WO2021227989A1 - 具有双受体激动作用的多肽衍生物及其用途 - Google Patents

具有双受体激动作用的多肽衍生物及其用途 Download PDF

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WO2021227989A1
WO2021227989A1 PCT/CN2021/092406 CN2021092406W WO2021227989A1 WO 2021227989 A1 WO2021227989 A1 WO 2021227989A1 CN 2021092406 W CN2021092406 W CN 2021092406W WO 2021227989 A1 WO2021227989 A1 WO 2021227989A1
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derivative
polypeptide
salt
gly
group
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PCT/CN2021/092406
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French (fr)
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韩英梅
刘巍
刘冰妮
孔维苓
赵娜夏
夏广萍
商倩
靳京
孔晓华
李玉荃
王松会
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天津药物研究院有限公司
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Priority to JP2022567566A priority Critical patent/JP2023525260A/ja
Priority to KR1020227043172A priority patent/KR20230008846A/ko
Priority to US17/923,936 priority patent/US20230174608A1/en
Priority to EP21803512.9A priority patent/EP4148064A4/en
Publication of WO2021227989A1 publication Critical patent/WO2021227989A1/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
    • C07K14/435—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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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

Definitions

  • the invention belongs to the field of medical biotechnology, and specifically relates to a glucagon-derived peptide with a glucagon/glucagon-like peptide-1 dual receptor agonistic effect and its use.
  • Obesity is a risk factor for many diseases and has become a global public health problem.
  • diseases such as metabolic syndrome, cardiovascular disease, and non-alcoholic fatty liver, including type 2 diabetes mellitus (T2DM)
  • T2DM type 2 diabetes mellitus
  • a number of large-sample clinical studies have found that compared with people of normal weight, the incidence of cardiovascular and metabolic multiple diseases is overweight with BMI 25.0-29.9kg/m 2 , 30.0-34.9kg/m 2 and BMI>35.0kg/m 2
  • People with obesity, obesity or severe obesity are 2 times, 5 times, and 15 times higher respectively (Lancet 2, e277–e285, 2017).
  • Studies have shown that 80 to 90% of T2DM patients are overweight or obese.
  • a moderate weight loss (4 to 5 kg) is beneficial to the prevention and control of the disease, including reducing the prevalence rate, controlling blood sugar and disabling (death) rate (Curr.Med. Res. Opin. 2011, 27(7), 1431-1438).
  • Pro-glucagon is a precursor polypeptide with 158 amino acids, which is processed in different tissues to produce glucagon (GC), glucagon-like peptide-1, 2 (GLP -1, 2) and a variety of proglucagon derived peptides such as gastrin.
  • GC glucagon
  • GLP -1, 2 glucagon-like peptide-1, 2
  • gastrin proglucagon derived peptides
  • These hormones are involved in the regulation of various physiological functions such as glucose homeostasis, insulin secretion, gastric emptying, intestinal growth and food intake. Therefore, the treatment of intestinal hormones based on proglucagon has become a research direction in the field of metabolic diseases.
  • GC is a 29 amino acid derivative peptide composed of amino acids at positions 33 to 61 corresponding to proglucagon. It is processed by pancreatic alpha cells and acts on the liver under stress conditions such as starvation and cold. Gluconeogenesis raises blood sugar levels to the normal range. In addition to the effect of raising blood sugar, animal and human test results show that GC also has the effects of fever, increased satiety, lipolysis, fat oxidation, ketogenesis, etc. Long-term administration can improve energy metabolism, including weight loss, but these effects have an impact on energy metabolism. The beneficial effect of saccharomyces cerevisiae has not been applied due to its inherent glycemic effect.
  • GLP-1 is a 37 amino acid residue derived peptide corresponding to amino acids 72 to 108 of proglucagon. It is secreted by intestinal L cells in response to meals and acts on pancreatic ⁇ -cells to promote insulin secretion. , At the same time antagonize the GC receptor to inhibit the increase in blood sugar. GLP-1 receptor agonists have been developed as hyperglycemia therapeutics for diabetic patients. They protect and proliferate islet cells while lowering blood sugar, slow down gastric emptying and inhibit food intake, which can effectively reduce weight.
  • GLP-1 receptor agonists There are already 7 GLP-1 receptor agonists on the market, including short-acting exenatide, liraglutide, lixisenatide (1 to 2 times/day), and long-acting abilutide, duo Laglutide, Byuderon, and Somaglutide (1 time/week).
  • GLP-1 receptor agonist drugs have a safe and unique hypoglycemic effect, they generally need to be used in large doses when used to reduce weight. These drugs are prone to gastrointestinal side effects at large doses, and are poorly tolerated. The window is narrow. Therefore, there is still a need for more tolerated therapeutic agents that can effectively control blood sugar and reduce weight.
  • Oxintomodulin is a hormone produced in the intestine during the post-translational modification and processing of proglucagon. It is secreted simultaneously from ileum L-cells and hormones such as GLP-1 in the meal response.
  • the acute effects of OXM include inhibition of gastric emptying, gastric and pancreatic exocrine and food intake, resting energy expenditure, etc., which can reduce body weight.
  • the specific receptor of OXM has not yet been clarified, but studies have found that OXM is an endogenous GCGR/GLP-1R dual agonist, and its activity on both receptors is weaker than the natural ligands of each receptor.
  • peripheral administration of OXM can reduce food intake and weight loss, and increase the metabolic rate and especially energy expenditure related to activities in obese subjects.
  • high-dose peripheral administration of OXM can reduce weight while reducing the incidence of common gastrointestinal side effects such as nausea and vomiting. Therefore, treatments based on OXM or GLP-1/GCGR dual agonists have shown potential application value for obesity and obese diabetes, but so far, no relevant drugs have been marketed.
  • An object of the present invention is to provide a glucagon polypeptide derivative, which is a variant designed based on the natural sequence of GC, which has a synergistic effect on energy metabolism through the dual agonism of the GC/GLP-1 receptor, It can effectively reduce blood sugar, reduce weight, improve body fat levels, and can be used for the treatment of diabetes, obesity, metabolic syndrome, and non-alcoholic fatty liver disease.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the glucagon polypeptide derivative of the present invention.
  • Another object of the present invention is to provide a use of the glucagon polypeptide derivative of the present invention.
  • the present invention provides a polypeptide derivative, a modified derivative thereof, or a salt thereof, which comprises a polypeptide having the sequence of the following general formula I:
  • X 2 is Ser, D-Ser or Aib
  • X 10 is Tyr
  • X 15 is Asp or Glu
  • X 17 is Arg, Gln or Lys
  • X 18 is Ala
  • X 20 is Lys with modified side chain
  • X 21 is Asp or Glu
  • X 23 is Val or Ile
  • X 28 is Ala, Gly or Ser
  • X 29 is Gly or Glu
  • X 30 is Gly or does not exist
  • the C-terminal carboxyl group is free or amidated.
  • X 17 is Arg or Lys
  • X 28 is Ala or Ser
  • X 29 is Gly
  • X 30 is Gly
  • X 2 is Aib.
  • sequence of the general formula I is selected from:
  • K* is Lys whose side chain ⁇ -amino group is modified, and the C-terminal carboxyl group is amidated.
  • X 17 is Arg or Lys
  • X 28 is Gly
  • X 29 is Gly
  • sequence of the general formula I is selected from:
  • K* is Lys whose side chain ⁇ -amino group is modified, and the C-terminal carboxyl group is amidated.
  • X 17 is Arg or Lys
  • X 28 is Gly
  • X 29 is Glu
  • X 30 is Gly
  • sequence of the general formula I is selected from:
  • K* is Lys whose side chain ⁇ -amino group is modified, and the C-terminal carboxyl group is amidated.
  • the side chain modified Lys means that the side chain ⁇ -amino group of the Lys is modified by coupling a fatty acyl group through a hydrophilic linking fragment.
  • the hydrophilic linking fragment used to modify the ⁇ -amino group of the side chain of Lys is selected from one or more of Glu, ⁇ Glu, Gly and Ado (8-amino-3,6 dioxyoctanoic acid) Fragments.
  • the hydrophilic linking fragments are preferably - ⁇ Glu-, - ⁇ Glu- ⁇ Glu-, -Glu- ⁇ Glu-, - ⁇ Glu-Gly-Gly-, - ⁇ Glu-Gly- ⁇ Glu-, - ⁇ Glu-Ado-Ado-,- Ado-Ado- ⁇ Glu- or - ⁇ Glu-Ado-Ado- ⁇ Glu-.
  • the fatty acyl group is a C 14-20 fatty acyl group, including a C 14-20 mono-fatty acyl group or a fatty diacid mono-acyl group; more preferably C 16-18 fatty acyl group, most preferably C 16 Monofatty acyl (palmitoyl).
  • the present invention provides a pharmaceutical composition, which contains the polypeptide derivative of the present invention, its modified derivative or its salt and optionally one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable auxiliary materials include carriers, diluents, water-soluble fillers, pH regulators, stabilizers, water for injection, osmotic pressure regulators and the like.
  • the water-soluble filler includes but is not limited to mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose and galactose;
  • the pH adjusting agent includes but not limited to citric acid, phosphoric acid, Organic or inorganic acids such as lactic acid, tartaric acid, hydrochloric acid, and physiologically acceptable such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate, etc.
  • the stabilizer includes but not limited to EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid , Aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxy cellulose or its derivatives such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sulfate Sodium or tris; the osmotic pressure regulator includes but is not limited to sodium chloride or potassium chloride.
  • the pharmaceutical composition of the present invention can be administered in the form of intravenous, intramuscular or subcutaneous injection or oral, rectal, or nasal administration.
  • the therapeutic dose range depends on the object to be treated, the method of administration, indications, and other factors.
  • the present invention provides the use of the polypeptide derivatives, modified derivatives or salts thereof of the present invention in the preparation of drugs for the treatment of metabolic diseases.
  • the metabolic diseases are diabetes, obesity, Fatty liver, hyperlipidemia and/or metabolic syndrome; more preferably, the fatty liver is non-alcoholic fatty liver.
  • the present invention provides a method for treating metabolic diseases, comprising administering to a patient in need a therapeutically effective amount of the polypeptide derivative of the present invention, its modified derivative or salt thereof, or the pharmaceutical combination of the present invention ⁇ ;
  • the metabolic disease is diabetes, obesity, fatty liver, hyperlipidemia and/or metabolic syndrome; more preferably, the fatty liver is non-alcoholic fatty liver.
  • endogenous GLP-1 is a derivative peptide containing 37 amino acid residues (7-36/37) corresponding to amino acids 72 to 108 of proglucagon, its amino acids
  • the sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR(7-36)(SEQ ID NO.1), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(7-37), and its C-terminal carboxyl group is free or amidated.
  • Endogenous GC is a 29-amino acid derivative peptide corresponding to amino acids 33 to 61 of proglucagon.
  • the amino acid sequence is: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO.
  • GLP-1 pair Its receptor is highly selective, and GC is also a weak agonist of GLP-1 receptor.
  • Oxinthomodulin is an endogenous GC/GLP-1R dual agonist, its amino acid sequence is HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO.3), containing the original sequence of GC (1-29) and corresponding to pancreas Insert peptide-1 (IP-1, 30-37) of the amino acid sequence 82-89 of proglucagon.
  • CN201680036771.3 is a design scheme that selects the C-terminal insertion peptide (KRNRNNIA) of the OXM sequence and replaces it with the GGPSSG peptide.
  • the existing public information When designing dual agonists based on the GC sequence, in order to obtain the expected titer balance, the existing public information often adopts the replacement of the 16-23 fragments of the GC sequence similar to the corresponding GLP-1 sequence, or replaces 16, 17, 18, Mutations at individual sites such as 20 and 23.
  • the C-terminal changes of GC sequence are extremely sensitive to the recognition of GC/GLP-1 receptors, especially to the activity of GC receptors. Therefore, the existing technical solutions generally choose to retain the original sequence of the C-terminal 27-29 fragment, and only a few technical solutions take conservative mutation measures, such as replacing the sensitive amino acid 27Met with Leu and 28Asn with Glu or Ala.
  • the long-chain fatty acyl modification on the polypeptide sequence is generally to extend the in vivo half-life of the polypeptide, but when applied to the design of GC/GLP-1 dual receptor agonists, the modification at the appropriate site can adjust the receptor selectivity and activity balance.
  • the general measure taken in the prior art data is to modify the palmitoyl group on the ⁇ -side chain of the 10 Lys mutant residue to obtain a strong agonist with balanced potency (EC50pM).
  • E50pM balanced potency
  • the design of the polypeptide backbone is based on rational mutations at individual sites of the 1-26 peptide segment of the GC sequence, and then the C-terminus is appropriately extended, and then side chain modification measures are taken, such as using 20-position fat.
  • Acyl modification yields a GC/GLP-1 dual receptor agonist with balanced activity and titer ratio and good solubility and stability.
  • positions 16-20 of the GC(1-26) sequence are reasonably replaced, such as replacing 16Ser with Glu, and replacing 18Arg with Ala or Lys, etc., which is beneficial to increase GLP-1.
  • Receptor-selective mutations Different from the conservative substitution schemes for the C-terminus of the GC sequence in most of the disclosed technical schemes, the preferred technical scheme of the present invention replaces 27Met with Glu, and then replaces positions 28 and 29 with electrically neutral amino acids such as Gly and Ala. It is generally believed that the C-terminal carboxyl group of the GC sequence is free to maintain the activity.
  • replacing 27Met with Glu retains the negative charge of the C-terminal and reduces the impact on the activity, and further amidates the C-terminal to improve the stability of the peptide sequence.
  • the N-terminal dipeptide is easily affected by the two in the body.
  • Peptide kininase recognizes and is hydrolyzed and inactivated, resulting in a short plasma half-life ( ⁇ 12min); its physical properties are unstable, that is, its isoelectric point (pI) is 7.6, which is neutral, hydrophobic, and has poor solubility, and it is easy to aggregate in solution
  • pI isoelectric point
  • Precipitation Met, Asp, Asn and other amino acids that are easy to oxidize or racemize in the sequence, resulting in unstable chemical properties.
  • the 2Ser of the polypeptide sequence is usually replaced with Aib or D-Ser to improve metabolic stability and ensure the continuous exertion of polypeptide activity.
  • the sensitive amino acids in the GC sequence are further replaced, for example, Asp at positions 15, 21 is replaced with Glu.
  • the adjusted polypeptide sequence increases the number of negative charges at the C-terminus of the sequence, reduces the pI value, and helps to improve the solubility of the peptide under physiological conditions and improve chemical stability.
  • a fatty acyl group is modified by a hydrophilic linker on the side chain of Lys at position 20 of the peptide sequence.
  • the hydrophilic linking arm is - ⁇ -Glu-, - ⁇ -Glu- ⁇ -Glu- or in other embodiments, -Ado-Ado- ⁇ -Glu- .
  • the fatty acyl group is a C 14-20 fatty acyl group, preferably a C 14-20 mono-fatty acyl group or a fatty diacid mono-acyl group. In certain preferred embodiments, the fatty acyl group is a C 16 or C 18 mono fatty acyl group.
  • the present invention provides a peptide with GLP-1/glucagon dual receptor agonism.
  • the peptide has the following active characteristics: the polypeptide provided by the present invention and the receptors of GLP-1R/GCGR Compared with the natural ligands, it has at least 0.1% receptor agonistic activity, such as the activity represented by EC 50 (nM) in the examples of the present invention.
  • the EC 50 (nM) value of the compound provided by the present invention includes but is not limited to 10 -2 to 10 2 times that of the endogenous ligand (GLP-1).
  • the preferred activity intensity is equivalent to that of the endogenous ligand (GLP-1).
  • the intensity of the agonistic activity on the glucagon receptor (GCGR) is expressed in EC 50 (nM) value, which is equivalent to or equivalent to 1 to 103 times that of the endogenous ligand (GC).
  • GCGR glucagon receptor
  • nM EC 50
  • the agonistic effect of the compound of the present invention on the GLP-1 receptor is stronger than the agonistic effect on the glucagon receptor, or the agonistic effect on the two receptors is comparable.
  • the relative activity intensity of GLP-1 and glucagon receptor can be expressed by the titer ratio (GLP-1R EC50 /GCGR EC50 ), that is, the polypeptide comprising the sequence of general formula I provided by the present invention has a positive effect on GLP-1/pancreas.
  • the potency ratio of the glucagon receptor includes but is not limited to 10:1 to 1:100. In a preferred embodiment of the present invention, the ratio is in the range of 10:1 to 1:10, and a more preferred range is 5:1 to 1:10.
  • the basic peptide chain of the polypeptide derivative with the sequence of the general formula I provided by the present invention can be prepared by a method known in the art:
  • the target peptide is prepared using the Fmoc solid-phase synthesis method, which is well known to those skilled in the art.
  • Substituents can be synthetically introduced step by step through the above-mentioned peptide synthesis steps.
  • Use appropriate protecting group substituents such as Fmoc-8-amino-3,6-dioxaoctanoic acid, and Fmoc- ⁇ -Glu-OtBu.
  • the introduction of the fatty chain part, especially the fatty diacid monoacyl group can be achieved by using, but not limited to, C 18 , C 20 alkanoic acid mono-tert-butyl ester.
  • the unreacted intermediate can be blocked with excess acetic anhydride and pyridine.
  • the ⁇ -amino group of the modifiable Lys can be protected with Mtt or Dde.
  • the target product can be separated by a suitable method known in the art. Applicable methods include but are not limited to ultrafiltration, dialysis or chromatography. In the embodiment of the present invention, preparative high performance liquid chromatography is preferably used for purification.
  • Receptor activity determination In the embodiment of the present invention, the effect of in vitro cAMP production mediated by GLP-1/GC receptor is evaluated for the effect of the polypeptide on GLP-1/GC receptor.
  • a single administration of a normal glucose load mouse model and continuous administration of a high-fat diet obese diabetic mouse (Dio) model are used to evaluate the effect of the polypeptide provided by the present invention on blood glucose. And weight.
  • the experimental results of the present invention show that the compound provided by the technical scheme of the present invention has the same activity on GLP-1 receptor (GLP-1R) as endogenous GLP-1, and retains certain activity on GC receptor (GCGR). , The potency ratio is in the appropriate range. Compared with a simple GLP-1 receptor agonist, the compound provided by the technical scheme of the present invention can effectively reduce blood sugar while more significantly promoting weight loss and preventing weight gain, and can reverse or alleviate liver fatty disease and insulin resistance. , Has shown unexpected multiple benefits for complex metabolic syndrome.
  • GLP-1R GLP-1 receptor
  • GCGR GC receptor
  • Figure 1 shows the effects of compounds dgc016, dgc005, and dgc020 on the body weight of DIO mice;
  • Figure 2 shows the effects of compounds dgc005, dgc016, and dgc020 administered continuously for 23 days on liver lesions in DIO model mice;
  • Figure 2A shows the liver pathological examination of the blank control group
  • Figure 2B shows the model control group
  • Figure 2C shows the liver pathological section of the positive control drug group
  • Figure 2D shows the liver pathological section of the compound dgc005 group
  • Figure 2E shows the liver pathological section of the compound dgc016 group Inspection chart
  • Fig. 2F shows the liver pathological biopsy chart of the compound dgc020 group;
  • Figure 3 shows the effect of compounds dgc003, dgc004, dgc010, and dgc017 on the body weight of DIO model mice after continuous administration for 13 days.
  • Figure 4 shows the effect of compound dgc005 and dgc016 administered in divided doses for 21 days on the body weight of DIO model mice.
  • Trp Tryptophan (W)
  • HATU O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-Tetramethyl-uronium hexafluorophosphate
  • FBS Fetal Bovine Serum
  • the basic linear sequence of the polypeptide provided by the present invention and the side chain modified derivative peptide are prepared according to the following general methods:
  • Fmoc-amino acid-resin is obtained by coupling the resin solid phase carrier and the C-terminal amino acid protected by Fmoc in the presence of the activator system; among them, the C-terminal amidated peptides are synthesized using amino resins, such as Rink Amide AM, Rink Amide , Rink MBHA, etc.; Fmoc-amino acid to resin ratio (mol/mol) is 3 ⁇ 5:1, coupling activator is HOBT/DIC or HOBT/HBTU/DIEA.
  • the coupling activators used were HOBT/DIC, HOBT/HBTU/DIEA and HOBT/HATU/DIEA, the ninhydrin method was used to detect the end of the reaction, and the deprotecting agent was a NMP (DMF) solution containing 20% piperidine.
  • the effect of the polypeptide on the GLP-1/GC receptor was evaluated by the effect on the GLP-1/GC receptor-mediated cAMP production in vitro.
  • the HEK293 cell line stably expressing GLP-1R and GCGR is used for the screening of GLP-1R agonists and GCGR agonists.
  • test sample The compound was prepared into a 100 ⁇ M stock solution and gradually diluted to working concentrations of 1 ⁇ M, 100nM, 10nM, 1nM, 10 -1 nM, 10 -2 nM, 10 -3 nM, H 2 O.
  • Working concentration 1 ⁇ M, 100nM, 10nM, 1nM, 10 -1 nM, 10 -2 nM, 10 -3 nM and H 2 O (each well contains 0.1% BSA).
  • Working concentration 1 ⁇ M, 100nM, 10nM, 1nM, 10 -1 nM, 10 -2 nM, 10 -3 nM and DMSO (each well contains 1% DMSO).
  • DMEM medium (GIBCO, Cat No: 12800017);
  • test compound 1) Mix the test compound with serum-free culture solution (containing 0.1% BSA, 0.5 mM IBMX) to 2 times the working concentration.
  • Example 3 The hypoglycemic effect of the compound of Example 1
  • the hypoglycemic effect of the compound of Example 1 was evaluated by using a single-dose glucose load test in normal mice.
  • Kunming mice male, weight range 20-22g were adaptively reared in a barrier environment animal room for 3 days and then divided into a blank control group, a model control group, a positive control group and a test compound group, each with 5 mice , Fasting for 6h.
  • the blank control group and the model control group were given physiological saline, the test compound group and the positive control group were administered at a dose of 30 nmol/kg, and the positive control drug was semaglutide.
  • the model control group, the positive control group, and the test compound group were given glucose by gavage at 2.5 g/kg (0.01 mL ⁇ g -1 body weight), and the blank control group was given distilled water. Blood sugar level.
  • Table 3 The results are shown in Table 3.
  • test compound had a significant inhibitory effect on the increase of glucose load blood glucose in normal mice at the administered dose (P ⁇ 0.01).
  • the inhibitory effect of the compounds dgc005, dgc016 and dgc006 was comparable to that of the positive control drug. quite.
  • the model group was randomly divided into 5 groups according to body weight (weight range 39.5-49.5g), each with 5 animals, namely the model control group, the positive control group, and the test compound group (dgc005, dgc016, dgc020) is administered subcutaneously every day, the blank control group and model control group are injected subcutaneously with saline every day for 23 days.
  • the dose of the positive control drug and the test compound are both 30nmol/kg, and the volume of each group is 0.005mL/g body weight.
  • the body weight was weighed before each administration, and the food intake was weighed every 2 days for 72 hours. Fasting for 16 hours before the last administration, the fasting blood glucose level was measured, blood was collected from the orbital venous plexus to determine triglycerides and total cholesterol; the liver was taken for pathological observation.
  • the compound dgc005 group showed a certain effect of reducing total cholesterol (TC) (Compared with the model control group, P ⁇ 0.05, compared with the positive control group, P ⁇ 0.05, compared with the blank control group, there is no statistical difference), and it can significantly reduce Triglycerides (TG) (P ⁇ 0.01 compared with the model control group, P ⁇ 0.05 compared with the positive control group, P ⁇ 0.01 compared with the blank control group).
  • TC total cholesterol
  • TG Triglycerides
  • the compound dgc016 group showed the effect of reducing triglycerides (P ⁇ 0.01 compared with the model control group, no statistical difference compared with the positive control group, P ⁇ 0.01 compared with the blank control group).
  • the livers of C576L/6 mice in each group were fixed with 12% formaldehyde solution to prepare specimens.
  • the tissues were repaired, dehydrated by gradient alcohol, embedded in paraffin, HE stained, and examined under light microscope.
  • Blank control group 2 animals with complete liver lobule structure, neat arrangement of hepatocyte cords, clear liver cell morphology, no obvious pathological changes in the portal area and interstitium (see Figure 2A).
  • Model control group Severe lesions were seen in 2 animals: diffuse vacuolar degeneration of hepatocytes, and some perivascular mononuclear cell infiltration (2/2, severe) (see Figure 2B).
  • Positive control group Mild to moderate lesions were seen in 2 animals: few to more vacuolar degeneration of hepatocytes, one animal was partially infiltrated by mononuclear cells around blood vessels, and local clear hepatocyte foci (1/2, (Slight, 1/2, Moderate) (see Figure 2C).
  • Compound dgc005 group 2 animals had complete liver lobules, neatly arranged hepatocyte cords, clear liver cell morphology, and no obvious pathological changes in the portal area and interstitium (see Figure 2D).
  • Compound dgc016 group 2 animals had complete liver lobules, neatly arranged hepatocyte cords, clear liver cell morphology, and no obvious pathological changes in the portal area and interstitium (see Figure 2E).
  • Compound dgc020 group 2 animals had complete liver lobules, neatly arranged hepatocyte cords, clear liver cell morphology, and no obvious pathological changes in the portal area and interstitium (see Figure 2F).
  • liver of this experimental model control group can be seen: different degrees of hepatocyte vacuolar degeneration, some peripheral mononuclear cells infiltration, showing severe disease, the positive control group liver can also see the above Lesions, the degree of lesions were significantly reduced, and showed mild to moderate lesions, indicating that this model has a certain therapeutic effect on liver lesions.
  • the structure of liver lobules is complete, the hepatocyte cords are arranged neatly, the morphology of hepatocytes is clear, and no obvious pathological changes are seen in the portal area and interstitium, which is indistinguishable from the blank control group. The effect of treatment of lesions is obvious.
  • the day before the first administration the model group was randomly divided into 6 groups according to body weight (weight range 40.5-50.5g), each with 5 animals, namely the model control group, the positive control group, and the test compound group (dgc003, dgc004, dgc010, dgc017) were administered by subcutaneous injection once a day.
  • the blank control group and the model control group were injected subcutaneously with saline daily for 14 days.
  • the doses of the positive control drug and the test compound were both 30nmol/kg, and each group was given
  • the medicine volume is 0.005mL/g body weight.
  • the body weight was weighed before each administration, and the food intake was weighed every 2 days for 72 hours.
  • mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • 40 mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • H10060 high-fat diet to induce obesity (DIO)
  • the model group was randomly divided into 7 groups according to body weight (weight range 45-60g), each with 5 animals, namely the model control group and the positive control group (semaglutide, semaglutide)-10nmol/kg , Positive control group -30nmol/kg, dgc005-10nmol/kg, dgc005-30nmol/kg, dgc016-10nmol/kg, dgc016-30nmol/kg, the positive control group and the test compound group are injected subcutaneously every day, the blank control group
  • the model control group was injected with saline subcutaneously every day for 22 days (the volume of administration in each group was 0.005 mL/g body weight).
  • the body weight was weighed before each administration, and the food intake was weighed every 2 days for 72 hours. Fasting for 16 hours before the last administration, and intragastric administration of glucose solution (1 g/kg, administration volume of 0.01 mL/g body weight) 30 min after the administration for glucose tolerance test, and the blood glucose value was measured 0.5 h after the administration.
  • each dose group of the test compound can significantly reduce the fasting blood glucose value of the animal before the administration.
  • each dose group of the test compound has a significant inhibitory effect on the increase of the animal glucose load blood glucose value (with the model Compared with the control group (P ⁇ 0.01, there is no statistical difference compared with the positive control group and the blank control group), indicating that the long-term administration of the test compound still has a blood sugar lowering effect equivalent to that of the positive control drug.
  • the results are shown in Table 8.
  • ⁇ P ⁇ 0.01 compared with the blank control group; ⁇ : P ⁇ 0.05 compared with the blank control group.

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Abstract

本发明提供一种多肽衍生物、其修饰衍生物或其盐，以及所述多肽衍生物、其修饰衍生物或其盐的用途。所述多肽衍生物、其修饰衍生物或其盐包含具有以下通式Ⅰ的序列的多肽：通式Ⅰ：HX2QGTFTSDX10SKYLX15EX17X18AX20X 21FX23AWLEX28X29X30，其中，X2、X10、X15、X17、X18、X20、X21、X23、X28、X29、X30的定义与权利要求书和说明书的定义一致。本发明的多肽衍生物是GC/GLP-1受体的双重激动剂，对能量代谢产生协同影响，能够有效降低血糖同时减轻体重、改善体脂水平，在糖尿病、肥胖等代谢性疾病的治疗领域具有潜在的应用价值。

Description

具有双受体激动作用的多肽衍生物及其用途 技术领域
本发明属于医药生物技术领域，具体涉及一种具有胰高血糖素/胰高血糖素样肽-1双重受体激动作用的胰高血糖素衍生肽以及其用途。
背景技术
肥胖是引起多种疾病的风险因素，已成为全球性的公众健康问题。尤其是包括2型糖尿病(T2DM)的代谢综合征、心血管病、非酒精性脂肪肝等常见疾病，其发病率与病程进展与肥胖密切相关。多项大样本临床研究发现，与正常体重人群相比，心血管代谢多重疾病的发病率在BMI 25.0–29.9kg/m 2、30.0–34.9kg/m 2和BMI>35.0kg/m 2的超重、肥胖或严重肥胖人群分别高出2倍、5倍、15倍(Lancet 2,e277–e285，2017)。研究表明，80～90％的T2DM患者超重或肥胖，适度的减重(4～5kg)有利于预防和控制病情，包括减少患病率、控制血糖和致残(死)率(Curr.Med.Res.Opin.2011,27(7),1431-1438)。
饮食控制和锻炼是最理想的减轻体重手段，但一般收效不佳。肥胖的药物干预疗效有限或存在多种风险，包括严重的心血管影响和因中枢神经作用引起的精神症状等副作用。至目前为止，少有药物单独使用能够达到超过5-10％的体重减轻幅度。T2DM治疗药物中只有SGLT2抑制剂和GLP-1受体激动剂类对体重控制有良性效果。减肥手术效果显著，但手术风险较大，而且长期效应仍不确定。因此，用于控制体重的药物仍存在巨大的临床需求，兼具原发病症治疗作用并能够安全有效地控制体重的药物是理想的选择。
机体的血糖和能量调节信号系统是由多种因子维持精细的平衡，包括不同的多肽类激素。胰高血糖素原(pro-glucagon)是一种具有158个氨基酸的前体多肽，其在不同组织中被加工生成胰高血糖素(GC)、胰高血糖素样肽-1、2(GLP-1，2)及胃泌素等多种胰高血糖素原的衍生肽，这些激素参与葡萄糖体内平衡、胰岛素分泌、胃排空、肠道生长以及食物摄取等多种生理功能的调节。因此，基于胰高血糖素原的肠道激素的治疗已成为代谢病领域深受关注的研究方向。
GC是对应于胰高血糖素原的33至61位氨基酸组成的含29个氨基酸的衍生肽，在胰腺α细胞加工生成，在机体饥饿、寒冷等应激状态下作用 于肝脏，通过糖分解和糖异生作用使血糖水平升至正常范围。除升血糖作用外，动物和人体试验结果表明GC还具有发热、增加饱腹感、脂解、脂肪氧化、生酮等作用，长期给药可以改善能量代谢，包括减轻体重，但这些对能量代谢的有益作用因其固有的升糖作用未能得以应用。
GLP-1是对应于胰高血糖素原的72至108位氨基酸组成的含37个氨基酸残基的衍生肽，在机体进餐响应中由肠道L细胞分泌，作用于胰腺β-细胞促进胰岛素分泌、同时拮抗GC受体抑制血糖升高。GLP-1受体激动剂被开发为糖尿病患者的高血糖治疗剂，降血糖的同时保护和增殖胰岛细胞，而且减缓胃排空和抑制食物摄入，可有效减轻体重。已经有7个GLP-1受体激动剂上市，包括短效的艾塞那肽、利拉鲁肽、利西拉来(1～2次/日)、以及长效的阿必鲁泰、杜拉鲁肽、Byuderon、以及索玛鲁肽(1次/周)。GLP-1受体激动剂类药物虽具有安全独特的降血糖作用，但用于减轻体重时，一般需要使用大剂量，而这些药物在大剂量下易产生胃肠道副作用，耐受性差，治疗窗较窄。因此，仍然需要更为耐受的，可有效控制血糖和减轻体重的治疗剂。
胃泌酸调节素(Oxintomodulin，OXM)是胰高血糖素原翻译后修饰加工过程中在肠道产生的激素，在进餐反应中从回肠L-细胞与GLP-1等激素同时分泌。OXM的急性影响包括对胃排空、胃和胰腺的外分泌以及摄食的抑制作用、静息能量消耗等，可产生减轻体重作用。OXM特异的受体至今尚未明确，但研究发现OXM是内源性GCGR/GLP-1R双重激动剂，而且对两个受体的活性效力弱于各受体的天然配体。在动物和人体试验中发现，外周给予OXM可减少摄食量和减轻体重，在肥胖对象中增加代谢率以及特别是与活动相关的能量消耗。尤其是在临床试验中大剂量外周给予OXM，减轻体重的同时恶心、呕吐等常见胃肠道副作用发生概率较低。因此，基于OXM或GLP-1/GCGR双重激动剂的治疗对肥胖症和肥胖型糖尿病显示了潜在的应用价值，但至目前为止，尚无有关药物上市。
发明内容
本发明的一个目的是提供一种胰高血糖素多肽衍生物，所述多肽是基于GC天然序列设计的变体，其通过GC/GLP-1受体的双重激动作用对能量代谢产生协同影响，可有效降低血糖、减轻体重、改善体脂水平，可用于糖尿病、肥胖、代谢综合征以及非酒精性脂肪肝等疾病的治疗。
本发明的另一个目的是提供一种包含本发明的胰高血糖素多肽衍生 物的药物组合物。
本发明的又一个目的是提供一种本发明的胰高血糖素多肽衍生物的用途。
本发明的目的是通过以下技术方案来实现的。
一方面，本发明提供一种多肽衍生物、其修饰衍生物或其盐，其包含具有以下通式Ⅰ的序列的多肽：
HX 2QGTFTSDX 10SKYLX 15EX 17X 18AX 20X 21FX 23AWLEX 28X 29X 30
通式Ⅰ
其中：
X 2为Ser、D-Ser或Aib；
X 10为Tyr；
X 15为Asp或Glu；
X 17为Arg、Gln或Lys；
X 18为Ala；
X 20为侧链被修饰的Lys；
X 21为Asp或Glu；
X 23为Val或Ile；
X 28为Ala、Gly或Ser；
X 29为Gly或Glu；
X 30为Gly或不存在；
C-末端羧基游离或酰胺化。
在一些优选的实施方案中，所述通式Ⅰ的序列中，
X 17为Arg或Lys；
X 28为Ala或Ser；
X 29为Gly；
X 30为Gly；
并且，C-末端羧基酰胺化。
在优选的实施方案中，通式Ⅰ的序列中，X 2为Aib。
在优选的实施方案中，所述通式Ⅰ的序列选自：
SEQ.ID.NO.4 HSQGTFTSDYSKYLDERAAK*EFIAWLEAGG
SEQ.ID.NO.5 HdSQGTFTSDYSKYLEEKAAK*EFIAWLEAGG
SEQ.ID.NO.6 HdSQGTFTSDYSKYLEERAAK*EFIAWLEAGG
SEQ.ID.NO.7 HAibQGTFTSDYSKYLDERAAK*EFIAWLEAGG
SEQ.ID.NO.8 HAibQGTFTSDYSKYLEERAAK*EFIAWLEAGG
SEQ.ID.NO.9 HAibQGTFTSDYSKYLDERAAK*EFVAWLEAGG
SEQ.ID.NO.10 HSQGTFTSDYSKYLDERAAK*EFIAWLESGG
SEQ.ID.NO.11 HdSQGTFTSDYSKYLEERAAK*EFVAWLESGG
SEQ.ID.NO.12 HAibQGTFTSDYSKYLDERAAK*EFIAWLESGG
SEQ.ID.NO.13 HAibQGTFTSDYSKYLEERAAK*EFIAWLESGG
SEQ.ID.NO.14 HAibQGTFTSDYSKYLEEKAAK*EFVAWLESGG
SEQ.ID.NO.15 HAibQGTFTSDYSKYLDEKAAK*EFIAWLESGG
在SEQ.ID.NOs.4～15中，K*为侧链ε-氨基被修饰的Lys，C-末端羧基酰胺化。
在一些优选的实施方案中，所述通式Ⅰ的序列中，
X 17为Arg或Lys；
X 28为Gly；
X 29为Gly；
X 30不存在；
并且，C-末端羧基酰胺化。
在优选的实施方案中，所述通式Ⅰ的序列选自：
SEQ.ID.NO.16 HSQGTFTSDYSKYLDERAAK*EFIAWLEGG
SEQ.ID.NO.17 HdSQGTFTSDYSKYLEERAAK*EFVAWLEGG
SEQ.ID.NO.18 HAibQGTFTSDYSKYLDERAAK*EFIAWLEGG
SEQ.ID.NO.19 HAibQGTFTSDYSKYLEERAAK*EFIAWLEGG
SEQ.ID.NO.20 HAibQGTFTSDYSKYLEEKAAK*EFVAWLEGG
SEQ.ID.NO.21 HAibQGTFTSDYSKYLDEKAAK*EFIAWLEGG
在SEQ.ID.NOs.16～21中，K*为侧链ε-氨基被修饰的Lys，C-末端羧基酰胺化。
在一些优选的实施方案中，所述通式Ⅰ的序列中，
X 17为Arg或Lys；
X 28为Gly；
X 29为Glu；
X 30为Gly；
并且，C-末端羧基酰胺化。
在优选的实施方案中，所述通式I的序列选自：
SEQ.ID.NO.22 HSQGTFTSDYSKYLDERAAK*EFIAWLEGEG
SEQ.ID.NO.23 HdSQGTFTSDYSKYLEEKAAK*EFVAWLEGEG
SEQ.ID.NO.24 HAibQGTFTSDYSKYLDERAAK*EFIAWLEGEG
SEQ.ID.NO.25 HAibQGTFTSDYSKYLEERAAK*EFIAWLEGEG
SEQ.ID.NO.26 HAibQGTFTSDYSKYLEEKAAK*EFVAWLEGEG
SEQ.ID.NO.27 HAibQGTFTSDYSKYLDEKAAK*EFIAWLEGEG
在SEQ.ID.NOs.22～27中，K*为侧链ε-氨基被修饰的Lys，C-末端羧基酰胺化。
在本发明的一些优选的实施方案中，所述侧链被修饰的Lys是指所述Lys的侧链ε-氨基通过亲水性连接片段偶联脂肪酰基而被修饰。
优选地，所述用于修饰Lys的侧链ε-氨基的亲水性连接片段选自由Glu、γGlu、Gly和Ado(8-氨基-3,6二氧辛酸)中的一种或多种组成的片段。所述亲水性连接片段优选为-γGlu-、-γGlu-γGlu-、-Glu-γGlu-、-γGlu-Gly-Gly-、-γGlu-Gly-γGlu-、-γGlu-Ado-Ado-、-Ado-Ado-γGlu-或-γGlu-Ado-Ado-γGlu-。
在本发明优选的实施方案中，所述脂肪酰基为C 14-20的脂肪酰基，包括C 14-20的单脂肪酰基或脂肪二酸单酰基；更优选C 16-18脂肪酰基，最优选C 16单脂肪酰基(棕榈酰基)。
另一方面，本发明提供一种药物组合物，其含有本发明所述的多肽衍生物、其修饰衍生物或其盐和任选的一种或多种药学上可接受的辅料。
优选地，所述药学上可接受的辅料包括载体、稀释剂、水溶性填充剂、pH调节剂、稳定剂、注射用水、渗透压调节剂等。
优选地，所述水溶性填充剂包括但不限于甘露醇、低分子右旋糖酐、山梨醇、聚乙二醇、葡萄糖、乳糖和半乳糖；所述pH调节剂包括但不限于枸橼酸、磷酸、乳酸、酒石酸、盐酸等有机或无机酸以及氢氧化钾、氢氧化钠、氢氧化铵、碳酸钠、碳酸钾、碳酸铵、碳酸氢钾、碳酸氢钠、碳酸氢铵盐等生理上可接受的无机碱或盐；所述稳定剂包括但不限于EDTA-2Na、硫代硫酸钠、焦亚硫酸钠、亚硫酸钠、磷酸氢二钾、碳酸氢钠、碳酸钠、精氨酸、赖氨酸、谷氨酸、天冬氨酸、聚乙二醇、聚乙烯醇、聚乙烯吡咯烷酮、羧基/羟基纤维素或其衍生物如HPC、HPC-SL、HPC-L或HPMC、环糊精、十二烷基硫酸钠或三羟甲基氨基甲烷；所述渗透压调节剂包括但不限于氯化钠或氯化钾。
优选地，本发明所述的药物组合物可以静脉、肌肉或皮下注射剂形式或口服、直肠、鼻腔给药。治疗剂量范围取决于治疗对象、给药方式、适 应症以及其他因素等。
又一方面，本发明提供本发明所述的多肽衍生物、其修饰衍生物或其盐在制备用于治疗代谢性疾病的药物中的应用，优选地，所述代谢性疾病为糖尿病、肥胖、脂肪肝、高血脂症和/或代谢综合征；更优选地，所述脂肪肝为非酒精性脂肪肝。
另一方面，本发明提供一种治疗代谢性疾病的方法，包括给予需要的患者治疗有效量的本发明所述的多肽衍生物、其修饰衍生物或其盐，或本发明所述的药物组合物；优选地，所述代谢性疾病为糖尿病、肥胖、脂肪肝、高血脂症和/或代谢综合征；更优选地，所述脂肪肝为非酒精性脂肪肝。
胰高血糖素衍生肽的结构：内源性GLP-1是对应于胰高血糖素原的72至108位氨基酸组成的含37个氨基酸残基(7-36/37)的衍生肽,其氨基酸序列为HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR(7-36)(SEQ ID NO.1),HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(7-37)，其C-端羧基游离或酰胺化。内源性GC是对应于胰高血糖素原的33至61位氨基酸组成的含29个氨基酸的衍生肽，氨基酸序列为：HSQGTFTSDYSKYLDSRRAQDFVQWLMNT(SEQ ID NO.2)，C-端羧基游离。天然GLP-1和GC的氨基酸序列有47％的同源性(Andreas Evers等，J.Med.Chem.2017,60,4293-4303)，二者的N-端序列高度保守，GLP-1对其受体具有高度选择性，而GC同时是GLP-1受体的弱激动剂。
胃泌酸调节素(Oxinthomodulin，OXM)是内源性GC/GLP-1R双重激动剂，其氨基酸序列是HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA(SEQ ID NO.3),包含GC的原序列(1-29)和对应于胰高血糖素原氨基酸序列82-89的插入肽-1(IP-1，30-37)。以OXM为先导序列的现有技术文件，如CN200980132562.9、CN201080027026.5、CN201680062196.4中是将GC原序列(1-29)的变体，以Exendin-4的C-末端10肽(GPSSGAPPPS缩写为Cex)序列延长C-端。而CN201680036771.3是选择将OXM序列的C-末端插入肽段(KRNRNNIA)以GGPSSG肽段替换的设计方案。这些已有的技术均采取了在保留GC全长序列(1-29)的基础上进行若干位点突变，再以不同肽段延长的设计思路。
基于GC序列设计双重激动剂时，为得到预期的效价平衡，现有公开资料中多采取对GC序列的16—23片段进行类似GLP-1相应序列的替换，或对16、17、18、20、23等个别位点的突变。而GC序列C-末端变化对 GC/GLP-1受体的识别极为敏感，尤其是对GC受体的活性极为敏感。因此，现有技术方案中普遍选择保留C-末端27-29片段的原序列，仅有个别技术方案采取保守的突变措施，例如，将敏感氨基酸27Met替换为Leu，28Asn替换为Glu或Ala。多肽序列上的长链脂肪酰修饰一般是为了延长多肽的体内半衰期，但在应用于GC/GLP-1双重受体激动剂的设计时，适当位点上的修饰可以调节受体选择性和活性平衡。现有技术资料中采取的普遍措施为 10Lys突变残基的ε-侧链上修饰棕榈酰基，得到效价平衡的强激动剂(EC50pM)。但本发明人发现，这些强激动剂在模型动物体内药效评价时长期给药后出现糖耐量异常现象，并不能达到预期的代谢调控目的。
综观本领域公开的技术资料可以发现，由于长链多肽的结构复杂性和与其受体结合机制的特殊性，适当活性强度和效价比平衡的双重激动剂的获得是需要通过不同技术措施的合理组合来实现的，对多肽结构的改变，如不同位点氨基酸残基的突变、修饰位点、基团的改变都可以导致活性和效价比平衡的变化，其结果一般是难以预料的。
本发明提供的技术方案中，多肽主链的设计针对GC序列的1-26肽段的个别位点进行合理突变的基础上再适当延长C末端，后采取侧链修饰措施，例如用20位脂肪酰修饰，得到了活性效价比平衡而且溶解性、稳定性均佳的GC/GLP-1双重受体激动剂。
在本发明的一些特定实施方案中，对GC(1-26)序列的位点16-20进行了合理替换，如，将16Ser替换为Glu，18Arg替换为Ala或Lys等有利于提高GLP-1受体选择性的突变。与公开的多数技术方案中对GC序列C-末端的保守替换方案不同，本发明优选技术方案中是以Glu替换27Met，再以Gly、Ala等电中性氨基酸替换位点28、29。一般认为，GC序列的C-末端羧基游离有利于保持活性。本发明实施方案中用Glu替换27Met保留了C-端的负电性而减少对活性的影响，进而将C-末端酰胺化提高了肽序列的稳定性。
一般地，对天然序列进行尽可能少的改变有利于保持同源性，但基于内源性胰高血糖素原序列的设计存在较多的成药性难题：N-端二肽易被体内的二肽激肽酶识别而被水解失活，导致血浆半衰期短(≤12min)；物理性质不稳定，即等电点(pI)7.6，呈中性，而且疏水，溶解性差，很容易在溶液中聚集沉淀；序列中有Met、Asp、Asn等易于氧化或消旋的氨基酸，导致化学性质不稳定。
在本发明优选实施方案中，所述多肽序列的2Ser通常被替换为Aib 或D-Ser，以提高代谢稳定性，保证多肽活性的持续发挥。与此同时，在本发明优选的实施方案中，进一步对GC序列中的敏感氨基酸进行了替换，如15，21位的Asp替换为Glu。
经过上述调整后的多肽序列增加了序列C-端的负电荷数量，降低了pI值，有助于提高肽在生理条件下的溶解性并提高化学稳定性。
脂肪酰基团和连接子的结构以及修饰位点同时影响化合物的活性。在本发明的实施方案中，在肽序列20位Lys的侧链上通过亲水性连接臂修饰了脂肪酰基。在本发明的某些实施方案中，所述亲水性连接臂是-γ-Glu-、-γ-Glu-γ-Glu-或在另一些实施方案中是-Ado-Ado-γ-Glu-。所述脂肪酰基是C 14-20的脂肪酰基，优选为C 14-20的单脂肪酰基或脂肪二酸单酰基。在某些优选的实施方案中，所述脂肪酰基是C 16或C 18单脂肪酰基。
本发明提供具有GLP-1/胰高血糖素双重受体激动作用的肽，通过本发明前述的结构设计，所述肽具有以下活性特点：本发明提供的多肽与GLP-1R/GCGR各受体的天然配体相比至少具有0.1％的受体激动活性，如本发明实施例中以EC 50(nM)表示的活性。本发明提供的化合物的EC 50(nM)值包括但不限于内源性配体(GLP-1)的10 -2～10 2倍，优选的活性强度相当于内源性配体(GLP-1)、比内源性配体(GLP-1)弱或强1-10倍，如2、3、6或10倍，或强10-100倍。对胰高血糖素受体(GCGR)的激动活性强度以EC 50(nM)值表示，与内源性配体(GC)相当或相当于内源性配体的1～10 3倍。在优选的实施方案中，本发明的化合物对GLP-1受体的激动作用强于对胰高血糖素受体的激动作用，或对两个受体的激动作用强度相当。对GLP-1和胰高血糖素受体的相对活性强度可以用效价比(GLP-1R EC50/GCGR EC50)表示，即本发明提供的包含通式Ⅰ的序列的多肽对GLP-1/胰高血糖素受体的效价比包括但不限于10:1至1:100。在本发明优选实施方案中该比例在10:1-1:10范围，而更优选范围是5:1-1:10。
本发明提供的具有通式Ⅰ的序列的多肽衍生物的基本肽链可以通过本领域公知的方法制备得到：
1)通过常规固相或液相方法逐步或通过片段组装合成；
2)在宿主细胞中表达编码多肽的核酸构建体，并从宿主细胞培养物回收表达产物；
3)影响编码多肽的核酸构建体的无细胞体外表达，并回收表达产物；
或通过方法1)、2)或3)的任意组合来获得肽片段，随后连接这些片段以获得目标肽。
本发明提供的实施方案中优选地，使用Fmoc固相合成方法制备得到目标肽，该技术是本领域技术人员所熟知的。
取代基可以通过上述肽合成步骤逐步合成引入。使用适当保护基的取代基，如，Fmoc-8-氨基-3,6二氧杂辛酸，和Fmoc-γ-Glu-OtBu。脂肪链部分的引入，尤其是脂肪二酸单酰基，可以使用但不限于C 18、C 20烷酸单叔丁酯来实现。在每个偶联步骤后，未反应的中间物可以使用过量的乙酸酐和吡啶进行封闭。可修饰的Lys的ε-氨基可以使用Mtt或Dde保护。
纯化：缀合反应之后，可以通过本领域公知的合适方法将目标产物分离。适用的方法包括但不限于超滤法、透析法或色谱法等。本发明实施方案中优选采用制备型高效液相色谱法纯化。
受体活性测定：本发明的实施方案中通过GLP-1/GC受体介导的体外cAMP产生的影响评价了所述多肽对GLP-1/GC受体的作用。
对体重和血糖的调节作用：本发明的实施方案中采用糖负荷正常小鼠模型单次给药和高脂饮食肥胖型糖尿病小鼠(Dio)模型连续给药评价了本发明提供多肽的对血糖和体重的影响。
本发明的实验结果表明，通过本发明技术方案提供的化合物对GLP-1受体(GLP-1R)的活性与内源性GLP-1相当，对GC受体(GCGR)则保留了一定的活性，效价比在适当范围内。与单纯的GLP-1受体激动剂相比，通过本发明技术方案提供的化合物在有效降低血糖的同时更为显著地促进减重和防止增重，可以逆转或缓解肝脏脂肪性病变和胰岛素抵抗，对复杂的代谢综合征显示了预料不到的多重益处。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了化合物dgc016、dgc005、dgc020对DIO鼠体重的影响；
图2示出了化合物dgc005、dgc016、dgc020连续给药23天对DIO模型鼠肝脏病变的影响；其中，图2A示出了空白对照组的肝脏病理切片检查图，图2B示出了模型对照组的肝脏病理切片检查图，图2C示出了阳性对照药组的肝脏病理切片检查图，图2D示出了化合物dgc005组的肝脏病理切片检查图，图2E示出了化合物dgc016组的肝脏病理切片检查图，图2F示出了化合物dgc020组的肝脏病理切片检查图；
图3示出了化合物dgc003、dgc004、dgc010、dgc017连续给药13天对DIO模型鼠体重的影响。
图4示出了化合物dgc005、dgc016分剂量连续给药21天对DIO模型鼠体重的影响。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。本实施例仅为解释本发明，不意味以任何方式限制本发明内容。
氨基酸缩写的说明：
Gly:甘氨酸(G)
Ala:丙氨酸(A)
Val:缬氨酸(V)
Leu:亮氨酸(L)
Phe:苯丙氨酸(F)
Trp:色氨酸(W)
Ser:丝氨酸(S)
Thr:苏氨酸(T)
Glu:谷氨酸(E)
Gln:谷氨酰胺(Q)
Asp:天冬氨酸(D)
Asn:天冬酰胺(N)
Tyr:酪氨酸(Y)
Arg:精氨酸(R)
Lys:赖氨酸(K)
His:组氨酸(H)
Aib:α-氨基异丁酸
Ado:8-氨基-3,6-二氧杂辛酸
试剂缩写的说明
Boc:叔丁氧基羰基
Tert-Bu:叔丁基
DCM:二氯甲烷
DIC：二异丙基碳二亚胺
Fmoc:9-芴甲氧基羰基
HoBt:1-羟基苯并三唑
HBTU:2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基-脲鎓六氟磷酸酯
HATU:O-(7-氮杂苯并三唑-1-基)-N,N,N’,N’-四甲基-脲鎓六氟磷酸酯
Mtt:4-甲基三苯甲基
NMP:N-甲基吡咯烷酮
DMF:二甲基甲酰胺
Pbf:2，2，4，6，7-五甲基二氢苯并呋喃
Dde:1-(4,4-二甲基-2,6-二氧代亚环己基)-3-甲基-丁基
Trt:三苯基甲基
EDT:乙二硫醇
TFA:三氟乙酸
TIS:三异丙基硅烷
FBS:胎牛血清
实施例1
本发明提供的多肽基本线性序列以及侧链修饰衍生肽按照以下通用方法制备：
1)合成：采用Fmoc策略，用PSI200型多肽合成仪，按照如下步骤逐步合成：
a)在活化剂系统存在下由树脂固相载体和Fmoc保护的C-端氨基酸偶联得到Fmoc-氨基酸-树脂；其中，合成C-端酰胺化多肽采用氨基树脂，如Rink Amide AM，Rink Amide，Rink MBHA等；Fmoc-氨基酸和树脂比(mol/mol)为3～5:1,偶联活化剂为HOBT/DIC或HOBT/HBTU/DIEA。
b)肽链的延长：通过固相合成法按照肽序列氨基酸顺序连接氨基酸，得到N-端和侧链保护的肽－树脂偶联物；带侧链氨基酸采取如下保护措施：色氨酸用Boc，谷氨酸用OtBu，赖氨酸用Boc，谷氨酰胺用Trt，酪氨酸用tBu，丝氨酸用Trt或tBu，天冬氨酸用OtBu，苏氨酸用tBu，半胱氨酸用Trt，精氨酸用Pbf保护，组氨酸的α-氨基用Boc保护，侧链用Trt或Boc保护，可修饰的赖氨酸的ε-氨基用Dde保护。使用的偶联活化剂为HOBT/DIC、HOBT/HBTU/DIEA和HOBT/HATU/DIEA，茚三酮法检测反应终点，脱保护剂为含20％哌啶的NMP(DMF)溶液。
c)赖氨酸的ε-氨基脱保护：
上述步骤中合成完成的全保护多肽-树脂以NMP-DCM(1：1V/V)洗涤3次，加入新鲜制备的2.0％水合肼的NMP溶液，在室温下搅拌12.0分钟，过滤，重复两次，用DCM和NMP各洗涤树脂三次。
d)赖氨酸侧链的修饰：
赖氨酸的ε-氨基脱保护完成后，按比例(树脂：连接基1:4-5(mol/mol))加入Fmoc-Ado或Fmoc-γ-Glu(tBu)以及HOBt/HBTU,DIEA，搅拌反应2.0-4.0小时，脱Fmoc保护，同法继续连接所需链长的连接臂和脂肪酰基。重复两次后反应仍未完全，加过量的乙酸酐/吡啶封闭，继续下一步反应。
e)多肽的裂解：全保护肽-树脂先用NMP洗涤，后用DCM洗涤3-6次去除NMP，加入TFA/EDT/TIS/H2O(92.5:2.5:2.5:2.5v/v)溶液，置室温氮气保护下搅拌90min，脱保护和脱树脂。抽滤得滤液，用过量冰乙醚沉淀粗多肽，离心，收集沉淀，再用少量乙醚洗涤沉淀，真空下干燥，得到多肽粗品。
2)纯化：将多肽粗品溶解于水或10-15％乙腈(10-50mg/ml)，采用制备型HPLC法，C8或C18色谱柱，乙腈－水－三氟乙酸系统分离纯化，浓缩，冻干，得多肽纯品(纯度≥97％)。
以上述方法制备，并经质谱确证得到以下表1中所示结构的多肽衍生物。
表1 合成的化合物
化合物编号 序列号 K *
dgc001 SEQ ID NO.4 -γE-OC 16H 31
dgc002 SEQ ID NO.6 -Ado-Ado-γE-OC 17H 32COOH
dgc003 SEQ ID NO.7 -γE-OC 16H 31
dgc004 SEQ ID NO.8 -γE-OC 16H 31
dgc005 SEQ ID NO.8 -γE-γE-OC 16H 31
dgc006 SEQ ID NO.8 -Ado-Ado-γE-OC 17H 31COOH
dgc007 SEQ ID NO.12 -γE-OC 16H 31
dgc008 SEQ ID NO.13 -γE-OC 16H 31
dgc009 SEQ ID NO.13 -Ado-Ado-γE-OC 16H 31
dgc010 SEQ ID NO.13 -γE-γE-OC 16H 31
dgc011 SEQ ID NO.8 -Ado-Ado-γE-OC 16H 31
dgc012 SEQ ID NO.17 -Ado-Ado-γE-OC 17H 32COOH
dgc013 SEQ ID NO.18 -γE-γE-OC 16H 31
dgc014 SEQ ID NO.18 -γE-OC 16H 31
dgc015 SEQ ID NO.19 -γE-OC 16H 31
dgc016 SEQ ID NO.19 -γE-γE-OC 16H 31
dgc017 SEQ ID NO.21 -γE-OC 16H 31
dgc018 SEQ ID NO.24 -γE-OC 16H 31
dgc019 SEQ ID NO.25 -γE-OC 16H 31
dgc020 SEQ ID NO.25 -γE-γE-OC 16H 31
dgc021 SEQ ID NO.25 -Ado-Ado-γE-OC 16H 31
dgc022 SEQ ID NO.27 -γE-OC 16H 31
注：所有化合物C-末端羧基酰胺化
实施例2 对GLP-1/GC受体的作用
通过对GLP-1/GC受体介导的体外cAMP产生的影响评价所述多肽对GLP-1/GC受体的作用。
稳定表达GLP-1R、GCGR的HEK293细胞系，用于GLP-1R激动剂和GCGR激动剂的筛选。
受试样品配制：将化合物配制成100μM储存液，逐步稀释成1μM、100nM、10nM、1nM、10 -1nM、10 -2nM、10 -3nM、H 2O的工作浓度。
阳性对照品配制：
GLP-1：
贮存方法：0.1％BSA超纯水溶解的1mM储存液，-80℃密闭保存。
工作浓度：1μM、100nM、10nM、1nM、10 -1nM、10 -2nM、10 -3nM和H 2O(每孔都含有0.1％的BSA)。
胰高血糖素(GC)：
贮存方法：1mM DMSO溶液，-80℃密闭保存。
工作浓度：1μM、100nM、10nM、1nM、10 -1nM、10 -2nM、10 -3nM和DMSO(每个孔都含有1％的DMSO)。
试剂与仪器：
主要试剂：DMEM培养基(GIBCO,Cat No:12800017)；
cAMP检测试剂盒(PerkinElmer,Cat No:TRF0264)。
主要仪器：Envision 2104多功能微孔板酶标仪(PerkinElmer)。
实验步骤：
1)将待测化合物用无血清培液(含0.1％BSA,0.5mM IBMX)配成工作浓度的2倍。
2)消化细胞，将细胞用无血清培液悬浮(含0.5mM IBMX)，并且进行计数，按照1000细胞/5μl/孔加入384孔板，然后加入5μl待测化合物，避光反应30min。
3)反应结束后，加入cAMP检测底物，室温避光反应60min。
4)反应结束后，在Envision2104多功能微孔板酶标仪检测，最终读取数值为OD665nm处与OD615nm处的比值。
5)用Origin软件计算50％有效浓度(EC 50)。
表2 多肽对GLP-1/GC受体的激动作用
Figure PCTCN2021092406-appb-000001
Figure PCTCN2021092406-appb-000002
表2显示的体外受体激动活性结果表明，具有本发明的氨基酸序列的多肽衍生物具有GLP-1/GC受体的双重激动活性，多肽衍生物的效价比在适当范围内。
实施例3 实施例1化合物的降血糖作用
采用正常小鼠单次给药糖负荷试验评价了实施例1化合物的降血糖作用。
方法：昆明种小鼠(雄性，体重范围20-22g)，于屏障环境动物房内适应性饲养3天后分为空白对照组、模型对照组、阳性对照组以及受试化合物组，每组5只，禁食6h。空白对照组和模型对照组给予生理盐水，受试化合物组和阳性对照组给药剂量为30nmol/kg，阳性对照药为索马鲁肽(semaglutide)。给药60min后模型对照组、阳性对照组以及受试化合物组分别以2.5g/kg(0.01mL·g -1体重)灌胃给予葡萄糖，空白对照组给予蒸馏水，测定糖负荷后第30min时的血糖值。结果见表3。
表3 化合物对正常小鼠糖负荷的影响(n＝5，mmol/L)
分组 空腹血糖值 糖负荷后血糖值 血糖降低率(％)
空白对照组 4.08±0.83 4.48±0.83 -
模型对照组 3.43±0.66 17.00±2.96 -
阳性对照组 3.78±1.28 5.58±1.55 ** 67.18
dgc005 3.96±0.62 6.70±1.88 ** 60.59
dgc016 3.66±0.77 6.82±1.78 ** 59.88
dgc020 4.24±0.63 9.82±2.06 **## 42.24
dgc003 4.20±0.97 8.82±1.63 **# 48.12
dgc004 3.42±0.85 8.46±2.28 **# 50.24
dgc013 3.92±0.51 9.26±1.76 **## 45.53
dgc010 3.78±0.84 8.10±1.13 **# 52.35
dgc017 3.72±0.37 10.36±1.87 **## 39.06
dgc006 3.80±0.51 8.02±2.92 ** 52.82
dgc008 3.64±1.00 8.72±1.99 **# 48.70
**：与模型对照组相比P＜0.01；
##：与阳性对照组相比P＜0.01； #：与阳性对照组相比P＜0.05
结果显示，与模型对照组相比，受试化合物在给药剂量下对正常小鼠糖负荷血糖升高有显著抑制作用(P＜0.01)，化合物dgc005、dgc016和dgc006的抑制作用与阳性对照药相当。
实施例4
化合物dgc005、dgc016、dgc020多次给药对肥胖模型鼠的治疗作用
试验方法：
C57BL/6J Gpt小鼠(购自江苏集萃药康生物科技有限公司)(6-8周龄)，用H10060高脂饲料(购自北京华阜康生物科技有限公司)饲养饮食诱导肥胖(DIO)，空白对照组(n＝5)采用标准饲料饲养，购入后饲养11周。首次给药前一天，模型组按照体重随机分组(体重范围39.5-49.5g)，分为5组，每组5只，分别为模型对照组、阳性对照组、受试化合物组(dgc005、dgc016、dgc020)每天皮下注射给药，空白对照组、模型对照组每天皮下注射生理盐水，共给药23天，阳性对照药和受试化合物给药剂量均为30nmol/kg，各组给药容积均为0.005mL/g体重。每次给药前称量体重，每隔2天称量72h进食量。末次给药前禁食16h，测定空腹血糖值，眼眶静脉丛采血测定甘油三酯、总胆固醇；摘取肝脏进行病理切片观察。
结果：
1、对体重的影响：见图1。给药第3天开始所有受试化合物体重降幅与阳性对照药相比区分明显，并持续降低。与给药第一天相比，受试化合物dgc005、dgc016、dgc020末次给药前未禁食体重降幅分别为27％、29.3％、 25.0％，而阳性对照药降幅为20.9％。说明在给药剂量下受试化合物降体重作用优于阳性对照药。
2、多次给药对空腹血糖的影响：见表4。
表4 连续给药23天后各样品对动物空腹血糖的影响(n＝5)
Figure PCTCN2021092406-appb-000003
**：与模型对照组相比P＜0.01
#：与阳性对照组相比P＜0.05
△△：与空白对照组相比P＜0.01
表4结果显示，受试化合物连续给药23天，对空腹血糖改善作用与阳性对照药相当。
3、对摄食量的影响：结果见表5。受试样品组72h进食量相对于模型对照组存在不同程度的减少，第7天开始72h进食量呈逐渐增加趋势。
表5 多次给药对动物72h进食量的影响(g/只，n＝5)
给药天数 空白对照组 模型对照组 阳性对照组 dgc005 dgc016 dgc020
4 11.30 5.60 3.50 2.50 3.30 3.50
7 9.30 6.00 5.80 3.80 4.80 4.50
10 10.40 8.20 5.40 4.20 6.10 5.70
13 9.20 7.80 5.50 4.60 5.70 5.50
4、对血脂的影响：结果见表6。化合物dgc005组显示一定的降低总胆固醇(TC)作用(与模型对照组相比P＜0.05，与阳性对照组相比P＜0.05，与空白对照组相比无统计学差异)，同时可显著降低甘油三酯(TG)(与模型对照组相比P＜0.01，与阳性对照组相比P＜0.05，与空白对照组相比P＜0.01)。化合物dgc016组显示了降低甘油三酯的作用(与模型对照组相比P＜0.01，与阳性对照组相比无统计学差异，与空白对照组相比P＜0.01)。
表6 多次给药23天后受试化合物对动物血脂的影响(mmol/L，n＝5)
Figure PCTCN2021092406-appb-000004
**：与模型对照组相比P＜0.01； *：与模型对照组相比P＜0.05
#：与阳性对照组相比P＜0.05
△△：与空白对照组相比P＜0.01； △：与空白对照组相比P＜0.05
5、肝脏病理切片检查：
将各组C576L/6小鼠肝脏用12％甲醛溶液固定制成标本，组织经修块，梯度酒精脱水，石蜡包埋，HE染色，光镜下检查。
结果：
空白对照组：2只动物肝小叶结构完整，肝细胞索排列整齐，肝细胞形态清晰，汇管区及间质未见明显病理改变(见图2A)。
模型对照组：2只动物可见重度病变：弥漫性肝细胞空泡变性，部分血管周围单个核细胞浸润(2/2、重度)(见图2B)。
阳性对照药组：2只动物可见轻微至中度病变：少量至较多肝细胞空泡变性，其中1只动物部分血管周围单个核细胞浸润，另见局部透明型肝细胞灶(1/2、轻微，1/2、中度)(见图2C)。
化合物dgc005组：2只动物肝小叶结构完整，肝细胞索排列整齐，肝细胞形态清晰，汇管区及间质未见明显病理改变(见图2D)。
化合物dgc016组：2只动物肝小叶结构完整，肝细胞索排列整齐，肝细胞形态清晰，汇管区及间质未见明显病理改变(见图2E)。
化合物dgc020组：2只动物肝小叶结构完整，肝细胞索排列整齐，肝细胞形态清晰，汇管区及间质未见明显病理改变(见图2F)。
结论：
由镜下检查结果可见：与空白对照组比较，本试验模型对照组肝脏可见：不同程度的肝细胞空泡变性，部分血管周围单个核细胞浸润，呈重度病变，阳性对照药组肝脏亦可见上述病变，病变程度明显减轻，呈轻微至中度病变，表明对本模型肝脏病变有一定的治疗效果。本发明受试化合物组则肝小叶结构完整，肝细胞索排列整齐，肝细胞形态清晰，汇管区及间质未见明显病理改变，与空白对照组无差别，表明本发明受试化合物对本模型肝脏病变治疗效果明显。
实施例5
化合物dgc003、dgc004、dgc010、dgc017给药2周对DIO模型鼠体重的影响
方法：用H10060高脂饲料饲养饮食诱导肥胖(DIO)C57BL/6N小鼠，对照组(n＝5)采用标准饲料饲养。首次给药前一天，模型组按照体重随机分组(体重范围40.5-50.5g)，分为6组，每组5只，分别为模型对照组、阳性对照组、受试化合物组(dgc003、dgc004、dgc010、dgc017)每天皮下注射给药1次，空白对照组、模型对照组每天皮下注射生理盐水，共给药14天，阳性对照药和受试化合物给药剂量均为30nmol/kg，各组给药容积均为0.005mL/g体重。每次给药前称量体重，每隔2天称量72h进食量。
结果：见图3。连续给药2周，阳性对照药给药1-5天体重降幅明显，其后下降趋势平缓，受试化合物组5天之后与阳性对照药的降幅区分明显，并呈持续下降趋势，到末次给药dgc003、dgc004、dgc010、dgc017组降幅分别达到16.7％、19.9％、16.4％、15.9％，大于阳性对照药的12.9％。
实施例6
化合物dgc005、dgc016分剂量多次给药对DIO鼠体重、血糖的影响
方法：
C57BL/6N小鼠(购自北京维通利华实验动物技术有限公司)，40只，模型组(n＝35)用H10060高脂饲料饲养饮食诱导肥胖(DIO)，空白对照组(n＝5)采用标准饲料饲养，均饲养36周。首次给药当天，模型组按照体重随机分组(体重范围45-60g)，分为7组，每组5只，分别为模型对照组、阳性对照组(索马鲁肽，semaglutide)-10nmol/kg、阳性对照组-30nmol/kg、dgc005-10nmol/kg、dgc005-30nmol/kg、dgc016-10nmol/kg、dgc016-30nmol/kg，阳性对照组和受试化合物组每天皮下注射给药，空白对照组、模型对照组每天皮下注射生理盐水，共给药22天(各组给药容积均为0.005mL/g体重)。每次给药前称量体重，每隔2天称量72h进食量。于末次给药前禁食16h，给药后30min灌胃给予葡萄糖溶液(1g/kg，给药容积为0.01mL/g体重)进行糖耐量试验，于给糖后0.5h测定血糖值。
结果：
1)对体重的影响：结果见图4。与模型对照组相比，阳性对照组和受 试化合物组从给药第二天开始出现持续的体重降低，受试化合物大剂量组从第3天开始，低剂量组从第4天开始与阳性对照药降低幅度区分明显，到给药第21天受试化合物各剂量组未禁食体重降幅均优于相应剂量的阳性对照组。结果如下表7所示。
表7 各组动物给药第21天未禁食体重与第0天相比降低率(％，n＝5)
Figure PCTCN2021092406-appb-000005
2)对血糖的影响：
末次给药前，受试化合物各剂量组均能明显降低动物给药前空腹血糖值，糖负荷后0.5h，受试化合物各剂量组对动物糖负荷血糖值升高有明显抑制作用(与模型对照组相比P＜0.01，与阳性对照组、空白对照组相比无统计学差异)，说明受试化合物长期给药仍具有与阳性对照药相当的降血糖作用。结果见表8。
表8 末次给药(第22天)后各样品对动物糖负荷的影响(n＝5)
Figure PCTCN2021092406-appb-000006
**：与模型对照组相比P＜0.01； *：与模型对照组相比P＜0.05
△△：与空白对照组相比P＜0.01； △：与空白对照组相比P＜0.05。

Claims (14)

  1. 一种多肽衍生物、其修饰衍生物或其盐，其包含具有以下通式Ⅰ的序列的多肽：
    HX 2QGTFTSDX 10SKYLX 15EX 17X 18AX 20X 21FX 23AWLEX 28X 29X 30
    通式Ⅰ
    其中：
    X 2为Ser、D-Ser或Aib；
    X 10为Tyr；
    X 15为Asp或Glu；
    X 17为Arg、Gln或Lys；
    X 18为Ala；
    X 20为侧链被修饰的Lys；
    X 21为Asp或Glu；
    X 23为Val或Ile；
    X 28为Ala、Gly或Ser；
    X 29为Gly或Glu；
    X 30为Gly或不存在；
    C-末端羧基游离或酰胺化。
  2. 如权利要求1所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述通式Ⅰ的序列中，
    X 17为Arg或Lys；
    X 28为Ala或Ser；
    X 29为Gly；
    X 30为Gly；
    并且，C-末端羧基酰胺化。
  3. 如权利要求1或2所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述通式Ⅰ的序列中，X 2为Aib。
  4. 如权利要求1所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述通式Ⅰ的序列中，
    X 17为Arg或Lys；
    X 28为Gly；
    X 29为Gly；
    X 30不存在；
    并且，C-末端羧基酰胺化。
  5. 如权利要求1所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述通式Ⅰ的序列中，
    X 17为Arg或Lys；
    X 28为Gly；
    X 29为Glu；
    X 30为Gly；
    并且，C-末端羧基酰胺化。
  6. 如权利要求1所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述多肽的序列选自SEQ ID NOs.4-27中的任一个。
  7. 如权利要求1-6中任一项所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述侧链被修饰的Lys是指所述Lys的侧链ε-氨基通过亲水性连接片段偶联脂肪酰基而被修饰。
  8. 如权利要求7所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述亲水性连接片段选自由Glu、γGlu、Gly和Ado(8-氨基-3,6二氧辛酸)中的一种或多种组成的片段。
  9. 如权利要求8所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述亲水性连接片段为-γGlu-、-γGlu-γGlu-、-Glu-γGlu-、-γGlu-Gly-Gly-、-γGlu-Gly-γGlu-、-γGlu-Ado-Ado-、-Ado-Ado-γGlu-或-γGlu-Ado-Ado-γGlu-。
  10. 如权利要求7或8所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述脂肪酰基为C 14-20的脂肪酰基，优选为C 14-20的单脂肪酰基或脂肪二酸单酰基。
  11. 如权利要求10所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，所述脂肪酰基为C 16-18脂肪酰基。
  12. 一种药物组合物，其含有权利要求1-11中任一项所述的多肽衍生物、其修饰衍生物或其盐和任选的一种或多种药学上可接受的辅料。
  13. 权利要求1-11中任一项所述的多肽衍生物、其修饰衍生物或其盐在制备用于治疗代谢性疾病的药物中的应用，优选地，所述代谢性疾病为糖尿病、肥胖、脂肪肝、高血脂症和/或代谢综合征；更优选地，所述脂肪肝为非酒精性脂肪肝。
  14. 一种治疗代谢性疾病的方法，包括给予需要的患者治疗有效量的 权利要求1-11中任一项所述的多肽衍生物、其修饰衍生物或其盐，或权利要求12所述的药物组合物；优选地，所述代谢性疾病为糖尿病、肥胖、脂肪肝、高血脂症和/或代谢综合征；更优选地，所述脂肪肝为非酒精性脂肪肝。
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