WO2020046002A1 - Nouvelle utilisation d'un peptide pour inhiber des fonctions et des expressions de biomarqueurs de maladies multiples - Google Patents

Nouvelle utilisation d'un peptide pour inhiber des fonctions et des expressions de biomarqueurs de maladies multiples 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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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/ko
Application filed by 주식회사 나이벡 filed Critical 주식회사 나이벡
Priority to CN201980063987.2A priority Critical patent/CN112912093A/zh
Priority to EP23151983.6A priority patent/EP4218788B1/fr
Priority to EP19855142.6A priority patent/EP3862015B1/fr
Priority to US17/272,010 priority patent/US20210340182A1/en
Priority to DK19855142.6T priority patent/DK3862015T3/da
Publication of WO2020046002A1 publication Critical patent/WO2020046002A1/fr
Priority to US18/385,640 priority patent/US20240166688A1/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

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  • 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.

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  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une nouvelle utilisation d'un peptide pour inhiber les fonctions et les expressions de biomarqueurs de maladie multiples et, plus spécifiquement, une composition pharmaceutique pour traiter ou prévenir une inflammation, des maladies métaboliques ou des maladies fibrotiques, la composition comprenant, en tant que principe actif, un ou plusieurs peptides quelconques parmi des peptides qui sont représentés par les séquences d'acides aminés de SEQ ID NO : 1 à 8, et qui, pour l'histone désacétylase 5 (HDAC5), GDF15 et ATF3 qui sont des biomarqueurs associés à une inflammation, des maladies métaboliques ou des maladies fibrotiques, ont les fonctions d'inhibition de la phosphorylation de HDAC5, inhibition de l'expression de la protéine GDF15 et inhibition de l'expression de la protéine ATF3. Le peptide, selon la présente invention, permet le triple blocage des biomarqueurs associés à une inflammation, des maladies métaboliques ou des maladies fibrotiques par inhibition de la phosphorylation de HDAC, l'inhibition de l'expression de la protéine GDF15 et l'inhibition de l'expression de la protéine ATF3 et, par conséquent, la production de cytokine inflammatoire peut être réduite et, par conséquent, le peptide est efficace dans la prévention et le traitement de maladies inflammatoires, métaboliques et fibrotiques.
PCT/KR2019/011037 2018-08-31 2019-08-29 Nouvelle utilisation d'un peptide pour inhiber des fonctions et des expressions de biomarqueurs de maladies multiples WO2020046002A1 (fr)

Priority Applications (6)

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CN201980063987.2A CN112912093A (zh) 2018-08-31 2019-08-29 肽用于抑制多种疾病生物标志物的功能和表达的新用途
EP23151983.6A EP4218788B1 (fr) 2018-08-31 2019-08-29 Peptides pour l'utilisation dans le traitement de la stéatohépatite non-alcoolique
EP19855142.6A EP3862015B1 (fr) 2018-08-31 2019-08-29 Peptides pour l'utilisation dans le traitement de la colite inflammatoire
US17/272,010 US20210340182A1 (en) 2018-08-31 2019-08-29 Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers
DK19855142.6T DK3862015T3 (da) 2018-08-31 2019-08-29 Peptider til anvendelse ved behandling af inflammatorisk colitis
US18/385,640 US20240166688A1 (en) 2018-08-31 2023-10-31 Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers

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KR20180103615 2018-08-31
KR10-2018-0103615 2018-08-31
KR1020190105917A KR102244161B1 (ko) 2018-08-31 2019-08-28 다중의 질환 바이오마커의 기능 및 발현을 억제하는 펩타이드의 신규한 용도
KR10-2019-0105917 2019-08-28

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US18/385,640 Division US20240166688A1 (en) 2018-08-31 2023-10-31 Novel use of peptide for inhibiting functions and expressions of multiple disease biomarkers

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CN115737616A (zh) * 2021-11-29 2023-03-07 南方医科大学南方医院 Lmk-235在医学中的新应用
CN115737616B (zh) * 2021-11-29 2024-06-07 南方医科大学南方医院 Lmk-235在医学中的新应用

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