WO2016045400A1 - 胃泌酸调节素类似物 - Google Patents

胃泌酸调节素类似物 Download PDF

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WO2016045400A1
WO2016045400A1 PCT/CN2015/080042 CN2015080042W WO2016045400A1 WO 2016045400 A1 WO2016045400 A1 WO 2016045400A1 CN 2015080042 W CN2015080042 W CN 2015080042W WO 2016045400 A1 WO2016045400 A1 WO 2016045400A1
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ser
lys
gly
asp
seq
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PCT/CN2015/080042
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French (fr)
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蒋先兴
陈元文
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蒋先兴
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Priority to KR1020167027899A priority Critical patent/KR101963202B1/ko
Priority to AU2015321161A priority patent/AU2015321161B2/en
Priority to EP15844187.3A priority patent/EP3199546B1/en
Priority to US15/300,069 priority patent/US10479819B2/en
Priority to DK15844187.3T priority patent/DK3199546T3/da
Priority to JP2017516944A priority patent/JP6895883B2/ja
Publication of WO2016045400A1 publication Critical patent/WO2016045400A1/zh

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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
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/06—Preparations for care of the skin for countering cellulitis
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones

Definitions

  • the invention belongs to the technical field of biochemistry and relates to a polypeptide of a class of oxyntomodulin analogs.
  • the invention also relates to the therapeutic use of the above novel oxyntomodulin analogs.
  • Diabetes mellitus is a metabolic disease characterized by relative or absolute deficiency of insulin. Relative or absolute deficiency of insulin causes hyperglycemia, which leads to three major nutrient metabolism disorders, which ultimately affect the normal physiological function of patients and cause complications.
  • the number of people with diabetes worldwide is increasing. The number of people with diabetes in the age of 20-79 has reached 382 million in 2013 and is expected to reach 439 million in 2030. Among them, China is the world's largest diabetes country. At present, China has about 110 million, and it is still growing rapidly. The increase in obesity leads to an increase in diabetes, and about 90% of people with type 2 diabetes can be classified as obese. There are 246 million people worldwide with diabetes, and 380 million people are expected to have diabetes by 2025.
  • Peptide drugs Smaller molecular chemical drugs and macromolecular protein drugs have their own advantages as follows: First, most of them are derived from endogenous peptides or other natural peptides, with clear structure and clear mechanism of action; secondly, they are related to general small molecule drugs. Higher activity, smaller dosage, lower toxic side effects, and metabolic end products are amino acids (no toxic side effects); third, they are less immunogenic than foreign proteins and can be chemically synthesized. The product has high purity and controllable quality. Fourthly, peptide drugs can often avoid digestion of the gastrointestinal tract and overcome the drawbacks that protein molecules are destroyed by digestive enzymes and cannot be taken orally.
  • GLP-1 Glueagon-like peptide
  • GLP-1- (7-37) GLP-1-(7-36)-amide.
  • GLP-1 exerts anti-diabetic effects by binding to the specific receptor glucagon-like peptide 1 receptor (GLP1R).
  • GLP1R glucagon-like peptide 1 receptor
  • the main physiological functions are: improving islet ⁇ -cell function, promoting insulin secretion, reducing postprandial blood glucose and maintaining blood glucose. Steady state, strengthen insulin biosynthesis, inhibit glucagon secretion, inhibit gastrointestinal motility, especially gastric emptying, thereby increasing satiety, reducing appetite and controlling body weight.
  • GLP-1 Compared with traditional antidiabetic drugs, GLP-1 has the advantages of maintaining blood glucose homeostasis and controlling body weight (Cardiovascular effects of glucagonlike peptide-1agonists, Am J Cardiol.2011; 108:33B-41B. Cardiovascular effects of the DPP-4inhibitors , Diab Vasc Dis Res. 2012; 9: 109-16. Incretin mimetics as a novel therapeutic option for hepatic steatosis, Liver Int. 2006; 26: 1015-7.). GLP-1 has a very short half-life and is rapidly degraded by dipeptidyl-peptidase-IV (DPP-IV) or Neutral endopeptidase (NEP) 24.11 after secretion. Therefore, GLP-1 cannot be directly used for clinical purposes, but an anti-enzymatic GLP-1 receptor agonist needs to be developed.
  • DPP-IV dipeptidyl-peptidase-IV
  • NEP Neutral endopeptidase
  • GLP-1 receptor agonists are also obvious, mainly in the following aspects:
  • Oxyntomodulin a short peptide hormone secreted by intestinal epithelial L-cells, composed of 37 amino acids, which is endogenous to glucagon.
  • OXM Oxyntomodulin
  • body OXM is a balanced GLP-1R/GCGR dual-target agonist that is twice as potent at the glucagon receptor as the GLP-1 receptor and is more potent than the native membrane glucagon and GLP-1 Their respective receptors are less potent.
  • clinical studies have shown that subcutaneous injection of natural OXM for four weeks can significantly reduce the body weight of the patient and reduce food intake.
  • OXM has a short half-life, dipeptidyl peptidase IV (DPP-IV) is rapidly inactivated and has poor stability in vivo.
  • DPP-IV dipeptidyl peptidase IV
  • GLP-1R/GCGR co-agonists have been shown to significantly reduce body weight and fat mass in obese rats (DIO rat) induced by high-fat diets, surpassing any simple GLP-1R agonist and improving blood glucose control. . These changes are associated with reduced food intake and substantial increases in energy expenditure. It has also been reported that long-acting GLP-1R/GCGR dual-target agonists with protease resistance can significantly reduce body weight, lower triglycerides and higher resistance than long-acting GLP-1R agonists with the same potency. blood sugar.
  • long-acting GLP-1R/GCGR dual-target agonists can improve metabolic indicators such as plasma, insulin, leptin and adiponectin (Unimolecular dual incretins maximum metabolic benefits in rodents, monkeys, and humans, Sci Transl Med. 2013; 5:209.).
  • GLP-1R/GCGR dual-target agonists are currently the main direction for the development of peptide drugs for the treatment of diabetes.
  • OXM analogs that are injected once a day and once a week by chemical modification.
  • the most widely used modifier is methoxypolyethylene glycol (mPEG), which increases the molecular clearance capacity of OXM and reduces the renal filtration clearance of drug molecules, thereby prolonging the drug plasma after mPEG modification.
  • mPEG methoxypolyethylene glycol
  • mPEG is a molecule that the human body cannot metabolize, and the polypeptide protein derived from it can cause vacuolation of the kidney (Short communication: renal tubular vacuolation in animals with Polyethylene-glycol-conjugated proteins, Toxicol Sci. 1998; 42: 152-7; Safety assessment on polyethylene glycols (PEGs) and their derivatives as used in cosmetic products. Toxicology, 2005; 214: 1-38.). The vast majority of mPEG-modified drug molecules are used in cancer therapy, so the toxicity of PEG is often neglected to a great extent.
  • Oxyntomodulin OXM
  • the inventors have carried out a large number of experimental studies to modify the oxyntomodulin molecule, and the results show that the oxyntomodulin analogue has a longer half-life, has insulinotropic activity, no adverse reactions occur, and is used for diabetes, etc. Treatment of the disease.
  • Another object of the present invention is to provide a similar therapeutic use of the above-mentioned oxyntomodulin, which is potentially useful as a new generation of drugs for treating diabetes, or for reducing blood sugar, or for reducing body weight. .
  • the present invention is improved based on the parent peptide of oxyntomodulin (OXM), glucagon-like peptide (GLP-1), Exenatide, and glucagon.
  • OXM oxyntomodulin
  • GLP-1 glucagon-like peptide
  • Exenatide Exenatide
  • glucagon glucagon-like peptide
  • the parent peptide of oxyntomodulin (OXM) is as follows (SEQ ID NO. 25):
  • GLP-1 The sequence of the glucagon-like peptide (GLP-1) is as follows (SEQ ID NO. 26):
  • glucagon is as follows (SEQ ID NO. 28):
  • a first aspect of the invention provides an oxyntomodulin analog comprising a parent peptide represented by the following amino acid sequence:
  • R 1 -OH or -NH 2 ;
  • Xaa2 Aib, Ser or D-Ser
  • Xaa10 Lys or Tyr
  • Xaa13 Lys or Tyr
  • Xaa16 Ser, Aib, Lys or Glu
  • Xaa17 Lys or Arg
  • Xaa18 Arg or Ala
  • Xaa20 His, Gln or Lys
  • Xaa21 Asp or Glu
  • Xaa23 IIe, Leu or Val
  • Xaa24 Glu or Gln
  • Xaa27 Met, Leu or not present
  • Xaa28 Ser, Asn, Asp, Arg or not present;
  • Xaa29 Ala, Gly, Thr or not present;
  • Xaa30 Gly or does not exist
  • Xaa31 Gly or does not exist
  • Xaa33 Ser or does not exist
  • Xaa34 Ser or does not exist
  • Xaa35 Gly or does not exist
  • Xaa36 Ala or does not exist
  • Xaa40 Ser or does not exist
  • At least one of Xaa10, Xaa16, Xaa17 or Xaa20 is Lys, and the side chain of the at least one Lys or the 12th Lys of the sequence is linked to a lipophilic substituent in a manner of a lipid substituent having an amide bond with an amino group of a bridging group, the carboxyl group of the amino acid residue of the bridging group forming an amide bond with the N-terminal residue of the Lys of the parent peptide to the parent peptide;
  • the bridging group is Glu, Asp and/or (PEG) m , wherein m is an integer from 2 to 10; the lipophilic substituent is selected from CH 3 (CH 2 ) n CO- or HOOC (CH 2 ) An acyl group of n CO-, wherein n is an integer from 10 to 24.
  • a preferred bridging group can be Glu-(PEG) m or Asp-(PEG) m or (PEG) m in the following manner:
  • Preferred compounds of the invention are parent peptides having the following amino acid sequences:
  • R 1 -NH 2 ;
  • Xaa2 Aib or D-Ser
  • Xaa10 Lys or Tyr
  • Xaa13 Lys or Tyr
  • Xaa16 Ser, Aib, Glu or Lys
  • Xaa17 Lys or Arg
  • Xaa18 Arg or Ala
  • Xaa20 His, Gln or Lys
  • Xaa21 Asp or Glu
  • Xaa24 Glu or Gln
  • Xaa27 Met or Leu
  • Xaa28 Asn, Arg, Asp or absent;
  • Xaa29 Gly, Thr or not present
  • Xaa30 Gly or does not exist
  • Xaa31 Gly or does not exist
  • Xaa33 Ser or does not exist
  • Xaa34 Ser or does not exist
  • Xaa35 Gly or does not exist
  • Xaa36 Ala or does not exist
  • Xaa40 Ser or does not exist.
  • More preferred compounds are compounds 1 to 24, which contain the parent peptide having the amino acid sequences shown in SEQ ID NOS.
  • a conventional three-letter code is used to represent a natural amino acid
  • a recognized three-letter code is used to represent other amino acids, such as Aib (aminoisobutyric acid), Orn (ornithine).
  • the compounds of the invention are capable of stabilizing the helical structure of the molecule based on a theoretical intramolecular bridge, thereby increasing the potency and/or selectivity for the GLP-1R or GCGR receptor.
  • the compounds of the invention carry one or more intramolecular bridges in the sequence.
  • Such a bridge is formed between the side chains of two amino acid residues that are typically separated by three amino acids in a linear sequence.
  • the bridge can be formed between residue pairs 12 and 16, 16 and 20, 17 and 21 or 20 and 24 side chains.
  • the two side chains can be linked to each other by ionic interaction or by covalent bonds.
  • these pairs of residues may comprise oppositely charged side chains to form a salt bridge by ionic interaction.
  • one residue may be Glu or Asp
  • the other residue may be Lys or Arg, Lys paired with Glu, and Lys and Asp paired, respectively, can also react to form a lactam ring.
  • the compounds of the present invention bind to albumin in the blood based on theoretical lipophilic substituents, thereby protecting the compounds of the invention from enzymatic degradation, thereby increasing the half-life of the compounds.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the oxyntomodulin analog of the present invention, wherein the oxyntomodulin is similarly added as a active ingredient with a pharmaceutically acceptable carrier and/or adjuvant to prepare a drug combination.
  • a further aspect of the present invention provides a pharmaceutical use of the oxyntomodulin analog of the present invention, and cell and animal experiments show that the oxyntomodulin analog of the present invention has hypoglycemic action and is useful as a medicament for treating diabetes
  • the oxyntomodulin analog of the present invention can also reduce body weight and has potential use as a medicament for treating obesity.
  • the GLP-1R/GCGR dual-target agonist mentioned in the present invention is a homologous polypeptide
  • the homologous polypeptide in the present invention means that the polypeptide originally has a GLP-1, OXM, Glucagon or Exenatide amino acid sequence, but wherein One or more amino acid residues have been conservatively substituted with different amino acid residues, and the resulting polypeptides are useful in the practice of the invention.
  • the polypeptides of the invention can be used to prevent weight gain or to promote weight loss.
  • the polypeptide can cause a decrease in food intake and/or an increase in energy expenditure, resulting in an observable effect on body weight.
  • the compounds of the invention Independent of its effect on body weight, the compounds of the invention have a beneficial effect on circulating cholesterol levels (to reduce circulating LDL levels and increase HDL/LDL ratio).
  • the polypeptides of the invention may be used to directly or indirectly treat any condition caused by or characterized by overweight, such as treating and/or preventing obesity, sick fat Fatty, obesity-related inflammation, obesity-related gallbladder disease, and sleep apnea caused by obesity.
  • the compounds of the invention are also useful for preventing metabolic syndrome, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease or stroke.
  • the effect of a polypeptide of the invention in these conditions may be due to or associated with the effect of the polypeptide on body weight or may be independent of its effect on body weight.
  • compositions of the present invention are suitable for use in a variety of modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, and the like.
  • the polypeptide pharmaceutical compositions of the present invention may be formulated into a variety of suitable dosage forms comprising at least one effective amount of a polypeptide of the present invention and at least one pharmaceutically acceptable pharmaceutically acceptable carrier, depending on the mode of administration employed.
  • 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 pharmaceutical composition containing the polypeptide of the present invention may be formulated into a solution or a lyophilized powder for parenteral administration, and the powder may be reconstituted by adding a suitable solvent or other pharmaceutically acceptable carrier before use, and the liquid formulation is generally a buffer. Isotonic solution and aqueous solution.
  • the dosage of the polypeptide of the drug group of the present invention can be varied within a wide range, and those skilled in the art can easily according to some objective factors such as the type of the disease, the severity of the disease, the patient's body weight, the dosage form, the administration route and the like. Add to determine.
  • the invention relates to a gastric acid analog having the sequence:
  • the modification of Lys may be one of the following structures:
  • Boc is tert-butoxycarbonyl
  • Fmoc is fluorenylmethoxycarbonyl
  • t-Bu is tert-butyl
  • ivDDe is 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3- Base-butyl removal and lipophilic substituent
  • resin is resin
  • TFA is trifluoroacetic acid
  • EDT is 1,2-ethanedithiol
  • Phenol is phenol
  • FBS is fetal bovine serum
  • BSA bovine serum albumin
  • HPLC is a high-performance liquid phase
  • GLP-1R is a glucagon-like peptide 1 receptor
  • GCGR is a glucagon receptor
  • GLP-1 is a glucagon-like peptide
  • mPEG is a monomethoxy polyethylene.
  • OXM is oxyntomodulin
  • His is histidine
  • Ser is serine
  • D-Ser is D-serine
  • Gln is glutamine
  • Gly is glycine
  • Glu is glutamic acid
  • Ala is alanine.
  • Thr is threonine
  • Lys is lysine
  • Arg is arginine
  • Tyr is tyrosine
  • Asp is aspartic acid
  • Trp tryptophan
  • Phe is phenylalanine
  • IIe is is isoleucin Acid
  • Leu is leucine
  • Cys is cysteine
  • Pro is proline
  • Val is valine
  • Met is methionine
  • Asn is asparagine.
  • HomoLys is high lysine
  • Orn is ornithine
  • Dap is diaminopimelic acid
  • Dab is 2,4-diaminobutyric acid.
  • Figure 1 Stimulation curves of Exenatide and Compounds 4, 5, 6, and 7 on GLP-1R;
  • Figure 2 Stimulation curve of Glucagon and compound 4, 5, 6, 7 on GCGR;
  • Figure 3 Effect of Exenatide (Ex-4), Compounds 4, 5, 6 and 7 on insulin release after oral administration of glucose in male C57B1/6J mice, compared with the control group, p ⁇ 0.05;
  • Figure 5 Effect of polypeptide compounds 4, 5, 6 and 7 on body weight of obese mice, compared with the control group, p ⁇ 0.05.
  • polypeptides of 0.25 mol scale were synthesized on a CS336X peptide synthesizer (C S Bio, USA) according to the Fmoc/t-Bu strategy:
  • Step 2 using Rink amide resin as carrier, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N
  • the mixture of N-diisopropylethylamine in a ratio of 1:1:1 is used as a coupling agent, and N,N-dimethylformamide is used as a solvent to carry out a process reaction, followed by condensation.
  • the reaction is linked to Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH(2x), Fmoc-Trp(Boc)-OH,
  • the ratio of the amount of the first amino acid Fmoc-Ser(t-Bu)-OH to the amount of the resin is 1:1 to 6:1; 2) the next condensation Fmoc protects amino acids in the reaction, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N,N-diisopropylethylamine (DIEPA) is used in excess of 2 ⁇ 8 times, the reaction time is 1 to 5 hours.
  • HCTU 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate
  • DIEPA organic base N,N-diisopropylethylamine
  • the present invention comprises the following polypeptide compounds (Table 1):
  • the cAMP Assay Kit manufactured by Cisbo was used to detect the stimulation of glucagon-like peptide 1, GLP-1 receptor and glucagon receptor, and the positive was detected.
  • HEK-293 cells stably expressing human GCGR or GLP-1R for the CRE-luciferase system were seeded into 384-well plates at 98 L DMEM/10% FBS medium per well and 5000 cells per well. On the second day after inoculation, 2 L of the sample to be tested was transferred in a gradient, mixed with the cells and incubated for 12 hours.
  • the compounds of the present invention Compared with oxytomodulin (Oxyntomodulin, purchased from Shanghai Yinggong Industrial Co., Ltd., Cat. No. Es-1240), the compounds of the present invention have higher relative GLP-1R selectivity to the membrane glucagon receptor and GLP.
  • the -1 receptor is more potent.
  • Figure 1 is a stimulation curve of Exenatide and Compounds 4, 5, 6, and 7 for GLP-1R;
  • Figure 2 is a graph showing the stimulation of GCGR by Glucagon and Compounds 4, 5, 6, and 7.
  • mice Male CD1 male mice (Nanjing University Model Animal Research Center), the doses of 250 ⁇ g/kg were respectively administered by the intravenous (IV) or subcutaneous (SC) routes using the compounds prepared in the above examples, 4, 5, 6, 7, 19 , 20, 21, 22, 23 and 24 were compared to commercially purchased exenatide pure (Ex-4). Animals were bled at various times within 0-24 hours after dosing, plasma from each sample was collected and analyzed by LC-MS/MS assay. Pharmacokinetic parameters were calculated using a model dependent (data obtained for IV) and a model independent (for SC derived data).
  • the elimination half-lives of compounds 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 used by the IV route are about 4-6 hours, and their peak time (T max ) is about 12 hours.
  • the elimination half-life of sernatide is approximately 0.5 hours.
  • the peak times (T max ) of the compounds 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 used by the SC route are about 12 hours or more, and the Ex-4 elimination half-life is about 2 hours. No clinical adverse reactions occurred with compounds 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 by IV or SC route, respectively.
  • Example 5 Compounds 4, 5, 6, 7, 19, 20, 21, 22, 23, 24 and Ex-4 for oral glucose tolerance use
  • the polypeptides 4, 5, 6, 7, 19, 20, 21, 22, 23 and 24 (the compound was administered in an amount of 250 ⁇ g/kg) significantly reduced oral glucose tolerance.
  • Using the Software GraphPadPrism processes the data to make a blood glucose change line graph and calculates the area under the curve to obtain an AUC map ( Figures 3 and 4).
  • exenatide 250 ⁇ g/kg had a significant decrease in AUC during the first OGTT curve period (1-120 min) (P ⁇ 0.05), and in the subsequent three OGTT curve periods. (120-480 min), its AUC was close to the blank control group (P>0.05).
  • polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 19, polypeptide 20, polypeptide 21, polypeptide 22, polypeptide 23 and polypeptide 24 are in 4 OGTT curve periods (0-480 min). AUC was significantly decreased (P ⁇ 0.05).
  • the experimental results suggest that polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 19, polypeptide 20, polypeptide 21, polypeptide 22, polypeptide 23 and polypeptide 24 have long-lasting hypoglycemic effects.
  • the oxyntomodulin analog of the present invention exhibits double agonistic activity of GCGR and GLP-1R, has a long half-life and high enzymatic stability, high biological activity, and no adverse hair growth.
  • the compound of the invention has high synthesis yield, good stability, easy to scale up production, low cost, and can be used for preparing a medicament for treating overeating, obesity and overweight, elevated cholesterol and diabetes.

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Abstract

提供了一类胃泌酸调节素类似物，所述类似物具有GCGR和GLP-1R双激动活性，提高的酶解稳定性和生物活性，无不良反应。所述类似物可用于制备治疗摄食过量、肥胖和糖尿病的药物。

Description

胃泌酸调节素类似物 技术领域
本发明属于生物化学技术领域，涉及一类胃泌酸调节素类似物的多肽。
本发明还涉及上述新的胃泌酸调节素类似物的治疗用途。
背景技术
糖尿病(Diabetes mellitus，DM)是以胰岛素相对或绝对不足为特征的代谢性疾病，胰岛素相对或绝对不足引发高血糖，进而导致三大营养物质代谢紊乱，最终影响患者正常生理功能并且引起并发症。全球糖尿病患者与日俱增，在20-79岁的成人中，糖尿病患者人数在2013年已达3.82亿，预计2030年将达到4.39亿。其中，中国是世界第一糖尿病大国，当前我国约有1.1亿，而且还在快速增长。肥胖症的增多导致糖尿病的增多，约90％的患II型糖尿病的人群可划归为肥胖。全世界有2亿4千6百万人患有糖尿病，预计到2025年将会有3亿8千万人患糖尿病。许多人还有其他心血管风险因素，包括高/异常LDL及甘油三脂和低HDL。糖尿病目前不能根治，患者只能终生依赖药物。传统抗糖尿病药物各有缺点，迫切需要研发新型抗糖尿病药物。
肽类药物较小分子化学药物和大分子蛋白药物具有自身的优点如下：首先，它们多数源于内源性肽或其他天然肽，结构清楚，作用机制明确；其次，它们与一般小分子药物相比，活性更高、用药剂量更小、毒副作用更低，而且代谢终产物为氨基酸(无毒副作用)；第三，它们与外源蛋白质相比，免疫原性较低，而且可以化学合成，产品纯度高，质量可控；第四，多肽药物往往能规避胃肠道消化，克服蛋白质分子被消化酶破坏从而不能口服的弊端。
胰高血糖素样肽(Glueagon-like peptide-1，GLP-1)属于肠促胰岛素(Incretin)家族，是一种主要由小肠粘膜L细胞分泌的多肽，有两种活性形式：GLP-1-(7-37)和GLP-1-(7-36)-酰胺。GLP-1通过与特异性受体胰高血糖素样肽1受体(GLP1R)结合而发挥抗糖尿病作用，主要生理功能有：改善胰岛β细胞功能，促进胰岛素分泌，降低餐后血糖而维持血糖稳态，加强胰岛素生物合成，抑制胰高血糖素分泌，抑制胃肠蠕动尤其是胃排空从而增加饱足感、降低食欲并控制体重。与传统抗糖尿病药物相比，GLP-1的优点在于维持血糖稳态和有效控制体重(Cardiovascular effects of glucagonlike peptide-1agonists,Am J Cardiol.2011；108:33B-41B.Cardiovascular effects of the DPP-4inhibitors,Diab Vasc Dis Res.2012；9:109-16.Incretin mimetics as a novel therapeutic option for hepatic steatosis,Liver Int.2006；26:1015-7.)。GLP-1半衰期极短，分泌后很快被二肽基肽酶-IV(Dipeptidyl-peptidase-IV，DPP-IV)或中性肽链内切酶(Neutral endopeptidase，NEP)24.11降解。所以不能直接将GLP-1用于临床，而是需要研发抗酶解的GLP-1受体激动剂。
然而，临床试验证明GLP-1受体激动剂的缺点也很明显，主要有以下几个方面：
首先，半衰期较短导致注射频率密集，给病人带来不便；其次，药动学及安全性均不明确，引入的外源化学基团究竟如何代谢、如何排泄、对人体有何影响，均不明确，尚待进一步研究。最新临床前研究表明，均衡的胰高血糖素样肽1受体和胰高血糖素受体(GLP-1R/GCGR)双靶点激动剂表现出了相对于单纯GLP-1R激动剂更有效、更安全的治疗老鼠肥胖的作用，同时在血糖控制上也有所改进(A new glucagon and GLP-1co-agonist eliminates obesity in rodents,Nat Chem Biol.2009；5:749-57.)。与此相关的是关于胃泌酸调节素(Oxyntomodulin,OXM)的研究，OXM是肠上皮L-细胞分泌的一种短肽激素，由37个氨基酸组成，是胰高血糖素的内源性前体物。OXM是均衡的GLP-1R/GCGR双靶点激动剂，其对膜高血糖素受体的效力比对GLP-1受体的效力低2倍，并且比天然膜高血糖素和GLP-1对它们各自受体的效力低。尽管天然的OXM生物活性较低，临床研究表明持续四周皮下注射天然OXM仍然能够显著减轻病患的体重，减少摄食。(Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects:a double-blind,randomized,controlled trial,Diabetes.2005；54:2390-5.)OXM具有很短的半衰期，可被细胞表面的二肽基肽酶IV(DPP-IV)迅速失活，体内稳定性差。
GLP-1R/GCGR共激动剂已证实可明显减少高脂饮食诱导肥胖大鼠(DIO rat)体重和脂肪量，超过了任何一种单纯的GLP-1R激动剂，并在血糖控制上也有所改善。这些变化与减少食物摄入和大量增加能量消耗有关。另有报道指出：具有蛋白酶抗性的长效GLP-1R/GCGR双靶点激动剂，与具有相同效力的长效GLP-1R激动剂相比，可显著减轻体重、降低甘油三酯和抗高血糖。除此以外，长效GLP-1R/GCGR双靶点激动剂可改善代谢指标，例如血浆胰岛素、瘦素和脂联素(Unimolecular dual incretins maximize metabolic benefits in rodents,monkeys,and humans,Sci Transl Med.2013；5:209.)。
综上所述，研发GLP-1R/GCGR双靶点激动剂是目前研制治疗糖尿病的多肽药物的主要方向。近年来，研究人员通过化学修饰开发出了几种潜在的一天注射一次以及一周注射一次的OXM类似物。其中应用最为广泛的修饰剂是单甲氧基聚乙烯二醇(methoxypoly ethylene glycol，mPEG)，通过增加OXM的分子排阻体积，降低药物分子的肾脏过滤清除率，从而延长mPEG修饰后的药物血浆半衰期，以实现每周注射的目标。尽管这种方法能够显著提高多肽药物的半衰期，但大部分蛋白的生物活性也有不同程度降低。更危险的是，mPEG是一种人体不能代谢的分子，用其衍生的多肽蛋白类药物可导致肾脏的空泡化(Short communication:renal tubular vacuolation in animals treated with  polyethylene-glycol-conjugated proteins,Toxicol Sci.1998；42:152-7；Safety assessment on polyethylene glycols(PEGs)and their derivatives as used in cosmetic products.Toxicology,2005；214:1-38.)。绝大多数经mPEG修饰的药物分子被用于肿瘤治疗，因此PEG的毒性往往被极大限度的忽视。从化学的本质出发，只有通过优化多肽序列，才能从根本上解决多肽药物的生化和生理特性，这也是生物化学家公认的事实。并在此基础上，通过生物学手段进一步提高多肽药物的各种活性和稳定性指标。
发明内容
本发明的目的在于提供一种胃泌酸调节素(Oxyntomodulin,OXM)类似物。发明人经过大量的实验研究，对胃泌酸调节素分子进行了改造，结果表明该类胃泌酸调节素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，用于糖尿病等疾病的治疗。
本发明的另一个目的在于提供上述胃泌酸调节素类似的治疗用途，新的胃泌酸调节素类似物潜在地可作为新一代治疗糖尿病的药物，或用于降糖，或用于降低体重。
本发明是基于胃泌酸调节素(OXM)、胰高血糖素样肽(GLP-1)、艾塞那肽(Exenatide)及胰高血糖素(Glucagon)的母体肽而改进的。胃泌酸调节素(OXM)的母体肽如下(SEQ ID NO.25)：
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala-OH
胰高血糖素样肽(GLP-1)的序列如下(SEQ ID NO.26)：
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH
艾塞那肽(Exenatide)的序列如下(SEQ ID NO.27)：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
胰高血糖素(Glucagon)的序列如下(SEQ ID NO.28)：
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
本发明的第一个方面是提供一种胃泌酸调节素类似物，该胃泌酸调节素类似物含有以下氨基酸序列表示的母体肽:
His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xa a17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa 30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
其中，R1＝-OH或-NH2；
Xaa2＝Aib,Ser或D-Ser；
Xaa10＝Lys或Tyr；
Xaa13＝Lys或Tyr；
Xaa16＝Ser,Aib,Lys或Glu；
Xaa17＝Lys或Arg；
Xaa18＝Arg或Ala；
Xaa20＝His,Gln或Lys；
Xaa21＝Asp或Glu；
Xaa23＝IIe,Leu或Val；
Xaa24＝Glu或Gln；
Xaa27＝Met,Leu或不存在；
Xaa28＝Ser,Asn,Asp,Arg或不存在；
Xaa29＝Ala,Gly,Thr或不存在；
Xaa30＝Gly或不存在；
Xaa31＝Gly或不存在；
Xaa32＝Pro或不存在；
Xaa33＝Ser或不存在；
Xaa34＝Ser或不存在；
Xaa35＝Gly或不存在；
Xaa36＝Ala或不存在；
Xaa37＝Pro或不存在；
Xaa38＝Pro或不存在；
Xaa39＝Pro或不存在；
Xaa40＝Ser或不存在；
所述氨基酸序列中，Xaa10,Xaa16,Xaa17或Xaa20中至少一个为Lys，所述至少一个Lys或所述序列的第12位Lys的侧链与亲脂性的取代基相连，连接方式为所述亲脂性的取代基以其羧基与一个桥接基团的氨基形成酰胺键，桥接基团的氨基酸残基的羧基与母体肽的Lys的N-末端残基上形成一个酰胺键连接到母体肽上；
所述桥接基团为Glu，Asp和/或(PEG)m，其中m为2-10的整数；所述亲脂性取代基为选自CH3(CH2)nCO-或HOOC(CH2)nCO-的一个酰基，其中n是10-24的整数。
优选的桥接基团可以为Glu-(PEG)m或Asp-(PEG)m或(PEG)m，连接方式如下：
Figure PCTCN2015080042-appb-000001
本发明优选的化合物是含有下述氨基酸序列的母体肽：
His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xa a17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
其中，R1＝-NH2；
Xaa2＝Aib或D-Ser；
Xaa10＝Lys或Tyr；
Xaa13＝Lys或Tyr；
Xaa16＝Ser,Aib,Glu或Lys；
Xaa17＝Lys或Arg；
Xaa18＝Arg或Ala；
Xaa20＝His,Gln或Lys；
Xaa21＝Asp或Glu；
Xaa23＝IIe,Val；
Xaa24＝Glu或Gln；
Xaa27＝Met或Leu；
Xaa28＝Asn,Arg,Asp或不存在；
Xaa29＝Gly,Thr或不存在；
Xaa30＝Gly或不存在；
Xaa31＝Gly或不存在；
Xaa32＝Pro或不存在；
Xaa33＝Ser或不存在；
Xaa34＝Ser或不存在；
Xaa35＝Gly或不存在；
Xaa36＝Ala或不存在；
Xaa37＝Pro或不存在；
Xaa38＝Pro或不存在；
Xaa39＝Pro或不存在；
Xaa40＝Ser或不存在。
更优选的化合物为化合物1～24，其含有母体肽具有SEQ ID NO.1～24所示的氨基酸序列。
在通篇本申请的说明书中，采用常规的三字母代码代表天然氨基酸，并采用公认的三字母代码代表其他氨基酸，如Aib(氨基异丁酸)，Orn(鸟氨酸)。
本发明化合物基于理论分子内桥可以稳定分子的螺旋结构，从而提高了针对GLP-1R或GCGR受体的效力和/或选择性。本发明化合物在序列中携带一个或更多个分子内桥。这样的桥是由两个氨基酸残基的侧链之间形成，所述两个氨基酸残基通常被线性序列中的三个氨基酸所分隔。例如所述桥可在残基对12与16、16与20、17与21或者20与24侧链之间形成。两个侧链可通过离子相互作用或通过共价键彼此相连接。因此，这些残基对可包含带相反电荷的侧链，从而通过离子相互作用形成盐桥。例如，一个残基可以是Glu或Asp，而另一残基可以是Lys或Arg，Lys与Glu配对以及Lys与Asp配对分别还能够反应形成内酰胺环。
本发明化合物基于理论亲脂取代基可以结合血液中的白蛋白，从而保护本发明化合物免受酶降解，从而提高化合物的半衰期。
本发明的另一方面是提供含有本发明的胃泌酸调节素类似物的药物组合物，以所述胃泌酸调节素类似为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。
本发明的再一方面是提供本发明的胃泌酸调节素类似物的医药用途，细胞和动物实验显示，本发明的胃泌酸调节素类似物具有降糖作用，可用作治疗糖尿病的药物，本发明的胃泌酸调节素类似物还可以降低体重，具有作为治疗肥胖症药物的潜在用途。
本发明中提到的GLP-1R/GCGR双靶点激动剂为同源性多肽，本发明中的同源性多肽是指，多肽本来具有GLP-1,OXM，Glucagon或Exenatide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保守的取代，并且所得到的多肽可用于实施本发明。
本发明多肽可用于防止体重增长或促进体重减轻。所述多肽可引起摄食量降低和/或能量消耗升高，使得对体重产生可观察到的作用。独立于其对体重的作用，本发明化合物可对循环胆固醇水平具有有益作用(能够降低循环LDL水平以及提高HDL/LDL比值)。因此，本发明多肽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任何病症，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。本发明化合物还可用于预防代谢综合征、高血压、致动脉粥样化性异常脂肪血症、动脉粥样硬化、动脉硬化、冠心病或中风。本发明多肽在这些病症中的作用可以是由多肽对体重的作用所致或与之相关，或者可以独立于其对体重的作用。
本领域技术人员可以理解，本发明的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。
含有本发明多肽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，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。
本发明药物组物种多肽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，给药途径等因素很容易的加以确定。
本发明的优点:
1)在分子数相同时与天然OXM相比，具有更好的生物学活性；
2)在药物的药代实验中显示出显著延长的半衰期和稳定性；
3)合成产率高，稳定性好，易于放大生产，成本低。
在具体的实施方案中，涉及下述胃泌酸类似物，其具有序列：
化合物1(SEQ ID NO.1)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-N H2
化合物2(SEQ ID NO.2)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH2
化合物3(SEQ ID NO.3)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物4(SEQ ID NO.4)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物5(SEQ ID NO.5):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Ly s-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物6(SEQ ID NO.6)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Ser-L ys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物7(SEQ ID NO.7)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Se r-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-G ly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物8(SEQ ID NO.8)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物9(SEQ ID NO.9)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PE G2-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物10(SEQ ID NO.10)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PE G2-Glu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物11(SEQ ID NO.11)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PE G2-Glu-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物12(SEQ ID NO.12)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly- Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物13(SEQ ID NO.13)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-G ly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物14(SEQ ID NO.14)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物15(SEQ ID NO.15)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2)14CH3)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-G ly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物16(SEQ ID NO.16)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物17(SEQ ID NO.17)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物18(SEQ ID NO.18)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物19(SEQ ID NO.19)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Ser-Ly s-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-P ro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物20(SEQ ID NO.20)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gl y-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物21(SEQ ID NO.21)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Ser-Ly  s-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-P ro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物22(SEQ ID NO.22)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gl y-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物23(SEQ ID NO.23)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-CO(CH2)14CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pr o-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
化合物24(SEQ ID NO.24)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-Glu-CO(CH2)14CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-G ly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
上述序列中，Lys的修饰可以为下述结构之一：
Figure PCTCN2015080042-appb-000002
Figure PCTCN2015080042-appb-000003
上述与亲脂性取代基连接的Lys可替换为：
Figure PCTCN2015080042-appb-000004
本发明中所用缩写具体含义如下:
Boc为叔丁氧羰基，Fmoc为芴甲氧羰基，t-Bu为叔丁基，ivDDe为1-(4,4-二甲基-2,6-二氧代亚环己基)-3-甲基-丁基的脱除与亲脂取代基，resin为树脂，TFA为三氟乙酸，EDT为1,2-乙二硫醇，Phenol为苯酚，FBS为胎牛血清，BSA为牛血清白蛋白，HPLC为高效液相，GLP-1R为胰高血糖素样肽1受体，GCGR为胰高血糖素受体，GLP-1为胰高血糖素样肽，mPEG为单甲氧基聚乙烯二醇，OXM为胃泌酸调节素，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。HomoLys为高赖氨酸,Orn为鸟氨酸,Dap为二氨基庚二酸,Dab为2,4-二氨基丁酸。
附图说明
图1：Exenatide和化合物4,5,6,7对GLP-1R的刺激曲线；
图2：Glucagon和化合物4,5,6,7对GCGR的刺激曲线；
图3：艾塞那肽(Exenatide,Ex-4)，化合物4，5，6和7对雄性C57B1/6J小鼠口服葡萄糖后胰岛素释放的影响，和对照组比较，p<0.05；
图4：艾塞那肽(Exenatide,Ex-4)，化合物19，20，21，22，23和24对雄性 C57B1/6J小鼠口服葡萄糖后胰岛素释放的影响，和对照组比较，p<0.05；
图5：多肽化合物4，5，6和7对肥胖小鼠体重的影响，和对照组比较，p<0.05。
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。
实施例1单叔丁酯十八烷二酸的合成：
十八烷二酸(31.4克，100毫摩尔)悬浮在甲苯(250毫升)，将混合物加热至回流。N，N-二甲基甲酰胺二叔丁基缩醛(50.9克，250毫摩尔)逐滴加入在4小时内。将混合物回流过夜。将溶剂在真空中除去，在50℃下，将粗物质悬浮于DCM/乙酸乙酯(500毫升，1:1)中，搅拌15分钟。将固体通过过滤收集并研磨在DCM(200毫升)中。过滤，真空蒸发，得到粗单叔丁基十六烷20克。将其用庚烷(200ml)中重结晶，得到单叔丁酯十八烷二酸12.9克(33％)。初了重结晶，该单酯可以通过在AcOEt/庚烷的二氧化硅色谱纯化。1H-NMR(400MHz,CDCl3)δ：2.35(t，2H)，2.20(t，2H)，1.65-1.55(m，4H)，1.44(s，9H)，1.34-1.20(m，22H)。
实施例2多肽化合物的合成
以多肽化合物1和多肽化合物5的合成为举例
材料:
所有的氨基酸购自NovaBiochem公司。如果没有特别说明，其他所有试剂均为分析纯，购自Sigma公司。Protein Technologies PRELUDE 6通道多肽合成仪。Phenomenex Luna C18制备柱(46mm x 250mm)用来纯化多肽。高效液相色谱仪为Waters公司产品。质谱分析采用Agilent质谱仪进行测定。
方法:
1.多肽化合物1的合成：
结构结构序列：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH2
1a)主肽链组装：
按照Fmoc/t-Bu策略在CS336X多肽合成仪(美国C S Bio公司)上合成0.25mol规模的如下多肽：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O  tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(ivDde)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide树脂
(1)第一步：将0.75克Rink amide树脂在二氯甲烷中溶胀，用N，N-二甲基甲酰胺洗涤树脂三次；
(2)第二步：以Rink amide树脂为载体，以由苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐，1-羟基苯并三唑和N，N-二异丙基乙胺三者按物质的量比1:1:1配制的混合液为偶联剂，以N，N-二甲基甲酰胺为溶剂，进行程序反应，依次进行缩合反应连接Fmoc-Arg(pbf)-OH,Fmoc-Leu-OH(2x),Fmoc-Trp(Boc)-OH,
Fmoc-Glu(OtBu)-OH,Fmoc-Val-OH,Fmoc-Phe-OH,Fmoc-Asp(OtBu)-OH,
Fmoc-His(Boc)-OH,Fmoc-Ala-OH(2x),Fmoc-Lys(ivDde)-OH,Fmoc-Ser(t-Bu)-OH,
Fmoc-Asp(OtBu)-OH,Fmoc-Leu-OH,Fmoc-Tyr(t-Bu)-OH,Fmoc-Lys(Boc)-OH,
Fmoc-Ser(t-Bu)-OH,Fmoc-Tyr(t-Bu)-OH,Fmoc-Asp(OtBu)-OH,
Fmoc-Ser(t-Bu)-OH,Fmoc-Thr(t-Bu)-OH,Fmoc-Phe-OH,Fmoc-Thr(t-Bu)-OH,
Fmoc-Gly-OH,Fmoc-Gln(OtBu)-OH,Fmoc-D-Ser(t-Bu)-OH和Boc-His(Boc)-OH得
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(ivDde)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide树脂，其中每次缩合反应中Fmoc保护氨基酸的投料量与树脂用量的物质的量比为1:1～6:1，每次缩合反应中以由苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐用量与Fmoc保护氨基酸用量的物质的量比为3:1。
1b)1-(4，4-二甲基-2，6-二氧代亚环己基)-3-甲基-丁基(ivDde)的脱除与亲脂取代基的引入：
在N-甲基吡咯烷酮：二氯甲烷＝1:1(体积比)的溶液中，将由1a)中合成的保护的肽基树脂洗涤两次，加入新鲜制备的2.0％的肼水合物N-甲基吡咯烷酮溶液，将该反应混合物在室温下振摇12.0分钟，然后过滤。将肼处理步骤重复两次得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys-Ala-Al a-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide树脂。此后用二氯甲烷和N-甲基吡咯烷酮充分洗涤树脂。向其中加入FmocNH-PEG2-OH,苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐，1-羟基苯并三唑,二异丙基乙基胺的N-甲基吡咯烷酮混合偶联液，振摇3.0小时后，过滤，洗涤将肼处理步骤重复两次得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG2-OH)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Ar g(pbf)-Ala-rink amide树脂，哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团，重复一次Fmoc-PEG2-OH偶连反应得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG2-PEG2-OH)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-L eu-Arg(pbf)-Ala-rink amide树脂。哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团，然后按照常规条件依次偶连Fmoc-Glu-OtBu,tBu单保护的十八碳脂肪酸得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG2-PEG2-Glu-CO(CH2)16CO2tBu)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide树脂。
1c)多肽全保护的脱除：
将
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Ser(OtBu)-Lys(Fmoc-PEG2-PEG2-Glu-CO(CH2)16CO2tBu)-Ala-Ala-His(Boc)-Asp(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Arg(pbf)-Ala-rink amide树脂加入至圆底烧瓶中,冰浴下，加入切割液TFA/EDT/Phenol/H2O(88/2/5/5，体积比),升温，控制裂解液温度25℃,反应120分钟。过滤，滤饼用少量三氟乙酸洗涤3次，合并滤液。滤液在搅拌下缓慢倒入冰乙醚中。静置1.0小时以上,待沉淀完全。倾去上清液，沉淀离心，用冰乙醚洗涤6次，得到粗品化合物：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-Glu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH2。
1d)多肽化合物的精制纯化：
将1c中所得粗品溶于5.0％乙酸在乙腈：H2O＝1:1(体积比)的溶液中，通过5.0m反相C18的填充的50mm x 250mm柱上进行2次半制备型HPLC而纯化。用30-60％乙腈-0.1％三氟乙酸/H2O梯度以40mL/min将该柱洗脱45.0分钟，收集含有肽的组分，浓缩除去乙腈后冻干。得到HPLC纯度大于95％的纯品。用液质联用分析分离出的产物，发现质子化分子离子峰的m/z值为:4116.0,理论值为4116.6。
2.多肽化合物5的合成：
结构序列：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Ly s-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
2a)主肽链组装：
按照Fmoc/t-Bu策略在CS336X多肽合成仪(美国CS Bio公司)上合成0.25毫摩尔规模的如下多肽：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Lys(ivDde)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(P bf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂(1)第一步：将0.75克Rink amide MBHA-LL树脂(Novabiochem，loading 0.34毫摩尔/g)在二氯甲烷(DCM)中溶胀一个小时，用N，N-二甲基甲酰胺(DMF)充分洗涤树脂三次；
(2)第二步：以Rink amide树脂为载体，以偶联剂由6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)，有机碱N，N-二异丙基乙胺(DIEPA)两者按物质的量比1:1，以N，N-二甲基甲酰胺(DMF)为溶剂，进行程序反应，依次进行缩合反应连接Fmoc-Ser(t-Bu)-OH,Fmoc-Pro-OH(3x),Fmoc-Ala-OH,
Fmoc-Gly-OH,Fmoc-Ser(t-Bu)-OH(2x),Fmoc-Pro-OH,Fmoc-Gly-OH(2x),
Fmoc-Thr(t-Bu)-OH,Fmoc-Asn(Trt)-OH,Fmoc-Met-OH,Fmoc-Leu-OH,
Fmoc-Trp(Boc)-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Val-OH,Fmoc-Phe-OH,
Fmoc-Asp(OtBu)-OH,Fmoc-Gln(Trt)-OH,Fmoc-Ala-OH,Fmoc-Arg(Pbf)-OH(2x),
Fmoc-Aib-OH,Fmoc-Asp(OtBu)-OH,Fmoc-Leu-OH,Fmoc-Tyr(t-Bu)-OH,
Fmoc-Lys(Boc)-OH,Fmoc-Ser(t-Bu)-OH,Fmoc-Lys(ivDde)-OH,
Fmoc-Asp(OtBu)-OH,Fmoc-Ser(t-Bu)-OH,Fmoc-Thr(t-Bu)-OH,Fmoc-Phe-OH,
Thr(t-Bu)-OH,Fmoc-Gly-OH,Fmoc-Gln(Trt)-OH,Fmoc-D-Ser(t-Bu)-OH,
Boc-His(Boc)-OH得到：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Lys(ivDde)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(P bf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲醇(Methanol)，二氯甲烷(DCM)，N，N-二甲基甲酰胺(DMF)充分洗涤树脂各三次。
需要说明的是：1)其中第一个氨基酸Fmoc-Ser(t-Bu)-OH的投料量与树脂用量的物质的量比为1:1～6:1；2)接下来的每次缩合反应中Fmoc保护氨基酸， 6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)，有机碱N，N-二异丙基乙胺(DIEPA)的用量均过量2～8倍，反应时间为1～5个小时。
2b)1-(4，4-二甲基-2，6-二氧代亚环己基)-3-甲基-丁基(ivDde)的脱除与亲脂取代基的引入：
用N，N-二甲基甲酰胺(DMF)/二氯甲烷(DCM)＝1:1(体积比)的溶液中将树脂洗涤两次，加入新鲜制备的3.0％的肼水合物N，N-二甲基甲酰胺(DMF)溶液，将该反应混合物在室温下振摇10～30分钟，然后过滤。将肼处理步骤重复5次得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pb f)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲醇(Methanol)，二氯甲烷(DCM)，N，N-二甲基甲酰胺(DMF)充分洗涤树脂各三次。
加入FmocNH-PEG2-OH(Quanta BioDesign),2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，二异丙基乙基胺(DIEPA)的N，N-二甲基甲酰胺(DMF)混合偶联液(均过量5倍)，振摇2小时后，过滤。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲醇(Methanol)，二氯甲烷(DCM)，N，N-二甲基甲酰胺(DMF)充分洗涤树脂各三次得：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(Fmoc-PEG2)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(T rt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲醇(Methanol)，二氯甲烷(DCM)，N，N-二甲基甲酰胺(DMF)充分洗涤树脂各三次。
20％的哌啶(Piperidine)/N,N-二甲基甲酰胺(DMF)溶液脱除Fmoc基团(30分钟，重复脱除两次)，加Fmoc-PEG2-OH，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，二异丙基乙基胺(DIEPA)的N，N-二甲基甲酰胺(DMF)混合偶联液(均过量5倍)，偶连反应得到：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Lys(Fmoc-PEG2-PEG2)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(T rt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲 醇(Methanol)，二氯甲烷(DCM)，N，N-二甲基甲酰胺(DMF)充分洗涤树脂各三次。
20％的哌啶(Piperidine)/N,N-二甲基甲酰胺(DMF)溶液脱除Fmoc基团(30分钟，重复脱除两次)，加十六酸(棕榈酸)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，二异丙基乙基胺(DIEPA)的N，N-二甲基甲酰胺(DMF)混合偶联液(均过量5倍)，偶连反应得到：
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Lys(PEG2-PEG2-C16)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂。此后依次用N，N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，甲醇(Methanol)，二氯甲烷(DCM)充分洗涤树脂各三次后，真空抽干。
2c)多肽全保护的脱除：
将
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(O tBu)-Lys(PEG2-PEG2-C16)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Aib-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide树脂，加入切割液TFA/Phenol/thioanisole/EDT/H2O(82.5:5:5:2.5:5，体积比)，升温，控制裂解液温度25℃,反应2.5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乙醚中。静置2个小时以上，待沉淀完全，沉淀离心，用冰乙醚洗涤3次，得到粗品化合物：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Ly s-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2。
2d)多肽化合物的精制纯化：
将上2c中所得粗品溶于乙腈(ACN)/H2O＝1:2(体积比)的溶液中，通过5.0 m反相C18的填充的46mm x 250mm柱上进行制备型HPLC纯化。用30％乙腈(含0.05％三氟乙酸)/H2O(含0.05％三氟乙酸)为起始，以梯度(1.33％/min的速度增加乙腈的比例)，流速为15mL/min将该柱洗脱30分钟，收集含有肽的组分，冷冻抽干，得到HPLC纯度大于95％的纯品(如果纯度没有达到要求，可重复一次HPLC纯化)。用液质联用分析分离出的产物。基于以上合成步骤，本发明合成包括如下多肽化合物(表1)：
表1.本发明实施例中所合成的多肽化合物结构：
Figure PCTCN2015080042-appb-000005
Figure PCTCN2015080042-appb-000006
Figure PCTCN2015080042-appb-000007
实施例3GCGR和GLP-1R体外活性测定
利用Cisbo公司生产的cAMP Assay Kit来检测对胰高血糖素样肽1受体(glucagon-like peptide 1，GLP-1receptor)和胰高血糖素受体(glucagon receptor)的刺激作用，同时检测了阳性对照化合物胰高血糖素样肽1(GLP-1),艾塞那肽(Exenatide)以及胰高血糖素(glucagon)的剂量效应曲线。
将用于CRE-荧光素酶系统的，稳定表达人GCGR或GLP-1R的HEK-293 细胞，按照每孔98L DMEM/10％FBS培养基、5000个细胞接种到384孔板中。接种后第二天，将2L待测样品梯度转移，与该细胞混合并孵育12小时。在加入细胞以获得剂量应答曲线(从该曲线测定EC50值)之前，一般为待测化合物制备包含从0.005nM到100.0nM的10种稀释液，和艾塞那肽标准品(购买于杭州湃肽生化科技有限公司，纯度>98％，醋酸艾塞那肽，Cas No.:141732-76-5)准备0.005nM到100.0nM的10种标准溶液以及胰高血糖素标准品(购买于Sigma-Aldrich公司，货号和规格：1294036-2X2.94MG，Cas No.:16941-32-5)准备0.01nM到100nM的10种标准溶液。在孵育之后，将10.0L荧光素酶试剂直接加入每块平板中并轻轻混合2分钟。将平板放入Perkin Elmer读板仪读数。利用作图软件Prism 5制作化合物浓度曲线，计算EC50值。
与胃泌酸调节素(Oxyntomodulin,购于上海盈公实业有限公司，货号：Es-1240)相比，本发明化合物具有更高的相对GLP-1R选择性，对膜高血糖素受体和GLP-1受体效力更高。
表2.本发明代表多肽化合物的平均EC50值
Figure PCTCN2015080042-appb-000008
Figure PCTCN2015080042-appb-000009
图1为Exenatide和化合物4,5,6,7对GLP-1R的刺激曲线；
图2为Glucagon和化合物4,5,6,7对GCGR的刺激曲线。
实施例4化合物4,5,6,7,19,20,21,22,23,24和Ex-4药物代谢动力学分析
对于雄性CD1雄性小鼠(南京大学模式动物研究中心)250μg/kg的剂量通过静脉注射(IV)或皮下注射(SC)途径分别使用上述实施例中制备的化合物4,5,6,7,19,20,21,22,23和24与商业购买的艾塞那肽纯品(Ex-4)进行对照试验。在给药后的0-24小时内不同时间将动物进行放血处理，收集每个样本的血浆并用LC-MS/MS测定法分析。使用模型依赖(对于IV所得的数据)和模型非依赖(对于SC所得的数据)的方法计算药物代谢动力学参数。通过IV途径所使用的化合物4,5,6,7,19,20,21,22,23和24的消除半衰期大约为4-6小时，其达峰时间(Tmax)约为12小时，艾塞那肽的消除半衰期大约为0.5小时。而通过SC途径所使用的化合物4,5,6,7,19,20,21,22,23和24的其达峰时间(Tmax)约为12小时以上，而Ex-4消除半衰期大约为2小时。通过IV或SC途径分别使用化合物4,5,6,7,19,20,21,22,23和24均没有临床不良反应发生。
实施例5化合物4,5,6,7,19,20，21,22,23,24和Ex-4对口服葡萄糖耐量的作 用
将12到16周龄的雄性C57B1/6J小鼠(南京大学模式动物研究中心)按照相似的血糖(从尾尖获取的血液样品评估的)随机分组，每组8只。禁食(6小时)后对动物给药，约4小时后，获取初始血液样品(禁食血液葡萄糖水平)。随后，给予口服剂量的葡萄糖，并将动物放回其饲养笼中(t＝0)。在t＝15分钟、t＝30分钟、t＝60分钟、t＝90分钟和t＝120分钟时测量血糖。然后重复给予口服剂量的葡萄糖，跟踪血糖直到8个小时。多肽4,5,6,7,19,20,21,22,23和24(化合物的给药量为：250μg/kg)显著地降低了口服葡萄糖耐量。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(图3和4)。
与载剂(PBS)相比，艾塞那肽(250μg/kg)在第一个OGTT曲线时段(1-120min)其AUC可显著降低(P<0.05)，而在随后的三个OGTT曲线时段(120-480min)，其AUC接近空白对照组(P>0.05)。与载剂(PBS)相比，多肽4，多肽5，多肽6，多肽7，多肽19，多肽20，多肽21，多肽22，多肽23和多肽24在4个OGTT曲线时段(0-480min)其AUC均显著降低(P<0.05)，实验结果提示多肽4，多肽5，多肽6，多肽7，多肽19，多肽20，多肽21，多肽22，多肽23和多肽24具有长效降糖效果。
实施例6化合物4，5，6,7对肥胖小鼠体重的影响，降体重研究
将40只25周大小的DIO鼠(南京大学模式动物研究中心)随机分成5组(化合物4,5,6,7和生理盐水组)，每组8只，基础体重无差异，每组老鼠每天分别皮下注射化合物4(250μg/kg),化合物5(250μg/kg),化合物6(250μg/kg),化合物7(250μg/kg)和生理盐水后称量体重，与生理盐水组比较，11天到30天，合物4,5,6,7组的老鼠体重显著低于生理盐水组(P<0.05)，体重下降约20％左右(图5)。
工业应用性
本发明的胃泌酸调节素类似物，显示出了GCGR和GLP-1R双激动活性，半衰期长且具有高酶解稳定性，高生物活性，无不良发应发生。本发明的化合物合成产率高，稳定性好，易于放大生产，成本低，可以用于制备治疗摄食过量、肥胖症和超重、胆固醇升高以及糖尿病药物。
以上对发明的详细描述并不限制本发明，本领域技术人可以根据本发明做出各种改变和变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (11)

  1. 一种胃泌酸调节素类似物，含有以下氨基酸序列表示的母体肽:
    His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
    其中，R1＝-OH或-NH2；
    Xaa2＝Aib,Ser或D-Ser；
    Xaa10＝Lys或Tyr；
    Xaa13＝Lys或Tyr；
    Xaa16＝Ser,Aib,Lys或Glu；
    Xaa17＝Lys或Arg；
    Xaa18＝Arg或Ala；
    Xaa20＝His,Gln或Lys；
    Xaa21＝Asp或Glu；
    Xaa23＝IIe,Leu或Val；
    Xaa24＝Glu或Gln；
    Xaa27＝Met,Leu或不存在；
    Xaa28＝Ser,Asp,Asn,Arg或不存在；
    Xaa29＝Ala,Gly,Thr或不存在；
    Xaa30＝Gly或不存在；
    Xaa31＝Gly或不存在；
    Xaa32＝Pro或不存在；
    Xaa33＝Ser或不存在；
    Xaa34＝Ser或不存在；
    Xaa35＝Gly或不存在；
    Xaa36＝Ala或不存在；
    Xaa37＝Pro或不存在；
    Xaa38＝Pro或不存在；
    Xaa39＝Pro或不存在；
    Xaa40＝Ser或不存在；
    所述母体肽的氨基酸序列中，Xaa10,Xaa16,Xaa17或Xaa20中至少一个为Lys，所述至少一个Lys或所述序列的第12位Lys的侧链与亲脂性的取代基相连，连接方式为所述亲脂性的取代基以其羧基与一个桥接基团的氨基形成酰胺键，桥接基团的氨基酸残基的羧基与母体肽的Lys的N-末端残基上形成一个酰胺键连接到母体肽上；
    所述桥接基团为Glu，Asp和/或(PEG)m，其中m为2-10的整数；所述亲脂性取代基为选自CH3(CH2)nCO-或HOOC(CH2)nCO-的一个酰基，其中n是10-24的整数。
  2. 根据权利要求1所述的胃泌酸调节素类似物，其特征在于所述桥接基团为Glu-(PEG)m或Asp-(PEG)m或(PEG)m。
  3. 根据权利要求1所述的胃泌酸调节素类似物，其特征在于所述桥接基团在所述氨基酸序列的残基对12与16、16与20、17与21或者20与24侧链之间形成分子桥。
  4. 根据权利要求1所述的胃泌酸调节素类似物，其特征在于与所述亲脂性取代基连接的Lys被HomoLys,Orn,Dap或Dab代替。
  5. 根据权利要求1所述的胃泌酸调节素类似物，其特征在于所述母体肽的氨基酸序列为:
    His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
    其中，R1＝-NH2；
    Xaa2＝Aib或D-Ser；
    Xaa10＝Lys或Tyr；
    Xaa13＝Lys或Tyr；
    Xaa16＝Ser,Aib,Glu或Lys；
    Xaa17＝Lys或Arg；
    Xaa18＝Arg或Ala；
    Xaa20＝His,Gln或Lys；
    Xaa21＝Asp或Glu；
    Xaa23＝IIe,Val；
    Xaa24＝Glu或Gln；
    Xaa27＝Met或Leu；
    Xaa28＝Asn,Asp,Arg或不存在；
    Xaa29＝Gly,Thr或不存在；
    Xaa30＝Gly或不存在；
    Xaa31＝Gly或不存在；
    Xaa32＝Pro或不存在；
    Xaa33＝Ser或不存在；
    Xaa34＝Ser或不存在；
    Xaa35＝Gly或不存在；
    Xaa36＝Ala或不存在；
    Xaa37＝Pro或不存在；
    Xaa38＝Pro或不存在；
    Xaa39＝Pro或不存在；
    Xaa40＝Ser或不存在。
  6. 根据权利要求5所述的胃泌酸调节素类似物，其特征在于所述母体肽的氨基酸序列为选自SEQ ID NO.1,SEQ ID NO.2，SEQ ID NO.，SEQ ID NO.3，SEQ ID NO.4，SEQ ID NO.5，SEQ ID NO.6，SEQ ID NO.7，SEQ ID NO.8，SEQ ID NO.9，SEQ ID NO.10，SEQ ID NO.11，SEQ ID NO.12，SEQ ID NO.13，SEQ ID NO.14，SEQ ID NO.15，SEQ ID NO.16，SEQ ID NO.17和SEQ ID NO.18,SEQ ID NO.19，SEQ ID NO.20，SEQ ID NO.21，SEQ ID NO.22，SEQ ID NO.23和SEQ ID NO.24的序列。
  7. 根据权利要求1或5所述的胃泌酸调节素类似物，其特征在于当所述氨基酸序列的第10、12、16、17或20位为Lys时，与所述Lys侧链连接的亲脂性取代基为下述结构之一：
    Figure PCTCN2015080042-appb-100002
  8. 一种药物组合物，含有权利要求1或5所述的胃泌酸调节素类似物。
  9. 权利要求1所述的胃泌酸调节素类似物在制备治疗糖尿病药物中的应用。
  10. 权利要求1所述的胃泌酸调节素类似物在制备降糖药物中的应用。
  11. 权利要求1所述的胃泌酸调节素类似物在制备降低体重药物中的应用。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019520314A (ja) * 2016-04-22 2019-07-18 深▲せん▼市図微安創科技開発有限公司Ｓｈｅｎｚｈｅｎ Ｔｕｒｉｅｒ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． 脂肪性肝疾患、高脂血症及び動脈硬化治療用のｇｌｐ−１ｒ／ｇｃｇｒデュアルアゴニストペプチド
CN110357959A (zh) * 2018-04-10 2019-10-22 鸿运华宁（杭州）生物医药有限公司 Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
WO2020109526A2 (en) 2018-11-30 2020-06-04 Opko Ireland Global Holdings, Ltd. Oxyntomodulin peptide analog formulations
CN111372945A (zh) * 2017-11-06 2020-07-03 中山大学 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗特发性肺间质纤维化
JP2020531442A (ja) * 2017-08-16 2020-11-05 ドン−ア エスティ カンパニー リミテッド アシル化オキシントモジュリンペプチド類似体
CN112898405A (zh) * 2021-01-22 2021-06-04 深圳市图微安创科技开发有限公司 多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用
CN114920818A (zh) * 2017-11-06 2022-08-19 深圳市图微安创科技开发有限公司 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗胆汁性肝硬化

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA116217C2 (uk) 2012-10-09 2018-02-26 Санофі Пептидна сполука як подвійний агоніст рецепторів glp1-1 та глюкагону
JP2016503771A (ja) 2012-12-21 2016-02-08 サノフイ エキセンジン−４誘導体
EP3080150B1 (en) 2013-12-13 2018-08-01 Sanofi Exendin-4 peptide analogues as dual glp-1/gip receptor agonists
EP3080149A1 (en) 2013-12-13 2016-10-19 Sanofi Dual glp-1/glucagon receptor agonists
WO2015086729A1 (en) 2013-12-13 2015-06-18 Sanofi Dual glp-1/gip receptor agonists
TW201609796A (zh) 2013-12-13 2016-03-16 賽諾菲公司 非醯化之艾塞那肽－４（ｅｘｅｎｄｉｎ－４）胜肽類似物
TW201625668A (zh) 2014-04-07 2016-07-16 賽諾菲公司 作為胜肽性雙重ｇｌｐ－１／昇糖素受體激動劑之艾塞那肽－４衍生物
TW201625670A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自ｅｘｅｎｄｉｎ－４之雙重ｇｌｐ－１／升糖素受體促效劑
TW201625669A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自艾塞那肽－４（Ｅｘｅｎｄｉｎ－４）之肽類雙重ＧＬＰ－１／升糖素受體促效劑
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
AR105319A1 (es) 2015-06-05 2017-09-27 Sanofi Sa Profármacos que comprenden un conjugado agonista dual de glp-1 / glucagón conector ácido hialurónico
AR105284A1 (es) 2015-07-10 2017-09-20 Sanofi Sa Derivados de exendina-4 como agonistas peptídicos duales específicos de los receptores de glp-1 / glucagón
CN108602894A (zh) * 2015-10-30 2018-09-28 纽约州立大学研究基金会 胃泌酸调节素类似物及其制备和使用方法
CN108299553B (zh) * 2017-01-13 2021-07-16 博瑞生物医药（苏州）股份有限公司 胃泌酸调节素修饰物
CN108299554B (zh) * 2017-01-13 2021-05-25 博瑞生物医药（苏州）股份有限公司 胃泌酸调节素类似物
CN106986924A (zh) * 2017-03-23 2017-07-28 中国药科大学 胃泌酸调节素（oxm）类似物及其应用
US20230183310A1 (en) * 2020-02-24 2023-06-15 Shenzhen Turier Biotech Co., Ltd. Polypeptide Compounds and Use Thereof in the Prevention or Treatment of Diabetes or Diabetic Complications
AR122579A1 (es) * 2020-06-12 2022-09-21 Lilly Co Eli Proceso para preparar un agonista dual glp-1 / glucagón
US20230364194A1 (en) * 2020-09-28 2023-11-16 Shenzhen Turier Biotech. Co., Ltd. GLP-1R/GCGR Dual Target Agonist Polypeptide Derivative for Treatment of Hepatic Fibrosis Associated with Viral Hepatitis
WO2022117044A1 (en) * 2020-12-03 2022-06-09 Sunshine Lake Pharma Co., Ltd. Glp-1/gcg dual receptor agonist polypeptide and fusion protein thereof
WO2022156189A1 (zh) * 2021-01-22 2022-07-28 深圳市图微安创科技开发有限公司 多肽化合物在预防或治疗炎症性肠病及其相关的肠纤维化中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101389648A (zh) * 2006-02-22 2009-03-18 默克公司 肽胃泌酸调节素衍生物
CN103764673A (zh) * 2011-06-10 2014-04-30 北京韩美药品有限公司 葡萄糖依赖性促胰岛素多肽类似物、其药物组合物及应用

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101349808B1 (ko) * 2005-06-13 2014-02-13 임페리얼 이노베이션스 리미티드 섭취행동에 관한 신규 화합물들 및 이들의 효능
GB0511986D0 (en) * 2005-06-13 2005-07-20 Imp College Innovations Ltd Novel compounds and their effects on feeding behaviour
TWI428346B (zh) * 2006-12-13 2014-03-01 Imp Innovations Ltd 新穎化合物及其等對進食行為影響
NZ593811A (en) 2008-12-15 2013-03-28 Zealand Pharma As Glucagon analogues
AU2008365556A1 (en) * 2008-12-15 2011-07-21 Zealand Pharma A/S Glucagon analogues
WO2010096052A1 (en) * 2009-02-19 2010-08-26 Merck Sharp & Dohme Corp. Oxyntomodulin analogs
AU2010272944B2 (en) 2009-07-13 2015-11-19 Zealand Pharma A/S Acylated glucagon analogues
EP2758426B1 (en) * 2011-09-23 2019-08-07 Novo Nordisk A/S Novel glucagon analogues
JP6290193B2 (ja) * 2012-06-04 2018-03-07 オプコ バイオロジクス リミテッド ペグ化ｏｘｍ変異体
JP6311708B2 (ja) * 2012-06-21 2018-04-18 インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションＩｎｄｉａｎａ Ｕｎｉｖｅｒｓｉｔｙ Ｒｅｓｅａｒｃｈ Ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ Ｃｏｒｐｏｒａｔｉｏｎ Ｇｉｐ受容体活性を示すグルカゴンアナローグ
TWI617574B (zh) * 2012-12-11 2018-03-11 梅迪繆思有限公司 用於治療肥胖之升糖素與ｇｌｐ－１共促效劑

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101389648A (zh) * 2006-02-22 2009-03-18 默克公司 肽胃泌酸调节素衍生物
CN103764673A (zh) * 2011-06-10 2014-04-30 北京韩美药品有限公司 葡萄糖依赖性促胰岛素多肽类似物、其药物组合物及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARALYN R. DRUCE ET AL.: "Investigation of Structure-Activity Relationships of Oxyntomodulin (Oxm) Using Oxm Analogs", ENDOCRINOLOGY, vol. 150, no. 4, 30 April 2009 (2009-04-30), XP009116621, DOI: doi:10.1210/en.2008-0828 *

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* Cited by examiner, † Cited by third party
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EP3447067A4 (en) * 2016-04-22 2020-01-01 Shenzhen Turier Biotech Co., Ltd. GLP-1R / GCGR-DUALTARGET AGONIST POLYPEPTIDE FOR THE TREATMENT OF FATTY LIVER, HYPERLIPIDEMIA AND ATHEROSCLEROSIS
JP2019520314A (ja) * 2016-04-22 2019-07-18 深▲せん▼市図微安創科技開発有限公司Ｓｈｅｎｚｈｅｎ Ｔｕｒｉｅｒ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． 脂肪性肝疾患、高脂血症及び動脈硬化治療用のｇｌｐ−１ｒ／ｇｃｇｒデュアルアゴニストペプチド
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JP7211659B2 (ja) 2017-08-16 2023-01-24 ドン－ア エスティ カンパニー リミテッド アシル化オキシントモジュリンペプチド類似体
JP2020531442A (ja) * 2017-08-16 2020-11-05 ドン−ア エスティ カンパニー リミテッド アシル化オキシントモジュリンペプチド類似体
JP2022023179A (ja) * 2017-08-16 2022-02-07 ドン－ア エスティ カンパニー リミテッド アシル化オキシントモジュリンペプチド類似体
CN111372945A (zh) * 2017-11-06 2020-07-03 中山大学 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗特发性肺间质纤维化
CN114920818A (zh) * 2017-11-06 2022-08-19 深圳市图微安创科技开发有限公司 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗胆汁性肝硬化
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