제제 및 좌제로 구성된 군에서 선택되는 어느 하나의 제형으로 제제화 할 수 있으나, 이에 한정되지는 않는다. 이들 제제는 당분야에서 제제화에 사용되는 통상의 방법 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA 에 개시되어 있는 방법으로 제조될 수 있으며 각 질환에 따라 또는 성분에 따라 다양한 제제로 제제화될 수 있다.Therefore, the composition of the present invention is formulated into any one formulation selected from the group consisting of injections, oral formulations, patches, solutions, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal administrations and suppositories. It may be, but is not limited thereto. These formulations may be prepared by conventional methods used in the art for formulation or by methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA and formulated into various formulations depending on the individual disease or component. Can be.
상기 치료용 약학적 조성물은 약제학적으로 허용가능한 보조제를 추가로 함유하도록 제제화 될 수 있으며, 상기 약제학적으로 허용가능한 보조제는 부형제, 희석제, 완충제, 항균성 보존제, 계면활성제, 산화방지제, 증점제 및 점도개질제로 구성된 군에서 선택된 하나 이상인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.The therapeutic pharmaceutical composition may be formulated to further contain a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable adjuvant is an excipient, diluent, buffer, antimicrobial preservative, surfactant, antioxidant, thickener and viscosity modifier. One or more selected from the group consisting of, but is not limited thereto.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명의 서열번호 1 내지 8의 펩타이드를 염증성 질환, 대사성 질환, 자가면역 질환 및/또는 섬유화 질환의 치료 용도로 사용할 때, 본 발명의 서열번호 1~8의 펩타이드의 일일 투여량은 약 1 내지 100 ㎎/㎏ 이고, 바람직하게는 5 내지 50 ㎎/㎏ 이며, 일일 일회 또는 주 2-3회에 나누어 투여할 수 있으나, 이에 한정되는 것은 아니다.The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. When the peptides of SEQ ID NOs: 1 to 8 of the present invention are used for the treatment of inflammatory diseases, metabolic diseases, autoimmune diseases and / or fibrotic diseases, the daily dosage of the peptides of SEQ ID NOS: 1 to 8 of the present invention is about 1 to 8 100 mg / kg, preferably 5 to 50 mg / kg, and may be administered once a day or divided into 2-3 times a week, but is not limited thereto.
이 경우, 투여 용법 및 투여 용량은 개별 환자의 연령, 체중 및 반응에 따라 다양할 것이다. 적합한 투여 용법 및 투여 용량은 이러한 인자를 당연히 고려하는 이 분야의 통상의 지식을 가진 자에 의해 쉽게 선택될 수 있다. In this case, the dosage regimen and dosage will vary depending on the age, weight and response of the individual patient. Appropriate dosage regimens and dosages can be readily selected by those of ordinary skill in the art that naturally consider such factors.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 펩타이드 합성Example 1: Peptide Synthesis
N 말단으로부터 순서대로 서열번호 1 내지 8의 펩타이드를 F-moc 고체상 화학합성방법(Solid phase peptide synthesis)으로 합성하였다. 합성된 펩타이드 서열은 레진으로부터 절단시켜 세척하고, 동결건조 후 액체크로마토그래피에 의해 분리 및 정제하였다. 정제된 펩타이드는 MALDI-TOF 분석을 이용하여 분자량을 확인하였다.Peptides of SEQ ID NOS: 1 to 8 were sequentially synthesized from the N terminus by F-moc solid phase peptide synthesis. The synthesized peptide sequences were cleaved from the resin, washed, lyophilized and separated and purified by liquid chromatography. Purified peptides were checked for molecular weight using MALDI-TOF analysis.
서열번호 1 GKCSTRGRKCCRRKK SEQ ID NO: 1 GKCSTRGRKCCRRKK
서열번호 2 (M1) GKCSTRGRKCMRRKKSEQ ID NO: 2 (M1) GKCSTRGRKCMRRKK
서열번호 3 (M2) GKCSTRGRKMCRRKKSEQ ID NO: 3 (M2) GKCSTRGRKMCRRKK
서열번호 4 (M3) GKCSTRGRKMMRRKKSEQ ID NO: 4 (M3) GKCSTRGRKMMRRKK
서열번호 5 (M4) GKMSTRGRKCCRRKK SEQ ID NO: 5 (M4) GKMSTRGRKCCRRKK
서열번호 6 (M5) GKMSTRGRKMCRRKK SEQ ID NO: 6 (M5) GKMSTRGRKMCRRKK
서열번호 7 (M6) GKMSTRGRKCMRRKK SEQ ID NO: 7 (M6) GKMSTRGRKCMRRKK
서열번호 8 (M7) GKMSTRGRKMMRRKKSEQ ID NO: 8 (M7) GKMSTRGRKMMRRKK
실험예 1. 합성 펩타이드의 HDAC5 인산화 저해 및 결합 친화성 확인Experimental Example 1. HDAC5 phosphorylation inhibition and binding affinity of the synthetic peptide
RAW 264.7 세포를 0.5% FBS에서 2시간 동안 스타베이션(starvation) 시키고, 200 μM 서열번호 1의 펩타이드를 1 시간 동안 처리한 후, LPS (1 μg/mL)로 30분, 37℃로 염증을 유발시켰다. 처리하지 않은 세포 (NT), LPS로 처리한 세포, 서열번호 1과 LPS로 처리한 세포의 라이세이트 (cell lysate)를 Full Moon Biosystems사의 포스포 마이크로 어레이 (Phospho Explorer Antibody Microarray)에 적용하였다. 해당 포스포 마이크로 어레이는 30개의 신호전달체계와 관련 있는 1318 개의 항체를 인식할 수 있다. 각각의 항체는 표준화된 사이즈의 글래스 마이크로스코프 슬라이드 (standard-size coated glass microscope slides)에 코팅되어 있고, 슬라이드의 스캔닝은 GenePix 4100A scanner (Axon Instrument, Foster City, CA, USA)로 실시하였다. 데이터는 Genowiz 4.0TM (Ocimum Biosolutions, Hyderabad, India)로 분석하였다. 인산화 비율은 아래와 같이 계산하였다. RAW 264.7 cells were starvated in 0.5% FBS for 2 hours, treated with 200 μM of SEQ ID NO: 1 peptide for 1 hour, followed by LPS (1 μg / mL) for 30 minutes at 37 ° C. I was. Cell lysates of untreated cells (NT), LPS-treated cells, SEQ ID NO: 1 and LPS-treated cells were applied to a Phospho Explorer Antibody Microarray from Full Moon Biosystems. The phosphor microarray can recognize 1318 antibodies related to 30 signaling systems. Each antibody was coated on standard-size coated glass microscope slides, and the slides were scanned with a GenePix 4100A scanner (Axon Instrument, Foster City, CA, USA). Data was analyzed with Genowiz 4.0TM (Ocimum Biosolutions, Hyderabad, India). Phosphorylation ratio was calculated as follows.
각 항체에서의 인산화 비율 계산식: Formulation of phosphorylation rate at each antibody:
phosphorylation ratio = (실험군의 인산화 수치/실험군의 비인산화 수치)/(대조군의 인산화 수치/대조군의 비인산화 수치) phosphorylation ratio = (phosphorylation level of the experimental group / non-phosphorylation level of the experimental group) / (phosphorylation level of the control group / non-phosphorylation level of the control group)
분석 후, 각 단백질의 정보는 UniPort 데이터베이스를 이용하여 검색하였다. 그 결과, 서열번호 1의 펩타이드와 LPS를 처리하였을 때, LPS 만 처리한 세포보다 HDAC5의 인산화가 가장 많이 저해되었고 (0.278), NF-κB p100/p52 (0.451), NF-κB p105/p50 (0.485), IκBα (0.509) 등 NF-κB신호와 관련된 단백질의 인산화가 저해된 것을 확인할 수 있었다(도 1a).After analysis, the information of each protein was searched using UniPort database. As a result, when the LPS-treated peptide and LPS were treated, HDAC5 phosphorylation was more inhibited than that of LPS-only cells (0.278), NF-κB p100 / p52 (0.451), and NF-κB p105 / p50 ( 0.485), IκBα (0.509), it was confirmed that the phosphorylation of proteins related to NF-κB signal was inhibited (Fig. 1a).
상기 마이크로 어레이 결과를 기초로, 웨스턴블럿을 실시하여 서열번호 1-3의 펩타이드에 의한 HDAC5 및 p65의 인산화의 변화를 검증하고자 하였다. 6-well plate 에 70%의 density로 RAW 264.7 cells을 plating한 후 16시간 뒤에 0.5% FBS가 포함된 DMEM 배지로 2시간 동안 cell starvation 하였다. 서열번호 1~3의 펩타이드 각각을 50~1000μM 농도로 포함한 배지를 1시간 동안 처리한 후, LPS를 1μg/ml 농도로 처리하여 30분 동안 염증 반응을 유도하였다. Protease inhibiotr와 phosphatase inhibitor를 포함한 RIPA lysis buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)를 사용하여 단백질을 lysis하였다. BCA protein assay를 통해 단백질을 정량하고, western blot 으로 HDAC 5와 p65 단백질의 발현량을 확인하였다. Western blot의 경우, 8% SDS PAGE gel 에 size marker와 함께 sample을 동량 loading하여 약 2시간 전기영동한 후, nitrocellulose membrane에 transfer하였다. Transfer 한 membrane은 5% skim milk로 1시간 blocking하고, 1st antibody를 1:1000 비율로 하룻밤 동안 반응시켰다. 이 후 membrane을 0.1% tween-20을 포함한 TBST로 세척 하고 HRP가 부착된 2nd antibody로 1시간 반응시킨 후, ECL substrate로 화학발광을 확인하였다.Based on the microarray results, Western blot was performed to verify the change in phosphorylation of HDAC5 and p65 by the peptides of SEQ ID NOs. 1-3. After plating RAW 264.7 cells at a density of 70% on a 6-well plate, 16 hours later, cell starvation was performed with DMEM medium containing 0.5% FBS for 2 hours. After treatment with a medium containing each of the peptides of SEQ ID NO: 1 to 50 ~ 1000μM concentration for 1 hour, LPS was treated at a concentration of 1μg / ml to induce an inflammatory response for 30 minutes. Proteins were lysed using RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibiotr and phosphatase inhibitor. Proteins were quantified by BCA protein assay and the expression levels of HDAC 5 and p65 protein were confirmed by western blot. In the case of Western blot, the sample was loaded in an 8% SDS PAGE gel with the size marker in the same amount and electrophoresed for about 2 hours, and then transferred to the nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and reacted with 1st antibody overnight at 1: 1000 ratio. Thereafter, the membrane was washed with TBST containing 0.1% tween-20, and reacted with HRP-attached 2nd antibody for 1 hour, and then chemiluminescence was confirmed by ECL substrate.
그 결과, LPS만 단독으로 처리한 경우에는 HDAC5의 인산화가 증가하였으나 LPS와 서열번호 1 내지 3의 펩타이드를 동시에 처리하였을 때, HDCA5의 인산화가 감소하였다. 이를 통해, 서열번호 1 내지 3의 펩타이드가 HDAC5를 통해 신호전달을 억제하는 효과가 있음을 확인할 수 있었다.  As a result, when only LPS alone was treated, HDAC5 phosphorylation increased, but when LPS and peptides of SEQ ID NOs: 1 to 3 were simultaneously treated, HDCA5 phosphorylation decreased. Through this, it was confirmed that the peptide of SEQ ID NO: 1 to 3 has the effect of inhibiting signal transduction through HDAC5.
또한, 서열번호 1 내지 3의 펩타이드와 기존에 알려진 HDAC 인산화 저해제인 SAHA, LMK와 비교하여 인산화된 HDAC5 단백질과 HDAC5 단백질의 인산화 정도를 Western blot으로 확인한 결과, 서열번호 1 내지 3의 펩타이드는 SAHA, LMK와 비교하여 HDAC5 인산화 저해가 더 우수한 것으로 확인되었다.In addition, the degree of phosphorylation of the phosphorylated HDAC5 protein and the HDAC5 protein was compared with the peptides of SEQ ID NOs: 1 to 3 and SAHA, LMK, which are known HDAC phosphorylation inhibitors. It was found that HDAC5 phosphorylation inhibition was better than LMK.
한편, HDAC5와 서열번호 1 내지 8의 펩타이드의 결합 친화력을 Biacore T200 (BIAcore AB, Uppsala, Sweden)으로 확인하였다. 이를 위하여 HDAC5 단백질 (Abcam, MA, USA)을 sodium acetate buffer (pH 4.5)에 녹이고, N-hydroxysuccinimide (NHS)와 N-ethyl-N0-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC)를 크로스링커로 사용하여 dextran-covered sensor chip surface (CM5 chip)에 결합시켰다. 덱스트란의 반응하지 않은 활성 그룹은 ethanolamine (1M)을 가하여 블록시켰다. 칩에 결합된 HDAC5의 수치는 4440RU (1000RU=1 ng/mm2)로 측정되었다. 서열번호 1 내지 8의 펩타이드를 각각 다양한 농도(15.625, 31.25, 62.5, 125, 250, 500nM)로 30μL/min 속도로 반응시켰고, 해리반응은 5분 동안 유지하였다. 1:1 binding model을 사용하여 BIAevaluation software로 rate constants for association (ka), dissociation (kd), dissociation constant (KD)를 계산하였다. On the other hand, the binding affinity between the HDAC5 and the peptides of SEQ ID NOS: 1 to 8 was confirmed by Biacore T200 (BIAcore AB, Uppsala, Sweden). To this end, HDAC5 protein (Abcam, MA, USA) is dissolved in sodium acetate buffer (pH 4.5), and N-hydroxysuccinimide (NHS) and N-ethyl-N0- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) are crosslinkers. To dextran-covered sensor chip surface (CM5 chip). Unreacted active groups of dextran were blocked by addition of ethanolamine (1M). The value of HDAC5 bound to the chip was measured at 4440 RU (1000 RU = 1 ng / mm 2 ). Peptides of SEQ ID NO: 1 to 8 were reacted at various concentrations (15.625, 31.25, 62.5, 125, 250, 500 nM) at a rate of 30 μL / min, and the dissociation reaction was maintained for 5 minutes. Rate constants for association (ka), dissociation (kd), and dissociation constant (KD) were calculated using the BIAevaluation software using a 1: 1 binding model.
하기 표 1은 서열번호 1 내지 8의 펩타이드와 HDAC5 단백질간의 결합친화성을 나타내는 Biacore의 분석결과이다. 미스매치 펩타이드 (mismatch peptide)와 비교했을 때, 서열번호 1 내지 8의 펩타이드는 KD값이 10-7 정도였고, 미스매치 펩타이드는 10-4으로 나왔다. KD값이 작을수록 결합친화력이 더 좋은 것이기 때문에, 서열번호 1 내지 8의 펩타이드는 HDAC5 단백질에 대한 결합력이 우수하며, HDAC5 단백질과 결합하면서 인산화를 감소시키는 것으로 예측할 수 있었다.Table 1 below shows the analysis results of Biacore showing the binding affinity between the peptides of SEQ ID NOs. 1 to 8 and the HDAC5 protein. Compared to the mismatch peptide, the peptides of SEQ ID NOs: 1 to 8 had KD values of about 10 −7 , and the mismatch peptides showed about 10 −4 . Since the smaller the KD value, the better the binding affinity, the peptides of SEQ ID NOs: 1 to 8 had excellent binding ability to the HDAC5 protein and could be predicted to reduce phosphorylation while binding to the HDAC5 protein.
Figure PCTKR2019011037-appb-T000001
Figure PCTKR2019011037-appb-T000001
실험예 2. 합성 펩타이드의 관절염 치료효과 확인 Experimental Example 2. Confirmation of arthritis treatment effect of synthetic peptide
본 발명에 따른 펩타이드가 in vivo 상에서 류마티스 관절염 치료 효과가 있는지 조사하기 위하여, 콜라겐 유도 관절염 동물 모델에서 관절염 치료 효과를 확인하고자 하였다. 이를 위하여 암컷 Wistar rats (105-135 g, n=67)을 Harlan Co. Ltd. (Indianapolis, IN, USA) 에서 구입하였다. 모든 랫트는 병원균이 없는 환경에서 사육되었고, 멸균된 표준 랫트사료(Harlan Co. Ltd, USA)와 물을 자유롭게 섭취하도록 하였다. 동물 실험은 서울대학교 IACUC(Institutional Animal Care and Use Committee)의 승인하에서 이루어졌다. 콜라겐 유도 관절염은 공지된 바에 따라 유도되었다(Nishikawa et al., Arthritis and rheumatism. 2003;48:2670-81).In order to investigate whether the peptides according to the present invention have a therapeutic effect on rheumatoid arthritis in vivo , an attempt was made to confirm the therapeutic effect of arthritis in a collagen-induced arthritis animal model. Female Wistar rats (105-135 g, n = 67) were used for this purpose in Harlan Co. Ltd. (Indianapolis, IN, USA). All rats were bred in a pathogen free environment and were given free intake of standard sterile rat feed (Harlan Co. Ltd, USA) and water. Animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC). Collagen induced arthritis was induced as known (Nishikawa et al., Arthritis and rheumatism. 2003; 48: 2670-81).
구체적으로, 랫트의 근육에 0.1 M acetic acid 0.5 mL와 Freund's incomplete adjuvant 0.5 mL 혼합액에 소 콜라겐 타입 II 500μg을 주사하였다. 7일째에 0.1 M acetic acid 0.5 mL와 Freund's incomplete adjuvant 0.5 mL에 소 콜라겐 타입 II 250㎍을 추가 접종하여 collagen induced arthritis (CIA) 랫트를 제작하였다. CIA 유발군 (n=10), 저농도의 서열번호 1의 펩타이드를 주사한 CIA 유발군(LD AIP, 15 mg/kg, n=10), 고농도의 서열번호 1의 펩타이드를 주사한 CIA 유발군 (HD AIP, 30 mg/kg, n=10), etanercept (Enbrel)을 주사한 CIA 유발군 (10 mg/kg, n=10), 정상군 (n=6)으로 나누었다. 최초 콜라겐을 주사한 28일 후부터 서열번호 1의 펩타이드를 주 2회 4주간 목 뒷덜미에 피하주사 하였다. etanercept는 주 3회 4주간 복강내에 주사하였다. 양쪽의 hind paw swelling (뒷발의 붓기)를 22, 29, 31, 35, 38, 42, 45, 49, 52, 56 일에 plethysmometer (LE7500 N, IWOO Scientific Corporation, KOREA)를 사용하여 2번씩 측정하였다. Plethysmometer는 3mL의 무게로 보정한 이후 사용하였다. 측정된 부위는 선을 그어 표시하고, 발목에 가깝게 측정하였다. Hind paw swelling increase (%) 와 hind paw swelling inhibition (%) 은 아래식을 사용하여 구하였다. Specifically, 500 μg of bovine collagen type II was injected into a rat muscle mixture of 0.5 mL of 0.1 M acetic acid and 0.5 mL of Freund's incomplete adjuvant. On day 7, collagen induced arthritis (CIA) rats were prepared by further inoculating 250 μg of bovine collagen type II in 0.5 mL of 0.1 M acetic acid and 0.5 mL of Freund's incomplete adjuvant. CIA-induced group (n = 10), CIA-induced group injected with low concentration of SEQ ID NO: 1 peptide (LD AIP, 15 mg / kg, n = 10), CIA-induced group injected with high concentration of SEQ ID NO: 1 peptide ( HD AIP, 30 mg / kg, n = 10), and CIA-induced group (10 mg / kg, n = 10) injected with etanercept (Enbrel) were divided into normal group (n = 6). From the 28th day after the first collagen injection, the peptide of SEQ ID NO: 1 was injected subcutaneously in the back of the neck twice a week for 4 weeks. etanercept was injected intraperitoneally three times a week for four weeks. Both hind paw swellings were measured twice using a plethysmometer (LE7500 N, IWOO Scientific Corporation, KOREA) at 22, 29, 31, 35, 38, 42, 45, 49, 52, 56 days. . Plethysmometer was used after calibrating to the weight of 3mL. The measured area was marked with a line and measured close to the ankle. Hind paw swelling increase (%) and hind paw swelling inhibition (%) were obtained using the following equation.
Figure PCTKR2019011037-appb-I000001
Figure PCTKR2019011037-appb-I000001
Figure PCTKR2019011037-appb-I000002
Figure PCTKR2019011037-appb-I000002
hind paw swelling (%) 측정 결과, 정상그룹과 비교하여 CIA 유발군은 hind paw swelling이 증가하였고, 양성 대조군인 Etanercept (Enbrelⓡ)를 주사한 CIA 유발군은 35 - 56일 사이에 hind paw swelling (%)가 감소하였다 (P<0.05). 서열번호 1을 주사한 CIA 유발군은 hind paw swelling (%)가 농도 의존적으로 감소하였다. 고농도 (30mg/kg)의 서열번호 1 의 펩타이드를 주사한 CIA 유발군은 38 -49일 사이에 hind paw swelling (%)가 감소하였다 (P<0.05). In hind paw swelling (%), the CIA-induced group increased hind paw swelling in comparison with the normal group, and CIA-induced group injected with Etanercept (Enbrel®), a positive control, showed hind paw swelling between %) Decreased ( P <0.05). In the CIA-induced group injected with SEQ ID NO: 1, hind paw swelling (%) decreased in a concentration-dependent manner. In the CIA-induced group injected with high concentration (30 mg / kg) of SEQ ID NO: 1 peptide, hind paw swelling (%) decreased between 38 and 49 days ( P <0.05).
hind paw swelling inhibition (%) 측정 결과, 고농도 (30mg/kg)의 서열번호 1의 펩타이드를 투여한 CIA 유발 그룹은 38일째 35%가 저해되었다. 저농도 (15mg/kg)의 서열번호 1의 펩타이드를 투여한 CIA 유발 그룹은 38일째에 32%의 저해를 보였고, etanercept를 투여한 CIA 유발 그룹은 37%의 저해율을 보여, 고농도의 서열번호 1의 펩타이드는 양성 대조군인 etanercept와 유사한 효과를 보여 주었다.As a result of hind paw swelling inhibition (%), 35% of CIA-induced groups receiving high concentration (30mg / kg) of SEQ ID NO: 1 peptide were inhibited at 38 days. The CIA-induced group receiving low concentration (15mg / kg) of the peptide of SEQ ID NO: 1 showed 32% inhibition on day 38, and the CIA-induced group receiving etanercept showed 37% inhibition rate. Peptides showed similar effects to etanercept, a positive control.
hind paw의 뼈 손상 정도를 관찰하기 위해 microcomputed tomography (micro-CT)를 SkyScan 1172 (SkyScan, Aartselaar, Belgium)을 이용하여 측정하였다. total volume (TV; mm3), bone volume (BV;mm3), bone volume fraction (BV/TV;%)을 CTAn software를 사용하여 분석하였다. 57일째 랫트를 희생하여 조직학적 분석을 진행하였다. 양 뒷발과 관절을 채취하여 10% buffered formalin에 고정하고, 30% citrate-buffered formic acid에 3일 동안 4℃에서 탈회하였다. 파라핀블럭을 만든 후 4 μm 두께로 섹션하고, 헤마톡실린-에오신 (hematoxylin and eosin, HE) 염색을 하였고, 광학현미경으로 병리학적 점수를 평가하였다. 관절의 염증지수는 하기와 같은 기준으로 측정하였다(Shiozawa et al., J Clin Invest. 1997;99(6):1210-1216). 0, normal synovium; 1, synovial membrane hypertrophy and cell infiltrates; 2, pannus and cartilage erosion; 3, major erosion of cartilage and subchondral bone; and 4, loss of joint integrity and ankylosis. Microcomputed tomography (micro-CT) was measured using SkyScan 1172 (SkyScan, Aartselaar, Belgium) to observe the degree of bone damage in the hind paw. Total volume (TV; mm 3 ), bone volume (BV; mm 3 ), bone volume fraction (BV / TV;%) were analyzed using CTAn software. Rats were sacrificed on day 57 for histological analysis. Both hind paws and joints were harvested and fixed in 10% buffered formalin and demineralized in 30% citrate-buffered formic acid at 4 ° C for 3 days. Paraffin blocks were made and then sectioned to a thickness of 4 μm, stained with hematoxylin and eosin (HE), and pathological scores were evaluated by light microscopy. Joint inflammation index was measured based on the following criteria (Shiozawa et al., J Clin Invest. 1997; 99 (6): 1210-1216). 0, normal synovium; 1, synovial membrane hypertrophy and cell infiltrates; 2, pannus and cartilage erosion; 3, major erosion of cartilage and subchondral bone; and 4, loss of joint integrity and ankylosis.
그 결과, CIA 유발군에서는 심한 골손상이 관찰되었으나. 서열번호 1의 펩타이드를 주사한 CIA 유발군에서는 골조직이 보존되었고, 특히 고농도의 서열번호 1의 펩타이드를 주사한 CIA 유발군은 etanercept를 주사한 CIA 유발군과 비교했을 때 유사한 효과를 나타내었다(도 3a).As a result, severe bone damage was observed in the CIA-induced group. Bone tissue was preserved in the CIA-induced group injected with the peptide of SEQ ID NO: 1, and in particular, the CIA-induced group injected with the high concentration of the peptide of SEQ ID NO: 1 showed a similar effect compared to the CIA-induced group injected with etanercept (Fig. 3a).
또한, bone volume (BV), bone volume/tissue volume (BV/TV), bone surface areas adjusted to BV (BS/BV), trabecular thickness (Tb.Th)를 분석한 결과, 고농도의 서열번호 1을 주사한 CIA 유발군은 높은 BV, BV/TV, Tb.Th를 보였고, 낮은 BS/BV를 보여주어, 서열번호 1의 펩타이드는 관절염을 경감시키면서 관절부위의 뼈손상을 감소시키는 기능이 있는 것을 확인할 수 있었다(도 3b). 여기서, BV와 BV/TV는 서로 다른 크기의 샘플에서 골이 보존되는 것을 비교할 수 있는 파라미터이다. BS/BV는 erosion에 의해 bone surface가 손실되는 것을 알려주는 파라미터이다. Tb.Th는 관절염증에 의해 periarticular osteopenia와 상반된 연관이 있는 파라미터이다 (Parfitt AM et al., J Bone Miner Res. 1987;2(6):595-610).In addition, bone volume (BV), bone volume / tissue volume (BV / TV), bone surface areas adjusted to BV (BS / BV), and trabecular thickness (Tb.Th) were analyzed. One CIA-induced group showed high BV, BV / TV, Tb.Th and low BS / BV, indicating that the peptide of SEQ ID NO: 1 has the function of reducing bone damage at the joint area while relieving arthritis. (FIG. 3B). Here, BV and BV / TV are parameters that can be compared to preserve the bone in samples of different sizes. BS / BV is a parameter that indicates the bone surface is lost by erosion. Tb.Th is a parameter that is contradictory to periarticular osteopenia by arthritis (Parfitt AM et al., J Bone Miner Res. 1987; 2 (6): 595-610).
또한, 조직을 현미경으로 관찰한 결과, CIA 유발군은 synovial tissue에서 만성염증을 보였고, pannus 형성, 관절 및 뼈의 파괴를 나타내는 반면, 서열번호 1의 펩타이드를 주사한 CIA유발군은 농도 의존적으로 염증의 감소를 확인할 수 있었다. Etanercept를 주사한 CIA 유발군은 저농도의 서열번호 1을 주사한 CIA 유발군과 유사한 결과를 나타내었다(도 4a). In addition, the microscopic examination of the tissues revealed that the CIA-induced group showed chronic inflammation in synovial tissue and showed pannus formation, destruction of joints and bones, whereas the CIA-induced group injected with the peptide of SEQ ID NO: 1 concentration-dependent inflammation. The decrease of was confirmed. The CIA-induced group injected with Etanercept showed similar results to the CIA-induced group injected with low concentration SEQID 1 (FIG. 4A).
염증지수에서도 서열번호 1의 펩타이드를 주사한 CIA 유발군(1.5±1.17) 에서 염증 지수가 CIA 유발군(3.3±0.67; p<0.01)보다 현저히 낮은 것으로 나타났다. 한편, Etanercept를 주사한 CIA 유발군 (2.3±1.16) 은 CIA 유발군보다 염증지수가 유의하게 낮지 않은 것으로 나타났다. 따라서 서열번호 1의 펩타이드는 Etanercept 보다 염증세포의 침윤을 저해하고 CIA 유발 랫트에서 관절염 증상은 감소시키는 효과가 더 높은 것으로 확인되었다(도 4b). In the inflammation index, the CIA-induced group (1.5 ± 1.17) injected with the peptide of SEQ ID NO: 1 showed significantly lower inflammation index than the CIA-induced group (3.3 ± 0.67; p <0.01). On the other hand, the CIA-induced group (2.3 ± 1.16) injected with Etanercept was not significantly lower than the CIA-induced group. Therefore, the peptide of SEQ ID NO: 1 was found to have a higher effect of inhibiting the infiltration of inflammatory cells and reducing arthritis symptoms in CIA-induced rats than Etanercept (FIG. 4B).
랫트를 희생시켜 전신혈액을 채취하여 혈액내 TNF-α 와 IL-6의 농도를 ELISA kit (R&D, Minneapolis, MN, USA)로 측정하였다. Systemic blood was collected at the expense of rats and the concentrations of TNF-α and IL-6 in the blood were measured by ELISA kit (R & D, Minneapolis, MN, USA).
그 결과, CIA 유발군은 TNF-α 농도는 128 pg/ml, IL-6의 농도는 109pg/ml이였으나, 고농도의 서열번호 1의 펩타이드를 주사하였을 때, TNF-α 농도는 40.3 pg/mL, IL-6의 농도는 35.2 pg/mL이었고, etanercept를 주사하였을 때, TNF-α 농도는 39 pg/mL, IL-6의 농도는 24 pg/mL로 확인되었다(도 5). 이로써, 서열번호 1의 펩타이드는 etanercept과 유사한 정도로 염증성 사이토카인인 TNF-α와 IL-6의 혈중 농도를 감소시키는 효과가 있음을 알 수 있었다.As a result, in the CIA-induced group, the concentration of TNF-α was 128 pg / ml and IL-6 was 109 pg / ml. However, when the high concentration of the peptide of SEQ ID NO: 1 was injected, the concentration of TNF-α was 40.3 pg / mL. IL-6 concentration was 35.2 pg / mL, and when etanercept was injected, TNF-α concentration was 39 pg / mL and IL-6 concentration was 24 pg / mL (Fig. 5). As a result, the peptide of SEQ ID NO: 1 was found to have an effect of reducing blood levels of inflammatory cytokines TNF-α and IL-6 to a similar degree to etanercept.
상기 결과를 종합하면, 서열번호 1 내지 8의 펩타이드는 HDAC5의 인산화를 저해하고, 그에 따라 염증성 사이토카인의 생성을 억제하여 항염 활성 및 관절염 치료 효과를 나타내는 것으로 예측할 수 있다.Taken together, the peptides of SEQ ID NOs: 1 to 8 can be predicted to inhibit the phosphorylation of HDAC5, thereby inhibiting the production of inflammatory cytokines, thereby exhibiting anti-inflammatory activity and therapeutic effect of arthritis.
실험예 3: 합성 펩타이드의 염증성 대장염 치료효과 확인 Experimental Example 3: Confirming the Inflammatory Colitis Treatment Effect of Synthetic Peptides
본 발명에 따른 펩타이드가 in vivo 상에 염증성 대장염 치료 효과가 있는지 조사하기 위하여, 대장염 유도 동물 모델에서 대장염 치료 효과를 확인하고자 하였다. 대장염 유도 전, 랫트는 24시간 동안 음식 섭취는 제한하였으나 물은 자유롭게 마실 수 있도록 하였다. 랫트를 에테르로 가볍게 마취시켰다. 고무 캐뉼라(rubber cannula; o.d., 2 mm)를 결장내로 삽입하였는데, 그 끝은 항문(anus)에서 8 cm 정도 떨어진 부위로서 대략 비장굴곡부(splenic flexture) 근처에 올 수 있도록 하였다. 50% (v/v) 에탄올 수용액에 녹인 2,4,6-trinitro benzene sulfonic acid (TNBS; Sigma Chemical Co, St Louis, Missouri, USA)를 고무 캐뉼라(15 mg/0.3 mL/rat)를 통해 결장 내로 주입하였다. 대장염 유도 72시간 후, 서열번호 1의 펩타이드 (30mg/kg)을 하루에 한번 씩 복강주사 하였고, 7일 동안 투약 후, 랫트를 희생시켰다. 랫트의 체중은 매일 오전 9시에 측정되었고, 대장의 길이는 랫트를 희생시키고, 맹장에서 항문까지 vernier caliper (Mitutoyo, Japan)를 이용하여 측정하였다. In order to investigate whether the peptide according to the present invention has an effect of treating inflammatory colitis on in vivo , it was intended to confirm the effect of colitis treatment in a colitis-induced animal model. Before colitis induction, rats restricted their food intake for 24 hours, but allowed water to drink freely. Rats were lightly anesthetized with ether. A rubber cannula (od, 2 mm) was inserted into the colon, with the tip about 8 cm away from the anus so that it could be near the splenic flexture. 2,4,6-trinitro benzene sulfonic acid (TNBS; Sigma Chemical Co, St Louis, Missouri, USA) dissolved in a 50% (v / v) aqueous ethanol solution is colonized via a rubber cannula (15 mg / 0.3 mL / rat). Injected into. After 72 hours of colitis induction, the peptide of SEQ ID NO: 1 (30mg / kg) was intraperitoneally injected once a day, and after administration for 7 days, the rats were sacrificed. Rat body weight was measured at 9:00 am daily, and colon length was measured using a vernier caliper (Mitutoyo, Japan) from the cecum to the anus, at the expense of the rat.
실험 8일째 렛트로부터 대장을 적출하여 육안으로 관찰해 본 결과, TNBS 주입으로 대장염을 유발시킨 군(TNBS)의 대장은 정상군(Normal)에 비하여 현저히 그 길이가 단축되고 대장 조직의 충혈이 관찰되었으나, 대장염을 유발시킨 후 서열번호 1의 펩타이드를 주사한 군은 정상군(CON)과 유사하게 관찰되어 대장염이 뚜렷하게 억제되었음을 알 수 있었다(도 6a). On the 8th day of the experiment, colons were removed from the rats and visually observed, and the colon of the TNBS-induced colitis (TNBS) was significantly shorter in length than the normal group and hyperemia of the colon tissue was observed. , The group injected with the peptide of SEQ ID NO: 1 after inducing colitis were observed similarly to the normal group (CON), indicating that colitis was clearly inhibited (FIG. 6A).
또한, 적출한 대장의 길이를 측정한 결과에서도, TNBS 주입으로 대장염을 유발시킨 군(TNBS)의 대장은 정상군(Normal)에 비하여 현저히 감소하였으나, 서열번호 1의 펩타이드를 주사한 군은 대장의 길이가 정상군과 유사하게 증가하였다.In addition, even in the result of measuring the length of the colon, the colon of the group (TNBS) that induced colitis by TNBS injection was significantly reduced compared to the normal group, but the group injected with the peptide of SEQ ID NO: 1 Length increased similarly to normal group.
적출된 대장은 -70℃에서 냉동저장 된 후, 대장의 근육층(muscle layer)에서 점막조직을 채취하여 점막 조직의 무게를 측정하고, 10mg의 점막조직을 삼중세척완충액(50mM Tris-HCl, pH 8.0, 150mM NaCl, 0.1% sodium deoxycholate and 1mM phenylmethysulfonyl fluoride)에 용해시켜서 균질화하였다. 이후 ELISA kit (R&D, Minneapolis, MN, USA)를 이용하여 p40, TNF-α, IL-12을 정량하였다.After the colon is frozen and stored at -70 ℃, the mucosal tissue is collected from the muscle layer of the large intestine and the mucosal tissue is weighed, and 10 mg of the mucosal tissue is triple-washed with a buffer (50 mM Tris-HCl, pH 8.0). , Homogenized by dissolving in 150 mM NaCl, 0.1% sodium deoxycholate and 1 mM phenylmethysulfonyl fluoride). Then, p40, TNF-α, IL-12 were quantified using an ELISA kit (R & D, Minneapolis, MN, USA).
그 결과, TNBS를 처리한 군에서 p40, TNF-α, IL-12의 발현이 증가하였으나, 서열번호 1의 펩타이드를 주사한 군은 정상군과 유사하게 p40, TNF-α, IL-12가 감소하는 것을 확인할 수 있었다(도 6c).As a result, the expression of p40, TNF-α and IL-12 increased in the TNBS-treated group, but the p40, TNF-α and IL-12 decreased in the group injected with the peptide of SEQ ID NO: 1, similar to the normal group. It was confirmed that (Fig. 6c).
실험예 4: 합성 펩타이드에 의한 간질환 마커 GDF15와 ATF3 단백질의 발현 감소 효과 확인 Experimental Example 4: Confirmation of the effect of reducing the expression of liver disease markers GDF15 and ATF3 protein by the synthetic peptide
웨스턴블럿을 실시하여 서열번호 1 내지 4의 펩타이드에 의해 유도되는 간질환 마커인 GDF15와 ATF3 단백질의 발현의 변화를 확인하고자 하였다. 6-well plate 에 70%의 density로 HepG2 cells을 plating 한 후 16시간 뒤에 0.5% FBS가 포함된 DMEM 배지로 2시간 동안 cell starvation 하였다. 서열번호 1 내지 4의 펩타이드를 각각 100, 200μM 농도로 포함한 배지를 2시간 동안 처리한 후, 팔미틱 액시드 (palmitic acid)를 200μM 농도로 처리하여 24시간 동안 염증 반응을 유도하였다. Protease inhibitor와 phosphatase inhibitor를 포함한 RIPA lysis buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)를 사용하여 단백질을 lysis하였다. BCA protein assay를 통해 단백질을 정량하고, western blot으로 GDF15, ATF3, FAS, tubulin 단백질의 발현량을 확인하였다. Western blot의 경우, 8% SDS PAGE gel 에 size marker와 함께 sample을 동량 loading하여 약 2시간 전기영동 한 후, nitrocellulose membrane에 transfer하였다. Transfer 한 membrane은 5% skim milk로 1시간 blocking하고, 1st antibody를 1:1000 비율로 하룻밤 동안 반응시켰다. 이후 membrane을 0.1% tween-20을 포함한 TBST로 세척 하고 HRP가 부착된 2nd antibody로 1시간 반응시킨 후, ECL substrate로 화학발광을 확인하였다.Western blot was performed to confirm the change in expression of GDF15 and ATF3 proteins, which are liver disease markers induced by the peptides of SEQ ID NOS: 1-4. HepG2 cells were plated at 70% density on a 6-well plate, followed by cell starvation for 2 hours with DMEM medium containing 0.5% FBS after 16 hours. After treating the medium containing the peptide of SEQ ID NOS: 1 to 4 at 100 and 200 μM concentrations for 2 hours, the palmitic acid was treated at 200 μM concentration to induce an inflammatory response for 24 hours. Proteins were lysed using RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Proteins were quantified by BCA protein assay and the expression levels of GDF15, ATF3, FAS and tubulin proteins were confirmed by western blot. In the case of Western blot, the sample was loaded in an 8% SDS PAGE gel with the size marker in the same amount and electrophoresed for about 2 hours, and then transferred to the nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and reacted with 1st antibody overnight at 1: 1000 ratio. After the membrane was washed with TBST containing 0.1% tween-20 and reacted with HRP-attached 2nd antibody for 1 hour, the chemiluminescence was confirmed by ECL substrate.
그 결과, 팔미틱액시드 (PA)만 단독으로 처리한 경우에는 간질환 마커인 GDF15와 ATF3 단백질의 발현이 증가하였으나, 서열번호 1 내지 4의 펩타이드를 처리하였을 때, 각각 GDF15와 ATF3 단백질의 발현이 감소하였다. 이를 통해, 서열번호 1-4의 펩타이드가 간질환 마커인 GDF15와 ATF3 발현을 억제하는 것으로 보아 팔미틱 액시드에 의해 유도된 간질환을 치료하는 효과가 있음을 확인할 수 있었다(도 7). As a result, the expression of GDF15 and ATF3 proteins, which are liver disease markers, increased when palmitic acid (PA) alone was treated. However, when the peptides of SEQ ID NOS: 1 to 4 were treated, the expression of GDF15 and ATF3 proteins was decreased. Decreased. Through this, it was confirmed that the peptides of SEQ ID NOS: 1-4 inhibit the expression of GDF15 and ATF3, which are liver disease markers, and thus have an effect of treating liver disease induced by palmitic acid (FIG. 7).
실험예 5: 합성 펩타이드에 의한 간염 치료효과 및 비만 억제효과 확인 Experimental Example 5: Confirmation of hepatitis treatment effect and obesity inhibitory effect by synthetic peptide
본 발명에 따른 펩타이드가 in vivo 상에 간염 치료 효과 및 비만 억제효과가 있는지 조사하기 위하여, 고지방식이로 유도된 비알코올성 간염 유발 동물 모델을 이용하였다. 수컷 C57BL/6종 마우스(7주령)를 1주간 순화시키고, 20마리의 동물을 각각 정상식이군(control), 고지방식이군(DIO: HFD induced obesity), 서열번호 1의 펩타이드 저용량 (LD 서열번호1, 15mg/kg) 또는 고용량 (HD 서열번호 1, 30mg/kg)을 주사한 고지방식이군 등 실험군 당 5 마리씩으로 분리하고 7주간 고지방식이 또는 정상식이로 사육하였다. 고지방식이는 60% high fat diet(Dyets, Inc)를 식이하였다. 7주간 사육하는 동안 매주 2회씩 체중과 사료섭취량을 측정하였으며, 7주간 사육한 후 50일째에 동물을 희생시켜, 간 및 지방조직을 채취하여 크기를 비교하였다.In order to investigate whether the peptides according to the present invention have a hepatitis therapeutic effect and an obesity inhibitory effect in vivo , a non-alcoholic hepatitis-induced animal model induced by a high fat diet was used. Male C57BL / 6 mice (7 weeks old) were acclimated for 1 week, and 20 animals were controlled in normal diet, HFD induced obesity (DIO), and peptide low dose (LD SEQ ID NO: 1), respectively. , 15mg / kg) or high doses (HD SEQ ID NO: 1, 30mg / kg) were injected into each of the 5 groups per experimental group, such as the high-fat diet group was injected and fed to a high-fat diet or normal diet for 7 weeks. The high-fat diet was fed a 60% high fat diet (Dyets, Inc). During the seven-week breeding, body weight and feed intake were measured twice a week. After breeding for seven weeks, animals were sacrificed on the 50th day, and liver and adipose tissues were collected and compared in size.
그 결과, 고지방식이군은 간과 피하지방조직의 크기가 정상식이군에 비해 크게 증가하였으나, 고지방식이에 의해 증가된 간과 피하지방의 크기가 서열번호 1의 펩타이드를 주사한 실험군에서는 감소하는 효과를 확인할 수 있었다(도 8).As a result, in the high fat diet group, the size of liver and subcutaneous fat tissue was significantly increased compared with the normal diet group, but the size of liver and subcutaneous fat increased by the high fat diet was decreased in the experimental group injected with the peptide of SEQ ID NO: 1. Could be (FIG. 8).
이로써, 서열번호 1 펩타이드가 간 비대증 및 지방간을 치료하는 효과를 발휘하고, 또한 피하지방 증가를 감소시켜 비만을 억제하는 효과가 있음을 알 수 있었다.As a result, it was found that the SEQ ID NO: 1 peptide has an effect of treating liver hypertrophy and fatty liver, and also has an effect of suppressing obesity by reducing an increase in subcutaneous fat.
실험예 6: 합성 펩타이드의 DSS유도 염증성 장질환 동물모델에서의 치료효과 확인Experimental Example 6: Confirmation of therapeutic effect of synthetic peptides in animal model of DSS-induced inflammatory bowel disease
동물수준에서 합성 펩타이드의 치료효과를 확인하기 위해, DSS로 ICR 마우스에 염증성 장질환을 유도하며 동시에 합성 펩타이드를 주입하였다. 10일 동안 5% DSS를 식용수로 사용하면서 마우스에 염증을 유발 하였고, 동시에 합성 펩타이드를 IP injection 하였다. Normal, Defect, 양성대조군(SAHA), 면역조절대조군(anti-TNF-a antibody), 서열번호 1-4 펩타이드를 처리하였다. 10일 후, CO2 과호흡에 의한 희생 후, Colon 확보하여 길이 비교하였다 (도 9a). 조직을 고정 및 처리과정을 거쳐 파라핀 블록을 제작하고, H&E 염색을 진행하여 장내 융털 손상여부를 확인하였다.In order to confirm the therapeutic effect of the synthetic peptide at the animal level, DSS was induced with inflammatory bowel disease and simultaneously injected with the synthetic peptide. Inflammation was induced in mice using 5% DSS as drinking water for 10 days, and at the same time, IP injection of synthetic peptides was performed. Normal, Defect, positive control (SAHA), immunoregulatory control (anti-TNF-a antibody), SEQ ID NO: 1-4 peptides were treated. After 10 days, after sacrifice by CO2 over-breathing, colon was secured and the length was compared (FIG. 9A). The tissue was fixed and treated to prepare a paraffin block, and H & E staining was performed to check whether the intestinal vill was damaged.
그 결과, DSS로 염증을 유도한 군에서 염증세포가 증가하고, 장내 융모가 손상되었으나, 서열번호 1-4의 펩타이드를 주사한 군은 정상군과 비슷하게 염증세포와 융모 손상이 감소한 것을 확인할 수 있었다.(도 9b).As a result, inflammatory cells increased and intestinal villi were damaged in the DSS-induced group, but the group injected with the peptides of SEQ ID NOS: 1-4 showed a decrease in inflammatory cells and villi in the same group as the normal group. (FIG. 9B).
실험예 7: 합성 펩타이드의 MCD 다이어트 마우스 모델을 이용한 비알콜성 지방간염 치료효과 확인Experimental Example 7: Confirmation of non-alcoholic steatohepatitis treatment effect using MCD diet mouse model of the synthetic peptide
본 발명에 따른 동물수준에서 펩타이드의 지방 간염 치료 효과를 확인하기 위하여, MCD(Methionine/Choline Deficient) 다이어트 마우스 모델을 이용하였다. 수컷 C57BL/6J 마우스를 이용하였고, Methionine/Choline Deficient(MCD) 사료를 8주간 자율급이 하는 방식으로 비알콜성 지방간염을 유도하였다. 유도 후, 서열번호 1-6의 펩타이드를 피하투여 방법으로 1일 1회 투여, 3일 간격으로 총 12회를 투여하였다. 매주 2회 모든 동물에 대하여 체중 측정하였다. 투여 종료 후 isoflurane으로 호흡 마취 하에 후대정맥으로 혈액 채취한 후, 방혈시키는 방식으로 안락사를 진행하였다. 동물 부검을 하고 간을 확보하여 중성 완충 포르말린 용액에 고정하였다. 파라핀 블록을 제작하여 H&E 염색을 통해 간조직 내에 축적된 지방을 확인하고, 섬유화 된 정도를 확인하여, NAS score를 평가하였다.In order to confirm the effect of the peptide hepatitis on the animal level according to the invention, MCD (Methionine / Choline Deficient) diet mouse model was used. Male C57BL / 6J mice were used and non-alcoholic steatohepatitis was induced in a manner of self-leveling for 8 weeks with Methionine / Choline Deficient (MCD) diet. After induction, the peptides of SEQ ID NOS: 1-6 were administered once daily by subcutaneous administration, and a total of 12 administrations were performed every three days. All animals were weighed twice weekly. After the end of the administration, blood was collected by posterior vena cava under respiratory anesthesia with isoflurane, and euthanasia was performed by bleeding. Animal necropsy and liver were secured and fixed in neutral buffered formalin solution. Paraffin blocks were prepared to confirm fat accumulation in liver tissues through H & E staining, and to determine the degree of fibrosis, and NAS score was evaluated.
그 결과, 도 10a에 나타난 바와 같이, MCD 다이어트를 유도한 군에서 염증 및 지방입자의 크기와 수가 증가하였고, 펩타이드를 주입한 군에서는 염증과 지방입자가 크게 감소된 것을 확인할 수 있었다. 도 10b와 도 10c에 나타난 바와 같이, MCD 다이어트를 유도한 군에서 섬유화(도 10b는 붉은색, 도 10c는 푸른색)가 크게 진행 되었고, 펩타이드를 주입한 군에서는 섬유화가 크게 감소된 것을 확인할 수 있었다. As a result, as shown in Figure 10a, the size and number of inflammation and fat particles in the MCD diet-induced group was increased, it was confirmed that the inflammation and fat particles were significantly reduced in the group injected with the peptide. As shown in Figure 10b and Figure 10c, fibrosis (Fig. 10b is red, Fig. 10c is blue) in the MCD diet-induced group was greatly progressed, it was confirmed that the fibrosis was significantly reduced in the group injected with the peptide. there was.
NAS Score는 Ballooning degeneration, Lobular inflammation, Steatosis의 합계를 측정한 것이다. NAS Score에서도 펩타이드를 처리한 군에서 50% 이상 감소하였기 때문에 비알콜성 지방간이 개선된 것을 확인할 수 있었다 (도 10d)NAS Score is a measure of ballooning degeneration, lobular inflammation, and steatosis. NAS score was also reduced by more than 50% in the peptide-treated group was confirmed that the non-alcoholic fatty liver is improved (Fig. 10d)
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 펩타이드는 염증 및 대사성 질환에 관계된 바이오마커에 대해 Histone deacetylase (HDAC)의 인산화 저해, GDF15 단백질의 발현저해, ATF3 단백질의 발현저해 등으로 삼중 차단함으로써, 염증유발 사이토카인 (inflammatory cytokine)의 생성을 감소시킬 수 있는바, 대사성 질환의 예방 및 치료에 효과가 있다. Peptides according to the present invention by inhibiting the phosphorylation of Histone deacetylase (HDAC), GDF15 protein expression, ATF3 protein expression inhibition, etc. to the biomarkers related to inflammatory and metabolic diseases, inflammatory cytokine (inflammatory cytokine) It can reduce the production of, it is effective in the prevention and treatment of metabolic diseases.
전자파일 첨부하였음.Electronic file attached.

Claims (10)

  1. 서열번호 1 내지 서열번호 8로 구성된 군 중 선택되는 어느 하나의 아미노산 서열로 표시되는 펩타이드를 유효성분으로 포함하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물. A pharmaceutical composition for treating or preventing inflammation, metabolic or fibrotic disease, comprising as an active ingredient a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  2. 제1항에 있어서, 상기 질환은 HDAC5 단백질의 과인산화, GDF15 단백질의 과발현 및/또는 ATF3 단백질의 과발현에 의해 발병하는 질환인 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물. The pharmaceutical composition for treating or preventing inflammatory, metabolic or fibrotic diseases according to claim 1, wherein the disease is caused by hyperphosphorylation of HDAC5 protein, overexpression of GDF15 protein, and / or overexpression of ATF3 protein.
  3. 제1항에 있어서, 상기 펩타이드는 HDAC5 단백질의 인산화, GDF15 단백질의 발현 및/또는 ATF3 단백질의 발현을 억제하는 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물. The pharmaceutical composition of claim 1, wherein the peptide inhibits phosphorylation of HDAC5 protein, expression of GDF15 protein, and / or expression of ATF3 protein.
  4. 제1항에 있어서, 상기 대사성 질환은 관절염, 치주염, 아토피, 염증성 대장염, 궤양성 장염, 크론병, 간염, 지방간 및 비만으로 구성된 군에서 선택되는 하나 이상의 대사성 질환인 것을 특징으로 하는 대사성 질환 치료 또는 예방용 약학적 조성물. The method of claim 1, wherein the metabolic disease is at least one metabolic disease selected from the group consisting of arthritis, periodontitis, atopy, inflammatory colitis, ulcerative enteritis, Crohn's disease, hepatitis, fatty liver, and obesity. Preventive pharmaceutical composition.
  5. 제4항에 있어서, 상기 간염은 당뇨 및/또는 비만에 의해 발생하는 비알코올성 지방간염인 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물.The method of claim 4, wherein the hepatitis is a non-alcoholic steatohepatitis caused by diabetes and / or obesity, characterized in that the pharmaceutical composition for the treatment or prevention of inflammatory, metabolic or fibrotic diseases.
  6. 제 1항에 있어서, 상기 섬유화 질환은 간경화, 폐섬유증 폐쇄성 폐질환, 심부전, 동백경화, 만성신부전, 당뇨병 및 수술후 후유증으로 발생하는 켈로이드로 구성된 군에서 선택되는 하나 이상의 질환인 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물.The method according to claim 1, wherein the fibrosis disease is at least one disease selected from the group consisting of liver cirrhosis, pulmonary fibrosis obstructive pulmonary disease, heart failure, camellia hardening, chronic kidney failure, diabetes and postoperative sequelae. Pharmaceutical compositions for treating or preventing metabolic or fibrotic diseases.
  7. 제 1항에 있어서, 상기 약학적 조성물은 주사제, 경구투여용 제제, 패치제, 액제, 캡술, 과립, 정제, 산제, 분무제, 연고제, 겔제, 점막투여제제 및 좌제로 구성된 군에서 선택되는 어느 하나의 제형으로 제제화되는 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물.The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is any one selected from the group consisting of injections, oral dosage forms, patches, solutions, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal dosages, and suppositories. A pharmaceutical composition for the treatment or prevention of inflammatory, metabolic or fibrotic diseases, characterized in that it is formulated in the formulation of.
  8. 제 7항에 있어서, 약제학적으로 허용가능한 보조제를 추가로 함유하는 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 또는 예방용 약학적 조성물.8. The pharmaceutical composition for treating or preventing inflammatory, metabolic or fibrotic disease according to claim 7, further comprising a pharmaceutically acceptable adjuvant.
  9. 제 8항에 있어서, 상기 약제학적으로 허용가능한 보조제는 부형제, 희석제, 완충제, 항균성 보존제, 계면활성제, 산화방지제, 증점제 및 점도개질제로 구성된 군에서 선택된 하나 이상의 보조제인 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 및 예방용 약학적 조성물.9. The method of claim 8, wherein the pharmaceutically acceptable adjuvant is at least one adjuvant selected from the group consisting of excipients, diluents, buffers, antimicrobial preservatives, surfactants, antioxidants, thickeners and viscosity modifiers. Pharmaceutical compositions for treating and preventing fibrotic diseases.
  10. 제 1항에 있어서, 상기 펩타이드의 일일 투여량은 1 내지 100 ㎎/㎏ 이며, 일일 일회 또는 주 2-3회 투여하는 것을 특징으로 하는 염증, 대사성 또는 섬유화 질환 치료 및 예방용 약학적 조성물.The method of claim 1, wherein the daily dose of the peptide is 1 to 100 mg / kg, a pharmaceutical composition for the treatment and prevention of inflammation, metabolic or fibrotic disease, characterized in that administered once or 2-3 times a week.
PCT/KR2019/011037 2018-08-31 2019-08-29 Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers WO2020046002A1 (en)

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US17/272,010 US20210340182A1 (en) 2018-08-31 2019-08-29 Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers
CN201980063987.2A CN112912093A (en) 2018-08-31 2019-08-29 Novel use of peptides for inhibiting the function and expression of multiple disease biomarkers
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