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WO2019212279A1 - Composition pour l'excrétion de sucre - Google Patents

Composition pour l'excrétion de sucre Download PDF

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Publication number
WO2019212279A1
WO2019212279A1 PCT/KR2019/005314 KR2019005314W WO2019212279A1 WO 2019212279 A1 WO2019212279 A1 WO 2019212279A1 KR 2019005314 W KR2019005314 W KR 2019005314W WO 2019212279 A1 WO2019212279 A1 WO 2019212279A1
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Prior art keywords
sugar
composition
egfr
growth factor
releasing
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English (en)
Korean (ko)
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구철룡
조응혁
강찬우
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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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
    • A61K38/16—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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to a composition for excreting sugar into bowel movements.
  • Diabetes is a disease that is derived from various triggers and is characterized by elevated levels of plasma glucose (hyperglycemia) after the administration of glucose in the fasting state or during oral glucose tolerance tests.
  • type 1 diabetes or insulin-dependent diabetes (IDDM) patients produce little or no insulin, a hormone that regulates glucose utilization.
  • IDDM insulin-dependent diabetes
  • NIDDM non-insulin-dependent diabetes mellitus
  • hyperinsulinemia elevated plasma insulin levels
  • Insulin resistance is primarily caused by a reduced number of insulin receptors, but rather by post-insulin receptor binding defects that are not yet fully understood. This lack of responsiveness to insulin results in insufficient insulin-mediated activation of glucose uptake, oxidation and storage in muscle, and inadequate insulin-mediated inhibition of glucose production and secretion in the liver and lipolysis in adipose tissue.
  • a patient suffering from metabolic syndrome is characterized by having at least three symptoms selected from the group of five symptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) low high density lipoprotein cholesterol (HDL); (4) hypertension; And (5) increased fasting glucose, which may be within the characteristic range of type 2 diabetes if the patient is also diabetic.
  • Each of these symptoms is described in Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III, or ATP III), National Institutes of Health, 2001, NIH Publication No. 01-3670, clinically defined.
  • An object of the present invention is to first release the collagen, wheat protein, soy protein, skim milk powder, whey to the EGFR (epidermal growth factor receptor) ligand to pass through the stomach for the release of sugar to facilitate the excretion of sugar to the bowel It is to provide a composition.
  • an object of the present invention is to deliver a EGFR ligand material to the small intestine to promote the excretion of sugar in the bowel movement, and to provide a composition for the release of sugar as an active ingredient EGFR ligand material.
  • the radiotracer "18-fluorodeoxyglucose (FDG)" is an analog of glucose, which is used for positron emission tomography (PET).
  • FDG intake reflects local glucose uptake and excretion in tissues. Increased FDG uptake reflects high glycolytic activity and is associated with various diseases with high cellular metabolic activity such as cancer, local infection, and inflammation. However, high FDG levels are normally observed in organs associated with FDG excretion, such as kidneys and bladder. As described above, the FDG ingestion is performed at the lesion location of various diseases such as cancer and inflammation, but it is difficult to accurately diagnose whether the FDG ingestion is caused by cancer or inflammation.
  • EGFR epidermal growth factor receptor
  • EGFR ligand includes a type 1 transmembrane precursor that cleaves extracellular domains to release soluble ligands that bind to and activate EGFR.
  • the EGFR ligand substance may be EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor, betacellulin, amphiregulin, epiregulin ( epiregulin and epigen.
  • “Amphiregulin” is known to bind to the epidermal growth factor receptor, activate the epidermal cell receptor pathway (EGFR pathway), is involved in cell proliferation, and by the empyregulin-specific siRNA It is possible to inhibit the expression of MPregulin, which has been shown to have therapeutic effects in certain types of breast cancer (Cancer Res. 2008; 68: 225-2265).
  • shRNA against MPregulin can be used to inhibit cell infiltration in inflammatory breast cancer (J Cell Physiol. 2011 226 (10): 2691-2701) and MPregulin specific shRNA using Inhibition of expression has been shown to inhibit pulmonary artery remodeling in rats exposed to tobacco smoke (Arch Biochem Biophys, 1; 508 (1): 93-100).
  • EGF epidermal growth factor
  • empyregulin which are oversecreted in tissue remodeling following acute asthma
  • the present inventors carry out a coating process comprising water-soluble polymers such as proteins and polysaccharides in order to reach the small intestine by ingestion of "MP Regulin". This is because the stomach passes through the stomach until it reaches the small intestine to prevent unwanted reactions caused by the stomach acid secreted by the stomach.
  • Primary protein coating step of coating a material containing empiregulin in protein deposits such as soybean isolate protein, sugars such as gelatin, cellulose, gums such as xanthan gum, guar gum, polyvinylpyrrolidone, levan (Levan) It is characterized in that it comprises a secondary coating step of coating and drying by mixing and homogenizing a polysaccharide solution containing a polysaccharide component such as a water-soluble polymer such as).
  • metabolic disease refers to a disease caused by excessive synthesis or accumulation of fat due to abnormal energy metabolism in the body due to various causes such as excessive energy intake or hormonal imbalance.
  • the metabolic disease may specifically be obesity, diabetes, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, fatty liver or arteriosclerosis.
  • diabetes or “diabetes” is a disease caused by increasing the blood glucose concentration, the longer the disease period, such as blindness due to chronic complications, end stage renal failure, neurological disease, lower extremity and infectious diseases, etc. Soaring.
  • cerebrovascular disease and cardiovascular disease account for the largest portion of diabetes complications. About 3/4 of the disease deaths are caused by diabetes complications, and the risk of death from cardiovascular complications also increases the duration of diabetes for 10 years. It is reported to increase by 24% each time. The prevalence of coronary artery disease is two times higher and the prevalence of peripheral vascular disease is three times higher in diabetic patients than in normal people.
  • the causes of atherosclerosis in diabetes are hyperglycemia, lipid metabolism, hyperinsulinemia , Hypertension, changes in blood coagulation mechanisms are known.
  • the etiology of type 2 (insulin-independent) diabetes which accounts for more than 95% of diabetes, is known as a combined disorder of two causes: insulin secretion disorder and insulin resistance. In other words, diabetes is a disease of chronic hyperglycemia due to this complex disorder.
  • "acute hyperglycemic complications” means diabetic acidosis, diabetic xanthoma, diabetic amyotrophy, diabetic ketosis, diabetic coma ), Diabetic gastric disorder, diabetic gangrene, diabetic ulcer, diabetic complications, diabetic diarrhea, diabetic microangiopathy, Diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic blisters, diabetic diabetic cataract, diabetic dermopathy, diabetic scleredema, diabetic retinopathy, diabetic lipoids Necrobiosis lipoidica diabeticorum, and diabetic blood circulation disorder, and the like.
  • diabetic ketoacidosis is the most important acute metabolic complication occurring in diabetic patients and is caused by excessive accumulation of acid metabolites in the bloodstream and loss of water and sugar caused by using fat rather than sugar for the body. It is a disease. Diabetic ketoacidosis is caused by resistance to insulin or absence of insulin. Low insulin prevents glucose from entering the cell and accumulates in the blood. As a result, the cells do not receive glucose and use fat as an energy source. Fat metabolism produces fatty acids and glycerol, which provides some energy to the cells, but fatty acids are metabolized to ketoacids, resulting in acidosis. Acidosis increases potassium transport into blood vessels in cells and leads to hyperkalemia due to diuresis resulting in systemic potassium depletion.
  • the present invention is the primary coating of any one of collagen, wheat protein, soy protein, skim milk powder, whey to pass the stomach to the EGFR (epidermal growth factor receptor) ligand to promote the excretion of sugar to the bowel movement
  • EGFR epidermal growth factor receptor
  • the composition for releasing sugar delivers the EGFR ligand material to the end of the small intestine.
  • said small intestine is a common limb.
  • the small intestine terminal is an Ileum site of the small intestine connected through gastrectomy.
  • the sugar-discharging composition improves metabolic disease.
  • the sugar-discharging composition is for a subject undergoing gastrectomy.
  • the EGFR ligand material is EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor, betacellulin, amphiregulin ), Epiregulin and epigen, but are not limited thereto.
  • the composition for releasing sugar improves acute hyperglycemic complications.
  • the sugar-discharging composition improves diabetes. More specifically, in another embodiment of the present invention, the composition for releasing glucose improves diabetic ketoacidosis.
  • the present invention is a method for producing a composition for releasing sugar for promoting the excretion of sugar as described above, collagen, wheat protein to pass the stomach to the EGFR (epidermal growth factor receptor) ligand It provides a method for producing a sugar composition comprising the step of first coating with any one of, soy protein, skim milk powder, whey.
  • the present invention is first coated with any one of collagen, wheat protein, soy protein, skim milk powder, whey to pass the stomach to the EGFR (epidermal growth factor receptor) ligand and facilitate the ejection from the small intestine It is coated with a secondary coating material to provide a composition for releasing sugar for promoting the discharge of sugar to bowel movements.
  • EGFR epidermal growth factor receptor
  • the composition for releasing sugar delivers the EGFR ligand material to the end of the small intestine.
  • the secondary coating material is a water soluble polymer.
  • the secondary coating material is selected from the group consisting of gelatin, cellulose, xanthan gum, guar gum, polyvinylpyrrolidone, and Levan, but is not limited thereto.
  • said small intestine is a common limb.
  • the small intestine terminal is an Ileum site of the small intestine connected through gastrectomy.
  • the sugar-discharging composition improves metabolic disease.
  • the sugar-discharging composition is for a subject undergoing gastrectomy.
  • the EGFR ligand material is EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor, betacellulin, amphiregulin ), Epiregulin and epigen, but are not limited thereto.
  • the composition for releasing sugar improves acute hyperglycemic complications.
  • the sugar-discharging composition improves diabetes. More specifically, in another embodiment of the present invention, the composition for releasing glucose improves diabetic ketoacidosis.
  • the present invention is a method for producing a composition for releasing sugar for promoting the excretion of sugar as described above, collagen, wheat protein to pass the stomach to the EGFR (epidermal growth factor receptor) ligand , Primary coating with soy protein, skim milk powder, whey; And coating with a secondary coating material to facilitate ejection from the small intestine.
  • EGFR epidermal growth factor receptor
  • the present invention comprises the steps of primary coating with any one of collagen, wheat protein, soy protein, skim milk powder, whey to allow the EGFR (epidermal growth factor receptor) ligand to pass through the stomach; Allowing the EGFR (epidermal growth factor receptor) ligand to act in the intestine; And it provides a method for reducing blood glucose, comprising the step of promoting the excretion of sugar into bowel movements.
  • the EGFR ligand is EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor, betacellulin, amphiregulin, Epiregulin and epigen.
  • the method for reducing blood sugar ameliorates acute hyperglycemic complications.
  • the method for reducing blood sugar improves diabetes.
  • the method for reducing blood glucose improves diabetic ketoacidosis.
  • the present invention comprises the steps of primary coating with any one of collagen, wheat protein, soy protein, skim milk powder, whey to allow the EGFR (epidermal growth factor receptor) ligand to pass through the stomach; Second coating with a water-soluble polymer to cause the EGFR (epidermal growth factor receptor) ligand to be ejected from the intestine; Allowing the EGFR (epidermal growth factor receptor) ligand to act in the intestine; And it provides a method for reducing blood glucose, comprising the step of promoting the excretion of sugar into bowel movements.
  • the EGFR ligand is EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor, betacellulin, amphiregulin, Epiregulin and epigen.
  • the method for reducing blood sugar ameliorates acute hyperglycemic complications.
  • the method for reducing blood sugar improves diabetes.
  • the method for reducing blood glucose improves diabetic ketoacidosis.
  • the secondary coating is coated with one or more selected from the group consisting of gelatin, cellulose, xanthan gum, guar gum, polyvinylpyrrolidone, and Levan.
  • composition for releasing sugar according to the present invention is first coated with any one of collagen, wheat protein, soy protein, skim milk powder, whey to pass the stomach to EGFR (epidermal growth factor receptor) ligand to promote the excretion of sugar into bowel movements. It is effective in reducing sugar levels in the body.
  • the composition for releasing sugar according to the present invention has the effect of delivering the EGFR ligand substance to the terminal of the small intestine when applied in a subject suffering from metabolic syndrome such as hyperglycemia.
  • FIG. 1A to 1C are graphs showing oral glucose loading test (OGTT) results before and after gastrectomy.
  • FIG. 1A Blood glucose change in OGTT for 2 hours
  • FIG. 1B Blood sugar change seen in the first 15 minutes of OGTT
  • FIG. 1C Area under the curve of blood glucose change in OGTT for 2 hours. * P ⁇ 0.001
  • Figure 2 is a photograph showing the results of FDG PET imaging 4 weeks after surgery, according to the gastrectomy method.
  • Figure 3 is a schematic diagram and pictures showing the results of autoradiography 4 weeks after surgery, according to the gastrectomy method.
  • Figure 3 (a) Schematic diagram of the simulation surgery and ReY surgery.
  • FDG Fluorodeoxyglucose
  • 4A-4E are graphs showing mRNA expression in a type 2 diabetic rat animal model.
  • 4A is a graph for AR, Epiregulin, HbEGF.
  • 4B and 4C are images in Ileum via western blot and immunohistochemical staining.
  • 4D and 4E are Western blot results images and graphs showing Western blot results analyzing the activity of EGFR proteins.
  • 5A to 5E are images and graphs showing expression patterns of EGFR, AKT, ERK / 12, etc. in Ileum.
  • FIG. 6 is an image showing the expression of GLUT1 in the cell membrane by immunohistochemical staining.
  • 7A to 7C are graphs showing the expression patterns after treatment of IEC-18 cells with AR.
  • FIG. 8 is a graph showing glucose absorption results taken with a gamma scanner.
  • 9A to 9E are graphs showing the results of oral glucose tolerance test and images showing FDG uptake in the small intestine.
  • FIG. 10 is an image showing the FDG PET test results when the AR coated in the diabetic mouse model (db / db) for one week.
  • 11 is a graph showing hormonal changes when the composition according to the present invention is applied.
  • composition for releasing sugar according to the present invention is first coated with any one of collagen, wheat protein, soy protein, skim milk powder, whey to pass the stomach to EGFR (epidermal growth factor receptor) ligand to promote the excretion of sugar into bowel movements. It is effective in reducing sugar levels in the body.
  • EGFR epidermal growth factor receptor
  • the composition for releasing sugar according to the present invention can be applied to a subject suffering from metabolic syndrome such as hyperglycemia can deliver the EGFR ligand material to the terminal of the small intestine.
  • the RYGB procedure was as follows: rats were sedated and shaved abdomen using 1.5% isoflurane gas. Iodine was applied to the abdominal centerline and the skin and peritoneum were opened. The esophageal junction was identified, cut and sutured. A jejunum site about 34 cm from the duodenum was measured, cut and anastomized in the esophagus. The blind end of gastric limb was then anatomized about 18 cm from the anastomotic jejunostomy. Three limbs, an esophagojejunostomy limb (about 18 cm), a gastric branch (glastric limb (about 34 cm) and a common limb (about 24 cm) were formed. The simulated surgery was to open the peritoneal cavity and open for the same time as the RYGB procedure.
  • the normal small intestine is anatomically divided into three parts, and then divided into Duodenum, Jejunum, and Ileum from the next position above.
  • the small intestine of the sham-operated rat is constructed in the order described above.
  • Duodenum is Roux limb
  • jejunum is Biliopancreatic limb
  • ileum corresponds to Common limb.
  • Oral glucose tolerance test was performed in the type 2 diabetic rat animal model (OLETF) before gastrectomy during high fat diet and 4 weeks after surgery. 2 g of glucose per Kg of rat was diluted orally in saline (* P ⁇ 0.001). Five rats per group.
  • OLETF type 2 diabetic rat animal model
  • the preoperative body weight was 736.15 ⁇ 57.52 g (mean ⁇ standard deviation) and preoperative fasting blood glucose was 258 ⁇ 95.92 mg / d. There was no difference in body weight, fasting blood glucose, and oral glucose loading test according to the preoperative methods.
  • FDG uPET was performed before and 4 weeks after surgery to analyze glucose absorption in the small intestine.
  • 2g / kg oral glucose loading test was performed before and 4 weeks after surgery, respectively, to analyze the change in blood glucose metabolism over 2 hours (FIGS. 1A to 1C).
  • FIG. 3 shows a schematic diagram of the simulated surgery and ReY surgery
  • Figure 3 (b) shows the glucose absorption rate in the small intestine by performing autoradiography to excise the small intestine 1 hour after administration of Fluorodeoxyglucose (FDG) Indicates that the change is confirmed.
  • FDG Fluorodeoxyglucose
  • RNA analysis Common branch bowel segments for RNA analysis were selected based on autoradiographic images. Two bowel segments were selected for RNA analysis, the bowel segment with the highest FDG uptake and the bowel segment with the lowest FDG uptake. Autoradiographic images were used to semi-quantitatively analyze the corresponding bowel segments (counts / pixel) to assess the high and low rates of FDG uptake. The segment of the simulation was also analyzed in the same way.
  • RNA sequencing Four weeks after gastrectomy, a comparison of gene expression patterns associated with increased glucose uptake in the small intestine ileum was performed by high throughput RNA sequencing.
  • OLETF model with simulated surgery and ReY gastrectomy, Ileum was collected from the sites with and without increased FDG uptake as shown on autoradiography (5 per group and 2 Ileum for each individual). ), High throughput Total RNA Sequencing was performed.
  • the simulated rats showed the overall minimum interval change in FDG intake (4 of 5 rats), but in the RYGB rat group all rats showed diffuse bowel absorption (5 of 5 rats). ).
  • FIGS. 4A-4E increased expression of Amphiregulin (Areg) and EGFR in common limbs (corresponding to Ileum) where FDG uptake of the RYGB surgical group occurred in type 2 diabetic rat animal models Signaling activation was confirmed.
  • Figure 4a through the RNA sequencing found a gene that is significantly increased only in the FDG (+) of ileum and AR, Epiregulin, HbEGF was selected as candidates. Reconfirmed via real time PCR.
  • Western blots and immunohistochemical staining confirmed a significant increase in AR in the plenum of the RYGB surgical group, especially at the sites where high FDG uptake occurred (see FIGS. 4B and 4C). As shown in FIGS.
  • IEC-18 cells For the cultivation of IEC-18 cells, placed in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin and 37 in a humidified atmosphere containing 10% CO 2 and 90% air. Maintained at ° C. Preservation cultures were subcultured every 3-4 days.
  • IEC-18 cells were seeded in 6-well dishes at a density of 2 ⁇ 10 5 cells / dish. The culture was used 6 days after smear when the cell density was 1.5 ⁇ 10 5 cells / cm 2 .
  • MPP Regulin was purchased from R & D Systems (Cat. No. 989-AR).
  • Plasmids expressing rat Empiregulin were subcloned into pCDNA3.1. IEC-18 cells were plated at a density of 0.5 ⁇ 10 6 per 6-well one day before transfection. Cells were transfected with the expression plasmid using Polyjet transfection reagent (SignaGen) according to the manufacturer's instructions. Transfected IEC-18 cells were incubated for 48 hours at 37 ° C. before lysis.
  • Hexokinase2 F rat TGATCGCCTGCTTATTCACGG R rat AACCGCCTAGAAATCTCCAGA Glucose 6 phosphatase F rat GCCTCCTCAGCTGCATAATGGTCT R rat GAATGCTTTCTCGAAGTCCTCTTCTG GLUT1 F rat GTGCTCGGATCCCTGCAGTTCG R rat GGGATGGACTCTCCATAGCGGTG GLUT2 F rat TAGTCAGATTGCTGGCCTCAGCTT R rat TTGCCCTGACTTCCTCTTCCAACT G6pc F rat ACTCCCAGGACTGGTTTGTC R rat CCAGATGGGAAAGAGGACAT PEPCK F rat AATCCGAACGCCATTAAGAC R rat ATGCCTTCCCAGTAAACACC 18s F rat TGTCAATCCTGTCCGTGTCC R rat ACGGACCAGCGAAAGCAT
  • the blot was washed three times with TBST (Tris-buffered saline containing 0.05% Tween 20) and then reacted with horseradish peroxidase (HRP) -conjugated secondary antibody at 25 ° C. for 1 hour.
  • HRP horseradish peroxidase
  • As secondary antibodies donkey anti-rabbit IgG-HRP antibodies (1: 5000, Santa Cruz) and donkey anti-mouse IgG-HRP antibodies (1: 5000, Santa Cruz) were used. Immune reactivity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Mass., USA).
  • IEC-18 cells were fixed with 4% para-formaldehyde, permeabilized with 1% Triton X-100, blocked with 10% normal donkey serum and reacted with GLUT1 primary antibody (abcam) overnight at 4 ° C. Sections or cells are reacted with fluorescence-conjugated secondary antibodies (1: 1000, Jackson ImmunoResearch, West Grove, Pa., USA), mounted in Vectashield medium (Vector Laboratories, Burlingame, Calif., USA), and LSM 780 laser Observation was made under scanning confocal microscopy (Carl Zeiss, Oberkochen, Germany).
  • IEC-18 glucose in the cell lysate was measured after treatment with recombinant mouse empiregulin for 24 hours in low glucose medium and serum-free DMEM. IEC-18 cell glucose levels were detected using a Glucose Assay Kit (Cayman Chemical) according to the manufacturer's instructions. Absorbance signals were measured with a Gen 5 emission spectrometer (BioTek, Winooski, VT, USA).
  • IEC18 was reacted with low glucose DMEM overnight after vehicle or empiregulin 50ng / ml treatment and then cells were administered with 5 ⁇ Ci FDG with high glucose DMEM medium in each well. Two hours after FDG administration, cells were washed three times with PBS, cytolyzed, scraped, and quantitatively counted FDG uptake using the Perkin Elmer Wizard gamma counter. Finally, cellular protein concentrations were measured to correct for cell differences.
  • IEC18 was reacted with low glucose DMEM overnight after vehicle or empiregulin 50ng / ml treatment and then cells were reacted with 5uCi FDG and glucose DMEM medium in each well. Two hours after FDG administration, cells were washed three times with PBS and incubated for two hours with PBS. PBS was recovered and cells were lysed and scraped off and counted using a Perkin Elmer Wizard gamma counter. Finally, cellular protein concentrations were measured to correct for cell differences.
  • AR was confirmed to increase the expression of HIF1a, Glucose transporter.
  • Treatment of recombinant AR with an IEC18 cell line was found to increase GLUT1 mRNA expression in a dose-dependent manner. It was confirmed that the expression of GLUT1 even at the protein level was increased dose-dependently of AR (FIG. 5C).
  • Expression of HIF1 transcription factors known to regulate GLUT1 expression in IEC-18 cells overexpressing AR was increased and expression of GLUT2, as well as GLUT2, and SGLT1 was increased (FIGS. 5D and 5E).
  • Immunofluorescence staining Immunofluorescence staining 30 minutes after the AR treatment was confirmed that GLUT1 is expressed in the cell membrane.
  • GLUT1 did not migrate to the cell membrane but remained in the area around the cell nucleus (FIG. 6).
  • Increased small intestinal glycolysis showed overexpression of glucose transporter 1 (GLUT1) in RYGB rats and confirmed that GLUT1 promotes serum glucose excretion into the small intestinal lumen.
  • AR, Epiregulin, and Hb-EGF were incubated for 24 hours in hypoglycemic DMEM medium at 100 ng / mL each and washed. 10 uCi FDG was mixed in High DMEM and incubated for 2 hours. As shown in FIG. 8, the difference in glucose absorption taken by the gamma scanner was shown.
  • Example 5 In the mouse animal model, the effect of Amphiregulin administration on FDG glucose uptake and excretion in the small intestine and blood glucose reduction were confirmed.
  • mice Eight-week-old C57BL6 (non-diabetic) mice and db / db (type 2 diabetes) mice were obtained from Jackson laboratories and fed normal dietary rodents under controlled conditions (12-hour / 12-hour contrast cycle in a temperature and humidity controlled environment). And free access to water. All experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Severance Hospital in Korea. C57BL6 mice were fasted for 16 hours and 10 ug of AR was treated for 30 minutes (2 g / kg) before oral glucose testing. These mice received AR for 1 week. Blood glucose concentrations were measured using a glucometer (Arkray, Minneapolis, MN, USA) using a tail nick blood sample. Each group consisted of 5 mice.
  • the primary coating is to pass through the stomach to protect the reaction with gastric acid, and the primary coating is selected from any one or more selected from the group consisting of collagen, wheat protein, soy protein, skim milk powder, whey, It is not limited to this.
  • the secondary coating is for facilitating the ejection from the small intestine, by selecting any one or more from the group consisting of gelatin, cellulose, xanthan gum, guar gum, polyvinylpyrrolidone, and Levan (2) Car coating is carried out, but is not limited thereto.
  • FIG. 10 a illustrates an FDG uPET result image before AR processing.
  • FIG. 10B is a result of FDG uPET when fasting after AR treatment
  • FIG. 10C is a result of FDG uPET after food intake after AR treatment.
  • small intestine FDG uptake was increased over FDG uPET prior to AR treatment (see arrow in FIG. 10 b).
  • Fig. 10c it was confirmed that the intake of FDG in the small intestine after food intake in the AR-treated rats.
  • the present inventors further conducted the following experiment to prove whether there is a hormonal change when applying the composition according to the present invention.
  • the diabetic rat was administered with a composition for releasing glucose according to the present invention to act at the end of the small intestine.
  • a composition for releasing glucose according to the present invention to act at the end of the small intestine.
  • the composition for excretion of sugar according to the present invention can be applied to a subject suffering from metabolic syndrome such as hyperglycemia to deliver the EGFR ligand substance to the terminal of the small intestine. there was.
  • Example 6 After gastrectomy in diabetic patients, the increase in FDG glucose uptake in the small intestine and the postoperative hypoglycemic effect were confirmed.
  • Inclusion criteria were patients who underwent RYGB surgery for gastric cancer and followed up FDG PET / CT one year for surveillance. Exclusion criteria were patients who received chemotherapy within 1 month after PET / CT or had recurred from PET / CT. Patient demographics such as BMI, body weight, glucose and cholesterol were obtained preoperatively and within 1 month of PET / CT. FDG uptake in the intestines was determined using VOI with a threshold of 2.5. VOI was located in the small intestine and recorded all metabolically significant FDG uptake values. One or more Hba1c improvements were used for reference.
  • FIG. 12 shows an image of increased small intestine uptake in patients with significantly improved Hba1c compared to patients with no improved Hba1c.
  • FIG. 12A illustrates a 67-year-old male diabetic patient who received RYGB due to gastric cancer. The preoperative Hba1c level was 6.7 and the initial blood glucose level was 147 mg / dl. FDG PET / CT was obtained with minimal FDG intake in the small intestine for one year after surgery. The patient showed no significant decrease in Hba1c levels (6.5) or glucose levels (109 mg / dl).
  • FIG. 12B is a 37-year-old male diabetic patient who underwent RYGB for early gastric cancer.
  • the initial Hba1c level was 8.1 and the glucose level was 214 mg / dl.
  • the patient received FDG PET / CT a year later and showed multiple small intestine absorptions.
  • the patient had reduced Hba1c levels to 7.1 and improved glucose levels to 137 mg / dl.
  • composition for releasing sugar according to the present invention is first coated with any one of collagen, wheat protein, soy protein, skim milk powder, whey to pass the stomach to EGFR (epidermal growth factor receptor) ligand to promote the excretion of sugar into bowel movements. It is effective in reducing sugar levels in the body.
  • EGFR epidermal growth factor receptor
  • the composition for releasing sugar according to the present invention can be applied to a subject suffering from metabolic syndrome such as hyperglycemia can deliver the EGFR ligand material to the terminal of the small intestine.

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Abstract

La présente invention concerne une composition pour l'excrétion de sucre dans laquelle un ligand du récepteur du facteur de croissance épidermique (EGFR) est principalement revêtu de collagène, de protéine de blé, de protéine de soja, de lait écrémé ou de lactosérum, le ligand passant par l'estomac pour favoriser l'excrétion de sucres pendant la défécation.
PCT/KR2019/005314 2018-05-03 2019-05-03 Composition pour l'excrétion de sucre Ceased WO2019212279A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049845A1 (fr) * 2000-01-06 2001-07-12 Gropep Limited Variant d'epissage de facteur de croissance
JP2002541117A (ja) * 1999-04-06 2002-12-03 ジェネンテック・インコーポレーテッド 糖尿病治療へのＥｒｂＢレセプターリガンドの使用
JP2004520345A (ja) * 2001-01-12 2004-07-08 ワラター・ファーマシューティカルズ・インク 糖尿病を有する被験者にガストリン／ｃｃｋ受容体リガンド及びｅｇｆ受容体リガンド組成物を用いた島細胞新生治療方法の効果持続
KR20080016786A (ko) * 2005-02-03 2008-02-22 주식회사 바이오텍 아이 당뇨병 치료용 조성물 및 당뇨병 치료 방법
JP2008509093A (ja) * 2004-08-06 2008-03-27 ノヴォザイムズ グロペップ リミテッド 糖尿病治療法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002541117A (ja) * 1999-04-06 2002-12-03 ジェネンテック・インコーポレーテッド 糖尿病治療へのＥｒｂＢレセプターリガンドの使用
WO2001049845A1 (fr) * 2000-01-06 2001-07-12 Gropep Limited Variant d'epissage de facteur de croissance
JP2004520345A (ja) * 2001-01-12 2004-07-08 ワラター・ファーマシューティカルズ・インク 糖尿病を有する被験者にガストリン／ｃｃｋ受容体リガンド及びｅｇｆ受容体リガンド組成物を用いた島細胞新生治療方法の効果持続
JP2008509093A (ja) * 2004-08-06 2008-03-27 ノヴォザイムズ グロペップ リミテッド 糖尿病治療法
KR20080016786A (ko) * 2005-02-03 2008-02-22 주식회사 바이오텍 아이 당뇨병 치료용 조성물 및 당뇨병 치료 방법

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