[go: up one dir, main page]

WO2018045872A1 - 一种多肽及其用途 - Google Patents

一种多肽及其用途 Download PDF

Info

Publication number
WO2018045872A1
WO2018045872A1 PCT/CN2017/098674 CN2017098674W WO2018045872A1 WO 2018045872 A1 WO2018045872 A1 WO 2018045872A1 CN 2017098674 W CN2017098674 W CN 2017098674W WO 2018045872 A1 WO2018045872 A1 WO 2018045872A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
polypeptide
mice
glp
pharmaceutical composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/098674
Other languages
English (en)
French (fr)
Inventor
金亮
高华山
赵茜
张艳峰
潘怡
邢芸
申育萌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Pharmaceutical University
Original Assignee
China Pharmaceutical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201610805193.7A external-priority patent/CN106279400A/zh
Priority claimed from CN201710539987.8A external-priority patent/CN109200273B/zh
Priority claimed from CN201710714470.8A external-priority patent/CN109395065A/zh
Application filed by China Pharmaceutical University filed Critical China Pharmaceutical University
Publication of WO2018045872A1 publication Critical patent/WO2018045872A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to a polypeptide and use thereof, and in particular to a polypeptide P8, the use thereof comprising: use for preparing a medicament for preventing or treating diabetes, use for preparing a medicament for preventing or treating fatty liver disease, and for preparing a slimming drug Its use belongs to the field of biochemical pharmacy technology.
  • diabetes is a metabolic disorder characterized by chronic hyperglycemia. It is currently believed that genetic and environmental interactions are the main causes, but the pathogenesis is not fully understood. Diabetes can be divided into 4 types: type 1 diabetes, type 2 diabetes, gestational diabetes and other types of diabetes.
  • Type 1 diabetes mellitus T1DM is an autoimmune disease. Because of the loss of tolerance to autoantibodies, the islet ⁇ cells are damaged, the body cannot produce enough insulin, and insulin is absolutely lacking, causing blood sugar to rise. Once a patient is ill, insulin is required for life.
  • Type 2 diabetes also known as non-insulin-dependent diabetes, is due to low beta cell function, relative insulin deficiency and insulin resistance.
  • type 2 diabetes has gradually increased. According to the World Health Organization, there will be 300 million patients with type 2 diabetes worldwide in 2030.
  • the main treatments for type 2 diabetes are oral hypoglycemic agents and insulin. Although these drugs are effective in lowering blood sugar and improving patient symptoms, they still suffer from defects such as hypoglycemia and weight gain. In addition, with the prolongation of the course of diabetes, these drugs can not prevent the deterioration of ⁇ cells in patients.
  • Fatty liver also known as fatty liver disease, refers to a disease caused by excessive accumulation of fat in liver cells due to various reasons. The disease is seriously threatening the health of Chinese people and becoming the second largest liver disease after viral hepatitis. Fatty liver is a common clinical phenomenon, not an independent disease, its clinical manifestations are asymptomatic, and the severe cases are fierce. In general, fatty liver is a reversible disease, which can be restored to normal after early diagnosis and timely treatment.
  • GLP-1 glucagon-like peptide-1
  • Incretin incretin
  • GLP-1 has two biologically active forms, GLP-1 (7-37) and GLP-1 (7-36) amide, which are mainly present in the blood circulation in the active form of GLP-1 (7-36) amide.
  • GLP-1 (7-37) GLP-1 (7-36) amide.
  • GLP-1 Natural human GLP-1 is poorly stable, easily degraded by dipeptidyl peptidase IV (DPPIV), and rapidly cleared by the kidneys, with a half-life of t1/2 ⁇ 2 min. GLP-1 cleaves the N-terminal His-Ala by DPPIV to generate GLP-1 (9-36) amino polypeptide.
  • DPPIV dipeptidyl peptidase IV
  • GLP-1R Receptor
  • GLP-1 The physiological role of GLP-1 mainly includes three parts. One is to exert physiological effects by binding to GLP-1R, including: 1 glucose-dependent insulin secretion; 2 inhibition of glucagon secretion; 3 delayed gastric emptying, lowering Appetite, reduce diet, control body weight; 4 inhibit ⁇ cell apoptosis, promote ⁇ cell proliferation and differentiation. Second, GLP-1 is degraded by DPPIV to produce GLP-1 (9-36) amino polypeptide, which can inhibit the expression of hepatic gluconeogenesis and fatty acid synthase, inhibit gluconeogenesis and liver fat synthesis, and play insulin-like action.
  • GLP-1(9-36) amino polypeptide is cleaved into GLP-1(32-36) amino 5 peptide by endopeptidase (NEP24.11), which increases energy expenditure and inhibits weight gain in obese mice. .
  • the inventors developed the polypeptide P8 in the study of diabetes, and further found in the subsequent studies that the polypeptide P8 also has the function of treating fatty liver and obesity.
  • the technical problem to be solved by the present invention is to provide a polypeptide, that is, a polypeptide P8, which can treat diabetes, fatty liver and obesity, and further provide various uses of the polypeptide on the basis of the problems in the prior art. .
  • the fatty liver disease is nonalcoholic fatty liver.
  • the pharmaceutical composition comprises the polypeptide itself or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition comprises a pharmaceutical carrier and/or a pharmaceutically active substance.
  • the wild type of GLP-1 was transformed: the amino acid at position 2 was replaced by Ala to Gly, and the end of GLP-1 was added with Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser.
  • the sequence is as follows:
  • GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
  • a hypoglycemic peptide P8 is constructed by amino acid substitution and addition of a small peptide at the end.
  • the experimental results show that P8 peptide can effectively exert glucose-dependent insulin secretion (IPTGG), reduce blood glucose in STZ diabetic mice, inhibit feeding in STZ diabetic mice, maintain good islet morphology (HE staining), and increase STZ
  • IPTGG glucose-dependent insulin secretion
  • the islet area (immuno staining, and cumulative area) of diabetic model mice increased the C-peptide level of STZ diabetic model mice, and its hypoglycemic effect was significantly better than exenatide and GLP-1; P8 peptide can effectively reduce db
  • the fasting blood glucose of /db mice was significantly better than GLP-1.
  • the inventors screened a number of polypeptides that have been developed to screen for a polypeptide that can be used to prepare a medicament for preventing or treating fatty liver disease, and found that the previously obtained hypoglycemic peptide P8 meets the requirements.
  • Peptide P8 has been experimentally confirmed to be effective for fatty liver disease, which not only develops new uses for peptide P8, but also finds new therapeutic drugs for fatty liver disease. Agent.
  • Figure 1 is a graph showing the results of sugar-dependent insulinotropic secretion (IPTGG) of polypeptide P8 in Example 1 of the present invention.
  • Figure 2 is a graph showing the effect of polypeptide P8 on cumulative eating in STZ diabetic model mice in Example 2 of the present invention.
  • Fig. 3 is a graph showing the effect of polypeptide P8 on blood glucose of STZ diabetic model mice in Example 2 of the present invention.
  • Figure 4 is a graph showing the effect of polypeptide P8 on triglyceride and free fatty acids in STZ diabetic model mice in Example 2 of the present invention.
  • Fig. 5 is a graph showing the effect of polypeptide P8 on islet morphology (HE staining) of STZ diabetic model mice in Example 2 of the present invention.
  • Figure 6 is a graph showing the effect of polypeptide P8 on the islet area of STZ diabetic model mice in Example 2 of the present invention.
  • Figure 7 is a graph showing the effect of polypeptide P8 on the level of C-peptide of STZ diabetic model mice in Example 2 of the present invention.
  • Figure 8 is a graph showing the effect of polypeptide P8 on blood glucose of db/db diabetic model mice in Example 3 of the present invention.
  • * is used to indicate the significance of the P8 group compared with the vehicle group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001; using # indicates the significance of Exendin-4 compared with the vehicle group, #P ⁇ 0.05, ##P ⁇ 0.01, ###P ⁇ 0.001; $ is used to indicate the significance of the P8 group compared with the GLP-1 group, $P ⁇ 0.05, $$P ⁇ 0.01, and $$$P ⁇ 0.001.
  • Figure 9 is a graph showing the effect of polypeptide P8 on insulin tolerance in db/db diabetic model mice in Example 3 of the present invention.
  • Fig. 10 is a view showing the results of serum detection of mice when the modeling is successful in Example 4 of the present invention.
  • NC was a standard normal diet-fed group of mice
  • HFD was a high-fat diet-fed group of mice.
  • Fig. 11 is a view showing the results of HE staining of mouse liver tissue when the modeling was successful in Example 4 of the present invention.
  • NC was a standard normal diet-fed group of mice
  • HFD was a high-fat diet-fed group of mice.
  • Fig. 12 is a view showing the results of serum detection of a solvent control group mouse and a P8 intervention group mouse in Example 4 of the present invention.
  • HFD was a solvent control group
  • P8 was a P8 intervention group.
  • Figure 13 is a schematic diagram showing the results of HE staining of liver tissues of a solvent control group and a P8 intervention group in Example 4 of the present invention. Among them, HFD was a solvent control group and P8 was a P8 intervention group.
  • 14 to 17 are graphs showing changes in body weight, body weight percentage, cumulative food intake, and fasting blood glucose of the mice in the course of the intervention according to Example 5 of the present invention.
  • FIG. 18 and FIG. 19 are graphs showing fasting glucose tolerance results and fasting insulin tolerance results of mice after intervention in Example 5 of the present invention.
  • Figure 20 is a graph showing oxygen consumption results of mice after intervention in Example 5 of the present invention.
  • FIG. 21 and FIG. 22 are the results of MRI scan image display of the mice after intervention in Example 5 of the present invention, and the statistical results calculated according to the MRI scan results.
  • Vehicle or Ve represents the solvent control group
  • P8 represents the P8 intervention group
  • Exendin-4 or Ex4 represents Exendin-4 intervention group.
  • FIG. 8 and FIG. 12 the significance of comparison with the vehicle group is indicated by *, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • c57 mice Forty male 8-year-old c57 mice (purchased from the Center for Comparative Medical Sciences, Yangzhou University, license number: SCXK (Su) 2012-0004). They were randomly divided into 4 groups, 10 in each group, which were solvent control group (negative control group), Exendin-4 group (positive control group), GLP-1 group (positive control group), and P8 group (experimental group).
  • the peritoneal glucose tolerance process was as follows: c57 mice were fasted for 12 hours, fasting blood glucose was measured, peptide or solvent was injected, glucose (1.5 g/kg) was injected for 30 minutes, and blood glucose was measured at 15, 30, 60, and 120 minutes after injecting sugar, respectively. And calculate the area under the curve separately.
  • P8 had a glucose-dependent insulin-promoting function, and was significantly different from the solvent group and the GLP-1 group, and had the same effect as the Exendin-4 group.
  • the dose was 25 nmol/kg twice a day, the solvent was physiological saline, 0.1 ml was injected subcutaneously, and the drug was administered at a fixed time point, AM 9:00-10:00, PM 8:00-9:00.
  • the negative control group was injected subcutaneously with 0.1 ml of physiological saline.
  • mice were fasted for 8 hours, AM was fasted at 8:00, PM 4:00, fasting blood glucose was detected in the tail vein, and a blood glucose meter (OHA-231 type of Omron blood glucose meter) was used for blood glucose administration for 0 weeks. Then, after 1, 2, 3, 4, and 5 weeks of administration, fasting blood glucose was measured, and the fasting time and detection time were the same as 0 weeks.
  • the results are shown in Figure 3.
  • the P8 group can significantly reduce the fasting of STZ diabetic mice. Blood glucose was significantly better than GLP-1 and Exendin-4. By week 5, compared with the vehicle control group, p ⁇ 0.01, there was a significant difference.
  • mice Five weeks after the administration, the mice were sacrificed, and the pancreas was taken and fixed with formalin. After HE staining, the islet morphology was observed, and the results are shown in Fig. 5. In the solvent control group, the islet injury was severe, and the intact islet structure was almost invisible. The islet morphology of GLP-1 group, Exendin-4 group and P8 group was significantly improved. It was observed that the cells were lightly colored and arranged in a group-like islet structure. It is indicated that GLP-1 group, Exendin-4 group and P8 group can improve islet morphology.
  • mice Five weeks after the administration, the mice were sacrificed, pancreas was taken, formalin was fixed, and immunostaining (insulin, ki67) was carried out, and the results are shown in Fig. 6. Immunostaining images showed that there were few islet ⁇ cells in the solvent control group (STZ injury induced ⁇ -cell death), while the islet ⁇ cells in the GLP-1 group, Exendin-4 group and P8 group increased, indicating that the P8 group can significantly increase islet ⁇ cells. The amount increases the islet beta cell area.
  • mice Twenty-four 6-week-old male db/db mice (purchased from Nanjing University, license number: SCXK (Su) 2015-00001) were adapted for 1 week. Blood glucose was checked from the 7th week, AM was fasted at 8:00, and blood glucose was detected at the tail vein of AM 12:00. The blood glucose tester (OHA-231 type) was used for the test. At the 9th week, the mice were randomly divided into 4 groups, 6 in each group, which were solvent control group (negative control group), Exendin-4 group (positive control group), GLP-1 group (positive control group), In the P8 group (experimental group), administration was started and blood glucose was detected. At the 10th week, the results are shown in Fig. 8.
  • the blood glucose levels of the Exendin-4 group and the P8 group were significantly decreased, p ⁇ 0.001 and p ⁇ 0.05, respectively, and maintained continuously. Low blood glucose levels; there was a significant difference between the P8 group and the solvent control group. The hypoglycemic effect was significantly better than the GLP-1 group, which was basically the same as the Exendin-4 group.
  • mice were fasted for 8 hours, AM was fasted at 8:00, PM 4:00, fasting blood glucose was detected in the tail vein, and a blood glucose tester (OHL-231 type) was used to detect fasting blood glucose as insulin.
  • OHL-231 type blood glucose tester
  • Tolerance of 0 point blood glucose subcutaneous injection of insulin at a dose of 1 IU / kg, followed by 15, 30, 60, 90, 120 minutes after insulin injection Blood glucose is measured and the area under the curve is calculated separately.
  • the polypeptide P8 significantly improved the insulin tolerance, and was significantly different from the solvent control group and the GLP-1 group, and had the same effect as the Exendin-4 group.
  • GLP-1 analogues can significantly reduce blood glucose, inhibit eating, and reduce glycated hemoglobin in patients with type 2 diabetes.
  • GLP-1 and its analogues can lower blood sugar and reduce glycated hemoglobin in patients with type 1 diabetes, but some Individuals have disadvantages of weight loss.
  • the amino acid at position 2 of GLP-1 is replaced by Ala to Gly, and Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser is added at the end of GLP-1, thereby reducing type 1 diabetes and type 2 The role of blood sugar in diabetic mice.
  • mice Forty male c57 mice were fed ad libitum for one week and randomly divided into two groups. The standard common diet was fed with 10 and the high-fat diet was fed with 30. Each group was given free access to water.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • TG triglyceride
  • TC total cholesterol
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • TG triglyceride
  • TC total cholesterol
  • mice in the normal feed-fed group continued to be fed as normal control group (NC group)
  • 20 mice in the rouge diet-fed group were randomly divided into 2 groups, 1
  • the group was the solvent control group and the first group was the P8 intervention group.
  • NC group normal feed feeding + normal saline 0.2ml/kg intraperitoneal injection for 6 weeks
  • solvent control group HFD group
  • high fat diet + normal saline 0.2ml/kg intraperitoneal injection The duration of 6 weeks
  • P8 intervention group HFD + P8 group
  • mice in each group were fasted for 12 hours.
  • Blood samples were taken from the eyelids to detect serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC). Liver pathological sections were examined by HE staining for hepatic steatosis.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • TG triglyceride
  • TC total cholesterol
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • TG triglyceride
  • TC total cholesterol
  • ALT, AST, TG, and TC in the serum of the P8 intervention group were significantly decreased (P ⁇ 0.001) after 6 weeks of administration.
  • hepatocytes in the solvent control group were diffuse vesicular fat vacuolar degeneration, heavy water Degeneration, the nucleus was partially deformed by extrusion, and the hepatocytes were disordered, but no obvious inflammation, fibrosis, necrosis and other lesions.
  • the liver tissue of the P8 intervention group was normal.
  • the structure of the hepatic lobule was obvious, the hepatic cell cord was arranged radially, and no balloon-like degeneration was observed in the hepatocytes, which was almost close to the liver structure of normal mice.
  • polypeptide P8 has the effect of preventing or treating fatty liver.
  • mice were fed ad libitum for 1 week and randomly divided into two groups.
  • the standard common diet was fed with 10 and the high-fat diet was fed with more than 30. All groups were given free access to water.
  • mice with a fasting body weight of 35 g or more that is, 20% of the average body weight of the standard normal diet-fed mice
  • mice with 30 were selected as models. group.
  • mice in the standard normal diet feeding group were given normal feeding as a normal control group (NC group), and 30 mice in the model group were randomly divided into 3 groups, 10 in each group, 1 of which The group was the solvent control group (HFD group), the first group was the P8 intervention group (HFD+P8 group), and the first group was the Exendin-4 intervention group (HFD+Exendin-4 group).
  • the group was the solvent control group (HFD group)
  • the first group was the P8 intervention group
  • HFD+Exendin-4 group the Exendin-4 intervention group
  • mice 10 rats in the normal control group (NC group) were fed with standard normal diet for 6 weeks; 10 patients in the solvent control group (HFD group) were fed with high fat diet + normal saline 0.2 ml/kg intraperitoneally for 6 weeks; P8 intervention group 10 (HFD+P8 group), high fat diet + P8 peptide 50nmol/kg, 0.2ml/kg intraperitoneal injection for 6 weeks; Exendin-4 intervention group (HFD+Exendin-4 group) 10, high fat diet Feed +Exendin-4 peptide 50nmol/kg, 0.2ml/kg intraperitoneal injection for 6 weeks.
  • This example employs the polypeptide P8: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS. During the intervention, attention was paid to the changes in hair, appetite, bowel movements, activity, and body weight of the mice.
  • mice in (b) For the mice in (b), the fasting blood glucose and body weight of each mouse were measured weekly, and the food intake was recorded.
  • mice in (2) fasting glucose tolerance and fasting insulin tolerance were measured in the solvent control group, P8 intervention group and Exendin-4 intervention group after the intervention.
  • the fasting glucose tolerance test procedure was as follows: obese mice were fasted for 12 hours, fasting blood glucose was measured, and glucose (1.0 g/kg) was immediately injected. Then, blood glucose was measured at 15, 30, 45, 60, and 120 minutes after glucose injection, and blood glucose levels were recorded. Draw a curve and calculate the area under the curve.
  • the fasting insulin tolerance test procedure was as follows: obese mice were fasted for 4 hours, fasting blood glucose was measured, and insulin (0.5 U/kg) was injected immediately. Then, blood glucose was measured at 15, 30, 45, and 60 min after glucose injection, blood glucose values were recorded, and a curve was drawn. , calculate the area under the curve.
  • mice significantly improved glucose tolerance and insulin tolerance after treatment with P8 and Exendin-4, indicating that peptide P8 can improve glucose tolerance and insulin tolerance, and increase insulin sensitivity.
  • the open Oxymax indirect calorimetric system (Columbus, USA) was used to collect and analyze mouse metabolic indicators to assess the overall metabolic status of the mice.
  • the feeding conditions during the whole experiment were as follows: 1 / cage; the mouse high fat feed was installed in the feeding device of the system; the drinking water of the mouse was the same as the conventional water, and it was installed in the drinking bottle provided by the system; :00-18:00 is the daylight time (lights on), 18:00-6:00 is the night time (lights off); try to keep the test room quiet after the program starts.
  • mice after the intervention in (2), 3 in the solvent control group, P8 intervention group and Exendin-4 intervention group were used to detect the change of oxygen consumption.
  • mice were transferred from daily cages to indirect calori cages 3 days before the experiment was officially started. The time period is used as the adaptation period, and the data is discarded. The data on the first day of the official experiment was discarded, and the data on the 2nd day and the 3rd day were collected, the average value was included in the statistical analysis, and the resting metabolic rate was measured on the 4th day.
  • Food intake The system automatically collects the weight change of the feeder in each cycle, and uses the cumulative weight change value within 12h or 24h as the day/night or whole-day food intake to correct the mouse body weight.
  • the unit is g/kg body weight/day (g/kg per day).
  • Oxygen consumption (VO 2 ): The oxygen consumption of the mice within 24 hours was automatically collected by the system and corrected by the mouse lean body weight (1ean mass) in milliliters/lean body weight (kg). /hour (ml/kg lean mass/h).
  • mice in (b) After the intervention, the solvent control group, the P8 intervention group, and the Exendin-4 intervention group were respectively Perform the following experimental procedure.
  • mice The body composition of the mice was analyzed using a Lunar PIXImus density meter (Lunar) and a dual-energy X-ray absorptiometry (DEXA). Respiratory gating was used for all image analyses.
  • Mouse body temperature was maintained at 37 °C throughout the test using an animal incubation system (SA Instruments). Each mouse was collected with 20 coronal images and 20 frames of axial images with a layer thickness of 1.0 mm.
  • mice underwent magnetic resonance (MRI) analysis using an Echo MRI-100 Whole Body Composition Analyzer (Echo Medicine Systems) to obtain mouse fat mass and lean body mass by whole body scanning. (1ean mass) content results, and calculate their ratio to body weight. Each mouse was tested at least three times and the mean was taken into statistical analysis.
  • MRI magnetic resonance
  • Echo Medicine Systems Echo Medicine Systems
  • the results showed that compared with the solvent control group, the intra-abdominal fat and subcutaneous fat of the P8 group and the Exendin-4 group were significantly reduced.
  • the MRI scan image showed the results as shown in Fig. 21, calculated according to the MRI scan results.
  • the statistical results are shown in Figure 22.
  • the anatomical results of the three groups of mice also support the above results.
  • mice in the P8 group was significantly less than that in the Vehicle group, and it was mainly related to the decrease in body fat content.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Diabetes (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Endocrinology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Obesity (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Emergency Medicine (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种多肽，其氨基酸序列为：HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS。该多肽的用途为：用于制备治疗或预防糖尿病药物或药物组合物的用途，用于制备减肥药物或药物组合物的用途，以及用于制备预防或治疗脂肪肝病药物或药物组合物的用途。该多肽能够有效地发挥糖依赖性促胰岛素分泌作用；针对STZ糖尿病模型小鼠，能降低其血糖，抑制其进食，使其保持良好的胰岛形态，增加其胰岛面积，增加起C肽水平，且降糖作用明显优于艾塞那肽和GLP-1；能有效地降低db/db小鼠的空腹血糖，作用明显优于GLP-1；对脂肪肝病和肥胖症具有疗效。

Description

一种多肽及其用途 技术领域
本发明涉及一种多肽及其用途，具体而言涉及多肽P8，其用途包括：用于制备预防或治疗糖尿病药物的用途、用于制备预防或治疗脂肪肝病药物的用途、以及用于制备减肥药物的用途，属于生化药学技术领域。
背景技术
据发明人了解，糖尿病是一种以慢性高血糖为特征的代谢紊乱综合征，目前认为遗传和环境的相互作用是主要诱因，但是其发病机理尚未完全明确。糖尿病可分为4型：1型糖尿病、2型糖尿病、妊娠期糖尿病和其他类型糖尿病。1型糖尿病(Type 1diabetes mellitus,T1DM)是一种自身免疫性疾病，由于对自身抗体耐受性的丧失使胰岛β细胞损伤，机体无法产生足够的胰岛素，胰岛素绝对缺乏，引起血糖升高，故患者一旦发病需终生注射胰岛素。2型糖尿病(Type 2diabetes mellitus,T2DM)，又被称之为非胰岛素依赖糖尿病，缘于β细胞功能低下，胰岛素相对缺乏和胰岛素抵抗。近年来，2型糖尿病的患病率逐渐增加，据世界卫生组织预测，2030年，全球将有3亿2型糖尿病患者。目前，针对2型糖尿病的治疗药物主要是口服降糖药和胰岛素。虽然该类药物能有效降低血糖，并改善患者症状，但仍然存在低血糖和体重增加等缺陷。此外，随着糖尿病病程的延长，该类药物均不能阻止患者β细胞的恶化。
脂肪肝又称脂肪性肝病，是指由于各种原因引起的肝细胞内脂肪堆积过多的病变，该病正严重威胁国人的健康，成为仅次于病毒性肝炎的第二大肝病。脂肪肝是一种常见的临床现象，而非一种独立的疾病，其临床表现轻者无症状，重者病情凶猛。一般而言，脂肪肝属可逆性疾病，早期诊断并及时治疗常可恢复正常。
肥胖症在全球范围内已成为一种严重威胁健康的流行病，无论是发达国家还是发展中国家均面临着肥胖症加速发展的困扰，随之带来巨大的医疗费用和社会负担。据统计，目前全球已有15亿超重和5亿肥胖患者。对于这些BMI指数高的人群来说，肥胖会引发其他一些疾病发病率的上升，其中最为显著的是心血管疾病、糖尿病和癌症。
关于肥胖的医学治疗手段主要有外科手术和药物治疗两种，通过减肥手术减少胃的大小、增加饱胀感、减少食物摄取的量，从而达到减肥的效果，但是减肥手术有严重的手术和代谢并发症的风险，而且手术非常昂贵，因此药物治疗是更主要的选择。
在针对糖尿病的研究成果中，胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是由胰高血糖素原基因编码，后经加工修饰得到的30个氨基酸的肠降血糖素(incretin)；主要由小肠黏膜的L细胞合成并分泌，在胰岛的α细胞也有表达。GLP-1具有两种生物活性形式，GLP-1(7-37)和GLP-1(7-36)酰胺，血液循环中主要以GLP-1(7-36)酰胺的活性形式存在，两者具有共同的生物学活性。
天然人GLP-1的稳定性较差，易被二肽基肽酶Ⅳ(DPPⅣ)降解，并快速被肾脏清除，其半衰期t1/2≤2min。GLP-1经DPPⅣ切割掉N末端His-Ala，生成GLP-1(9-36)氨基多肽，最初研究认为，GLP-1(9-36)氨基多肽不具有生物学活性，而且对GLP-1受体(GLP-1R) 还具有一定的拮抗作用，近些年发现GLP-1(9-36)氨基多肽具有类胰岛素的作用。
GLP-1的生理作用主要包括三个部分，一是通过与GLP-1R结合，发挥生理学效应，包括：①葡萄糖依赖性促胰岛素分泌；②抑制胰高血糖素分泌；③延迟胃排空，降低食欲，减少饮食,控制体重；④抑制β细胞凋亡，促进β细胞增值和分化。二是，GLP-1经过DPPⅣ降解，生成GLP-1(9-36)氨基多肽，能够抑制肝脏糖异生酶和脂肪酸合酶的表达，抑制糖异生和肝脏脂肪合成，发挥类胰岛素作用。三是GLP-1(9-36)氨基多肽经过肽链内切酶(NEP24.11)切割成GLP-1(32-36)氨基5肽，在肥胖型小鼠上增加能量消耗，抑制体重增长。
发明人在针对糖尿病的研究中研制出了多肽P8，并在后续研究中进一步发现多肽P8还具有治疗脂肪肝和肥胖症的功能。
发明内容
本发明所要解决的技术问题是：针对现有技术存在的问题，提供一种多肽，即多肽P8，能治疗糖尿病、脂肪肝和肥胖症；并在此基础上，进一步提供该多肽的多种用途。
本发明的技术方案如下：
一种多肽，其特征是，该多肽的氨基酸序列为：
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS。
上述多肽用于制备治疗或预防1型糖尿病药物或药物组合物的用途。
上述多肽用于制备治疗或预防2型糖尿病药物或药物组合物的用途。
上述多肽用于制备减肥药物或药物组合物的用途。
上述多肽用于制备预防或治疗脂肪肝病药物或药物组合物的用途。
上述用途中，所述脂肪肝病为非酒精性脂肪肝。
上述用途中，所述药物组合物包括该多肽自身或其药用盐。
上述用途中，所述药物组合物包括药物载体和/或药物活性物质。
发明人在实践研究中，将GLP-1的野生型进行改造：第2位的氨基酸由Ala替换成Gly，在GLP-1的末尾添加Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser，序列如下：
GLP-1：HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
P8：H
Figure PCTCN2017098674-appb-000001
EGTFTSDVSSYLEGQAAKEFIAWLVKGRG
Figure PCTCN2017098674-appb-000002
本发明通过氨基酸替换和末尾添加小肽，构建了一种降糖肽P8。实验结果证明P8肽能够有效地发挥糖依赖性促胰岛素分泌作用(IPTGG)，降低STZ糖尿病模型小鼠的血糖，抑制STZ糖尿病模型小鼠的进食，保持良好的胰岛形态(HE染色)，增加STZ糖尿病模型小鼠的胰岛面积(免疫染色，并累计面积)，增加STZ糖尿病模型小鼠的C肽水平，其降糖作用明显优于艾塞那肽和GLP-1；P8肽能够有效地降低db/db小鼠的空腹血糖，作用明显优于GLP-1。
在进一步地实践研究中，发明人为筛选出能用于制备预防或治疗脂肪肝病药物的多肽，尝试了若干已研发出的多肽，并发现先前获得的降糖调脂肽P8符合要求。多肽P8经实验确证对脂肪肝病具有疗效，这不仅为多肽P8开发出新的用途，也为脂肪肝病找到了新的治疗药 剂。
在进一步地实践研究中，发明人为筛选出能用于制备减肥药物的多肽，尝试了若干已研发出的多肽，并发现先前获得的降糖调脂肽P8符合要求。多肽P8经实验确证对肥胖症具有疗效，这不仅为多肽P8开发出新的用途，也为肥胖症找到了新的治疗药剂。
附图说明
图1为本发明实施例1中多肽P8的糖依赖性的促胰岛素分泌作用(IPTGG)结果图。
图2为本发明实施例2中多肽P8对STZ糖尿病模型小鼠累计进食的影响结果图。
图3为本发明实施例2中多肽P8对STZ糖尿病模型小鼠血糖的影响结果图。
图4为本发明实施例2中多肽P8对STZ糖尿病模型小鼠甘油三酯和游离脂肪酸的影响结果图。
图5为本发明实施例2中多肽P8对STZ糖尿病模型小鼠胰岛形态(HE染色)的影响结果图。
图6为本发明实施例2中多肽P8对STZ糖尿病模型小鼠胰岛面积的影响结果图。
图7为本发明实施例2中多肽P8对STZ糖尿病模型小鼠的C肽水平的影响结果图。
图8为本发明实施例3中多肽P8对db/db糖尿病模型小鼠的血糖的影响结果图。图中，用*表示P8组与vehicle组比较的显著性，*P<0.05，**P<0.01，***P<0.001；用#表示Exendin-4与vehicle组比较的显著性，#P<0.05，##P<0.01，###P<0.001；用$表示P8组与GLP-1组比较的显著性，$P<0.05，$$P<0.01，$$$P<0.001。
图9为本发明实施例3中多肽P8对db/db糖尿病模型小鼠的胰岛素耐量的影响结果图。
图10为本发明实施例4中判断造模是否成功时小鼠的血清检测结果示意图。其中，NC为标准普通饲料喂养组小鼠，HFD为高脂饲料喂养组小鼠。
图11为本发明实施例4中判断造模是否成功时小鼠肝组织HE染色的结果示意图。其中，NC为标准普通饲料喂养组小鼠，HFD为高脂饲料喂养组小鼠。
图12为本发明实施例4中溶剂对照组小鼠和P8干预组小鼠的血清检测结果示意图。其中，HFD为溶剂对照组小鼠，P8为P8干预组小鼠。
图13为本发明实施例4中溶剂对照组小鼠和P8干预组小鼠的肝组织HE染色结果示意图。其中，HFD为溶剂对照组小鼠，P8为P8干预组小鼠。
图14至图17分别为本发明实施例5在干预过程中小鼠体重重量、体重百分比、累积进食量、空腹血糖的变化曲线。
图18、图19分别为本发明实施例5干预后小鼠的空腹糖耐量结果曲线、空腹胰岛素耐量结果曲线。
图20为本发明实施例5干预后小鼠的耗氧量结果曲线。
图21和图22分别为本发明实施例5干预后小鼠的MRI扫描图像显示结果、根据MRI扫描结果计算得出的统计结果。
以上各图中，Vehicle或Ve表示溶剂对照组，P8表示P8干预组，Exendin-4或Ex4表示 Exendin-4干预组。除图8、图10以及图12外，用*表示与vehicle组比较的显著性，*P<0.05，**P<0.01，***P<0.001。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1：多肽P8的糖依赖性的促胰岛素分泌作用(IPTGG)
40只8周龄的雄性c57小鼠(购自扬州大学实比较医学中心，许可证号：SCXK(苏)2012-0004)。随机分为4组，每组10只，分别为溶剂对照组(阴性对照组)、Exendin-4组(阳性对照组)、GLP-1组(阳性对照组)、P8组(实验组)。腹腔糖耐量过程如下：c57小鼠空腹12小时，测空腹血糖，分别注射肽或者溶剂，30分钟注射葡萄糖(1.5g/kg)，之后分别在注射糖之后15、30、60、120分钟检测血糖，并分别计算曲线下面积。结果如图1所示，P8具有葡萄糖依赖性的促胰岛素分泌功能，与溶剂组和GLP-1组均具有显著性差异，与Exendin-4组效果一样。
实施例2：多肽P8的药效学评价一
1、STZ糖尿病模型小鼠造模，及分组
80只6周龄的雄性c57小鼠(购自扬州大学实比较医学中心，许可证号：SCXK(苏)2012-0004)，适应性饲养1周。实验前空腹12小时，按50mg/kg的剂量腹腔注射STZ溶液(pH＝5.2的柠檬酸缓冲液)，连续注射5天，注射后1周、2周分别检测空腹血糖，血糖值均超过11.0mmol/L，为造模成功。将成模小鼠随机分成4组，每组12只，分别为溶剂对照组(阴性对照组)、Exendin-4组(阳性对照组)、GLP-1组(阳性对照组)、P8组(实验组)。给药剂量为25nmol/kg，每天2次，溶剂为生理盐水，皮下注射0.1ml，固定时间点给药，AM 9：00—10:00，PM 8:00—9:00。阴性对照组，皮下注射0.1ml生理盐水。
2、对STZ糖尿病模型小鼠累计进食的影响
从给药开始，每组分别记录初始添食量，隔1天或2天检测剩食量，并继续添加新的鼠粮，并记录，依次类推，到5周给药结束，记录小鼠的总进食量，并绘制累计进食曲线，结果如图2所示，P8组累计进食明显少于溶剂对照组、GLP-1组和Exendin-4组，具有明显的抑制进食效果。
3、对STZ糖尿病模型小鼠血糖的影响
小鼠空腹8小时，AM 8:00开始禁食，PM 4:00，尾静脉检测空腹血糖，检测用血糖检测仪(欧姆龙血糖仪HEA-231型)，作为给药0周血糖。之后分别在给药1、2、3、4、5周，检测空腹血糖，禁食时间和检测时间同0周方法，结果如图3所示，P8组能够显著降低STZ糖尿病模型小鼠的空腹血糖，明显优于GLP-1组和Exendin-4组，到第5周，与溶剂对照组比较，p≤0.01，具有显著性差异。
4、对STZ糖尿病模型小鼠甘油三酯和游离脂肪酸的影响
给药5周后，眼眶取血，静置收集血清，试剂盒分别检测甘油三酯(TG)和游离脂肪酸 (FFA)水平，结果如图4所示。结果显示，血清甘油三酯水平，P8组与溶剂对照组比较，p≤0.001，具有极显著性差异；Exendin-4组与溶剂对照组比较具有显著性差异，p≤0.01；GLP-1与溶剂对照组比较，无明显差异性；血清游离脂肪酸水平，P8组与溶剂对照组比较具有显著性差异，p≤0.01；GLP-1组、Exendin-4组与溶剂对照组比较，无显性著差异。结果说明，多肽P8处理后能够明显降低STZ糖尿病模型小鼠的甘油三酯和游离脂肪酸水平，优于GLP-1组、Exendin-4组。
5、对STZ糖尿病模型小鼠胰岛形态(HE染色)的影响
给药5周后，处死小鼠，取胰腺，用福尔马林固定，经HE染色后，观察胰岛形态，结果如图5所示。溶剂对照组胰岛损伤严重，几乎看不到完整的胰岛结构，GLP-1组、Exendin-4组和P8组胰岛形态得到明显改善，可以观察到细胞着色浅、排列呈团索状的胰岛结构。说明GLP-1组、Exendin-4组和P8组均能改善胰岛形态。
6、对STZ糖尿病模型小鼠胰岛面积的影响
给药5周后，处死小鼠，取胰腺，福尔马林固定，免疫染色(insulin、ki67)，结果如图6所示。免疫染色图片显示，溶剂对照组胰岛β细胞很少(STZ损伤诱导β-细胞死亡)，而GLP-1组、Exendin-4组和P8组胰岛β细胞增多，说明P8组能够明显增加胰岛β细胞量，增加胰岛β细胞面积。
7、对STZ糖尿病模型小鼠的C肽水平的影响
给药5周后，眼眶取血，静置收集血清，试剂盒检测C肽水平，结果如图7所示。结果显示，P8组与溶剂对照组存在显著差异，p≤0.05；GLP-1组、Exendin-4组与溶剂对照组比较，无明显差异，说明P8处理后能够明显降低STZ糖尿病模型小鼠的C肽水平，优于GLP-1组、Exendin-4组。
实施例3：多肽P8的药效学评价二
1、对db/db糖尿病模型小鼠血糖的影响
24只6周龄的雄性db/db小鼠(购自南京大学，许可证号：SCXK(苏)2015-00001)，适应性饲养1周。从第7周开始检查血糖，AM 8:00开始禁食，AM 12:00尾静脉检测血糖，检测用血糖检测仪(欧姆龙血糖仪HEA-231型)。第9周时，将小鼠随机分为4组，每组6只，分别为溶剂对照组(阴性对照组)、Exendin-4组(阳性对照组)、GLP-1组(阳性对照组)、P8组(实验组)，开始给药，并检测血糖。第10周时，结果如图8所示，与溶剂对照组、GLP-1组相比，Exendin-4组和P8组的血糖显著降低，分别为p≤0.001和p≤0.05，并持续维持较低的血糖水平；P8组与溶剂对照组比较存在显著差异，降糖明显优于GLP-1组，与Exendin-4组基本持平。
2、对db/db糖尿病模型小鼠胰岛素耐量的影响
第16周，小鼠空腹8小时，AM 8:00开始禁食，PM 4:00，尾静脉检测空腹血糖，检测用血糖检测仪(欧姆龙血糖仪HEA-231型)，检测空腹血糖，作为胰岛素耐量的0点血糖，按1IU/kg的剂量皮下注射胰岛素，之后分别在注射胰岛素之后15、30、60、90、120分钟检 测血糖，并分别计算曲线下面积。结果如图9所示，多肽P8显著改善胰岛素耐量，与溶剂对照组和GLP-1组均具有显著性差异，与Exendin-4组效果一样。
临床证实GLP-1类似物能够明显的降低2型糖尿病病人的血糖，抑制进食，降低糖化血红蛋白等，同时，GLP-1及其类似物能够降低1型糖尿病病人的血糖，降低糖化血红蛋白，但是有些个体存在体重减轻的不利因素。本发明将GLP-1的第2位的氨基酸由Ala替换成Gly，在GLP-1的末尾添加Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser，起到了降低1型糖尿病和2型糖尿病小鼠血糖的作用。
实施例4：多肽P8对脂肪肝模型小鼠的影响
40只雄性c57小鼠适应性喂养1周，随机分为两组，标准普通饲料喂养10只，高脂饲料喂养30只，各组均自由饮水。
(1)判断造模是否成功
在第12周末，两组分别随机抽取1只小鼠，禁食12h后，眼眶取血检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和总胆固醇(TC)，取血后迅速取出肝脏病理切片，HE染色观察肝组织脂肪变情况。
结果如图10和图11所示，高脂饲料喂养组小鼠的血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和总胆固醇(TC)均异常升高，且肝组织HE染色有大量脂滴形成，由此可确定脂肪肝模型小鼠造模成功。因此，将高脂饲料喂养组定为模型组。
(2)多肽P8给药后对模型组小鼠的影响
自第13周开始，将普通饲料喂养组9只小鼠继续予普通饲料喂养作为正常对照组(NC组)，将髙脂饲料喂养组中取20只小鼠并随机分为2组，其中1组为溶剂对照组，1组为P8干预组。
各组分别按下述实验方案进行干预：
正常对照组(NC组)9只，普通饲料喂养+生理盐水0.2ml/kg腹腔注射，时长6周；溶剂对照组(HFD组)10只，高脂饲料喂养+生理盐水0.2ml/kg腹腔注射，时长6周；P8干预组(HFD+P8组)10只，高脂饲料喂养+P8肽50nmol/kg，0.2ml/kg腹腔注射，时长6周。
干预结束后，将各组小鼠禁食12h后，眼眶取血检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和总胆固醇(TC)，取血后迅速取出肝脏病理切片，HE染色观察肝组织脂肪变情况。
结果表明，正常对照组小鼠的血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)和总胆固醇(TC)与图10中NC组保持一致，肝组织HE染色结果与图11中NC组保持一致。
如图12所示，与溶剂对照组相比，在给药6周后，P8干预组小鼠血清中的ALT、AST、TG、TC均显著降低(P＜0.001)具有显著性差异。
如图13所示，在干预6周后，溶剂对照组的肝细胞弥漫小泡性脂肪空泡变性、重度水 样变性，细胞核被挤压变形部分消失，肝细胞索素乱，但未见明显炎症、纤维化、坏死等病灶；与溶剂对照组相比，P8干预组大鼠的肝脏组织肝脏组织结构正常，肝小叶结构明显，肝细胞索呈放射状排列，肝细胞内未见气球样变性，几乎接近正常小鼠肝脏结构。
由此可见，多肽P8具有预防或治疗脂肪肝的作用。
实施例5：多肽P8对肥胖模型小鼠的影响
若干雄性c57小鼠适应性喂养1周，随机分为两组，标准普通饲料喂养10只，高脂饲料喂养数量大于30只，各组均自由饮水。
(一)选出造模成功的小鼠
在第12周末，高脂饲料喂养组小鼠中，空腹体重大于等于35g(即超过标准普通饲料喂养组小鼠平均体重的20％)的小鼠即为肥胖模型小鼠，挑选30只作为模型组。
(二)以多肽P8、Exendin-4分别对模型组小鼠进行干预
自第13周开始，将标准普通饲料喂养组10只小鼠继续给予普通饲料喂养作为正常对照组(NC组)，将模型组30只小鼠随机分为3组，每组10只，其中1组为溶剂对照组(HFD组)，1组为P8干预组(HFD+P8组)，1组为Exendin-4干预组(HFD+Exendin-4组)。
各组分别按下述实验方案进行干预：
正常对照组(NC组)10只，标准普通饲料喂养，时长6周；溶剂对照组(HFD组)10只，高脂饲料喂养+生理盐水0.2ml/kg腹腔注射，时长6周；P8干预组(HFD+P8组)10只，高脂饲料喂养+P8肽50nmol/kg，0.2ml/kg腹腔注射，时长6周；Exendin-4干预组(HFD+Exendin-4组)10只，高脂饲料喂养+Exendin-4肽50nmol/kg，0.2ml/kg腹腔注射，时长6周。本实施例采用多肽P8：HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS。干预期间注意观察小鼠的毛发改变，食欲、大小便、活动、体重等变化情况。
(三)干预时小鼠空腹血糖、体重、进食量的变化
对(二)中的小鼠，每周检测各小鼠空腹血糖、体重，并对其进食量进行记录。
结果表明，如图14至图17所示，与对照组相比，P8组与Exendin-4组均能显著降低肥胖小鼠体重，并且其进食量较对照组略有下降；由其空腹血糖可知，肥胖小鼠有轻微高血糖，胰岛素抵抗，经治疗后，P8组与Exendin-4组均使小鼠血糖降至正常。
(四)干预后小鼠的空腹糖耐量及空腹胰岛素耐量
对(二)中的小鼠，在干预结束后，对溶剂对照组、P8干预组、Exendin-4干预组分别进行空腹糖耐量及空腹胰岛素耐量检测。
空腹糖耐量检测过程如下：肥胖小鼠空腹12h，测空腹血糖，并立即注射葡萄糖(1.0g/kg)，之后分别在注射葡萄糖之后15、30、45、60、120min检测血糖，记录血糖值，绘制曲线，计算曲线下面积。
空腹胰岛素耐量检测过程如下：肥胖小鼠空腹4h，测空腹血糖，并立即注射胰岛素(0.5U/kg)，之后分别在注射葡萄糖之后15、30、45、60min检测血糖，记录血糖值，绘制曲线，计算曲线下面积。
空腹糖耐量的结果如图18所示，从葡萄糖耐量实验曲线下面积(GTT AUC)来看，P8组与Exendin-4组的曲线下面积均明显小于对照组，且与对照组比较具有明显差异(P8：P<0.005；Exendin-4：P<0.001)。
空腹胰岛素耐量的结果如图19所示，从胰岛素耐量实验曲线下面积(ITT AUC)来看，P8组与Exendin-4组的曲线下面积均明显小于对照组，且与对照组比较具有明显差异(P8：P<0.005；Exendin-4：P<0.001)。
结果表明，肥胖小鼠经过P8及Exendin-4治疗后，显著改善葡萄糖耐量和胰岛素耐量，这说明多肽P8可以改善葡萄糖耐量和胰岛素耐量，增加胰岛素的敏感性。
(五)干预后小鼠的耗氧量变化
采用开放式Oxymax间接测热系统(美国Columbus公司)采集并分析小鼠代谢指标，评估小鼠的整体代谢状况。整个实验过程中的饲养条件如下：1只/笼；小鼠高脂饲料，装于系统自带的进食器中；小鼠饮用水与常规用水相同，装于系统自带的饮水瓶中；6：00-18：00为白昼时间(开启灯光)，18：00-6：00为夜晚时间(关闭灯光)；程序运行开始后尽量保持测试房间安静。
取(二)中干预结束后的小鼠共9只，溶剂对照组、P8干预组、Exendin-4干预组各3只，以备耗氧量变化检测。
为消除环境改变、单笼饲养、饲料性状改变导致的应激反应对小鼠整体代谢情况的影响，实验正式开始前3天，将9只小鼠从日常饲养笼转移至间接测热笼中，将该时间段作为适应期，数据弃去不用。正式实验第l天的数据弃去不用，收集第2天和第3天的数据，取其平均值计入统计分析，第4天进行静息代谢率的测定。
每个测定循环中由系统自带的计算机自动收集数据，测定顺序为l号笼、2号笼、3号笼……9号笼，每测定3个笼子后自动校正1次系统各参数，循环往复。主要数据收集和分析的简要方法如下：
(1).摄食量(food intake)：系统自动收集每个循环内进食器的重量变化，以12h或24h内的累积重量变化值作为昼/夜或全天摄食量，以小鼠体重进行校正，单位为克/公斤体重/天(g/kg per day)。
(2).氧气消耗量(oxygen consumption，VO2)：由系统自动收集小鼠24h内氧气消耗量，以小鼠瘦体质重量(1ean mass)进行校正，单位为毫升/瘦体质重量(公斤)/小时(ml/kg lean mass/h)。
结果表明，在整个测定期间，三组小鼠的摄食量无显著区别，这说明能量摄入减少并不是导致肥胖小鼠体重和脂肪含量降低的主要原因。如图20所示，与对照组小鼠相比，P8组和Exendin-4组小鼠昼夜氧气消耗量(VO2)显著增加(P<0.05)；在24小时内，P8组和Exendin-4组小鼠的能量消耗值始终高于对照组小鼠。这说明多肽P8可以提高小鼠代谢水平。
(六)干预后小鼠的脂肪量变化
对(二)中的小鼠，在干预结束后，对溶剂对照组、P8干预组、Exendin-4干预组分别 进行如下实验过程。
小鼠身体组成成分分析采用Lunar PIXImus密度测量仪(Lunar公司)和双能X线吸收测量仪(DEXA)。所有图像分析均采用呼吸门控法。在整个测试过程中，小鼠体温用动物保温系统(SA Instruments公司)维持在37℃。每只小鼠均采集20帧冠状面图像和20帧轴状面图像，层厚为1.0mm。为了得到更好的脂肪组织对比效果，采用T1-weighted spin-echo成像方法检测，成像参数为repetition time(TR)＝500ms，echo time(TE)＝10.7ms，field of view＝50.0×45.0mm(冠状面)，45.0×45.0mm(轴状面)，matrix size＝256×256.每只小鼠至少检测两次，取其均值进入统计分析。
此外，所有小鼠均进行了磁共振(MRI)分析，所用仪器为Echo MRI-100全身组成成分分析仪(Echo Medicine Systems公司)，通过全身扫描得到小鼠体脂含量(fat mass)和瘦体质(1ean mass)含量结果，并计算其与体重的比值。每只小鼠至少检测三次，取其均值进入统计分析。
结果表明，与溶剂对照组小鼠相比，P8组与Exendin-4组小鼠腹内脂肪及皮下脂肪均显著减少；其中，MRI扫描图像显示结果如图21所示，根据MRI扫描结果计算得出的统计结果如图22所示。三组小鼠的解剖结果也支持上述结果。
综上所述，P8组小鼠体重较Vehicle组小鼠显著减轻，且主要和体内脂肪含量减少有关。
以上实验结果表明，多肽P8对肥胖症具有疗效。
序列表
Figure PCTCN2017098674-appb-000003

Claims (8)

  1. 一种多肽，其特征是，该多肽的氨基酸序列为：
    HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS。
  2. 权利要求1述及多肽用于制备治疗或预防1型糖尿病药物或药物组合物的用途。
  3. 权利要求1述及多肽用于制备治疗或预防2型糖尿病药物或药物组合物的用途。
  4. 权利要求1述及多肽用于制备减肥药物或药物组合物的用途。
  5. 权利要求1述及多肽用于制备预防或治疗脂肪肝病药物或药物组合物的用途。
  6. 根据权利要求5述及的用途，其特征是，所述脂肪肝病为非酒精性脂肪肝。
  7. 根据权利要求2至6任一项述及的用途，其特征是，所述药物组合物包括该多肽自身或其药用盐。
  8. 根据权利要求2至6任一项述及的用途，其特征是，所述药物组合物包括药物载体和/或药物活性物质。
PCT/CN2017/098674 2016-09-06 2017-08-23 一种多肽及其用途 Ceased WO2018045872A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201610805193.7A CN106279400A (zh) 2016-09-06 2016-09-06 P8降糖肽的设计及其用途
CN201610805193.7 2016-09-06
CN201710539987.8A CN109200273B (zh) 2017-07-04 2017-07-04 一种多肽用于制备预防或治疗脂肪肝病药物的用途
CN201710539987.8 2017-07-04
CN201710714470.8A CN109395065A (zh) 2017-08-18 2017-08-18 一种多肽用于制备减肥药物的用途
CN201710714470.8 2017-08-18

Publications (1)

Publication Number Publication Date
WO2018045872A1 true WO2018045872A1 (zh) 2018-03-15

Family

ID=61562429

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/098674 Ceased WO2018045872A1 (zh) 2016-09-06 2017-08-23 一种多肽及其用途

Country Status (1)

Country Link
WO (1) WO2018045872A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111138552A (zh) * 2020-01-15 2020-05-12 中国药科大学 一种降脂多肽及其制药应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060205037A1 (en) * 2003-08-28 2006-09-14 Homayoun Sadeghi Modified transferrin fusion proteins
WO2009109927A2 (en) * 2008-03-05 2009-09-11 Tel Hashomer Medical Research Infrastructure And Services Ltd. Glp-1 receptor agonists and related active pharmaceutical ingredients for treatment of cancer
CN101712722A (zh) * 2000-12-07 2010-05-26 伊莱利利公司 Glp-1融合蛋白
CN105727261A (zh) * 2008-06-27 2016-07-06 杜克大学 包含弹性蛋白样肽的治疗剂
CN105801705A (zh) * 2014-12-31 2016-07-27 天视珍生物技术（天津）有限公司 含胰高血糖素样肽-1和免疫球蛋白杂合Fc的融合多肽及其用途
CN106279400A (zh) * 2016-09-06 2017-01-04 中国药科大学 P8降糖肽的设计及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101712722A (zh) * 2000-12-07 2010-05-26 伊莱利利公司 Glp-1融合蛋白
US20060205037A1 (en) * 2003-08-28 2006-09-14 Homayoun Sadeghi Modified transferrin fusion proteins
WO2009109927A2 (en) * 2008-03-05 2009-09-11 Tel Hashomer Medical Research Infrastructure And Services Ltd. Glp-1 receptor agonists and related active pharmaceutical ingredients for treatment of cancer
CN105727261A (zh) * 2008-06-27 2016-07-06 杜克大学 包含弹性蛋白样肽的治疗剂
CN105801705A (zh) * 2014-12-31 2016-07-27 天视珍生物技术（天津）有限公司 含胰高血糖素样肽-1和免疫球蛋白杂合Fc的融合多肽及其用途
CN106279400A (zh) * 2016-09-06 2017-01-04 中国药科大学 P8降糖肽的设计及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHAO, LIJUAN ET AL.: "Progress of Extrapancreatic Effects by GLP-1 and Its Analogues", MEDICINE AND PHILOSOPHY, vol. 36, no. 1B, 31 January 2015 (2015-01-31), pages 94 *
ZHOU, SAIJUN ET AL.: "Prevention and Treatment of Nonalcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus", WORLD CHINESE JOURNAL OF DIGESTOLOGY, vol. 23, no. 32, 18 November 2015 (2015-11-18), pages 5113 - 5122 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111138552A (zh) * 2020-01-15 2020-05-12 中国药科大学 一种降脂多肽及其制药应用

Similar Documents

Publication Publication Date Title
Wang et al. Transient receptor potential vanilloid 1 activation enhances gut glucagon-like peptide-1 secretion and improves glucose homeostasis
Cernea et al. Therapy in the early stage: incretins
Witteles et al. Dipeptidyl peptidase 4 inhibition increases myocardial glucose uptake in nonischemic cardiomyopathy
EP2763690B1 (en) Lixisenatide for use in the treatment of stenosis or/and obstruction in the pancreatic duct system
Rybicka et al. The dawn phenomenon and the Somogyi effect—two phenomena of morning hyperglycaemia
Hansen et al. Incretin mimetics: a novel therapeutic option for patients with type 2 diabetes–a review
Rameshrad et al. An overview of glucagon-like peptide-1 receptor agonists for the treatment of metabolic syndrome: A drug repositioning
JP2004508410A (ja) Ｇｌｐ−１及びｇｌｐ−２ペプチドの使用方法
CN106279400A (zh) P8降糖肽的设计及其用途
US20130096059A1 (en) Glp-1 agonist for use in the treatment of stenosis or/and obstruction in the biliary tract
CN104159598A (zh) 治疗糖尿病的方法和组合物
Samson et al. Prevention of type 2 diabetes mellitus: potential of pharmacological agents
Ryan et al. Review of the therapeutic uses of liraglutide
Dejgaard et al. Liraglutide for treating type 1 diabetes
Kim et al. Recurrent acute pancreatitis in a patient with type IIb hyperlipoproteinemia: a case report and review of the literature in Korea
Ruze et al. Central GLP-1 contributes to improved cognitive function and brain glucose uptake after duodenum-jejunum bypass on obese and diabetic rats
Reboredo et al. Oral administration of zein-based nanoparticles reduces glycemia and improves glucose tolerance in rats
Zafer et al. GLP‐1 Receptor Agonists and Glucagon/GIP/GLP‐1 Receptor Dual or Triple Agonists—Mechanism of Action and Emerging Therapeutic Landscape in MASLD
Koullias et al. Targeting Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): Available and Future Pharmaceutical Options
WO2018045872A1 (zh) 一种多肽及其用途
Osonoi et al. Canagliflozin increases postprandial total glucagon-like peptide 1 levels in the absence of α-glucosidase inhibitor therapy in patients with Type 2 diabetes: a single-arm, non-randomized, open-label study
Ishimura et al. Effects of patient age on the therapeutic effects of GIP/GLP-1 receptor agonists (tirzepatide)
Liu et al. The Clinical Application of GLP-1RAs and GLP-1/GIP Dual Receptor Agonists Based on Pharmacological Mechanisms: A Review
Wasilewska et al. Semaglutide-properties, action and chromatographic analysis
WO2018113340A1 (zh) 多肽p11及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17848045

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17848045

Country of ref document: EP

Kind code of ref document: A1