WO2021182927A1 - 신규 대사증후군 및 그와 관련된 질환 치료용 약학 조성물 - Google Patents

신규 대사증후군 및 그와 관련된 질환 치료용 약학 조성물 Download PDF

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WO2021182927A1
WO2021182927A1 PCT/KR2021/003127 KR2021003127W WO2021182927A1 WO 2021182927 A1 WO2021182927 A1 WO 2021182927A1 KR 2021003127 W KR2021003127 W KR 2021003127W WO 2021182927 A1 WO2021182927 A1 WO 2021182927A1
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glp
fusion protein
seq
analogue
region
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PCT/KR2021/003127
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English (en)
French (fr)
Korean (ko)
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양상인
안인복
이용호
배수한
이명식
김은란
박정수
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주식회사 에스엘메타젠
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Priority to CN202180034874.7A priority Critical patent/CN115996740A/zh
Publication of WO2021182927A1 publication Critical patent/WO2021182927A1/ko

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a novel pharmaceutical composition, and more particularly, to a pharmaceutical composition for the treatment of metabolic syndrome and related diseases such as nonalcoholic steatohepatitis.
  • Metabolic syndrome is a collection of various physical phenomena that increase the risk of heart disease, stroke, and type 2 diabetes simultaneously. These phenomena include increased blood pressure, high blood sugar, excessive body fat around the waist, abnormal blood cholesterol or triglyceride levels; and the like. These metabolic syndromes can be intimidating in that they do not accompany clear signs or symptoms. If these conditions are left unattended or worsened, they will eventually develop into cardiovascular diseases such as arteriosclerosis and stroke, or into intractable diseases such as type 2 diabetes and nonalcoholic fatty liver disease. have.
  • Non-alcoholic steatohepatitis (hereinafter, referred to as 'NASH') has a characteristic etiology depending on the stage of progression.
  • the etiology starting from insulin resistance, dysregulation of glucose/lipids, steatosis, inflammation and fibrosis, and apoptosis, are gradually related to non-alcoholic fatty liver disease (non-alcoholic fatty liver disease).
  • Liver disease hereinafter, abbreviated as 'NAFLD') progresses to NASH (classified into F0, F1, F2, F3, F4 stages) and further progresses to cirrhosis (compensated ⁇ decompensated).
  • Nonalcoholic fatty liver disease is a liver disease that is rapidly increasing along with metabolic syndrome accompanied by obesity, diabetes, hypertension, etc., and can progress to cirrhosis or liver cancer.
  • 'NASH' non-alcoholic steatohepatitis
  • NASH-related cirrhosis are rapidly increasing, many studies are being conducted to develop therapeutic agents for them.
  • NASH has a characteristic etiology depending on the stage of progression.
  • pathogenesis such as insulin resistance, dysregulation of glucose/lipids, steatosis, inflammation and fibrosis, and apoptosis are gradually related, and NASH (F0, NASH (F0, It progresses to F1, F2, F3, and F4 stages) and further progresses to cirrhosis (compensated ⁇ decompensated).
  • the intestine is anatomically and physiologically connected and has various effects on liver pathology, so proper management of the intestinal-liver axis is effective in preventing fibrosis in alcoholic and nonalcoholic steatohepatitis, which has been suggested as a fundamental treatment to reduce cirrhosis itself.
  • a leaky gut can be a cutting edge for toxins, antigens or bacteria to pass into the body and has been suggested to play a pathogenic role in progressive cirrhosis, and it has been suggested that gut microbiota and probiotics (probiotics, prebiotics) in the field of liver disease There is a lot of interest in the role of beneficial bacteria in the gut, such as biotics, etc.).
  • the intestinal flora has emerged as a mediator of the intestinal-liver axis, and the weakening of the intestinal barrier function due to excessive intake of high-fat diet produces a large amount of intestinal microorganisms (microbe-related molecular patterns: MAMPs such as LPS and LTA), and the intestinal flora itself may metastasize to the liver and promote liver diseases such as hepatitis and liver fibrosis.
  • MAMPs microbe-related molecular patterns
  • MAMPs microbe-related molecular patterns
  • the intestinal flora itself may metastasize to the liver and promote liver diseases such as hepatitis and liver fibrosis.
  • Bile acids are actively absorbed and enter the colonic epithelium. Secondary bile acids are toxic and cause DNA damage, producing senescence-associated secretory phenotype (SASP) secretory factors in HSC (hepatic stellate cell) cells.
  • SASP senescence-associated secretory phenotype
  • the intestinal flora is involved in choline metabolism and is converted to TMA (trimethylamine), which is transferred to the liver and converted to TAMO (trimethylamine oxide), causing hepatitis.
  • TMA trimethylamine
  • TAMO trimethylamine oxide
  • the intestinal flora plays a role in maintaining the homeostasis of the human body, and when the homeostasis is impaired, metabolites and components extracted from the intestinal flora move to the liver, causing pathological effects in the liver. cause.
  • Molecule-based therapeutics developed for the treatment of NASH have made it possible to understand the mechanism of NASH progression. This was presented.
  • WO2016043533A1 discloses a therapeutic agent for nonalcoholic fatty liver disease comprising an oxyntomodulin derivative, which is a dual agonist of GLP-1/glucagon receptor with increased half-life as an active ingredient
  • US9938335 also discloses dual glucagon receptor/GLP-1 receptor Disclosed are a glucagon-like peptide used as an agonist and a method for treating NAFLD and NASH using the same.
  • the present invention is to solve various problems including the above-mentioned problems, and an object of the present invention is to provide a novel pharmaceutical composition that can more efficiently treat various metabolic syndromes including NASH.
  • the protection scope of the present invention is not limited to the above purpose.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of metabolic syndrome comprising a second fusion protein linked to the Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of obesity comprising a second fusion protein linked to an Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of type 2 diabetes* comprising a second fusion protein linked to the Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition is provided.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue
  • a pharmaceutical composition for the treatment of liver fibrosis comprising a second fusion protein linked to an antibody Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient .
  • the step of administering to an individual suffering from metabolic syndrome a GLP-2 analog comprising a second fusion protein linked to an antibody Fc region, and a bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein A method for treating metabolic syndrome in the subject is provided, comprising.
  • a method for treating obesity in the subject is provided.
  • the GLP-2 analog comprises a second fusion protein linked to an antibody Fc region, and the bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein is administered to an individual with type 2 diabetes.
  • a method for treating non-alcoholic steatohepatitis or non-alcoholic steatohepatitis in the subject comprising administering to the subject.
  • the step of administering to an individual suffering from liver fibrosis a GLP-2 analog comprising a second fusion protein linked to an antibody Fc region, and a bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein A method for treating liver fibrosis in the subject is provided, comprising.
  • the dual specificity fusion protein according to an embodiment of the present invention binds to the GLP-1 receptor and the GLP-2 receptor in the small intestine to increase the villi length and crypt depth of the small intestine, and to improve the intestinal environment, such as improving the intestinal flora. have an effect
  • the dual specificity fusion protein according to an embodiment of the present invention has an effect of reducing insulin resistance and weight loss.
  • FIG. 1 is a schematic diagram showing the schematic structure of a dual specificity fusion protein according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the interaction relationship between various GLP-1 and/or GLP-2 analogs and GLP-1 receptors, GLP-2 receptors and other glucagon receptors.
  • 3A is a series of gel pictures showing the results of SDS-PAGE analysis under non-reducing (left) and reducing (right) conditions after purifying various bispecific fusion proteins according to an embodiment of the present invention:
  • Figure 3b shows the non-reduced (NR) and reduced (R) after purification of MG12-5 (right) containing the GLP-2 homodimer (GLP-2-Fc homodimer, left) and Knobs-into-Holes (KiH) structure. ) is a series of gel pictures showing the results of SDS-PAGE analysis under the conditions:
  • Figure 4a is a graph showing the results of analyzing the biological activity of the GLP-1-Fc homodimeric protein and GLP-1 peptide according to an embodiment of the present invention by a luciferase reporter assay.
  • Figure 4b is a graph showing the results of measuring the biological activity of MG12-1 according to an embodiment of the present invention compared to the GLP-1 peptide.
  • Figure 4c is a graph showing the results of measuring the biological activity of MG12-3 in comparison with the GLP-1 peptide according to an embodiment of the present invention.
  • 4d is a graph showing the results of measuring the biological activity of MG12-4 according to an embodiment of the present invention compared to the GLP-1 peptide.
  • Figure 4e is a graph showing the results of measuring the biological activity of MG12-5 according to an embodiment of the present invention compared to the GLP-1 peptide.
  • 5A is a graph showing the results of fluorescence analysis of the GLP-2 activity of the GLP-2-Fc homodimer protein according to an embodiment of the present invention compared to the GLP-2 peptide.
  • 5B is a graph showing the results of fluorescence analysis of the GLP-2 activity of the MG12-1 protein according to an embodiment of the present invention compared to the GLP-2 peptide.
  • FIG. 5c is a graph showing the results of fluorescence analysis comparing the GLP-2 activity of the MG12-3 protein with the GLP-2 peptide according to an embodiment of the present invention.
  • 5D is a graph showing the results of fluorescence analysis of the GLP-2 activity of the MG12-4 protein according to an embodiment of the present invention compared to the GLP-2 peptide.
  • 5e is a graph showing the results of fluorescence analysis of the GLP-2 activity of the MG12-5 protein according to an embodiment of the present invention compared to the GLP-2 peptide.
  • 6A is a graph showing the results of analyzing the pharmacokinetics (PK) profile of various dual specificity fusion proteins according to an embodiment of the present invention to animals (rat) by ELISA analysis using GLP-1-Fc. .
  • 6b is a graph showing the results of analyzing the pharmacokinetics (PK) profile of various dual specificity fusion proteins according to an embodiment of the present invention to animals (rat) by ELISA analysis using GLP-2-Fc. .
  • FIG. 7a to 7m are results of measuring various physiological effects in experimental animals of the dual specificity protein (GLP1/2-Fc) according to an embodiment of the present invention with increased half-life
  • FIG. 7a is an embodiment of the present invention. It is a graph showing the results of measuring the body weight over time after subcutaneous administration of various drugs including the fusion protein according to the example twice a week for 4 weeks
  • Figure 7b is a fusion protein according to an embodiment of the present invention
  • FIG. 7d is a graph showing the result of measuring the level of total cholesterol in the blood after the end of the experiment in each experimental group
  • FIG. 7e is a graph showing the result of measuring the level of HDL in the blood after the end of the experiment in each experimental group
  • FIG. 7f is a graph showing the results of measuring the blood triglyceride level after the end of the experiment in each experimental group
  • FIG. 7g is a graph showing the IPITT results performed for 3 weeks in each experimental group
  • FIG. 7h is the IPGTT results performed in each experimental group for 4 weeks
  • 7i is a graph showing the results of monitoring blood glucose levels for 0 to 4 weeks after peptide treatment in each experimental group
  • FIG. 7j is a graph showing the results of measuring insulin levels in each experimental group for 4 weeks
  • FIG. 7k is a graph showing the result of calculating HOMO-IR in each experimental group.
  • IPITT Intraperitoneal insulin tolerance test.
  • IPGTT Intraperitoneal glucose tolerance test.
  • HOMO-IR Homeostatic model evaluation for insulin resistance.
  • FIG. 8A to 8L show the effect of a dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention on body weight and glucose homeostasis in a dose-dependent manner.
  • Figure 8a shows the results of weekly body weight measurement during the administration period after subcutaneous administration of the dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention to experimental animals at various concentrations twice a week for 4 weeks; is a graph
  • FIG. 8b is a graph showing the result of monitoring the change in body weight of the experimental animal
  • FIG. 8c is a graph showing the result of measuring the epididymal fat mass after 4 weeks of the experimental animal
  • FIG. 8d is drug administration It is a graph showing the result of measuring the blood triglyceride concentration measured 4 weeks after the start
  • FIG. 8E is a graph showing the result of measuring the total cholesterol concentration in the blood
  • FIG. 8F is a graph showing the result of measuring the blood HDL concentration
  • 8g is a graph showing the results of measuring the total bilirubin concentration in the blood of each experimental group at 4 weeks after administration of various drugs including the dual specificity fusion protein according to an embodiment of the present invention
  • FIG. 8h is a graph showing various concentrations It is a graph showing the results of measuring the total bilirubin concentration in the blood at 4 weeks after administration of the dual specificity fusion protein (GLP-1/2-Fc) of the present invention
  • FIG. 8j is the IPGTT result performed up to 4 weeks after administration of the dual specificity fusion protein at various concentrations according to an embodiment of the present invention.
  • FIG. 8K is a graph showing the result of measuring the basal blood glucose level before drug administration
  • FIG. 8L is a graph showing the result of measuring the basal blood glucose level at the end of the experiment.
  • FIG. 9a to 9g show the effect of reducing hepatic lipid accumulation and inflammation of the dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention with improved half-life.
  • FIG. 9b is the H&E staining result of the liver tissue section excised from each experimental group
  • FIG. 9c is a graph showing the result of measuring the liver weight excised after the end of the test
  • FIG. 9d is the end of the test It is a graph showing the result of measuring the liver to body weight ratio
  • FIG. 9e is a graph showing the result of measuring the amount of triglyceride (TG) in the liver tissue after the end of the test
  • FIG. 9f is an indicator of liver damage after the end of the experiment It is a graph showing the result of measuring the amount of phosphorus serum ALT, and FIG. 9g is a graph showing the result of measuring the serum AST level, which is another indicator of liver damage, after the end of the test.
  • H&E hematoxylin and eosin
  • TG triglyceride
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase.
  • FIG. 10A to 10F show the effect of GLP1/2-Fc according to an embodiment of the present invention on liver lipid accumulation in a dose-dependent manner
  • FIG. A series of photographs Figure 10b is a series of H & E staining photographs of liver tissue sections taken to analyze liver lipid deposition for each experimental group
  • Figure 10c is a graph showing the results of measuring the liver weight after the end of the test
  • Figure 10d is a graph showing the result of measuring the liver TG level after the end of the test
  • Figure 10e is a graph showing the measurement result of the serum ALT level, which is an indicator of liver damage
  • Figure 10f is another indicator of liver damage
  • serum AST level It is a graph showing the measurement result. Data were analyzed by one-way ANOVA followed by Dunnett's test.
  • H&E hematoxylin and eosin
  • TG triglyceride
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • FIG. 11a to 11e show the results of analyzing whether the dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention can improve liver fibrosis
  • FIG. 11a shows liver fibrosis evaluation for each experimental group. It is a series of photos showing the results of performing Sirius red staining for Then, it was analyzed by Dunnett's test, and expressed as mean ⁇ SEM (*** p ⁇ 0.001 vs. negative control),
  • Figure 11c shows the correlation of liver TG and area of Sirius red staining by Pearson's correlation analysis on a per-individual basis It is a graph showing one result, and FIG.
  • FIG. 11D is a graph showing the results of evaluating the correlation of liver TG and the area of Sirius red staining by Pearson's correlation analysis for each experimental group
  • FIG. 11E is a representative photograph for each experimental group. It is a series of micrographs showing the results of analyzing the degree of type 3 collagen deposition by immunohistochemical analysis. Scale bar: 200 ⁇ m.
  • Figures 12a and 12b are the results of examining the concentration-dependent effect of the dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention on liver fibrosis
  • Figure 12a is a negative control group (vehicle) and treatment concentration Differently, it is a series of photographs showing the Sirius red staining results for liver tissues of animals administered with GLP1/2-Fc
  • FIG. 13A to 13M show the results of examining the effect of the dual specificity fusion protein (GLP1/2-Fc) on the intestine according to an embodiment of the present invention
  • FIG. 13A is a series of photographs of the intestines extracted from each experimental group. a photograph
  • FIG. 12b is a graph showing the result of measuring the total length of the intestine in each experimental group
  • FIG. 12c is a graph showing the result of measuring the total length of the intestine in each experimental group
  • FIG. 12d is the measurement of the length of the small intestine in each experimental group It is a graph showing the result
  • Figure 12e is a graph showing the result of measuring the length of the colon in each experimental group
  • Figure 12f is a series of photos taken of the results of H & E staining of the cross section of the intestine in each experimental group
  • Figure 12g is As a photograph showing the result of staining Ki-67 protein (green) with immunofluorescence staining in each experimental group, the nucleus was visualized by DAPI staining
  • FIG. 12h is a graph showing the result of measuring the duodenal Crypt depth in each experimental group
  • Figure 12i is a graph showing the measurement result of the Crypt depth of the jejunum in each experimental group
  • Figure 12j is a graph showing the measurement result of the Crypt depth of the ileum in each experimental group
  • Figure 12k is a measurement of the duodenal villi height in each experimental group It is a graph showing the result
  • Figure 12l is a graph showing the result of measuring the height of the villi of the factory in each experimental group
  • Figure 12m is a graph showing the result of measuring the height of the villi of the ileum in each experimental group.
  • FIG. 14a to 14e show the concentration-dependent effects of the dual specificity fusion protein (GLP1/2-Fc) according to an embodiment of the present invention on the gastrointestinal tract
  • FIG. 14a shows the administration of various doses of GLP1/2-Fc.
  • It is a graph showing the result of measuring the total intestinal weight in a mouse
  • Figure 14b is a graph showing the result of measuring the total length of the intestine
  • Figure 14c is a graph showing the result of measuring the length of the small intestine
  • Figure 14d is the length of the colon is a graph showing the measurement result
  • FIG. 14e is a graph showing the measurement result of the Crypt depth in the colon of each experimental group.
  • Data were analyzed by one-way ANOVA followed by Dunnett's test. ** p ⁇ 0.01, **** p ⁇ 0.0001 versus negative control.
  • FIG. 15A to 15G are the results of examining the effects of the dual specificity fusion protein (GLP1/2-Fc) on intestinal permeability and serum endotoxin levels according to an embodiment of the present invention
  • Figure 15b is a graph showing the result of measuring the level of endotoxin in the serum
  • Figure 15c is As a representative photograph selected from each experimental group, it is an immunofluorescence microscopy photograph showing the detection result of zo-i protein (green) and nucleus (blue) in the ileum
  • FIG. 15e is a graph showing the results of measuring the thickness of the mucin layer in the ileum in each experimental group
  • Fig. 15f is the number of goblet cells per villi of the ileum in each experimental group.
  • Data were analyzed by one-way ANOVA followed by Dunnett's test. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, **** p ⁇ 0.0001 versus negative control. Scale bar: 200 ⁇ m.
  • 16a to 16l are the results of examining the effect of the dual specificity fusion protein (GLP1/2-Fc) on the intestinal flora composition according to an embodiment of the present invention.
  • FIG. 16b is a comparative diagram of the UPGMA phylogenetic tree of the analyzed intestinal flora
  • FIG. 16c is a bar graph showing the relative abundance of the intestinal flora identified in each experimental group by measuring the phylogenetic relative abundance at the phylum level
  • 16D is a bar graph showing the intestinal flora identified in each experimental group by measuring the relative abundance of the phylogenetic at the genus level
  • FIG. 16E is a negative control group and 18 microbial species in the GLP1/2-Fc administration group.
  • FIGS. 16f to 16l are of Akkermansia muciniphila (16f), Mailhella massiliensis (16g), Alistipes senegalensis (16h), Lactobacillus intestinalis (16i), Prevotellamassilia timonensis (16j), Faecalibaculum rodentium (16k) and Acetatifactor muris.
  • FIGS. 16f to 16l are of Akkermansia muciniphila (16f), Mailhella massiliensis (16g), Alistipes senegalensis (16h), Lactobacillus intestinalis (16i), Prevotellamassilia timonensis (16j), Faecalibaculum rodentium (16k) and Acetatifactor muris.
  • GLP-1 is an abbreviation of “glucagon-like peptide-1” and is 30 or 31 amino acids in length derived by tissue-specific post-translational processing of proglucagon peptides. is a peptide hormone of GLP-1 is produced and secreted by specific neurons in the enteroendocrine L-cells of the small intestine and the solitary tract nucleus of the brain stem during food intake.
  • the initial product, GLP-1(1-37) is readily amidated and cleaved with two equivalent biological activities by cleavage (GLP-1(7-36) amide and GLP-1(7-37)).
  • Active GLP-1 contains two alpha-helical regions at amino acid positions 13-20 and 24-35 and a linker region connecting the two alpha-helical regions. Since GLP-1 acts to lower blood sugar levels in a glucose-dependent manner, it has been developed and used as a treatment for type 2 diabetes. However, since GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) in vivo, its in vivo half-life is only 2 minutes, so its effect is extremely limited as a natural peptide.
  • DPP-4 dipeptidyl peptidase-4
  • GLP-2 is a 33 amino acid-long peptide produced by a special post-translational cleavage process of proglucagon like GLP-1. It is produced by enteroendocrine L cells of the small intestine and various neurons of the central nervous system. becomes this GLP-2 is secreted together with GLP-1 when food is ingested. In the case of GLP-2, when administered, it improves small intestine growth and function, reduces bone destruction, and is known to have neuroprotective action.
  • GLP-1 analog refers to a protein that biologically performs the function of GLP-1 and is capable of mediating downstream signaling by binding to the GLP-1 / exendin-4 receptor, and other It is called "GLP-1 recteptor agonist”.
  • GLP-2 analog refers to a protein that biologically performs the function of GLP-2, and is capable of mediating downstream signaling by binding to the GLP-2 receptor.
  • fusion protein refers to a recombinant protein in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain performs its original function, in other words " It is called "GLP-2 receptor agonist”.
  • half-life increasing moiety refers to a functional group that is linked to a recombinant protein to improve the half-life of the recombinant protein in the body.
  • Such "half-life increasing moieties” include antibody Fc regions (Capon et al ., Nature. 337: 525-531, 1989), PEG (Caliceti and Veronese, Adv. Drug Delivery Rev. 55: 1261-1277, 2003), XTEN (Schellenberger et al ., Nat. Biotechnol . 27: 1186-1190, 2009), PAS (Pro-Ala-Ser, Schlapschy et al ., Protein Eng. Des. Sel.
  • ELP elastine-like peptide
  • glycine-rich HAP homo-amino-acid polymer, Schlapschy et al ., Protein Eng. Des. Sel. 20: 273- 284, 2007
  • gelatin-like protein GLK, Huang et al ., Eur. J. Pharm. Biopharm. 74(3): 435-441, 2010
  • serum albumin Sheffield et al ., Cell Physiol. Biochem.
  • antibody Fc region refers to a crystallized fragment among fragments generated when an antibody is cleaved with papain, and a cell surface receptor called Fc receptor and some of the complement system. interacts with proteins.
  • the Fc region represents a homodimeric structure in which fragments comprising the second and third constant regions (CH2 and CH3) of the heavy chain are linked by intermolecular disulfide bonds at the hinge region.
  • the Fc region of IgG has a number of N-glycan attachment sites, which are known to play an important role in Fc receptor-mediated action.
  • hybrid Fc region refers to an Fc region peptide produced by a combination of parts of an Fc region of various isotypes of an antibody, such as IgA, IgE, IgD, IgG, IgM, etc., such Fc region
  • an antibody such as IgA, IgE, IgD, IgG, IgM, etc.
  • Exendin is a peptide consisting of 39 amino acids isolated from the venom of the lizard Heloderma suspectum.
  • Exendin 4 is 50% identical in amino acid sequence to GLP-1, is a member of the glucagon peptide family, and is known to perform an equivalent role to GLP-1 as an agonist of the GLP-1 receptor.
  • Exendin-4 is also called recent and "extenatide”.
  • Exendin 3 is a mutant in which the second and third amino acids in Exendin 4 are substituted with serine and aspartic acid, respectively.
  • Lutisenatide is one of the GLP-1 receptor agonists, manufactured by Sanofi, under the trade name Lyxumia in Europe, and Adlyxin under the trade name in the United States as a daily injection for the treatment of type 2 diabetes. It is a drug that is sold.
  • Albiglutide used in this document is one of the GLP-1 receptor agonists sold by GSK under the trade name of Eperzan in Europe and Tanzeum in the United States as a treatment for type 2 diabetes.
  • Liraglutide is a subcutaneously injectable GLP-1 receptor agonist marketed by Novo Nordisk under the trade name “Victoza” for the treatment of type 2 diabetes and obesity.
  • Taspoglutide is a GLP-1 receptor agonist co-developed by Ipsen and Roche for the treatment of type 2 diabetes.
  • Alanine which is the 8th and 35th amino acids of the GLP-1(7-36) peptide This is a GLP-1 derivative that is methylated and the last amino acid is amidated.
  • the C-terminus is not amidated and may be a general carboxyl group.
  • XTEN as used in this document is an unstructured low immunogenic peptide containing 6 amino acids added to improve the in vivo half-life of a protein drug developed by Amunix, usually 144 aa units. and is composed of amino acids of multiples thereof (US20100239554A1).
  • Teduglutide as used in this document is a mutant in which alanine (A), the second amino acid of GLP-2, is substituted with glycine (G). It is a commercially available GLP-2 analogue.
  • Glepaglutide used in this document is a GLP-2 analogue with improved half-life, developed as a treatment for short bowel syndrome and is currently undergoing phase 3 clinical trials for short bowel syndrome.
  • GLP-2 analogue 10 is one of the GLP-2 analogues, and is an intramolecular crosslinking agent lipidated through the thiol groups of the two substituted cysteines by substituting cysteines for the 11th and 18th amino acids of GLP-2. It has a stabilized structure by linking, and is characterized by adding 9 amino acids at the C-terminus of Exendin 4 to the C-terminus (Yang et al ., J. Med. Chem . 61: 3218-3223, 2018). ).
  • linker peptide is an unstructured peptide used to prepare a fusion protein by linking two or more proteins or peptides having different biological activities.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of metabolic syndrome comprising a second fusion protein linked to the Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of obesity comprising a second fusion protein linked to an Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition for the treatment of type 2 diabetes comprising a second fusion protein linked to an Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient do.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue are an antibody
  • a pharmaceutical composition is provided.
  • a bispecific fusion protein in which a GLP-1 analogue and a GLP-2 analogue are fused or ii) a first fusion protein in which a GLP-1 analogue is linked to an antibody Fc region and a GLP-2 analogue
  • a pharmaceutical composition for the treatment of liver fibrosis comprising a second fusion protein linked to an antibody Fc region, and comprising a dual specificity fusion protein produced by dimerization of the first fusion protein and the second fusion protein as an active ingredient .
  • the double fusion protein in which the GLP-1 analog and the GLP-2 analog are fused is proglucagon or its Instead of an analog, it may be a fusion protein in which a GLP-1 analog and a GLP-2 analog are directly linked, or two peptides are linked by a linker peptide other than the intermediate peptide.
  • the GLP-1 analog is GLP-1, Exendin 3 (Exendin 3), Exendin 4 (Exendin 4), GLP-1 / Exendin 4 hybrid peptide, GLP-1-XTEN, Exendin 4 -XTEN, Lixisenatide, Albiglutide, Liraglutide, or Taspoglutide.
  • the GLP-1 analog may be a GLP-1 continuous repeat in which two GLP-1s are linked by a linker peptide.
  • the bispecific fusion protein of i) may further include a half-life increasing moiety, wherein the half-life increasing moiety is inserted between the GLP-1 analogue and the GLP-2 analogue or the entire It can be added to the N-terminus or C-terminus of the fusion protein, and the half-life increasing moiety is an antibody Fc region, PEG, XTEN, PAS (Pro-Ala-Ser), ELP (elastin-like peptide), glycine-rich It may be a homo-amino-acid polymer (HAP), gelatin-like protein (GLP), or serum albumin.
  • a half-life increasing moiety is inserted between the GLP-1 analogue and the GLP-2 analogue or the entire It can be added to the N-terminus or C-terminus of the fusion protein, and the half-life increasing moiety is an antibody Fc region, PEG, XTEN, PAS (Pro-Ala-Ser), ELP (elastin-like peptid
  • the GLP-1 may include the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the Exendin 3 may include the amino acid sequence set forth in SEQ ID NO: 3.
  • Exendin 4 may include the amino acid sequence set forth in SEQ ID NO: 4.
  • the GLP-1/Exendin 4 hybrid may include the amino acid sequence shown in SEQ ID NO: 5.
  • the Lixisenatide may include the amino acid sequence shown in SEQ ID NO: 6.
  • the Exendin 4-XTEN may include the amino acid sequence set forth in SEQ ID NO: 7.
  • the Albiglutide may include the amino acid sequence shown in SEQ ID NO: 8.
  • the Liraglutide may include the amino acid sequence shown in SEQ ID NO: 9.
  • the Taspoglutide may include the amino acid sequence shown in SEQ ID NO: 10.
  • the GLP-1 continuous repeat may include the amino acid sequence shown in SEQ ID NO: 11.
  • the antibody Fc region may be a hybrid antibody Fc region, and the hybrid antibody Fc region may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 16.
  • the hybrid antibody Fc region may be additionally mutated so as not to cause unwanted side effects when administered in vivo, such as antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Threonine (T) which is the 18th amino acid of the hybrid Fc region variant shown in SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 12 in which methionine (M) at the 196th amino acid is substituted with leucine (L)
  • the GLP-2 analogue may be GLP-2, Teduglutide, Glepaglutide, or GLP-2 analogue 10.
  • the GLP-2 may include an amino acid sequence described in any one of SEQ ID NOs: 17 to 20.
  • the peptide consisting of the amino acid sequence shown in SEQ ID NO: 18 is a human GLP-2 wild-type peptide
  • the human GLP-2 mutant composed of the amino acid sequence shown in SEQ ID NO: 17 is a product in which alanine, the second amino acid, is substituted with glycine, and Teduglutide also called
  • the GLP-2 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 alanine (A), the second amino acid, is mutated to glycine (G), and asparagine (N), the 16th amino acid, is converted to glycine (G).
  • GLP-2 A2G, N16G, L17Q GLP-2 A2G, N16G, L17Q
  • L leucine
  • Q glutamine
  • GLP-2 wild-type peptide alanine, the 2nd amino acid, is substituted with glycine, and the 17th amino acid, leucine, is substituted with glutamine (A2G, L17Q, SEQ ID NO: 20) is also composed of the amino acid sequence shown in SEQ ID NO: 19 Since it can exert an equivalent function to that of a GLP-2 analogue, it is possible to use it as a GLP-2 analogue in the present invention.
  • the Teduglutide may include the amino acid sequence shown in SEQ ID NO: 17.
  • the Glepaglutide may include the amino acid sequence shown in SEQ ID NO: 21.
  • the GLP-2 analogue 10 may include the amino acid sequence shown in SEQ ID NO: 22.
  • the first fusion protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 30.
  • the second fusion protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 36.
  • the dual specificity fusion protein used in the pharmaceutical composition of the present invention at least one of the GLP-1 analogue and the GLP-2 analogue is a tandem repeat, and the GLP-1 analogue and the GLP-2 analogue
  • the number of repetitions of the GLP-2 analog may be different from each other.
  • the structure as described above is introduced for the asymmetry of the dual specificity fusion protein. As such, if the size of the unit fusion protein forming a heterodimer is changed, it is easy to monitor the degree of homodimer generation, so the quality control (quality control) control) is possible, and it has the advantage of reducing production costs because it can simplify the manufacturing process compared to PEGylation, which is well known for its half-life increasing technology.
  • the number of repeating units of the GLP-1 analog and the GLP-2 analog can be adjusted according to the required binding affinity for GLP-1R and GLP-2R.
  • the 10th amino acid of the CH3 domain is substituted with cysteine (C), and the 22nd amino acid, threonine (T), is substituted with tryptophan (W) (Knob structure)
  • tyrosine (Y) the 5th amino acid of the CH3 domain in the Fc region, is cysteine (C), the 22nd amino acid, threonine, is serine (S), and the 24th amino acid
  • leucine (L) is substituted with alanine (A) and tyrosine (Y), the 63rd amino acid, is substituted with valine (V) (Hole structure).
  • the first fusion protein is a CH3 domain of the Fc region
  • the 5th amino acid, tyrosine (Y), is cysteine (C), the 22nd amino acid, threonine, is serine (S), the 24th amino acid, leucine (L), is alanine (A), and the 63rd amino acid, tyrosine (Y)
  • This may be one substituted with valine (V) (Hole structure)
  • serine, the 10th amino acid of the CH3 domain in the hybrid Fc region is substituted with cysteine (C), and the 22nd amino acid threonine ( T) may be substituted with tryptophan (W) (Knob structure).
  • threonine (T) which is the 22nd amino acid of the CH3 domain in the hybrid Fc region
  • the second fusion protein is the 63rd amino acid of the CH3 domain in the hybrid Fc region.
  • Tyrosine (Y) is substituted with threonine (T)
  • tyrosine (Y) which is the 63rd amino acid of the CH3 domain in the hybrid Fc region
  • T threonine
  • T threonine
  • the mutation of the 63rd amino acid is not based on the amino acid sequence of the CH3 domain of human IgG1 described in SEQ ID NO: 69, but according to the numbering rule of the International ImMunoGeneTics information system (IMGT) (Lefranc et al ., Dev. Comp. Immunol . , 27: 55-77, 2003), may be denoted as Y86T.
  • IMGT International ImMunoGeneTics information system
  • one or more linker peptides may be inserted between fusion partners of the fusion protein, that is, between peptides or domains. That is, in the case of a bispecific fusion protein in which the GLP-1 analogue and the GLP-2 analogue of i) are fused, a linker peptide may be inserted between the GLP-1 analogue and the GLP-2 analogue, and the first In the case of a bispecific fusion protein produced by dimerization of the fusion protein and the second fusion protein, a linker peptide may be inserted between the GLP-1 analogue and the antibody Fc region inside the first fusion protein, and similarly, the second fusion protein A linker peptide may be inserted between the GLP-2 analogue and the antibody Fc region in the .
  • the linker peptide may or may not include an N-glycan attachment site, and more preferably, the first fusion protein does not include an N-glycan attachment site in the linker peptide and the second fusion protein does not include an N-glycan attachment site.
  • the fusion protein may include an N-glycan attachment site to the linker peptide.
  • neither the first fusion protein nor the second fusion protein may include an N-glycan attachment site.
  • the linker peptide is EPKSSDKTHTCPPCP (SEQ ID NO: 37), EPKSCDKTHTCPPCP (SEQ ID NO: 38), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 39), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 40), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 41), GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 42), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 43), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 44), GGGGSGGGGSGGGGS (SEQ ID NO: 45), GGSGG (SEQ ID NO: 46), GGSGGSGGS (SEQ ID NO: 47), GGGSGG (SEQ ID NO: 48), SEQ ID NO:
  • n is an integer from 1 to 10
  • (GGS) n is an integer from 1 to 10
  • (GS) n is an integer from 1 to 10
  • (GSSGGS) n unit: SEQ ID NO: 50
  • n is an integer from 1 to 10
  • KESGSVSSEQLAQFRSLD SEQ ID NO: 51
  • EGKSSGSGSESKST SEQ ID NO: 52
  • GSAGSAAGSGEF SEQ ID NO: 53
  • EAAAK n (unit: SEQ ID NO: 54, n is an integer from 1 to 10)
  • CRRRRRREAEAC SEQ ID NO: 55
  • a (EAAAK) 4 ALEA EAAAK) 4 A
  • PAPAP SEQ ID NO: 59) 60
  • (Ala-Pro)n (Ala-
  • composition may include an pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.
  • the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans.
  • examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid, sustained or delayed release of the active ingredient when administered to a mammal.
  • Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.
  • composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration may be administered, and may also be administered using an implantable device for sustained release or continuous or repeated release.
  • the number of administration may be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.
  • composition according to an embodiment of the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes, for example, a carrier for parenteral administration such as water, a suitable oil, saline, aqueous glucose and glycol, and may further include a stabilizer and a preservative.
  • Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • composition according to the present invention can be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, Antioxidants and the like may be included as appropriate.
  • solubilizing agent stabilizer
  • isotonic agent preservative
  • adsorption inhibitor surfactant
  • diluent diluent
  • excipient pH adjuster
  • analgesic agent buffer
  • Antioxidants and the like may be included as appropriate.
  • Pharmaceutically acceptable carriers and agents suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • the dosage of the composition to a patient will depend on many factors, including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the pharmaceutically active protein may be administered in an amount of 100 ng/body weight (kg) - 10 mg/body weight (kg), more preferably 1 to 500 ⁇ g/kg (body weight), and most Preferably, it may be administered at 5 to 50 ⁇ g/kg (body weight), and the dosage may be adjusted in consideration of the above factors.
  • the step of administering to an individual suffering from metabolic syndrome a GLP-2 analog comprising a second fusion protein linked to an antibody Fc region, and a bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein A method for treating metabolic syndrome in the subject is provided, comprising.
  • a method for treating obesity in the subject is provided.
  • the GLP-2 analog comprises a second fusion protein linked to an antibody Fc region, and the bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein is administered to an individual with type 2 diabetes.
  • a method for treating non-alcoholic steatohepatitis or non-alcoholic steatohepatitis in the subject comprising administering to the subject.
  • the step of administering to an individual suffering from liver fibrosis a GLP-2 analog comprising a second fusion protein linked to an antibody Fc region, and a bispecific fusion protein produced by dimerization of the first fusion protein and the second fusion protein A method for treating liver fibrosis in the subject is provided, comprising.
  • the term "therapeutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type and severity of the subject; Age, sex, drug activity, sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.
  • the therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may vary depending on the age, sex and condition of the patient. can be appropriately adjusted.
  • a linker peptide having a generally flexible structure may be inserted between the two or more peptides or domains.
  • the linker peptide is EPKSSDKTHTCPPCP (SEQ ID NO: 37), EPKSCDKTHTCPPCP (SEQ ID NO: 38), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 39), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 40), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 41), GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 42), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 43), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 44), GGGGSGGGGSGGGGS (SEQ ID NO: 45), GGSGG (SEQ ID NO: 46
  • n is an integer from 1 to 10
  • (GGS) n is an integer from 1 to 10
  • (GS) n is an integer from 1 to 10
  • (GSSGGS) n unit: SEQ ID NO: 50
  • n is an integer from 1 to 10
  • KESGSVSSEQLAQFRSLD SEQ ID NO: 51
  • EGKSSGSGSESKST SEQ ID NO: 52
  • GSAGSAAGSGEF SEQ ID NO: 53
  • EAAAK n (unit: SEQ ID NO: 54, n is an integer from 1 to 10)
  • CRRRRRREAEAC SEQ ID NO: 55
  • a (EAAAK) 4 ALEA EAAAK) 4 A
  • PAPAP SEQ ID NO: 59) 60
  • (Ala-Pro)n (Ala-
  • the dual specificity fusion protein comprises a first gene construct comprising a polynucleotide encoding the first fusion protein and a second gene construct comprising a polynucleotide encoding the second fusion protein.
  • Production is possible by transducing a recombinant expression vector containing a gene construct into a host cell and then expressing it in a recombinant manner.
  • the first gene construct and the second gene construct may be expressed by being inserted into one expression vector or inserted into two separate expression vectors for expression.
  • a vector is designed so that each gene construct is operably linked to two separate control sequences, or two gene constructs are operably linked to one control sequence, and both gene constructs are operably linked to one control sequence.
  • a method in which an internal ribosome entry site (IRES) connects may be used.
  • operably linked to refers to the regulation of a nucleic acid sequence of interest (eg, in an in vitro transcription/translation system or in a host cell) in such a way that its expression can be achieved. It means that it is connected to the sequence.
  • regulatory sequence is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). Regulatory sequences include instructing that a target nucleic acid can be constitutively expressed in many host cells, instructing the expression of a target nucleic acid only in specific tissue cells (eg, tissue-specific regulatory sequences), and This includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence). It can be understood by those skilled in the art that the design of the expression vector may vary depending on factors such as the selection of the host cell to be transformed and the level of desired protein expression.
  • the expression vector of the present invention can be introduced into a host cell to express the fusion protein.
  • Regulatory sequences enabling expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, they usually contain regulatory sequences responsible for initiation of transcription and, optionally, poly-A signals responsible for termination and stabilization of transcripts. Additional regulatory sequences may include, in addition to transcriptional regulators, translation enhancers and/or natively-combined or heterologous promoter regions.
  • Possible regulatory sequences enabling expression in, for example, mammalian host cells are the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human kidney element 1 ⁇ -promoter, glucocorticoid-inducible MMTV- promoters (Moloni mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifying agents such as CMV amplifiers or SV40-amplifiers.
  • neurofilament-promoter For expression in neurons, it is contemplated that neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used.
  • Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995.
  • a number of promoters have been disclosed, including the lac-promoter, the tac-promoter or the trp promoter.
  • the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention.
  • suitable expression vectors are known in the art, examples of which are Okayama-Berg cDNA expression vectors pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27 (Patent No.
  • the vector may further comprise a polynucleotide encoding a secretion signal.
  • the secretion signals are well known to those skilled in the art.
  • a leader sequence capable of guiding the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, preferably the translated protein or its It is a leader sequence capable of directly secreting a protein into the periplasmic or extracellular medium.
  • the vector of the present invention can be prepared by, for example, standard recombinant DNA techniques, and standard recombinant DNA techniques include, for example, blunt-end and adherent ligation, treatment with restriction enzymes to provide appropriate ends, inappropriate In order to prevent binding, phosphate group removal by alkaline phosphatase treatment and enzymatic linkage by T4 DNA ligase are included.
  • the vector of the present invention can be prepared by recombination of a DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology and a DNA encoding a dual specificity fusion protein of the present invention into a vector containing an appropriate regulatory sequence.
  • the vector containing the control sequence can be purchased or prepared commercially, and in an embodiment of the present invention, pBispecific backbone vector (Genexine, Inc., Korea) or pAD15 vector was used as a backbone vector.
  • the expression vector may further include a polynucleotide encoding a secretion signal sequence, wherein the secretion signal sequence induces secretion of the recombinant protein expressed in the cell out of the cell, and a tissue plasminogen activator (tPA) signal sequence; It may be an HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence or a growth hormone signal sequence.
  • tPA tissue plasminogen activator
  • the expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector is a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human chromosome. It is free to show any form, etc.
  • the present inventors designed various dual specificity fusion proteins including both GLP-1 analogues and GLP-2 analogues as shown in FIG. 1 and Table 1 below.
  • the GLP-1 receptor GLP-1R
  • GLP-1R GLP-1 receptor
  • Exendin 4 Exendin 4
  • OXM oxyntomodulin
  • a dual specificity fusion in which a glycan linker is included in the second fusion protein including GLP-2 and, conversely, an unmodified linker that does not include a glycan attachment site is included in the first fusion protein including a GLP-1 analog A protein (MG12-6) was also designed.
  • the present inventors devised dual specificity fusion proteins MG12-7 and MG12-8 using a GLP-2 analog in which the 17th alanine of GLP-2 was further substituted with glutamine.
  • MG12-7 uses the A2G variant set forth in SEQ ID NO: 1 as GLP-1, whereas in MG12-8, the two A2G variants are linked by a (G 4 S) 6 linker. It uses tandem repeats.
  • Example designation GLP-1/2 analogues (SEQ ID NO:) Linker (SEQ ID NO:) Fc area Overall configuration (SEQ ID NO:) One MG12-1 1.
  • GLP-1(1) 2.
  • GLP-2(17) Glycan Linker (43) Undeformed(42) knob hole First fusion protein (24)
  • Second fusion protein (32) 2 MG12-2 1.
  • GLP-2(17) 2.
  • GLP-1(1) Glycan Linker (43) Undeformed(42) knob hole Second fusion protein (33) First fusion protein (25) 3 MG12-3 1.
  • Exendin 4(4) 2.
  • Second fusion protein (32) 4 MG12-4 1.
  • GLP-1/Exendin 4 hybrid(5) 2.
  • Knobs-into-holes technology was applied in order to preferentially generate a heterodimer. That is, in the first fusion protein, in the hybrid Fc region, serine (S), the 10th amino acid of the CH3 domain, is substituted with cysteine (C), and threonine (T), the 22nd amino acid, is substituted with tryptophan (W) (Knob) ), in the second fusion protein, tyrosine (Y), the 5th amino acid of the CH3 domain in the Fc region, is cysteine (C), the 22nd amino acid, threonine (T), is serine (S), and the 24th amino acid is Leucine (L) may be substituted with alanine (A), and tyrosine (Y), the 63rd amino acid, may be substituted with valine (V) (Hole).
  • the position of the amino acid at which the mutation has occurred is based on the reference sequence (the amino acid sequence of the human IgG1 CH3 domain of SEQ ID NO: 69). Even if additional mutations such as addition, deletion or substitution of amino acids occur at a site unrelated to the Knobs-into-Holes structure on the CH3 domain, the amino acid corresponding to the corresponding position is mutated based on the reference sequence. Do it.
  • the Knobs-into-Holes structure can be introduced through other amino acid mutations well known in the art. Such mutations are described in the prior literature (Wei et al ., Oncotarget 2017, 8(31): 51037-51049; Ridgway et al ., Protein Eng .
  • Such selective mutations include, for example, a combination of a Knob structure in which threonine, the 22nd amino acid of the CH3 domain of the first fusion protein, is substituted with tyrosine, and a Hole structure in which tyrosine, the 63rd amino acid, of the CH3 domain of the second fusion protein is substituted with threonine.
  • a dual specificity dimer fusion protein can be generated.
  • the Knobs-into-Holes structure may be formed by introducing a hole structure to the first fusion protein and introducing a Knob structure to the second fusion protein.
  • Dual specificity of Examples 1 to 8 designed as described above After synthesizing the gene constructs encoding the first and second fusion proteins of the fusion protein by amplifying them using PCR and site-directed mutagenesis primers, they were respectively synthesized in the pAD15 vector (Genexine, Inc., Korea). By inserting, an expression vector was prepared.
  • Transient expression of the vector constructs prepared as described above was performed using Thermo Fisher's ExpiCHO kit. Specifically, after mixing the vector construct prepared as above and the ExpiFectamine reagent included in the kit in ExpiCHO-S cell, incubate for 1 day in an incubator with 8% CO2 and 37°C conditions, the temperature was lowered to 32°C. Culture was continued until the 7th day.
  • the fusion proteins of Examples 1 to 5 purified through Protein A column and secondary column were appropriately diluted with 4X LDS sample buffer and water for injection to prepare a final 3-10 ⁇ g/20 ⁇ L.
  • 4X LDS sample buffer, 10X reducing agent, and water for injection were appropriately diluted to make a final 3-10 ⁇ g/20 ⁇ L, and heated in a heating block at 70° C. for 10 minutes. 20 ⁇ L of the prepared sample was loaded into each well of the gel fixed in the pre-installed electrophoresis equipment.
  • 3-5 ⁇ L/well were loaded. After setting the power supply to 120 V, 90 minutes, electrophoresis was performed. After the electrophoresis was completed, the gel was separated and stained using a staining solution and a de-staining solution, and the results were analyzed.
  • GLP-2-Fc homodimer SEQ ID NO: 25
  • Fc Knobs-into-Holes
  • MG12 containing the Knobs-into-Holes structure SDS-PAGE analysis was performed for -5 under the same reducing/non-reducing conditions, respectively.
  • the present inventors investigated the GLP-1 in vitro activity of the dual specificity fusion protein prepared in the above Example by cAMP assay. Specifically, in order to evaluate the degree of cAMP induction by the GLP-1 specific reaction, a transformed cell line (GLP1R_cAMP/luc) was used to express the GLP-1 receptor together with the cAMP-specific luciferin-expressing cell line. produced. After the cells were thawed and properly maintained, 0.05% TE (Trypsin EDTA) was added to dissociate the cells from the flask, and the number of viable cells was counted.
  • GLP1R_cAMP/luc a transformed cell line
  • 0.05% TE Trpsin EDTA
  • the number of cells required for activity evaluation was recovered, washed, diluted with 0.5% FBS, DMEM/high glucose medium, and seeded at 2x10 4 cells/80 ⁇ L/well. After culturing the cells in a 37°C, 5% CO 2 incubator for about 16 hours, 20 ⁇ L/well of various concentrations to be evaluated were treated and reacted in a 37°C, 5% CO 2 incubator for 5 hours.
  • the reaction plate was treated with Bright-Glo TM assay reagent at 100 ⁇ L/well, and then reacted at room temperature for 2 minutes. After the reaction was completed, the plate was inserted into a luminometer and the degree of bioluminescence was measured.
  • the GLP-1-Fc homodimer exhibited an activity of about 72% compared to the native GLP-1 peptide, and Examples 1, 3, and 4 of the present invention
  • the dual specificity fusion proteins according to and 5 (MG12-1, 3, 4 and 5, respectively) exhibited relative activities of 9%, 118%, 39%, and 35%, respectively.
  • An N-terminal mutation was introduced to prevent cleavage by the DPP-4 enzyme, and in the case of MG12-1 including a sugar chain in the hinge conjugated with GLP-1, the activity was reduced by about 11 times due to glycosylation.
  • MG12-3 in which Exendin 4 was introduced instead of GLP-1 in MG12-1, showed approximately 13-fold increased activity than MG12-1.
  • MG12-4 and MG12-5 introduced with a GLP-1/Exendin 4 hybrid containing GLP-1 and Exendin 4 instead of GLP-1 were similar at about 35-39% regardless of the presence or absence of glycosylation of the hinge. showed relative activity.
  • the present inventors investigated the GLP-2 in vitro activity of the dual specificity fusion protein prepared in the above Example by cAMP assay.
  • a transformed cell line in which the GLP-2 receptor was expressed in a cell line expressing a cAMP-specifically opened CNG channel (Human GLP2R ACTOne TM ) was secured. After the cells were thawed and properly maintained, the cells were separated from the flask under the same conditions as in the GLP-1 in vitro activity assay, and the number of cells required for activity evaluation was recovered and washed.
  • the washed cells were diluted with cell culture medium (DMEM/high glucose medium, 10% FBS, 5% G418, 0.01% puromycin) and seeded at 3 ⁇ 5x10 4 cells/100 ⁇ L/well, 37°C for 20 hours, 5 Cells were cultured in a % CO 2 incubator. CO 2 After removing the plate from the incubator, the cells were observed under a microscope, and when the cell saturation reached 80% or more, 1X dye loading solution (Elite TM fluorescent membrane potential dye kit, eEnzyme) was added at 100 ⁇ L/well. After blocking the light at room temperature, the reaction was carried out for 2 to 2.5 hours, and the fluorescence baseline (F 0 ) was measured using ELISA before adding the test solution.
  • cell culture medium DMEM/high glucose medium, 10% FBS, 5% G418, 0.01% puromycin
  • test solutions of various concentrations to be evaluated were treated at 50 ⁇ L/well, reacted for 0.5 hours, and then the fluorescence value (F t ) was measured using ELISA. The reactivity of each test solution was evaluated using the F t /F 0 ratio.
  • the GLP-2-Fc homodimer exhibited an activity of about 132% compared to that of the native GLP-2 peptide, and Examples 1, 3, 4 and 5 of the present invention
  • the dual specificity fusion proteins (MG12-1, 3, 4, and 5, respectively) according to Since all MG12 variants have an N-terminal mutation introduced to prevent cleavage by the DPP-4 enzyme, and unlike GLP-1, since GLP-2 does not contain a sugar chain in the conjugated hinge, all variants are almost similar. It was confirmed to exhibit GLP-2 activity.
  • each protein was administered subcutaneously (SC) at a content of 1 mg/kg to 3 male SD (Sprague Dawley) rats per group. Blood was obtained before injection and after 0.5, 1, 5, 10, 24, 48, 72, 120, and 168 hours after injection, and stored at room temperature for 30 minutes for agglutination. After centrifuging the aggregated blood at 3,000 rpm for 10 minutes, serum of each sample was obtained and stored in a cryogenic freezer. An assay designed to specifically detect the GLP-1 site and Fc in the administered protein (GLP-1-Fc ELISA) and an assay designed to specifically detect the GLP-2 site and Fc in the administered protein (GLP-2- Fc ELISA).
  • a method of loading a biological sample on a plate coated with an antibody that binds to mouse-derived human immunoglobulin G4 (IgG4), and detecting the target protein using a biotinylated anti-GLP-1 antibody (GLP-1- Fc ELISA) and a rabbit polyclonal antibody that reacts specifically to GLP-2 is coated with a biological sample, and a target protein is used using a secondary antibody that is HRP-conjugated to mouse-derived human immunoglobulin G4 (IgG4). was used (GLP-2-Fc ELISA).
  • the obtained and prepared serum samples were loaded with appropriate dilutions to be analyzed at the position on the straight line of the standard curve.
  • MG12-1, 3, 4, and 5 were generally similar to each other in the results analyzed by GLP-1-Fc ELISA and GLP-2-Fc ELISA. showed a PK profile.
  • C max MG12-5 showed the highest value in both methods, whereas MG12-3 showed the lowest value. This trend was almost similar in AUC last.
  • terminal half-life both methods showed the longest half-life in MG12-3, MG12-4 in GLP-1-Fc ELISA and MG12-5 in GLP-2-Fc ELISA showed the lowest half-life.
  • 'GLP1/2-Fc' the dual specificity fusion protein
  • carrier PBS
  • GLP1-Fc homodimer GLP2-Fc homodimer
  • GLP1/2-Fc twice weekly for 4 weeks the body weight, fat accumulation, serum cholesterol concentration, serum high-density lipoprotein (HDL), serum triglyceride (TG) concentration, blood glucose concentration and insulin concentration of the experimental animals were measured.
  • HDL serum high-density lipoprotein
  • TG serum triglyceride
  • the GLP1/2-Fc administration group decreased body weight after 2 weeks compared to the negative control group, but significantly decreased body weight after 4 weeks ( FIG. 7a ).
  • the weight loss after 2 weeks was significant in both GLP2-Fc and GLP1/2-Fc ( FIG. 7b ).
  • the effect of GLP1/2-Fc on body weight was confirmed in a dose-dependent manner ( FIGS. 8A and 8B ). Consistent with body weight changes, GLP1/2-Fc significantly reduced fat accumulation more effectively ( FIG. 7c ) than when administered at a high dose (20 nmol/kg) ( FIG. 8c ).
  • Total serum cholesterol and high-density lipoprotein (HDL) were statistically significantly reduced by treatment with GLP1-Fc and GLP1/2-Fc (10 and 20 nmol/kg) ( FIGS. 7D , 7E , 8E and 8F ).
  • Serum triglyceride (TG) levels were not different except for the high dose (20 nmol/kg) of GLP1/2-Fc ( FIGS. 7F and 8D ). Because GLP-2 is involved in the refilling of the gallbladder, exogenous GLP-2 delivery can accelerate the abnormal dilatation of the gallbladder. Therefore, we analyzed gallbladder size (data not shown) and total serum bilirubin levels.
  • the present inventors performed an insulin tolerance test (ITT) and a glucose tolerance test (GTT) to evaluate the role of the dual specificity fusion protein (GLP1/2-Fc) on glucose homeostasis according to an embodiment of the present invention.
  • ITT insulin tolerance test
  • GTT glucose tolerance test
  • both GLP1/2-Fc and GLP1-Fc significantly improved insulin sensitivity ( FIGS. 7G and 7H ).
  • ITT and GTT administration of GLP1/2-Fc at a high dose did not show an additive effect ( FIGS. 8i and 8j ).
  • the present inventors investigated the fat content of liver tissue to evaluate the effect of the bispecific fusion protein (GLP1/2-Fc) of the present invention on lipid accumulation in the liver.
  • GLP1/2-Fc bispecific fusion protein
  • the size of the liver was smaller in dark red compared to that observed in the negative control group ( FIGS. 9A and 10A ), indicating less fat deposition in the liver.
  • a decrease in fat droplets was confirmed in the GLP1/2-Fc administration group and the GLP1-Fc administration group ( FIGS. 9b and 10b ), and the liver weight was further reduced in the fusion protein administration group (Fig. 9c and 10c).
  • liver to body weight ratio was significantly decreased only in the GLP1/2-Fc administration group ( FIG. 9d ).
  • Actual liver TG levels were similar to H&E staining (Fig. 9e and Fig. 10d).
  • the GLP1/2-Fc-administered group had much lower liver TG levels and serum ALT levels compared to GLP1-Fc ( FIGS. 9E and 10D ) ( FIG. 9F ). and 9e), but not AST levels ( FIGS. 9g and 10f ).
  • GLP1/2-Fc has a synergistic effect on liver inflammation and fat accumulation over single moieties (GLP1-Fc and GLP2-Fc).
  • the present inventors performed Sirius red staining and immunized against collagen. Immunostaining was performed. As a result, only GLP1/2-Fc significantly reduced collagen deposition ( FIGS. 11A and 11B , FIGS. 12A and 12B ). In addition, the staining result showed a positive correlation with the liver TG level (FIG. 11c). In the correlation analysis between Sirius staining intensity and TG, the GLP1/2-Fc administration group showed the lowest degree of TG hepatic deposition and fibrosis (Fig. 11d).
  • the present inventors first investigated the form of GLP1/2-Fc to evaluate whether it can affect the intestine.
  • the GLP1/2-Fc administration group had a thicker intestine and increased length compared to the negative control group ( FIG. 13a ).
  • intestinal weight was significantly increased in all three groups ( FIGS. 13b to 13d ).
  • most of the GLP1/2-Fc administration group increased in a dose-dependent manner ( FIGS. 14A and 14B ).
  • this effect was not observed in the colon ( FIG. 13E ).
  • FIG. 14b A strong effect was confirmed at high dose administration (FIG. 14b).
  • the GLP1/2-Fc administered group had a thicker epithelial layer and was filled with villi (FIG. 13f).
  • To determine whether GLP1/2-Fc increases intestinal volume immunodetection against Ki-67 protein was performed in the ileum.
  • Ki-67 protein expression was mainly confirmed in the GLP1/2-Fc administration group among the experimental groups (FIG. 13g). All three sections of the GLP1/2-Fc administration group showed a greater small intestinal crypt depth than the negative control group ( FIGS. 13H to 13J ).
  • the GLP1/2-Fc group had Verrucomicrobia It significantly increased the phyla (25% difference from the negative control group, p ⁇ 0.0005), and decreased the phylum Proteobacteria (10.5% difference from the negative control group, p ⁇ 0.005).
  • Akkermansia, Prevotellamassilia , Mailhella and Faecalibaculun were most significantly altered in the GLP1/2-Fc-treated group compared to the negative control group (Figs. 16c and 16d).
  • the present inventors further analyzed the taxonomic composition of the microbiome at the species level, and confirmed that 18 species related to obesity or metabolic dysfunction were significantly changed (Fig. 16e).
  • FIG. 16f Akkermansia muciniphila is known to be the most abundant dominant species in the intestine, which is known for its metabolic health benefits ( FIG. 16f ).
  • Mailhella massiliensis was most inhibited in the GLP-1/2-Fc administration group (FIG. 16g).
  • Alistipes senegalensis , Lactobacillus intestinealis , and Prevotellamassilia timonensis increased and were found in a healthy intestinal environment ( FIGS. 16H to 16J ).
  • Faecalibaculum rodentium and Acetatifactor muris were significantly reduced ( FIGS. 16k and 16l ), indicating that GLP1/2-Fc alters the composition of the intestinal flora.
  • the dual specificity fusion protein according to an embodiment of the present invention binds to the GLP-1 receptor and the GLP-2 receptor in the small intestine to increase the length and crypt depth of the small intestine and improve the intestinal flora. It has had the effect of improving my environment. Furthermore, the present inventors found that improvement of the small intestine environment by the bispecific fusion protein of the present invention can lead to improvement of symptoms of nonalcoholic fatty liver or nonalcoholic steatohepatitis by improving the metabolic state of the liver by affecting the intestinal-liver axis. It has been proven through various experiments. In addition, the dual specificity fusion protein according to an embodiment of the present invention exhibited an effect of reducing insulin resistance and weight loss.
  • the dual specificity fusion protein according to an embodiment of the present invention can be used to treat not only nonalcoholic fatty liver, but also various diseases related to metabolic syndrome, such as obesity, type 2 diabetes, nonalcoholic steatohepatitis, and liver fibrosis according to the course of nonalcoholic fatty liver. It can be effectively used in the treatment of chronic metabolic liver disease such as
  • composition according to an embodiment of the present invention can be usefully used in the development of therapeutic agents for metabolic diseases such as type 2 diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and liver fibrosis.
  • metabolic diseases such as type 2 diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and liver fibrosis.

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KR20160032699A (ko) * 2014-09-16 2016-03-24 한미약품 주식회사 지속형 glp-1/글루카곤 수용체 듀얼 아고니스트의 비알콜성 지방간질환에 대한 용도
KR20160083810A (ko) * 2014-12-31 2016-07-12 주식회사 제넥신 GLP 및 면역글로불린 하이브리드 Fc 융합 폴리펩타이드 및 이의 용도

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KR20080064840A (ko) * 2005-09-22 2008-07-09 바이오컴페터블즈 유케이 리미티드 펩티다아제 저항성이 증가된 ｇｌｐ―1（글루카곤 유사펩티드―1） 융합 폴리펩티드
US20090186817A1 (en) * 2006-03-21 2009-07-23 Amylin Pharmaceuticals, Inc. Peptide-peptidase inhibitor conjugates and methods of using same
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