WO2023000240A1 - 长效glp-1多肽类似物及其制备方法和应用 - Google Patents

长效glp-1多肽类似物及其制备方法和应用 Download PDF

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WO2023000240A1
WO2023000240A1 PCT/CN2021/107770 CN2021107770W WO2023000240A1 WO 2023000240 A1 WO2023000240 A1 WO 2023000240A1 CN 2021107770 W CN2021107770 W CN 2021107770W WO 2023000240 A1 WO2023000240 A1 WO 2023000240A1
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ser
glu
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孟静
蒋秀苹
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青岛博睿精创科技有限公司
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    • 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
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of biochemistry, in particular, the invention relates to a class of long-acting GLP-1 polypeptide analogues which can be used for treating or preventing diabetes or obesity, their preparation method and application.
  • Diabetes is a chronic disease in which the body is in a high blood sugar level for a long time and causes the body's glucose metabolism disorder.
  • the main characteristics of the disease are: chronic hyperglycemia, accompanied by insulin secretion defects or insulin dysfunction, which further affects fat, carbohydrates and insulin. Protein metabolism causes chronic damage to various organs, leading to organ dysfunction and even organ failure.
  • T2DM type 2 diabetes
  • the 2013 Global Disease Risk Study identified diabetes (all forms) as the ninth leading cause of reduced life expectancy. Based on the results of many surveys, diabetes has not been effectively controlled in developed countries such as Europe and the United States, or in developing countries like China. Diabetes has become another important chronic non-communicable disease that seriously endangers human health after cardiovascular and cerebrovascular diseases and tumors.
  • Obesity and diabetes belong to metabolic diseases, and obesity and diabetes are closely related. Diabetes is mainly caused by the decline of pancreatic ⁇ -cell function and insulin resistance, and obesity is the key factor of insulin resistance. Obese patients are prone to insulin resistance due to overweight and high fat content. Insulin resistance makes insulin in the body unable to exert the corresponding hypoglycemic effect. Insulin is the only blood sugar-lowering hormone in the body. In order to control blood sugar, the body must increase the ability of pancreatic beta cells to secrete insulin. When insulin secretion is increased and blood sugar cannot be normalized, diabetes occurs. Therefore, obesity is the root cause of insulin resistance and diabetes It happens for a very important reason.
  • GLP-1 glucose-dependent insulinotropic peptide
  • GLP-1R GLP-1 receptor
  • GLP-1 drugs are unstable and will be degraded by gastric acid after oral administration. Basically, they can only be administered by subcutaneous injection, and their half-life is short.
  • GLP-1 receptor agonists such as liraglutide and semaglutide have been approved for marketing in recent years.
  • the increased carbon chain of semaglutide has a greatly enhanced affinity for albumin, greatly slowing down its clearance by the kidneys.
  • the modification of the two places prolongs the half-life of rats to about 8 hours, and it only needs to be injected subcutaneously once a week clinically.
  • the purpose of the present invention is to provide a new class of longer-acting GLP-1 polypeptide analogs.
  • Another object of the present invention is to provide a preparation method of the long-acting GLP-1 polypeptide analog.
  • Another object of the present invention is to provide a composition containing the above-mentioned long-acting GLP-1 polypeptide analogue.
  • Another object of the present invention is to provide the application of the above-mentioned GLP-1 polypeptide analogs.
  • the long-acting GLP-1 polypeptide analog has the following amino acid sequence:
  • Xaa2 is Aib or D-Ser
  • Xaa9 is Asp or Glu
  • Xaa21 is Glu or Gln or Asp
  • Z is ⁇ Glu or Asp
  • x is 1, 2, 3 or 4;
  • y is 1, 2, 3 or 4;
  • n is an integer of 12-20, that is, n is 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the “side arm” structure includes a “side arm” short peptide chain -(Gly)x-(Ser-Gly)yZ- and a fatty acid substituent -CO(CH 2 ) n CO 2 H connected thereto.
  • the side chain amino group of the 20th Lys in the amino acid sequence of the main peptide chain is connected to the "side arm" short peptide chain structure by forming an amide bond with the carboxyl group of the glycine residue at the other end;
  • amino group of the "Z" amino acid residue at the end of the short peptide chain of the "side arm” is connected to the fatty acid substituent by forming an amide bond with the carboxyl group.
  • the carboxyl terminal of the amino acid sequence of the main peptide chain is not modified, or is modified with an amino group to form a -CONH 2 group.
  • Z is ⁇ Glu
  • x is 2 and y is 1, 2 or 3.
  • x is 2 and y is 1, or x is 2 and y is 2, or x is 2 and y is 3.
  • n is any integer in 14-18, that is, n is 14, 15, 16, 17 or 18.
  • the long-acting GLP-1 polypeptide analog according to the specific embodiment of the present invention in its amino acid sequence, Z is ⁇ Glu, x is 2, y is 1, 2 or 3, n is 18, and its sequence is as follows:,
  • the GLP-1 polypeptide analog is any one of the following compounds:
  • HsEGTFTSDVSSYLEGQAAK (GGSGSGSG- ⁇ -E-CO(CH 2 ) 18 CO 2 H)EFIAWLVRGRG;
  • HsEGTFTSDVSSYLEGQAAK (GGSGSGSG- ⁇ -E-CO(CH 2 ) 18 CO 2 H)QFIAWLVRGRG;
  • HsEGTFTSEVSSYLEGQAAK (GGSGSGSG- ⁇ -E-CO(CH 2 ) 18 CO 2 H)DFIAWLVRGRG;
  • the preparation method of the long-acting GLP-1 polypeptide analog according to the specific embodiment of the present invention comprises the following steps:
  • Step 1 According to the Fmoc/t-Bu strategy, synthesize the main peptide resin corresponding to the main peptide chain of the long-acting GLP-1 polypeptide analog, wherein the main peptide chain is His-Xaa2-Glu-Gly-Thr-Phe -Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly ;
  • Step 2 On the basis of the main peptide resin, according to the Fmoc/t-Bu strategy, couple the "side arm" structure corresponding to the long-acting GLP-1 polypeptide analog to obtain the long-acting GLP-1 polypeptide analog Corresponding polypeptide resin; wherein, the "side arm” structure is (Gly) x -(Ser-Gly) y -Z-CO(CH 2 ) n CO 2 H;
  • Step 3 adding a lysate to the polypeptide resin, performing a cleavage reaction, removing the full protection of the polypeptide, and extracting the crude compound;
  • the crude compound is purified to obtain a long-acting GLP-1 polypeptide analog.
  • step 2 the coupling agent used is 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide, the solvent is N,N-dimethylformamide, with 20% piperidine/ N,N-dimethylformamide solution removes Fmoc group;
  • the lysate is composed of TFA, DODT, m-cresol, and H 2 O in a volume ratio of 92.5:2.5:2.5:2.5;
  • the crude compound extraction methods include filtration, precipitation and/or methyl tert-butyl base ether extraction.
  • step 4 the obtained long-acting GLP-1 polypeptide analog has a purity greater than 96%.
  • Another object of the present invention is to provide a composition comprising a long-acting GLP-1 polypeptide analog and a pharmaceutically acceptable carrier or adjuvant.
  • a pharmaceutically acceptable carrier or adjuvant for example, carriers that can reduce drug degradation and loss, and reduce side effects, such as micelles, microemulsions, gels, and other carriers; excipients refer to materials added to make drugs into suitable dosage forms, such as buffers, excipients for lyophilization etc.
  • the pharmaceutical composition containing the polypeptide analog of the present invention can be made into a solution or a freeze-dried powder for parenteral administration, and the freeze-dried powder can be added with a suitable solvent or other pharmaceutically acceptable carrier before use.
  • liquid formulations are generally buffered, isotonic and aqueous solutions.
  • the buffer solution can be phosphate buffer solution
  • the isotonic solution can be 0.9% sodium chloride solution
  • the aqueous solution can be directly dissolved in pure water.
  • the long-acting GLP-1 polypeptide analogue is used as an active ingredient to add pharmaceutically acceptable carriers and/or excipients to make a pharmaceutical composition, which is suitable for various administration methods, such as oral administration, Transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc.
  • the pharmaceutical composition of the polypeptide analog of the present invention can be made into various suitable dosage forms, comprising at least one effective dose of the polypeptide analog of the present invention and at least one pharmaceutically acceptable medicinal carrier.
  • suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions for injection.
  • the dosage of the pharmaceutical composition of the present invention can vary within a large range, and those skilled in the art can determine it according to some objective factors, such as the type of disease, the severity of the disease, the patient's body weight, dosage form, route of administration, etc. .
  • Another object of the present invention is to provide the application of the above-mentioned long-acting GLP-1 polypeptide analogs and compositions.
  • the invention obtains a series of GLP-1 polypeptide analogs, and conducts research on the pharmacological effects of the series of drugs. Evaluate the GLP-1 receptor agonistic activity, hypoglycemic, lipid-lowering, weight-lowering and diabetic nephropathy activities of the synthetic peptide analog drugs, and conduct a preliminary study on their pharmacokinetics. The results show that compared with semaglutide, its long-term drug effect is far superior to semaglutide (half-life is 2 times or more than that of semaglutide), and it is also superior in treating and improving diabetic nephropathy. in semaglutide.
  • the long-acting GLP-1 polypeptide analog of the present invention has a longer half-life, has insulin-stimulating activity, and has no adverse reactions. drug.
  • the application of the long-acting GLP-1 polypeptide analog of the present invention specifically includes:
  • the present invention develops a site-specific "side arm” structure modification technology, breaking through the "stuck neck” technology of ultra-long-acting polypeptide and protein drug molecule modification , which greatly prolongs the half-life of the synthetic peptide analogs and realizes the ultra-long-acting peptide drugs.
  • the parent peptide in the long-acting GLP-1 polypeptide analog of the present invention is a homologous polypeptide, and the homologous polypeptide in the present invention refers to that the polypeptide originally has glucagon-like peptide (GLP-1), somalu
  • GLP-1 glucagon-like peptide
  • the amino acid at a specific site and the short peptide chain of the "side arm” connected to the specific site described in the present invention that is: the 20th Lys in the amino acid sequence
  • the site-specific "side arm” "Modification technology successfully broke through the "stuck neck” technology of molecular modification of ultra-long-acting polypeptide drugs.
  • the polypeptide drug designed in the present invention greatly prolongs its half-life, realizes the ultra-long-acting polypeptide drug, and its overall drug effect is much better than Positive drug semaglutide.
  • the GLP-1 polypeptide analogs of the present invention use lipophilic substituents to bind albumin in blood, protecting it from enzymatic degradation, thereby increasing the half-life.
  • the GLP-1 polypeptide analog of the present invention stabilizes the helical structure of the molecule through an intramolecular bridge, thereby improving the efficacy and/or selectivity for glucagon-like peptide 1 receptor (GLP-1R).
  • the GLP-1 polypeptide analog of the invention has high synthetic yield, good stability, easy scale-up production and low cost.
  • the GLP-1 polypeptide analog of the present invention is also superior to semaglutide in treating and improving diabetes and nephropathy.
  • the GLP-1 polypeptide analog of the present invention has a better pharmacological effect on reducing body weight, and the GLP-1 polypeptide analog can be used to prevent weight gain or promote weight loss by causing a decrease in food intake and/or an increase in energy consumption Therefore, the GLP-1 polypeptide analogs of the present invention can also be used for direct or indirect treatment of other diseases caused by or characterized by overweight, such as treatment and/or prevention of obesity, morbid obesity, obesity-related inflammation , obesity-related gallbladder disease, obesity-induced sleep apnea, the effects of the present invention in these diseases may be due to the direct or indirect effects of GLP-1 polypeptide analogs on body weight, or the effects on other aspects of the body other than body weight .
  • compounds 2, 5, 6 and 7 compared with semaglutide, compounds 2, 5, 6 and 7 have more excellent and significantly improved glucose tolerance effects, longer drug efficacy and half-life, and can significantly reduce
  • the liver weight and epididymal fat content of mice are also superior to semaglutide in improving oral glucose tolerance (OGTT) and insulin resistance (ITT) in diabetic mice.
  • OGTT oral glucose tolerance
  • ITT insulin resistance
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • HOBt 1-hydroxybenzotriazole
  • Fmoc fluorenylmethoxycarbonyl
  • resin is resin
  • FBS fetal bovine serum
  • GLP-1R pancreatic Glucagon-like peptide 1 receptor
  • GLP-1 glucagon-like peptide
  • His is histidine
  • Ser is serine
  • D-Ser D-type serine
  • Gln glutamine
  • Gly glycine
  • Glu glutamic acid
  • Ala is alanine
  • Thr threonine
  • Lys is lysine
  • Arg arginine
  • Tyr tyrosine
  • Asp is aspartic acid
  • Trp is tryptophan
  • Phe Phenylalanine
  • IIe is isoleucine
  • Leu leucine
  • Cys cysteine
  • Pro proline
  • Fig. 1 is in embodiment 3 based on 24h and 48h blood glucose monitoring figure after administration of experimental animals;
  • Fig. 1A is the change of blood glucose concentration within 120 minutes of intragastric administration of glucose after 24 hours of administration;
  • Figure 1B is the area under the curve (AUC) calculated in Figure 1A;
  • Fig. 1C is the change of blood sugar concentration within 120 minutes of intragastric administration of glucose after 48 hours of administration;
  • Figure 1D is the area under the curve (AUC) calculated in Figure 1C;
  • Fig. 2 is based on 72h and 96h blood glucose monitoring figure after administration in experimental animals in embodiment 3; Wherein:
  • Fig. 2A is the change of blood glucose concentration within 120 minutes of intragastric administration of glucose after 72 hours of administration;
  • Figure 2B is the area under the curve (AUC) calculated in Figure 2A;
  • Fig. 2C is the change of the blood glucose concentration within 120 minutes after intragastric administration of glucose for 96 hours;
  • Figure 2D is the area under the curve (AUC) calculated in Figure 2C;
  • Fig. 3 is the physical monitoring chart of the male db/db diabetic mouse of 8 weeks old in embodiment 4 after administration, wherein:
  • Fig. 3 A is the mouse body weight monitoring figure in embodiment 4;
  • Fig. 3B is the mouse blood glucose monitoring figure in embodiment 4;
  • Fig. 3 C is the monitoring diagram of mouse food intake in embodiment 4.
  • Figure 4 is a graph of serological indicators measured after 6 weeks of drug administration to 8-week-old db/db diabetic mice, wherein:
  • Figures 4A and 4B are the results of mouse serum ALT and AST respectively, p ⁇ 0.05;
  • Figure 4C and 4D are the results of mouse serum TG and T-CHO respectively, p ⁇ 0.05;
  • Figure 4E, 4F and 4G are the results of mouse serum HDL-C, LDL-C and GHb respectively, p ⁇ 0.05;
  • Fig. 5 is the figure of physiological index of mouse administration in the 6th week in embodiment 4, wherein,
  • Figure 5A is a mouse liver weight (liver weight) diagram
  • Figure 5B is a mouse liver index (liver/body weight) graph
  • Figure 5C is a mouse body weight (body weight) figure
  • Figure 5D is a mouse body mass index (BMI) graph
  • Figure 5E is a mouse epididymal fat weight (EAM weight) figure
  • Figure 5F is a mouse epididymis fat index (EAM/body weight) figure
  • Fig. 6 is mouse oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) experimental figure;
  • Fig. 6A is the OGTT result figure of the 4th week of mouse administration in embodiment 4.
  • Fig. 6B is the AUC histogram corresponding to Fig. 6A;
  • Fig. 6C is the ITT result chart measured in the 5th week of mouse administration in Example 4.
  • Figure 6D is a histogram of the 5th week of administration of AUC corresponding to Figure 6C; p ⁇ 0.05;
  • Fig. 7 is the blood drug concentration curve of different time periods after a single administration of SD rats at the age of 8 weeks, wherein:
  • Figures 7A1 and 7A2 are respectively the plasma concentration curves of semaglutide injected into the tail vein and subcutaneous injection of SD rats;
  • Figures 7B1 and 7B2 are respectively the blood concentration curves of compound 2 injected into the tail vein and subcutaneous injection of SD rats;
  • Figures 7C1 and 7C2 are respectively the plasma concentration curves of compound 5 injected into the tail vein and subcutaneous injection of SD rats;
  • Figures 7D1 and 7D2 are respectively the plasma concentration curves of compound 6 injected into tail vein and subcutaneous injection of SD rats;
  • Figures 7E1 and 7E2 are respectively the plasma concentration curves of compound 7 injected into the tail vein and subcutaneous injection of SD rats;
  • Boc-His(Trt)-OH and Fmoc-Aib-OH were purchased from Shanghai Jier, and mono-tert-butyl eicosanedioic acid was self-made.
  • the remaining amino acids were purchased from Chengdu Zhengyuan Company, and the condensing agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were of analytical grade, and solvents were purchased from Shanghai Titan Company.
  • the centrifuge was purchased from Lu Xiangyi.
  • a 5.0 m reverse phase C 18 preparative column (46mm x 250mm) was used to purify the polypeptide.
  • the high-performance liquid chromatograph is a product of Thermo Fisher Scientific. Mass spectrometry was performed using a Waters mass spectrometer.
  • H-Aib-EGT FTSDV SSYLE GQAAK GGSGSGSG- ⁇ -E-CO (CH 2 ) 18 CO 2 H
  • EFIAW LVRGR G (acetate)
  • Fmoc protects amino acid, 1-hydroxybenzotriazole (HOBT), N, N-diisopropylcarbodiimide (DIC) feeding amount is 5 times, and the reaction time is 2 hours.
  • Fmoc/t-Bu strategy couple Fmoc-Gly-OH: add Fmoc-Gly-OH, HOBT, appropriate amount of N, N-dimethylformamide (DMF) to the main peptide resin product, stir well with nitrogen, add DIC, nitrogen stirring reaction for 2 hours, detection of coupling effect with ninhydrin, colorless and transparent, the reaction is over.
  • Remove the reaction solution wash 3 times with N,N-dimethylformamide (DMF), and remove the Fmoc group with 20% piperidine/N,N-dimethylformamide (DMF) solution (Twice 5min+7min), after removing Fmoc, wash with DMF 6 times, sample ninhydrin for detection, and develop color.
  • Lysis solution TFA, DODT, m-cresol, and H 2 O were prepared in advance at a volume ratio of 92.5:2.5:2.5:2.5, and frozen in the refrigerator for 2 hours.
  • the filter cake was washed 3 times with a small amount of lysate, and the filtrates were combined.
  • the filtrate was slowly poured into iced methyl tert-butyl ether with stirring. Let it stand for more than 2 hours until the precipitation is complete. The supernatant was removed, the precipitate was centrifuged, washed three times with methyl tert-butyl ether, centrifuged, and the solid was blown dry with nitrogen.
  • ACN acetonitrile
  • H 2 O 1:2 (volume ratio)
  • the isolated product polypeptide was identified by liquid chromatography-mass spectrometry, and it was found that the m/z value of the protonated molecular ion peak was 4397.16, which was the target compound 5, and the theoretical value of the molecular weight of compound 5 was 4397.87.
  • the purification and product identification methods of compound 2 were the same as compound 5, and the purity was greater than 97.66%, and the total yield was 12%.
  • the isolated product was identified by liquid chromatography-mass spectrometry, and it was found that the m/z value of the protonated molecular ion peak was: 4253.07, which was the target compound 2, and the theoretical value of the molecular weight of compound 2 was 4253.74.
  • GLP-1R-Luciferase-HEK293 cell model the cell line constructed in our laboratory
  • the agonistic activity of GLP-1R was determined for compounds 1-8 and semaglutide.
  • the digested cells were plated on a 96-well plate (medium containing 10% FBS, GLP-1R-Luciferase-HEK293: 20,000 cells/well, 100 ⁇ L); after 36 hours, the medium in the 96-well plate was discarded, and 90 ⁇ L of serum-free medium was added; After 6 hours, prepare peptide drugs (8 peptide analogs and semaglutide) with a series of concentrations (0.01, 0.1, 1, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000pM) in serum-free medium, Add 10 ⁇ L to each well (that is, dilute 10 times) and incubate the cells for 5 hours; add 100 ⁇ L of cell lysate to each well, lyse on ice for 10 minutes and shake evenly, take 2 ⁇ L of the lysate and add it to a 384 microplate reader white plate, first add 10 ⁇ L of Firefly luciferase reaction solution, read, and then add 10 ⁇
  • compounds 1-8 of the present invention can activate GLP-1R, and compounds 2, 5, 6 and 7 have better agonistic activity to GLP-1R (Table 2), wherein, compounds 2,
  • the half-maximal effect concentration of 5 (hereinafter referred to as EC 50 , referring to the concentration that can cause 50% of the maximum effect) is lower than that of semaglutide, indicating that the agonistic activity on GLP-1R is better than that of semaglutide, while compound 6,
  • the agonistic activity of 7 on GLP-1R is comparable to that of semaglutide.
  • Example 3 The effect of compound 1-8 and semaglutide on oral glucose tolerance (OGTT)
  • mice Experimental Animal Center of Sun Yat-Sen University mice (Experimental Animal Center of Sun Yat-Sen University) were bred for one week to adapt to the environment, and were randomly divided into groups according to similar blood sugar (evaluated by blood samples obtained from the tip of the tail), with 8 mice in each group.
  • compounds 1-8 (respectively corresponding to numbers 1-8 in the figure) and semaglutide (corresponding to semaglutide in the figure) were given by subcutaneous injection at a dose of 120ug/kg, and the control group was given the same dose of PBS.
  • Glucose was administered intragastrically to prepare a stock solution with a concentration of 0.5 g/mL with ddH 2 O and stored at room temperature.
  • compounds 1-8 of the present invention all have the effect of improving the glucose tolerance of mice to varying degrees, and compounds 2, 5, 6 and 7 are different in the four OGTT curve periods (24, 48, 72, 96h), exhibited a more excellent and significantly improved glucose tolerance effect, and the results also showed that, compared with semaglutide, compounds 1-8, especially compounds 2, 5, 6 and 7 In the 4 OGTT curve periods (24, 48, 72, 96h), it showed a more excellent and significant long-term hypoglycemic effect.
  • Example 4 Compounds 2, 5, 6, 7 and semaglutide have therapeutic effects on diabetes in diabetic mice
  • db/db mice Forty-eight 8-week-old male Lepr db/db mice (db/db) and eight littermate normal mice (WT) were purchased from Nanjing University-Nanjing Institute of Biomedicine.
  • db/db mouse diabetes model purchased from Nanjing University-Nanjing Institute of Biomedicine, about 8 weeks old, and measure the blood sugar and body weight to ensure the smooth progress of the follow-up experiment
  • db/db mice into 6 groups randomly according to the blood sugar (compounds 2, 5, 6, 7 and semaglutide, normal saline group), 8 rats in each group, there was no difference in basal body weight and blood glucose.
  • Mice in each group were subcutaneously injected with compounds 2, 5, 6, 7 (120 ⁇ g/kg), semaglutide (120 ⁇ g/kg) and saline (WT group and db/db group) every other day.
  • mice After each administration, the mice were fasted for 6 hours every other day to measure the blood sugar and body weight of the mice; the water intake and food intake were measured every 7 days. OGTT was measured at the 4th week of administration, insulin tolerance (ITT) was measured at the 5th week, and samples were collected at the 6th week to detect various serological and physiological indicators.
  • ITT insulin tolerance
  • the monitoring results of blood sugar, body weight, water intake and food intake of the mice are shown in Figure 3, the serological indicators after sampling are shown in Figure 4, and the physiological indicators are shown in Figure 5.
  • Type 2 diabetes model characterized by obesity, insulin resistance, hyperglycemia, dyslipidemia and hepatic adipose vacuolar degeneration.
  • OGTT oral glucose tolerance test
  • ITT insulin tolerance test
  • Fig. 3 is the figure of physical monitoring after administration of male db/db diabetic mice at the age of 8 weeks, wherein, respectively subcutaneous injection of normal saline, compound 2 (120 ⁇ g/kg), compound 5 (120 ⁇ g/kg), compound 6 (120 ⁇ g /kg), Compound 7 (120 ⁇ g/kg) and semaglutide (120 ⁇ g/kg), were administered every other day for 6 weeks; the body weight and blood glucose of the mice (3A and 3B), the food intake of the mice was measured every 7 days (3C), wherein, *: indicates p ⁇ 0.05; **: indicates p ⁇ 0.01; ***: indicates p ⁇ 0.001.
  • Figure 3 (A, C) results show that compared with the db/db group, compounds 2, 5, 6, and 7 can significantly reduce the food intake of mice, thereby reducing the weight of mice, and the effect of reducing body weight is better than that of Soma Glutide.
  • Figure 4 shows that compounds 2, 5, 6 and 7 are also comparable to semaglutide in hepatoprotective function.
  • Glycosylated hemoglobin can be used as an index to reflect the control of blood sugar in a long period of time (4-10) weeks in diabetic patients. If the blood sugar is poorly controlled for a long time, the glycosylated hemoglobin will increase, so the measurement of glycosylated hemoglobin is helpful. It plays an important role in the study of peripheral blood vessels and cardiovascular complications of diabetes.
  • the experimental results in Figure 4G show that compounds 2, 5, 6 and 7 all have significant effects on reducing glycosylated hemoglobin, and are better than Somalu peptide.
  • compounds 2, 5, 6 and 7 and semaglutide were all administered at a dose of 50ug/kg, with an administration volume of 2mL/kg, an administration concentration of 60ug/mL, and a vehicle of normal saline, respectively. medication.

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Abstract

提供一类用于治疗或预防糖尿病或肥胖的长效GLP-1多肽类似物及其制备方法和应用，解决了复杂多肽半衰期短及代谢稳定性不足的问题。所述长效GLP-1多肽类似物的氨基酸序列结构中：第20位Lys与"侧臂"短肽链相连，连接的方式为：第20位Lys的"侧臂"短肽链氨基与"侧臂"短肽链的甘氨酸的羧基形成酰胺键；所述"侧臂"短肽链末端"Z"氨基酸与脂肪酸取代基相连。所述长效GLP-1多肽类似物半衰期长、合成产率高、稳定性好、易于放大生产、成本低，具有良好的治疗糖尿病、降低体重的药效作用。

Description

长效GLP-1多肽类似物及其制备方法和应用 技术领域
本发明属于生物化学技术领域，具体而言，本发明涉及一类可用于治疗或预防糖尿病或肥胖的长效GLP-1多肽类似物及其制备方法和应用。
背景技术
糖尿病是机体长时间处于高血糖水平而引起机体糖代谢紊乱的慢性疾病，该疾病的主要特点是：慢性高血糖，并且同时伴随着胰岛素分泌缺陷或者胰岛素功能障碍，进一步通过影响脂肪、碳水化合物和蛋白质的代谢，而对多种器官造成慢性损伤，导致器官逐渐出现功能障碍，甚至造成器官衰竭。
过去三十年来，全球范围内的糖尿病患者人数增加了三倍。在世界范围内，将近9％的成年人患有2型糖尿病(T2DM)。T2DM及其并发症的出现极大地加剧了世界范围内的残疾和死亡风险。例如，《2013年全球疾病风险研究》将糖尿病(所有形式)确定为预期寿命降低的第九大原因。基于许多调查的结果表明，不管是欧美的发达国家，还是类似于中国这样的发展中国家，糖尿病均没有得到有效控制。糖尿病已经成为继心脑血管疾病、肿瘤之后另一个严重危害人体健康的重要慢性非传染性疾病。
肥胖与糖尿病同属于代谢性疾病，肥胖与糖尿病发生密切相关。糖尿病发生的原因主要是胰岛β细胞功能的衰退和胰岛素的抵抗，肥胖是胰岛素抵抗的关键因素。肥胖患者由于体重超标、脂肪含量高，很容易产生胰岛素抵抗，胰岛素抵抗使体内的胰岛素不能发挥相应的降糖作用。胰岛素是体内唯一的降血糖激素，机体为控制血糖，必须增加胰岛β细胞分泌胰岛素的能力，当增加胰岛素分泌依然不能使血糖正常时就发生了糖尿病，所以肥胖是胰岛素抵抗产生的根源，也是糖尿病发生很重要的原因。
20世纪60年代，McIntyre和Elrick等人观察到一种有趣的现象，口服葡 萄糖对胰岛素分泌的促进作用明显高于静脉注射，这种效应被称为“肠促胰素效应”，随后在小肠粘膜提取物中发现胰高血糖素样肽-1(GLP-1)和糖依赖性胰岛素释放肽(GIP)。GLP-1是一种诱导胰岛素分泌的激素，对包括胰腺、心脏和肝脏等在内的多种重要器官具有有益作用。GLP-1受体(GLP-1R)激动剂类药物的优点在于可以有效控制血糖，同时明显降低低血糖事件发生率，并且还有明显减轻体重、降低心血管事件风险的获益。但GLP-1药物由于其特定的多肽结构自身的特点，存在不稳定性，口服后会被胃酸降解，基本只能通过皮下注射给药，且半衰期较短。
随着糖尿病及其治疗的深入研究，近年来利拉鲁肽、索马鲁肽等GLP-1受体激动剂被批准上市。其中，索马鲁肽增长的碳链对白蛋白的亲和力大大增强，大大减缓了肾脏对其的清除。两处的改造使大鼠的半衰期延长为8h左右，临床上每周只需进行一次皮下注射。
尽管如此，如何突破性解决多肽，特别是复杂多肽半衰期短及代谢稳定性不足的问题，仍然是本领域重大科学和核心问题；其中，超长效多肽药物分子修饰技术的发展是关键，也是本领域研究国际上待突破的瓶颈。
发明内容
针对以上问题，本发明的目的在于提供一类新型的更长效的GLP-1多肽类似物。
本发明的再一目的在于提供所述长效的GLP-1多肽类似物的制备方法。
本发明的再一目的在于提供一种含有上述长效GLP-1多肽类似物的组合物。
本发明的再一目的在于提供上述GLP-1多肽类似物的应用。
根据本发明具体实施方式的长效GLP-1多肽类似物，其氨基酸序列如下：
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{(Gly) x-(Ser-Gly) y-Z-CO(CH 2) nCO 2H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
其中：
Xaa2为Aib或D-Ser；
Xaa9为Asp或Glu；
Xaa21为Glu或Gln或Asp；
Z为γGlu或Asp；
x为1、2、3或4；
y为1、2、3或4；
n为12-20的整数，即n为12、13、14、15、16、17、18、19或20。
其中，
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly为主肽链；
(Gly) x-(Ser-Gly) y-Z-CO(CH 2) nCO 2H为“侧臂”结构；
“侧臂”结构包括“侧臂”短肽链-(Gly)x-(Ser-Gly)y-Z-和与之连接的脂肪酸取代基-CO(CH 2) nCO 2H。
主肽链氨基酸序列中第20位Lys的侧链氨基通过与另一端甘氨酸残基的羧基形成酰胺键方式与“侧臂”短肽链结构相连；
进一步，“侧臂”短肽链末端“Z”氨基酸残基的氨基通过与羧基形成酰胺键方式与脂肪酸取代基相连。
所述主肽链氨基酸序列的羧基端不经修饰，或者经氨基修饰形成-CONH 2基团。
优选的，Z为γGlu。
优选的，x为2，y为1、2或3，具体的，上述氨基酸序列中，x为2，y为1，或x为2，y为2，或x为2，y为3。
优选的，所述氨基酸序列结构中：n为14-18中任一整数，即n为14、15、16、17或18。
根据本发明具体实施方式的长效GLP-1多肽类似物，其氨基酸序列中，Z为γGlu，x为2，y为1、2或3，n为18，其序列如下：，
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys{(Gly) 2-(Ser-Gly) y-γGlu-CO(CH 2) 18CO 2H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
优选的，所述GLP-1多肽类似物为如下化合物中的任一种：
化合物1(SEQ ID NO.1)：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：H-Aib-EGTFTSDVSSYLEGQAAK(GGSG-γ-E-CO(CH 2) 18CO 2H)EFIAWLVRGRG；
化合物2(SEQ ID NO.2)：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：H-Aib-EGTFTSDVSSYLEGQAAK(GGSGSG-γ-E-CO(CH 2) 18CO 2H)EFIAWLVRGRG；
化合物3(SEQ ID NO.3)：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：H-Aib-EGTFTSEVSSYLEGQAAK(GGSGSG-γ-E-CO(CH 2) 18CO 2H)EFIAWLVRGRG；
化合物4(SEQ ID NO.4)：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：H-Aib-EGTFTSEVSSYLEGQAAK(GGSGSG-γ-E-CO(CH 2) 18CO 2H)DFIAWLVRGRG；
化合物5(SEQ ID NO.5)：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：H-Aib-EGTFTSDVSSYLEGQAAK(GGSGSGSG-γ-E-CO(CH 2) 18CO 2H)EFIAWLVRGRG；
化合物6(SEQ ID NO.6)：
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：HsEGTFTSDVSSYLEGQAAK(GGSGSGSG-γ-E-CO(CH 2) 18CO 2H)EFIAWLVRGRG；
化合物7(SEQ ID NO.7)：
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Gln-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
简写：HsEGTFTSDVSSYLEGQAAK(GGSGSGSG-γ-E-CO(CH 2) 18CO 2H)QFIAWLVRGRG；
化合物8(SEQ ID NO.8)：
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
简写：HsEGTFTSEVSSYLEGQAAK(GGSGSGSG-γ-E-CO(CH 2) 18CO 2H)DFIAWLVRGRG；
根据本发明具体实施方式的长效GLP-1多肽类似物的制备方法，包括以下步骤：
步骤1：按照Fmoc/t-Bu策略，合成所述长效GLP-1多肽类似物主肽链 对应的主肽树脂，其中，所述主肽链为His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
步骤2：在所述主肽树脂的基础上，按照Fmoc/t-Bu策略，偶联所述长效GLP-1多肽类似物对应的“侧臂”结构，得到长效GLP-1多肽类似物对应的多肽树脂；其中，所述“侧臂”结构为(Gly) x-(Ser-Gly) y-Z-CO(CH 2) nCO 2H；
步骤3：向所述多肽树脂中加入裂解液，进行裂解反应，脱除多肽全保护，提取粗品化合物；
纯化所述粗品化合物，获得长效GLP-1多肽类似物。
根据本发明具体实施方式的GLP-1多肽类似物的制备方法，
步骤2中，使用的偶联剂为1-羟基苯并三唑和N,N-二异丙基碳二亚胺，溶剂为N，N-二甲基甲酰胺，以20％的哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团；
步骤3中，裂解液由TFA、DODT、间甲酚、H 2O按体积比为92.5：2.5：2.5：2.5的比例组成；所述粗品化合物提取方式包括过滤、沉淀和/或甲基叔丁基醚提取。
步骤4中，获得的获得长效GLP-1多肽类似物纯度大于96％。
本发明的再一目的在于提供一种组合物，其包含长效GLP-1多肽类似物，还包含药学上可接受的载体或辅料。例如，能够减少药物降解及损失、降低副作用的载体，例如胶束、微乳液、凝胶等载体；辅料是指使药物制成适宜的剂型而加入的物料，例如缓冲剂、冻干用赋形剂等，能够将含有本发明多肽类似物的药物组合物可以制成溶液或者冻干粉末以用于胃肠外给药，冻干粉末在使用前可加入适当溶剂或其他可药用的载体将粉末重新配置，液体配方一般是缓冲液、等渗溶液和水溶液。缓冲液可以是磷酸盐缓冲液，等渗溶液可以是0.9％的氯化钠溶液，水溶液即直接用纯净水溶解。
本领域技术人员可以理解，以所述长效GLP-1多肽类似物作为活性成分添 加药学上可接受载体和/或辅料制成药物组合物，适用于各种给药方式，例如口服给药、经皮给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。根据所采用的给药方式，可将本发明的多肽类似物的药物组合物制成各种合适的剂型，其中包含至少一种有效剂量的本发明的多肽类似物和至少一种药学上可接受的药用载体。适当剂型的实例为片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆，用于皮肤表面的油膏和药贴，气雾剂、鼻喷剂，以及可用于注射的无菌溶液。
本发明的药物组合物的用量可以在一个较大范围内变动，本领域技术人员可以根据一些客观的因素，如根据疾病的种类、病情严重程度、病人体重、剂型、给药途径等因素加以确定。
本发明的再一目的在于提供上述长效GLP-1多肽类似物以及组合物的应用。
本发明得到一系列GLP-1多肽类似物，并对这一系列药物的药效作用展开研究。对合成的系列多肽类似物药物的GLP-1受体激动活性、降糖、降脂、降体重及糖尿病肾病方面等活性进行评价，并对其药代动力学进行初步研究。结果显示，通过跟索马鲁肽进行比较，其发挥药效的长效性远远优于索马鲁肽(半衰期达到索马鲁肽2倍或以上)，在治疗和改善糖尿病肾病方面也优于索马鲁肽。
由此证明，本发明的长效GLP-1多肽类似物具有更长的半衰期，具有促胰岛素活性、没有不良反应发生，可用于糖尿病、肥胖的治疗，潜在地可作为新一代治疗糖尿病、肥胖的药物。
本发明的长效GLP-1多肽类似物的应用具体包括：
在制备预防或治疗糖尿病的药物方面，在制备预防或治疗肥胖的药物方面，在制备能降低体重的保健品方面。
本发明的有益效果：
多肽半衰期和稳定性的解决是多肽药物设计及是否能够成药的关键，也是本领域研究的重大科学和核心问题。其中，超长效多肽药物分子修饰技术的发 展是关键，也是本领域研究国际上待突破的瓶颈。本发明通过生物信息学、结构生物学、计算机辅助设计、构效关系研究等，开发出位点特异性“侧臂”结构修饰技术，突破超长效多肽及蛋白药物分子修饰“卡脖子”技术，大大延长了合成的多肽类似物的半衰期，实现了多肽药物的超长效化。
本发明的长效GLP-1多肽类似物中的母体肽为同源性多肽，本发明中的同源性多肽是指，多肽本来具有胰高血糖素样肽(GLP-1)、索马鲁肽(Semaglutide)的氨基酸序列，但其中一个或多个氨基酸残基己被不同的氨基酸残基取代，并且其结构序列中增加了“侧臂”短肽氨基酸残基序列，这些氨基酸残基彼此之间是保守的，并且所得到的多肽可用于实施本发明。
具体的，我们通过特定位点的氨基酸替换和本发明所描述的特定位点(所述氨基酸序列中第20位Lys)连接的“侧臂”短肽链，即：位点特异性“侧臂”修饰技术，成功突破超长效多肽药物分子修饰“卡脖子”技术。跟无“侧臂”修饰的阳性药索玛鲁肽相比，本发明所设计的多肽药物大大延长了其作用半衰期，实现了多肽药物的超长效化，其整体的药物作用效果大大优于阳性药索玛鲁肽。
另外，本发明GLP-1多肽类似物利用亲脂性取代基结合血液中的白蛋白，保护其免受酶降解，从而提高半衰期。本发明GLP-1多肽类似物通过分子内桥稳定分子的螺旋结构，提高了针对胰高血糖素样肽1受体(GLP-1R)的效力和/或选择性。
本发明的GLP-1多肽类似物合成产率高，稳定性好，易于放大生产，成本低。
通过跟索马鲁肽进行比较，其发挥药效的长效性和稳定性远远优于索马鲁肽(半衰期达到索马鲁肽2倍或以上)。
在质量数相同时，本发明的GLP-1多肽类似物在治疗和改善糖尿病、肾病方面也优于索马鲁肽。
同时，本发明GLP-1多肽类似物具有更好的降低体重的药效作用，GLP-1多肽类似物可以通过引起摄食量降低和/或能量消耗升高，用于防止体重增长或促进体重减轻，因此，本发明GLP-1多肽类似物还可用于直接或间接治疗由体 重超重所引起的或者以其为特征的其他病症，例如治疗和/或预防肥胖症、病态肥胖症、肥胖症相关炎症、肥胖症相关的胆囊疾病、肥胖症引起的睡眠呼吸暂停，本发明在这些病症中的作用可以是由于GLP-1多肽类似物直接或间接对体重的作用，或者对体重以外身体其他方面的作用。
本发明实施例合成的化合物中，化合物2、5、6和7和索马鲁肽相比，具有更为优秀和显著地改善葡萄糖耐受效果、更长的药效和半衰期，同时能显著降低小鼠的肝重以及附睾脂肪含量，在改善糖尿病小鼠口服葡萄糖耐受能力(OGTT)及胰岛素抵抗(ITT)方面，也优于索马鲁肽。
本发明中所用缩写具体含义如下：
DCM为二氯甲烷，DMF为N，N-二甲基甲酰胺，HOBt为1-羟基苯并三唑，Fmoc为芴甲氧羰基，resin为树脂，FBS为胎牛血清，GLP-1R为胰高血糖素样肽1受体，GLP-1为胰高血糖素样肽，His为组氨酸，Ser为丝氨酸，D-Ser为D-型丝氨酸，Gln为谷氨酰胺，Gly为甘氨酸，Glu为谷氨酸，Ala为丙氨酸，Thr为苏氨酸，Lys为赖氨酸，Arg为精氨酸，Tyr为酪氨酸，Asp为天冬氨酸，Trp为色氨酸，Phe为苯丙氨酸，IIe为异亮氨酸，Leu为亮氨酸，Cys为半胱氨酸，Pro为脯氨酸，Val为缬氨酸，Met为蛋氨酸，Asn为天冬酰胺。Aib为2-氨基异丁酸，Iva为异缬氨酸。
附图说明
图1为实施例3中基于实验动物给药后24h和48h血糖监测图；其中：
图1A为给药24h后灌胃葡萄糖120分钟内血糖浓度的变化；
图1B为图1A计算的曲线下面积(AUC)；
图1C为给药48h后灌胃葡萄糖120分钟内血糖浓度的变化；
图1D为图1C计算的曲线下面积(AUC)；
图2为实施例3中基于实验动物给药后72h和96h血糖监测图；其中：
图2A为给药72h后灌胃葡萄糖120分钟内血糖浓度的变化；
图2B为图2A计算的曲线下面积(AUC)；
图2C为给药96h后灌胃葡萄糖120分钟内血糖浓度的变化；
图2D为图2C计算的曲线下面积(AUC)；
图3为实施例4中8周龄的雄性db/db糖尿病小鼠给药后身体监测图，其中：
图3A为实施例4中小鼠体重监测图；
图3B为实施例4中小鼠血糖监测图；
图3C为实施例4中小鼠进食量监测图；
图4为8周龄的db/db糖尿病小鼠给药治疗6周后，取材测定的血清学指标图，其中：
图4A和4B分别为小鼠血清ALT和AST结果，p<0.05；
图4C和4D分别为小鼠血清TG和T-CHO结果，p<0.05；
图4E、4F和4G分别为小鼠血清HDL-C、LDL-C和GHb结果图，p<0.05；
图5为实施例4中小鼠给药第6周生理指标图，其中，
图5A为小鼠肝重(liver weight)图；
图5B为小鼠肝指数(liver/body weight)图；
图5C为小鼠体重(body weight)图；
图5D为小鼠体重指数(BMI)图；
图5E为小鼠附睾脂肪重量(EAM weight)图；
图5F为小鼠附睾脂肪指数(EAM/body weight)图；
图6为小鼠口服葡萄糖耐受实验(OGTT)和胰岛素耐受测试(ITT)实验图；其中，
图6A为实施例4中小鼠给药第4周的OGTT结果图；
图6B为图6A对应的AUC柱状图；
图6C为实施例4中小鼠给药第5周测得的ITT结果图；
图6D为图6C对应的AUC给药第5周柱状图；p<0.05；
图7为8周龄的SD大鼠单次给药后不同时间段采血的血药浓度曲线，其中：
图7A1、7A2分别为SD大鼠尾静脉和皮下注射索马鲁肽的血药浓度曲线；
图7B1、7B2分别为SD大鼠尾静脉和皮下注射化合物2的血药浓度曲线；
图7C1、7C2分别为SD大鼠尾静脉和皮下注射化合物5的血药浓度曲线；
图7D1、7D2分别为SD大鼠尾静脉和皮下注射化合物6的血药浓度曲线；
图7E1、7E2分别为SD大鼠尾静脉和皮下注射化合物7的血药浓度曲线；
图8脂肪酸取代基分子式和结构图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
材料：
所有的氨基酸购自上海吉尔生化公司，索马鲁肽购自浙江湃肽生物有限公司，CAS No.：910463-68-2；如果没有特别说明，其他所有试剂均为分析纯，购自Sigma公司。采用Protein Technologies PRELUDE 6通道多肽合成仪，Phenomenex Luna C 18制备柱(46mm x 250mm)用来纯化多肽，高效液相色谱仪为Thermo公司产品，质谱分析采用Thermo液相质谱仪进行测定。
材料与方法：
Boc-His(Trt)-OH，Fmoc-Aib-OH购自上海吉尔，二十烷二酸单叔丁酯自制。其余的氨基酸购自成都郑源公司，缩合剂购自苏州昊凡公司。如果没有特别说明，其他所有试剂均为分析纯，溶剂购自上海泰坦公司。离心机购自卢湘仪。5.0m反相C 18制备柱(46mm x 250mm)用来纯化多肽。高效液相色谱仪为赛默飞公司产品。质谱分析采用Waters质谱仪进行测定。
实施例1 化合物5的合成
化合物5的氨基酸序列：
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Glym-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp
-Leu-Val-Arg-Gly-Arg-Gly
简写为：H-Aib-EGT FTSDV SSYLE GQAAK(GGSGSGSG-γ-E-CO(CH 2) 18CO 2H)EFIAW LVRGR G(醋酸盐)，
方法：
步骤1.合成主肽树脂
按照Fmoc/t-Bu策略手工合成，合成规模：0.5mmol，合成如下主肽树脂：
Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。
(1)：称取1.47克Wang Resin树脂(loading 0.47mmol/g，西安蓝晓)，加入反应柱中，加入N，N-二甲基甲酰胺(DMF)中溶胀30min，称取Fmoc-Gly-OH：1.233g(6eq)，HOBT：0.672g(7.2eq)，DMAP：0.06g(0.72eq)，待用。抽掉DMF，用N，N-二甲基甲酰胺(DMF)充分洗涤树脂2次，把上述称好的物料加入到反应柱中。加入适量的DMF，氮气搅拌均匀，加入N,N-二异丙基碳二亚胺(DIC)0.83mL(7.8eq)。反应2h，反应结束。抽掉反应液，用DMF洗涤3次，加入乙酸酐/吡啶(7：6，v/v)封闭4h。抽掉封闭液，DMF洗涤6次，得Fmoc-Gly-Wang Resin。
(2)：以Fmoc-Gly-Wang Resin为载体，以1-羟基苯并三唑(HOBT)，N,N-二异丙基碳二亚胺(DIC)为偶联剂，以N，N-二甲基甲酰胺(DMF)为溶剂，以20％的哌啶(Piperidine)/N,N-二甲基甲酰胺(DMF)溶液脱除Fmoc基团(两次5min+7min)，偶联过程用水合茚三酮监测偶联效果。进行手工投料，依次从C端到N端进行缩合反应连接Fmoc-Arg(pbf)-OH,Fmoc-Gly-OH,Fmoc-Arg(pbf)-OH,Fmoc-Val-OH,Fmoc-Leu-OH,Fmoc-Trp(Boc)-OH,Fmoc-Ala-OH,Fmoc-Ile-OH,Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Lys(Alloc)-OH,Fmoc-Ala-OH,Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH,Fmoc-Ser(tBu)-OH,Fmoc-Ser(tBu)-OH,Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH,Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Aib-OH,Boc-His(Trt)-OH以上氨基酸投料(相对于合成规模5eq)得到Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。
有几点需要说明：
1)Fmoc-Gly-Wang Resin的合成，因Wang Resin本身替代度较低，Fmoc-Gly-OH的投料量要大点，不然替代度偏低，浪费物料。用乙酸酐/吡啶封闭，防止缺损肽的出现。
2)接下来的每次缩合反应中Fmoc保护氨基酸，1-羟基苯并三唑(HOBT)，N,N-二异丙基碳二亚胺(DIC)投料量均为5倍，反应时间为2个小时。
(3)：烯丙氧羰基(Alloc)的脱除
树脂中加入二氯甲烷(DCM)，加入吗啉0.5mL(12eq)，称取0.173g四三苯基膦钯(0.3eq)加入反应柱中，反应1h。反应结束，抽掉反应液，用N，N-二甲基甲酰胺(DMF)洗涤3次，DCM洗涤6次。得到主肽树脂：Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。
步骤2.偶联“侧臂”结构：
按照Fmoc/t-Bu策略，偶联Fmoc-Gly-OH：向主肽树脂产物中加入Fmoc-Gly-OH、HOBT、适量N，N-二甲基甲酰胺(DMF)，氮气搅拌均匀，加入DIC，氮气搅拌反应2小时，用水合茚三酮检测偶联效果，无色透明，反应结束。抽掉反应液，用N，N-二甲基甲酰胺(DMF)洗涤3次，以20％的哌 啶(Piperidine)/N,N-二甲基甲酰胺(DMF)溶液脱除Fmoc基团(两次5min+7min)，脱除Fmoc后用DMF洗涤6次，取样水合茚三酮检测，显色。
重复上述操作依次偶联Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Glu-OtBu，二十烷二酸单叔丁酯。得到Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Gly-Gly-Ser(tBu)Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-二十烷二酸单叔丁酯)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。用N，N-二甲基甲酰胺(DMF)洗3次，二氯甲烷(DCM)洗3次，甲醇(Methanol)收缩2次，真空抽干，得干燥多肽树脂。
步骤3.脱除多肽全保护
裂解液：TFA、DODT、间甲酚、H 2O按体积比为92.5：2.5：2.5：2.5的比例事先配好，冰箱中冷冻2h。
按10mL裂解液/g多肽树脂，往干燥的多肽树脂Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-二十烷二酸单叔丁酯)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin中加入裂解液，升温到室温,裂解反应3个小时。过滤，滤饼用少量裂解液洗涤3次，合并滤液。滤液在搅拌下缓慢倒入冰的甲基叔丁基醚中。静置2个小时以上，待沉淀完全。去除上清液，沉淀离心，用甲基叔丁基醚洗涤3次，离心，固体用氮气吹干。得到粗品化合物His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Glu--二十烷二酸)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
步骤4.精制纯化粗品化合物
将步骤2中所得粗品化合物溶于乙腈(ACN)：H 2O＝1：2(体积比)的溶液中，通过5.0m反相C 18的填充的46mm x 250mm柱上进行制备型HPLC纯化。用45％乙腈/H 2O(含1％三氟乙酸)为起始，以梯度(0.33％/min的速度增加乙腈的比例)，流速为10mL/min将该柱洗脱60分钟，收集含有多肽的组分，得到HPLC纯度大于90％的样品。重复一次HPLC纯化，用31％乙腈/20mM磷酸二氢钠水溶液用1M的氢氧化钠溶液调pH至6.5为起始，以梯(0.33％/min的增加乙腈的比例)，流速为10mL/min，将该柱洗脱60分钟，收集含有多肽的组分，冷冻抽干，得到纯度大于96.86％，总收率为15％。
步骤5.产物确认
用液质联用鉴定分离出的产物多肽，发现质子化分子离子峰的m/z值为：4397.16，为目标化合物5，化合物5分子量的理论值为4397.87。
化合物2的合成
由于化合物2与化合物5的区别尽在于“侧臂”短肽链的序列不同，因此化合物2与化合物5的合成步骤的区别在于步骤2.“侧臂”结构的偶联：
该步骤中，需要依次偶联Fmoc-Gly-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Glu-OtBu，二十烷二酸单叔丁酯。得Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Gly-Gly-Ser(tBu)Gly-Ser(tBu)-Gly-Glu-OtBu-二十烷二酸单叔丁酯)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。
化合物2的精制纯化与产物确认方法同化合物5，得到纯度大于97.66％，总收率为12％。用液质联用鉴定分离出的产物，发现质子化分子离子峰的m/z值为：4253.07为目标化合物2，化合物2的分子量理论值为4253.74。
基于以上合成步骤、精制纯化与产物确认方法，根据化合物1、3、4、6、7、8主肽链及侧臂”短肽链中氨基酸的不同，调整化合物合成步骤1和2中氨 基酸的偶联顺序，最终合成对应的目标产物，再通过液质联用鉴定分离出的产物，确认质子化分子离子峰的m/z值(表1中的实测值)，并将实测值与分子量理论值比较，确认合成纯化的产物为目标产物，表1分别给出了化合物1-8的分子量理论值、液质鉴定的实测值、序列、分子式。
表1长效GLP-1多肽类似物氨基酸序列表及液质联用鉴定结果
Figure PCTCN2021107770-appb-000001
Figure PCTCN2021107770-appb-000002
实施例2 化合物1-8和索马鲁肽对GLP-1R(胰高血糖素样肽1受体)的激动活性评价
在GLP-1R-Luciferase-HEK293细胞模型中(本实验室构建的细胞系)，对化合物1-8和索马鲁肽进行GLP-1R的激动活性测定。
首先，消化细胞铺96孔板(含10％FBS的培养基，GLP-1R-Luciferase-HEK293：20000cell/well，100μL)；36h后，弃96孔板中培养基，加入90μL无血清培养基；6h后，用无血清培养基配制系列浓度(0.01、0.1、1、10、100、1000、10000、100000、1000000、10000000、100000000pM)的多肽药物(8个多肽类似物以及索马鲁肽)，每孔加入10μL(即稀释10倍)，孵育细胞5h；向每孔中加入100μL的细胞裂解液，冰上裂解10min后震荡均匀，取2μL裂解液加入384酶标仪白板中，先加入10μL的萤火虫荧光素酶反应液，读数，再加入10μL的海肾荧光素酶反应液，读数；数据处理，用萤火虫荧光素酶的读数除以海肾荧光素酶的读数，再扣除空白组的数值可得到不同浓度下的激动倍数。
表2化合物1-8与索马鲁肽激动GLP-1R的EC 50
  EC 50
索马鲁肽 222.2pM
化合物1 699.1pM
化合物2 64.73pM
化合物3 1003.7pM
化合物4 1349.9pM
化合物5 146.3pM
化合物6 203.5pM
化合物7 302.4pM
化合物8 1599.2pM
从实验结果可以看出，本发明的化合物1-8均能激活GLP-1R，且化合物2、5、6和7对GLP-1R具有更好的激动活性(表2)，其中，化合物2、5的半最大效应浓度(以下简称EC 50，是指能引起50％最大效应的浓度)低于索马鲁肽，说明对GLP-1R的激动活性均优于索马鲁肽，而化合物6、7对GLP-1R 的激动活性与索马鲁肽相当。
实施例3 化合物1-8和索马鲁肽对口服葡萄糖耐量(OGTT)的作用
将8周龄左右的雄性C57BL/6J小鼠(中山大学实验动物中心)，饲养一周适应环境，按照相似的血糖(从尾尖获取的血液样品评估)随机分组，每组8只。
本发明中化合物1-8(分别对应图中编号1-8)和索马鲁肽(图中对应semaglutide)均按照120ug/kg的剂量，皮下注射给药，对照组给予同等剂量的PBS。灌胃葡萄糖用ddH 2O配成浓度为0.5g/mL的贮备液，常温保存。灌胃前将小鼠禁食8h，分别在给药后24h、48h、72h和96h，按照2.5g/kg的剂量灌胃葡萄糖，并在t＝0min、t＝15min、t＝30min、t＝60min、t＝90min和t＝120min时测量血糖。使用软件GraphPadPrism处理数据作出血糖变化折线图，并计算曲线下面积得到AUC图，结果见图1、图2。
根据OGTT曲线统计的AUC(area under concentration-time curve)，AUC大则生物利用度高，反之则低。其中图1和图2中，*：表示p<0.05；**：表示p<0.01；***：表示p<0.001。
由图1A、B可知，在给药24h后，与载剂(PBS)相比，索马鲁肽和化合物1-8能显著降低AUC，说明索马鲁肽和化合物1-8具有显著的葡萄糖耐受性。
由图1C、D以及图2可知，在给药48h、72h、96h后，索马鲁肽与载剂(PBS)相比，对AUC的影响没有显著的区别，说明索马鲁肽已经不能改善葡萄糖耐受。在给药48h、72h、96h后，与PBS组相比，而化合物2、5、6和7仍能显著降低AUC，因此能改善葡萄糖耐受，因此，化合物2、5、6和7对于改善小鼠糖耐受的效果均优于索马鲁肽。
综上可知，与载剂(PBS)相比，本发明的化合物1-8，均有不同程度地改善小鼠糖耐受的效果，其中化合物2、5、6和7在4个OGTT曲线时段(24、48、72、96h)，展现了更为优秀和显著地改善葡萄糖耐受效果，同时结果也 说明，相对于索马鲁肽，化合物1-8，尤其化合物2、5、6和7在4个OGTT曲线时段(24、48、72、96h)，展现了更为优秀和显著地长效降糖效果。
实施例4 化合物2、5、6、7和索马鲁肽对糖尿病小鼠的糖尿病治疗作用
从南京大学-南京生物医药研究院购买8周龄的雄性Lepr db/db小鼠(db/db)48只和同窝出生的正常小鼠(WT)8只。
获得db/db小鼠糖尿病模型(从南京大学-南京生物医药研究院购买，8周左右小鼠，并测定血糖和体重保证后续实验顺利进行)，对db/db小鼠按照血糖随机分成6组(化合物2、5、6、7和索马鲁肽，生理盐水组)，每组8只，基础体重和血糖无差异。每组小鼠分别隔天皮下注射化合物2、5、6、7(120μg/kg)、索马鲁肽(120μg/kg)和生理盐水(WT组和db/db组)。在小鼠每次给药之后，隔天禁食6h测小鼠的血糖和体重；每7天测一次进水量和进食量。在给药第4周测OGTT，第5周测胰岛素耐量(ITT)，第6周取材，检测各种血清学指标和生理学指标。小鼠的血糖、体重、进水量和进食量监测结果见图3，取材后的血清学指标见图4，生理指标见图5。
2型糖尿病模型，其表现特征为：肥胖，胰岛素抵抗，高糖血症，血脂异常和肝脏脂肪空泡样变性等。在化合物2、5、6、7和索马鲁肽治疗糖尿病小鼠4周和5周的时候，分别进行了口服葡萄糖耐受实验(OGTT)和胰岛素耐受测试(ITT)，结果见图6。
图3为中8周龄的雄性db/db糖尿病小鼠给药后身体监测图，其中，分别皮下注射生理盐水、化合物2(120μg/kg)，化合物5(120μg/kg)，化合物6(120μg/kg)，化合物7(120μg/kg)和索马鲁肽(120μg/kg)，隔天给药,持续给药6周；每3天禁食6h测得的小鼠体重和血糖(3A和3B)，每7天测定一次小鼠进食量(3C)，其中，*：表示p<0.05；**：表示p<0.01；***：表示p<0.001。
图3(A、C)结果显示，与db/db组相比，化合物2、5、6、7均有显著减少小鼠的进食量，从而降低小鼠体重，且降低体重效果优于索马鲁肽。
图3B中的实验结果显示，与db/db组相比，化合物2、5、6、7和阳性对照药物索马鲁肽均能显著降低db/db小鼠的空腹血糖，说明这些药物均有显著的降糖效果，在第1周的时候血糖已经降到正常水平，且此后血糖相对稳定并优于索马鲁肽。
图4(A、B)显示，化合物2、5、6和7在肝脏保护功能方面也与索马鲁肽相当。
糖化血红蛋白(GHb)可用作反映糖尿病患得到血糖在一段较长时间内(4-10)周的控制指标，血糖长期控制不良的，其糖化血红蛋白就会升高，因此糖化血红蛋白测定有助于控制，对糖尿病的周围血管和心血管并发症的研究有重要作用，图4G中的实验结果显示，化合物2、5、6和7均具有显著的降低糖化血红蛋白的作用，并且优于索马鲁肽。
图5E结果显示，化合物2、5、6和7均能显著降低小鼠的肝重以及附睾脂肪含量，且优于索马鲁肽。
图6结果显示，在改善糖尿病小鼠口服葡萄糖耐受能力(OGTT)及胰岛素抵抗(ITT)方面，化合物2、5、6和7均优于阳性对照药索马鲁肽，结果见。
实施例5 化合物2、5、6和7和索马鲁肽在SD大鼠体内的药代动力学研究
将8周龄左右的SD大鼠(中山大学实验动物中心)，饲养一周适应环境，雌雄各半，按照体重随机分组，每组6只(雌雄各半)，共5组。
本实施例中化合物2、5、6和7和索马鲁肽均按照50ug/kg的剂量，给药体积2mL/kg，给药浓度60ug/mL，溶媒为生理盐水，分别进行皮下和尾静脉给药。
单次尾静脉给药组大鼠在给药前(0min)和给药后5min、15min、30min、1h、2h、4h、6h、8h、24h、36h、48h、72h、4d、5d、6d、7d、8d、9d、10d通过颈静脉进行血样采集，每个时间点采集量约0.2mL。单次皮下 注射组大鼠在给药前(0min)和给药后10min、20min、30min、1h、2h、4h、6h、8h、24h、36h、48h、72h、4d、5d、6d、7d、8d、9d、10d通过颈静脉进行血样采集，每个时间点采集量约0.2mL。置于含抗凝剂K 2-EDTA的EP管中,并于采血后1小时内于2-8℃离心(3500g，10min)，离心后分离得到的血浆装于标记好的EP管中，采用LC-MS/MS分析方法测定血浆中化合物2、5、6、7和索马鲁肽的药物浓度。数据处理采用WinNonlin 6.4软件中的非房室模型计算药代动力学参数。
由图7实验结果可知，单次皮下注射化合物2、5、6和7(50μg/kg)后，大鼠体内吸收较慢，达峰时间T max均为24h左右，半衰期t 1/2平均分别为14.99h、15.4h、16.6h和15.42h，化合物2、5、6和7均高于索马鲁肽(7.87h)。单次静脉注射化合物2、5、6和7(50μg/kg)后，半衰期t 1/2平均分别为14.49h、15.2h、16.2h和15.1h，均优于索马鲁肽(平均为8h)。
以上，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。

Claims (10)

  1. 一种长效GLP-1多肽类似物，其特征在于，所述长效GLP-1多肽类似物的氨基酸序列如下：
    His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{(Gly) x-(Ser-Gly) y-Z-CO(CH 2) nCO 2H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
    其中：
    Xaa2为Aib或D-Ser；
    Xaa9为Asp或Glu；
    Xaa21为Glu或Gln或Asp；
    Z为γGlu或Asp；
    x为1-4的整数；y为1-4的整数；n为12-20的整数。
  2. 根据权利要求1所述的长效GLP-1多肽类似物，其特征在于，Z为γGlu。
  3. 根据权利要求1所述的长效GLP-1多肽类似物，其特征在于，x为2，y为1、2或3。
  4. 根据权利要求1所述的长效GLP-1多肽类似物，其特征在于，n为14-18中任一整数。
  5. 根据权利要求1～4任一项所述的长效GLP-1多肽类似物，其特征在于，Z为γGlu，x为2，y为1、2或3，n为18。
  6. 根据权利要求1所述的长效GLP-1多肽类似物，其特征在于，所述长效GLP-1多肽类似物为如下化合物中的任一种：
    化合物1：
    His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物2：
    His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物3：
    His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物4：
    His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物5：
    His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物6：
    His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物7：
    His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Gln-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    化合物8：
    His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2) 18CO 2H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
  7. 权利要求1～6任一所述的长效GLP-1多肽类似物的制备方法，其特征在于，包括以下步骤：
    步骤1：按照Fmoc/t-Bu策略，合成所述长效GLP-1多肽类似物的主肽链对应的主肽树脂，其中，所述主肽链为His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
    步骤2：在所述主肽树脂的基础上，按照Fmoc/t-Bu策略，偶联所述长效GLP-1多肽类似物对应的“侧臂”结构，得到长效GLP-1多肽类似物对应的多肽树脂；其中，所述“侧臂”结构为(Gly) x-(Ser-Gly) y-Z-CO(CH 2) nCO 2H；
    步骤3：向所述多肽树脂中加入裂解液，进行裂解反应，脱除多肽全保护，提取粗品化合物；
    步骤4：纯化所述粗品化合物，获得长效GLP-1多肽类似物。
  8. 根据权利要求7所述的长效GLP-1多肽类似物的制备方法，其特征在于，
    步骤2中，使用的偶联剂为1-羟基苯并三唑和N,N-二异丙基碳二亚胺，溶剂为N，N-二甲基甲酰胺，以20％的哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团；
    步骤3中，裂解液由TFA、DODT、间甲酚、H 2O按体积比为92.5：2.5：2.5：2.5的比例组成；所述粗品化合物提取方式包括过滤、沉淀和/或甲基叔丁 基醚提取。
  9. 一种组合物，其特征在于，所述组合物包含权利要求1～6任一项所述的长效GLP-1多肽类似物和药学上可接受的载体或辅料。
  10. 权利要求1～6中任一项所述的长效GLP-1多肽类似物在制备预防或治疗糖尿病的药物中的应用；
    或者，所述的长效GLP-1多肽类似物在制备预防或治疗肥胖的药物中的应用；
    或者，所述的长效GLP-1多肽类似物在制备能降低体重的保健品中的应用。
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