WO2020098657A1 - 具有双重受体激动作用的胰高血糖素衍生肽及其用途 - Google Patents

具有双重受体激动作用的胰高血糖素衍生肽及其用途 Download PDF

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WO2020098657A1
WO2020098657A1 PCT/CN2019/117614 CN2019117614W WO2020098657A1 WO 2020098657 A1 WO2020098657 A1 WO 2020098657A1 CN 2019117614 W CN2019117614 W CN 2019117614W WO 2020098657 A1 WO2020098657 A1 WO 2020098657A1
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derivative
polypeptide
modified
salt
γglu
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PCT/CN2019/117614
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French (fr)
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韩英梅
刘巍
刘冰妮
赵娜夏
商倩
夏广萍
孔维苓
靳京
孔晓华
李玉荃
高琪
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天津药物研究院有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 in particular relates to a polypeptide having a dual agonistic effect of glucagon / glucagon-like peptide-1 receptor and its use.
  • Obesity is a risk factor for many diseases and has become a global public health problem.
  • metabolic syndrome including type 2 diabetes (T2DM), cardiovascular disease, non-alcoholic fatty liver and other common diseases
  • the incidence and progression of the disease are closely related to obesity.
  • T2DM type 2 diabetes
  • a large number of clinical studies have found that compared with normal-weight people, the incidence of multiple cardiovascular metabolic diseases is BMI 25.0–29.9 kg / m 2 , 30.0–34.9 kg / m 2 and BMI> 35.0 kg / m 2 overweight , Obese or severely obese people were 2 times, 5 times, and 15 times higher (Lancet 2, e277–e285, 2017).
  • 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 gastrin and other derivatives of proglucagon, 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 of great interest in the field of metabolic diseases.
  • GC is a 29-amino acid derivative peptide composed of amino acids 33 to 61 corresponding to proglucagon. It is processed and produced in pancreatic alpha cells and acts on the liver under stress conditions such as starvation and cold. Gluconeogenesis raises blood glucose 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, ketogenic, etc. Long-term administration can improve energy metabolism, including weight loss, but these have an effect on energy metabolism The beneficial effects of sucrose have not been applied due to their inherent glycemic effect.
  • GLP-1 is a derivative peptide containing 37 amino acid residues corresponding to the 72 to 108 amino acids of proglucagon. It is secreted by intestinal L cells during the body's meal response and acts on pancreatic ⁇ -cells to promote insulin secretion. , At the same time antagonize the GC receptor to inhibit the rise of blood sugar. GLP-1 receptor agonist was developed as a hyperglycemic therapeutic agent for diabetic patients, protects and proliferates islet cells while lowering blood glucose, and slows gastric emptying and inhibits food intake, which can effectively reduce body 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 abirutai, du Larutide, Byuderon, and Somalutide (1 time / week). GLP-1 receptor agonist drugs have a safe and unique hypoglycemic effect, but when taking into account the effect of weight loss, large doses are generally required, and at high doses these drugs are prone to gastrointestinal side effects and poor tolerance leads to treatment The window is narrow. Therefore, there is still a need for more tolerated therapeutic agents that can effectively control blood sugar and reduce weight.
  • Oxytomodulin is a hormone produced in the intestine during the post-translational modification of proglucagon. It is secreted from ileal L-cells and other hormones such as GLP-1 during the meal reaction.
  • the acute effects of OXM include gastric emptying, exocrine secretion of the stomach and pancreas, as well as suppression of food intake, resting energy expenditure, etc., which can have a weight-reducing effect.
  • OXM-specific receptors have not yet been clarified, but studies have found that OXM is an endogenous GCGR / GLP-1R dual agonist, and its activity against two receptors is weaker than the natural ligands of each receptor.
  • peripheral administration of OXM can reduce food intake and weight loss, increase metabolic rate and especially energy expenditure related to activity in obese subjects.
  • high-dose peripheral administration of OXM reduces body weight and the common occurrence of common gastrointestinal side effects such as nausea and vomiting is low. Therefore, treatment based on OXM or GLP-1 / GCGR dual agonists shows potential application value for obesity and obesity-type diabetes.
  • An object of the present invention is to provide a polypeptide derivative having a dual agonistic effect of glucagon / glucagon-like peptide-1 receptor, the polypeptide is a variant designed based on the natural sequence of GC, and retains the energy of GC.
  • the beneficial effects of metabolism also have a hypoglycemic effect, which has a synergistic effect on energy metabolism. potential.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the polypeptide derivative of the present invention having a dual agonist effect of glucagon / glucagon-like peptide-1 receptor.
  • Another object of the present invention is to provide a use of the polypeptide derivative of the present invention having a dual agonist effect of glucagon / glucagon-like peptide-1 receptor.
  • the present invention provides a polypeptide derivative, a modified derivative thereof, or a salt thereof, which comprises a polypeptide having the following formula I:
  • X 2 is Ser, D-Ser or Aib
  • X 10 is Lys or Tyr
  • X 12 is Lys or Arg
  • X 15 is Asp or Glu
  • X 17 is Arg or Lys
  • X 18 is Lys, Ala or Arg
  • X 20 is Gln or Lys
  • X 23 is Val or Ile
  • X 24 is Ala or Glu
  • X 27 is Leu or Val
  • X 28 is Ala or Glu
  • X 29 is Gly or Glu
  • X 30 is Glu, Arg or Gly
  • X 31 is Gly, -NH 2 or does not exist
  • C-terminal carboxyl group is free or amidated
  • X 2 is Aib
  • X 17 is Arg
  • X 18 is Ala
  • X 10 and X 20 have only one side chain modified Lys; and, when X 20 is a side chain modified Lys, X 10 is Tyr.
  • X 27 is Leu
  • X 28 is Glu
  • X 29 is Gly
  • X 30 is Arg
  • X 31 is Gly, -NH 2 or absent
  • C-terminal carboxyl group is free or amide Change.
  • sequence of the polypeptide of the present invention is selected from any one of the following SEQ ID NOs. 4-26: SEQ ID NO. 4 HAibQGTFTSD ⁇ SKYLEERAAKEFVEWLLEGRG
  • X 27 is Leu
  • X 28 is Glu
  • X 29 is Gly
  • X 30 is Gly
  • X 31 is -NH 2 or does not exist.
  • sequence of the polypeptide of the present invention is selected from any one of the following SEQ ID Nos. 25-34:
  • Aib represents ⁇ -aminoisobutyric acid
  • ⁇ represents Lys whose side chain is modified.
  • the side chain ⁇ -amino group of Lys is modified by coupling a fatty acid through a hydrophilic linking fragment.
  • the hydrophilic linking fragment is selected from fragments consisting of one or more of Glu, ⁇ Glu, Gly, and Ado (8-amino-3,6 dioxyoctanoic acid), preferably selected from ⁇ Glu- ⁇ Glu-, Glu- ⁇ Glu-, Glu- ⁇ Glu-, ⁇ Glu-Gly-Gly, ⁇ Glu-Gly- ⁇ Glu-, ⁇ Glu-Ado-Ado-; Ado-Ado- ⁇ Glu-, and ⁇ Glu-Ado-Ado- ⁇ Glu-.
  • the fatty acid used for acylation is preferably selected from C14-20 fatty acids, and more preferably C16-20 fatty diacids.
  • the present invention provides a pharmaceutical composition containing the polypeptide derivative of the present invention, a modified derivative thereof, or a salt thereof.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable auxiliary materials include carriers, diluents, water-soluble fillers, pH adjusting agents, stabilizers, water for injection, osmotic pressure adjusting agents and the like.
  • the water-soluble filler includes but is not limited to mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, etc .
  • the pH adjusting agent includes but is not limited to citric acid, phosphoric acid , Lactic acid, tartaric acid, hydrochloric acid and other organic or inorganic acids and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate and other physiologically acceptable Inorganic base or salt
  • the stabilizer includes but is not limited to EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamine Acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrroli
  • the pharmaceutical composition of the present invention can be administered in the form of intravenous, intramuscular, or subcutaneous injection, orally, rectally, or nasally.
  • the dosage range may be 5 ⁇ g-10 mg / time, depending on the subject to be treated, administration method, indications, and other factors.
  • the present invention provides the use of the polypeptide derivative or its salt according to the present invention in the preparation of a medicament for the treatment of metabolic diseases.
  • the metabolic diseases are diabetes, obesity, fatty liver, and 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, including administering to a patient in need of the polypeptide derivatives, modified derivatives or salts thereof of the present invention.
  • the metabolic diseases are diabetes and obesity , Fatty liver, hyperlipidemia and / or metabolic syndrome; more preferably, the fatty liver is non-alcoholic fatty liver.
  • the polypeptide derivative of the present invention Compared with a simple GLP-1 receptor agonist, the polypeptide derivative of the present invention has a more effective effect of lowering blood sugar while promoting weight loss and preventing weight gain, reversing insulin resistance, and has a significant effect compared with existing drugs. Unexpected beneficial effects.
  • endogenous GLP-1 is a derivative peptide containing 37 amino acid residues (7-36 / 37) corresponding to amino acids 72 to 108 of pre-glucagon, and its amino acids
  • the sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (7-36) (SEQ ID NO. 1), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (7-37), and its C-terminus is free or amidated.
  • Endogenous GC is a derivative peptide containing 29 amino acids corresponding to amino acids 33 to 61 of proglucagon.
  • the amino acid sequence is: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO.
  • the amino acid sequences of natural GLP-1 and GC have 47% homology (Andreas Evers et al., J. Med. Chem. 2017, 60, 4293-4303), the N-terminal sequences of the two are highly conserved, and GLP-1 pairs Its receptor is highly selective, and GC is also a weak agonist of the GLP-1 receptor. Therefore, it is feasible to design GLP-1 / GCGR dual agonists based on GC sequences.
  • the amino acid sequence of OXM is HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO.
  • positions 16-20, 24 and specific positions of the C-terminal sequence of the sequence are replaced with appropriate charged amino acids to obtain a sequence with an appropriate activity-potency ratio.
  • 16 Ser is replaced with Glu
  • 18Arg is replaced with Ala
  • 20Gln is replaced with Lys
  • the site 24Gln is replaced with Glu
  • site 28 is replaced with Glu and site 30 is Lys or Arg.
  • position 28 is Glu and position 30 is Arg.
  • the N-terminal dipeptide is susceptible to dipeptide kininase in the body Identified and inactivated by hydrolysis, resulting in short plasma half-life ( ⁇ 12min); unstable physical properties, ie isoelectric point (pI) 7.6, neutral, and hydrophobic, poor solubility, easy to precipitate in solution; sequence There are amino acids that are easily oxidized or racemic, such as Met, Asp, Asn, resulting in chemical instability.
  • amino acids such as Asp, Met, Gln, and Asn in the sequence were replaced, for example, 15, 21 Asp was replaced with Glu; 27 Met was replaced with Leu or Val; 28 Asn was replaced with Glu.
  • the present inventors found that the introduction of the above-mentioned charged amino acids aimed at adjusting the C-terminal charge distribution helps to improve the solubility of the modified peptide (Example 4). Therefore, these adjustment measures have multiple beneficial effects on the activity balance and the improvement of physical and chemical properties.
  • 2Ser is usually replaced with Aib or D-Ser, and the embodiment of the present invention is preferably replaced with Aib.
  • Fatty acyl modification is a polypeptide long-acting technique known in the art.
  • the fatty acyl group is modified on the Lys side chain at a specific position in the active peptide sequence by a hydrophilic linking arm.
  • the lysine residue is located at position 10
  • the modification site is at position 20.
  • the hydrophilic linking arm is - ⁇ -Glu- ⁇ -Glu-, in other embodiments it is - ⁇ -Glu-Ado-Ado- ⁇ -Glu; or in another In some embodiments, it is -Ado-Ado- ⁇ -Glu.
  • the fatty acyl group is preferably selected from C16-20 fatty acids or fatty diacids. In certain embodiments, the fatty acyl group is a C16 or C18 acyl group; in certain specific embodiments, it is a C18, C20 diacid monoester acyl group.
  • the present invention provides a GLP-1 / glucagon dual agonist peptide.
  • the polypeptide provided by the present invention is compared with the natural ligands of GLP-1 / GCGR receptors At least 1% of receptor agonistic activity.
  • the agonistic effect on the GLP-1 receptor is equivalent to the endogenous natural ligand GLP-1 (7-36 / 37) or equivalent to 150%, 200%, 300 of the natural ligand %, 500%, 1000% or more.
  • the agonistic activity on the glucagon receptor is equivalent to the endogenous ligand (GC) or 10 to 1000% of the action intensity of the endogenous agonist.
  • the polypeptide provided by the present invention has receptor agonist activity equal to or stronger than that of the endogenous ligand. In other embodiments, the agonistic effect on the GLP-1 receptor is stronger than the glucagon receptor, or the agonistic effect on the two receptors is equal.
  • the relative activity intensity for GLP-1 and glucagon receptors can be expressed by the potency ratio, that is, the potency ratio of the polypeptide of the general formula I provided by the present invention to the GLP-1 / glucagon receptor includes But not limited to 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 3: 1, 1: 1 to 1:10.
  • the basic peptide chain of the polypeptide derivative having the structure of general formula I provided by the present invention can be prepared by a method known in the art:
  • the target peptide is preferably prepared by using the Fmoc solid phase synthesis method, which is well known to those skilled in the art.
  • the substituents can be introduced synthetically step by step through the above peptide synthesis steps.
  • Use substituents with appropriate protecting groups such as Fmo-8-amino-3,6 dioxocetanoic acid, and Fmoc- ⁇ -Glu-OtBu.
  • the introduction of fatty chain moieties, especially fatty diacid monoester acyl groups can be achieved using but not limited to C18, C20 mono-tert-butyl alkanoates.
  • the unreacted intermediate can be blocked with excess acetic anhydride and pyridine.
  • the epsilon-amino group of the modifiable Lys can be protected with Mtt or Dde.
  • the target product can be isolated by a suitable method known in the art. Suitable 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 assay In embodiments of the present invention, the effect of the polypeptide on the GLP-1 / GC receptor was evaluated by the effect of GLP-1 / GC receptor-mediated cAMP production in vitro.
  • Trp Tryptophan (W)
  • HATU O- (7-azabenzotriazol-1-yl) -N, N, N ’, N’-tetramethyl-ureium hexafluorophosphate
  • FBS fetal bovine serum
  • the substantially linear sequence of the polypeptide provided by the present invention and the side chain modification derived peptide are prepared according to the following general method:
  • Fmoc-amino acid-resin is obtained by coupling the C-terminal amino acid protected by the resin solid phase carrier and Fmoc; wherein, the synthetic C-terminal amidated peptide uses amino resin, such as Rink Amide AM, Rink Amide , Rink MBHA, etc .; Fmoc-amino acid to resin ratio (mol / mol) is 3 to 5: 1, coupling activator is HOBT / DIC or HOBT / HBTU / DIEA.
  • Elongation of the peptide chain amino acids are connected according to the amino acid sequence of the peptide sequence by solid phase synthesis to obtain a peptide-resin conjugate protected at the N-terminus and side chain; amino acid with side chain takes the following protective measures: Boc for tryptophan , OtBu for glutamic acid, Boc for lysine, Trt for glutamine, tBu for tyrosine, Trt or tBu for serine, OtBu for aspartic acid, tBu for threonine, and Trt for cysteine , Arginine is protected with Pbf, the histidine (Trt) ⁇ -amino group is protected with Boc, and the modifiable lysine ⁇ -amino group is protected with Dde.
  • the coupling activators used were HOBT / DIC, HOBT / HBTU / DIEA and HOBT / HATU / DIEA.
  • the ninhydrin method was used to detect the end point of the reaction.
  • the deprotecting agent was NMP (DMF) solution containing 20% piperidine.
  • AKP09 SEQ ID NO.25 HAibQGTFTSDK ** SKYLEERAAKEFIAWLLEGRG AKP10 SEQ ID NO.22 HAibQGTFTSDYKSKYLEERAAK ** EFIAWLLEGREGR AKP11 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK * EFVEWLLEGRG-NH 2 AKP12 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK * EFVEWLLEGRG AKP13 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK ** EFVEWLLEGRG AKP14 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK *** EFVEWLLEGRG AKP15 SEQ ID NO.25 HAibQGTFTSDK * SKYLEERAAKEFVEWLLEGG-NH 2 AKP16 SEQ ID NO.27 HAibQGTFTSDK * SKYLE
  • AKP33 SEQ ID NO.13 HAibQGTFTSDK *** SKYLEERAAKEFIEWLLEGR-NH 2 AKP34 SEQ ID NO.14 HAibQGTFTSDK ** SKYLEERAAQEFIEWLLEGR-NH 2 AKP35 SEQ ID NO.15 HAibQGTFTSDK ** SKYLEERAAQEFIAWLLEGR-NH 2 AKP36 SEQ ID NO.17 HAibQGTFTSDYSKYLEERAAK ** EFVAWLLEGR-NH 2 AKP37 SEQ ID NO.18 HAibQGTFTSDYSKYLEERAAK ** EFVEWLLEGR-NH 2 AKP38 SEQ ID NO.18 HAibQGTFTSDYSKYLEERAAK * EFVEWLLEGR-NH 2 AKP39 SEQ ID NO.20 HAibQGTFTSDYSKYLEERAAK ** EFVAWLLEGRG AKP40 SEQ ID NO.21 HAibQGT
  • the effect of the polypeptide on the GLP-1 / GC receptor was evaluated by its effect on GLP-1 / GC receptor-mediated cAMP production in vitro.
  • test samples were incubated with purified porcine DPP-IV (5 milliunits) in 50 mmol / L TEA-HCl (pH 7.8) at 37 ° C for 0, 2, 4, and 8 hours.
  • HPLC method chromatographic column: Aeris widepore XB-C18 3.6 ⁇ m, 4.6 ⁇ 150mm; mobile phase: A: 0.05% TFA, B: 95% acetonitrile; detection wavelength: 214nm
  • test results show that the test compounds of the present invention have a half-life of more than 8 hours in the DPP-IV enzyme-containing solution system, indicating that the structural modification strategy adopted can effectively prevent the compound from being hydrolyzed by the DPP-IV enzyme.
  • test compound of the present invention has significantly improved solubility under endogenous glucagon under the pH conditions of injections acceptable to the body, and has characteristics favorable to the preparation.
  • the model group was randomly divided into 6 groups (average weight 45.2g) according to body weight, with 5 animals in each group. They were model control group, positive control group (somalutide), and test sample group. .
  • the blank control group and model control group were given subcutaneous saline every day, and the positive control group and the test sample group were given subcutaneous injections every day for 14 days. Weigh and record the body weight of the animals every day, compare the last dose day with the initial body weight, and calculate the weight change rate (%). The results are shown in Table 5.
  • the positive value of the calculation result indicates a decrease, and the negative value indicates an increase.
  • the model animals were continuously administered for 2 weeks. Compared with the model group, both the test compound and the positive control drug showed a significant weight loss effect. Among them, the compound of the present invention had a stronger effect than the positive control drug. As shown in Table 6, in the single-dose glucose load test, the test compound showed a hypoglycemic activity equivalent to that of the positive drug. Combined with the effect on body weight and the results of hypoglycemic effect, it is shown that the compound of the present invention has the characteristics of GC / GLP-1 dual agonist, which is consistent with the results of in vitro receptor activity evaluation, compared with the simple GLP-1 receptor agonist, More application potential.

Abstract

提供具有双受体激动作用的多肽衍生物、其修饰衍生物或其盐，包含具有以下通式Ⅰ序列的多肽：通式Ⅰ： HX 2QGTFTSDX 10SX 12YLX 15EX 17X 18AX 20EFX 23X 24WLX 27X 28X 29X 30X 31，其中，X 2、X 10、X 12、X 15、X 17、X 18、X 20、X 23、X 24、X 27、X 28、X 29、X 30和X 31的定义与权利要求书和说明书的定义一致。还提供所述多肽衍生物、其修饰衍生物或其盐的用途。所述多肽是基于GC天然序列设计的变体，通过GC/GLP-1受体的双重激动对能量代谢产生协同影响，有效降低血糖的同时可减轻体重、改善体脂水平，比单一受体激动剂在糖尿病、肥胖、代谢综合征以及非酒精性脂肪肝等疾病状态的改善更具潜力。

Description

具有双重受体激动作用的胰高血糖素衍生肽及其用途 技术领域
本发明属于医药生物技术领域，具体涉及具有胰高血糖素/胰高血糖素样肽-1受体双重激动作用的多肽以及其用途。
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。前胰高血糖(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是对应于前胰高血糖素的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是对应于前胰高血糖素的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受体激动剂药物具有安全独特的降血糖作用，但在兼顾减轻体重作用时，一般需要使用大剂量，而在大剂量下这些药物易产生胃肠道副作用，耐受性差而导致治疗窗较窄。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)是前胰高血糖素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。基于GC或OXM序列的双重激动剂已公开的专利文件有：CN200880012086.2、CN200980132562.9、CN201080027026.5、CN201280037512.4、CN201280040883.3、CN201310158648.7、CN201480025780.3、CN201380062842.3、CN201780013643.1、CN201680021972.6、CN201380048137.8、CN20980158734.X、CN200780006185.5等。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具有胰高血糖素/胰高血糖素样肽-1受体双重激动作用的多肽衍生物，所述多肽是基于GC天然序列设计的变体，保留GC对能量代谢的有益作用同时兼具降血糖作用，从而对能量代谢产生协同影响，比单一受体激动剂在制备用于治疗糖尿病、肥胖、代谢综合征以及非酒精性脂肪肝等疾病的药物方面更具潜力。
本发明的另一个目的是提供一种包含本发明的具有胰高血糖素/胰高血糖素样肽-1受体双重激动作用的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具有胰高血糖素/胰高血糖素样肽-1受体双重激动作用的多肽衍生物的用途。
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序列的多肽：
通式Ⅰ：HX 2QGTFTSDX 10SX 12YLX 15EX 17X 18AX 20EFX 23X 24WLX 27X 28X 29X 30X 31，
其中：
X 2为Ser、D-Ser或Aib；
X 10为Lys或Tyr；
X 12为Lys或Arg；
X 15为Asp或Glu；
X 17为Arg或Lys；
X 18为Lys、Ala或Arg；
X 20为Gln或Lys；
X 23为Val或Ile；
X 24为Ala或Glu；
X 27为Leu或Val；
X 28为Ala或Glu；
X 29为Gly或Glu；
X 30为Glu、Arg或Gly；
X 31为Gly、-NH 2或不存在；
C-末端羧基游离或酰胺化；
其中，X 10、X 17和X 20中有且只有一个侧链被修饰的Lys。
本发明优选的实施方案中，所述通式Ⅰ中，
X 2为Aib；
X 17为Arg；
X 18为Ala；
其中，X 10和X 20中有且只有一个侧链被修饰的Lys；并且，当X 20为侧链被修饰的Lys时，X 10是Tyr。
更优选地，所述通式Ⅰ中，X 27为Leu，X 28为Glu，X 29为Gly，X 30为Arg，X 31为Gly、-NH 2或不存在，C-末端羧基游离或酰胺化。
优选地，本发明所述多肽的序列选自以下SEQ ID NOs.4-26中的任一个：SEQ ID NO.4 HAibQGTFTSDψSKYLEERAAKEFVEWLLEGRG
SEQ ID NO.5 HAibQGTFTSDψSKYLEERAAQEFVEWLLEGRG
SEQ ID NO.6 HAibQGTFTSDψSKYLEERAAQEFVAWLLEGRG
SEQ ID NO.7 HAibQGTFTSDψSKYLEERAAKEFIEWLLEGRG
SEQ ID NO.8 HAibQGTFTSDψSKYLEERAAQEFIEWLLEGRG
SEQ ID NO.9 HAibQGTFTSDψSKYLEERAAQEFIAWLLEGRG
SEQ ID NO.10 HAibQGTFTSDψSKYLEERAAKEFVEWLLEGR
SEQ ID NO.11 HAibQGTFTSDψSKYLEERAAQEFVEWLLEGR
SEQ ID NO.12 HAibQGTFTSDψSKYLEERAAQEFVAWLLEGR
SEQ ID NO.13 HAibQGTFTSDψSKYLEERAAKEFIEWLLEGR
SEQ ID NO.14 HAibQGTFTSDψSKYLEERAAQEFIEWLLEGR
SEQ ID NO.15 HAibQGTFTSDψSKYLEERAAQEFIAWLLEGR
SEQ ID NO.16 HAibQGTFTSDYSKYLEERAAψEFVEWLLEGR
SEQ ID NO.17 HAibQGTFTSDYSKYLEERAAψEFVAWLLEGR
SEQ ID NO.18 HAibQGTFTSDYSKYLEERAAψEFVEWLLEGRG
SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAψEFVEWLLEGRG
SEQ ID NO.20 HAibQGTFTSDYSKYLEERAAψEFVAWLLEGRG
SEQ ID NO.21 HAibQGTFTSDYSKYLEERAAψEFIEWLLEGRG
SEQ ID NO.22 HAibQGTFTSDYSKYLEERAAψEFIAWLLEGRG
SEQ ID NO.23 HAibQGTFTSDYSKYLEERAAψEFIEWLLEGR
SEQ ID NO.24 HAibQGTFTSDYSKYLEERAAψEFIAWLLEGR
SEQ ID NO.25 HAibQGTFTSDψSKYLEERAAQEFIAWLLEGRG
SEQ ID NO.26 HAibQGTFTSDψSKYLEERAAQEFIAWLLEGR
在另一些优选的实施方案中，所述通式Ⅰ中，X 27为Leu；
X 28为Glu；
X 29为Gly；
X 30为Gly；
X 31为-NH 2或不存在。
优选地，本发明所述多肽的序列选自以下SEQ ID NOs.25-34中任一个：
SEQ ID NO.27 HAibQGTFTSDψSKYLEERAAKEFVEWLLEGG
SEQ ID NO.28 HAibQGTFTSDψSKYLEERAAQEFVEWLLEGG
SEQ ID NO.29 HAibQGTFTSDψSKYLEERAAQEFVAWLLEGG
SEQ ID NO.30 HAibQGTFTSDψSKYLEERAAKEFIEWLLEGG
SEQ ID NO.31 HAibQGTFTSDψSKYLEERAAQEFIEWLLEGG
SEQ ID NO.32 HAibQGTFTSDψSKYLEERAAQEFIAWLLEGG
SEQ ID NO.33 HAibQGTFTSDYSKYLEERAAψEFVEWLLEGG
SEQ ID NO.34 HAibQGTFTSDYSKYLEERAAψEFVAWLLEGG
SEQ ID NO.35 HAibQGTFTSDYSKYLEERAAψEFIEWLLEGG
SEQ ID NO.36 HAibQGTFTSDYSKYLEERAAψEFIAWLLEGG。
其中，在本发明的SEQ ID NOs.4～36中，Aib表示α-氨基异丁酸，ψ表示侧链被修饰的Lys，。
在本发明优选的实施方案中，本发明的多肽衍生物中，所述Lys的侧链ε-氨基是通过亲水性连接片段偶联脂肪酸而被修饰。
优选地，所述亲水性连接片段选自由Glu、γGlu、Gly、Ado(8-氨基-3,6二氧辛酸)中的一种或多种组成的片段，优选选自γGlu-γGlu-、Glu-γGlu-、Glu-γGlu-、γGlu-Gly-Gly、γGlu-Gly-γGlu-、γGlu-Ado-Ado-；Ado-Ado-γGlu-、和γGlu-Ado-Ado-γGlu-。
所述用于酰化的脂肪酸，优选选自C14-20的脂肪酸，更优选为C16-20脂肪二酸。
另一方面，本发明提供一种药物组合物，其含有本发明所述多肽衍生物、其修饰衍生物或其盐。
优选地，所述药物组合物进一步包含一种或多种药学上可接受的辅料。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、半乳糖等；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等；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。剂量范围可以为5μg-10mg/次，这取决于治疗对象、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本发明所述的多肽衍生物、其修饰衍生物或其盐，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。
与单纯的GLP-1受体激动剂相比，本发明的多肽衍生物在更为有效地降低血糖的同时具有促进减重和防止增重作用，逆转胰岛素抵抗，与现有药物相比具有意想不到的有益作用。
胰高血糖素衍生肽的结构：内源性GLP-1是对应于前胰高血糖素的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-端游离或酰胺化。内源性GC是对应于前胰高血糖素的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。因此，基于GC序列设计GLP-1/GCGR双重激动剂是可行的。OXM的氨基酸序列是HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA(SEQ ID NO.3),包含GC的原序列(1-29)和对应于前胰高血糖素原氨基酸序列82-89的插入肽-1(IP-1，30-37)。OXM对GC/GLP-1R的双重激动作用以及其相对于内源性GC对两个受体的活性效价比的显著改变提示，对GC序列的C-端电荷分布进行调整可以得到适当比例的GC/GLP-1R双重激动剂。
在本发明的实施方案中，对序列的位点16-20，位点24和C端序列的特定位点以适当的带电荷氨基酸进行替换，得到适当活性效价比的序列。如，在一些特定实施方案中，将 16Ser替换为Glu,同时18Arg、替换为Ala；在一些实施方案中，20Gln被替换为Lys；在另一些实施方案中，位点24Gln被替换为Glu；或位点28被替换为Glu、位点30为Lys或Arg。在一些优选实施方案中位点28为Glu位点30是Arg。
一般的，对天然序列进行尽可能少的改变有利于保持同源性，但内源性胰高血糖素-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。
为提高肽序列的化学稳定性，对序列中的Asp，Met，Gln，Asn等氨基酸进行了替换，如15，21Asp替换为Glu；27Met替换为Leu或Val；28Asn替换为Glu。同时本发明人发现上述旨在调整C-末端电荷分布的带电荷氨基酸的引入，有助于改善修饰肽的溶解性(实施例4)。因此，这些调整措施对活性平衡和理化性质的改善具有多重有益作用。
本发明优选实施方案中，为提高所述多肽的代谢稳定性，2Ser通常被替换为Aib或D-Ser，本发明实施方案优选以Aib替换。
脂肪酰修饰是本技术领域公知的多肽长效化技术。本发明的实施方案中，在活性肽序列中特定位点的Lys侧链上通过亲水性连接臂修饰了脂肪酰基。在某些优选的实施方案中所述赖氨酸残基位于10位，在某些实施方案中修饰位点为20位。为改善修饰后多肽的溶解性和保持多肽结构的柔性，以亲水性连接臂连接多肽和脂肪酰修饰基团是必要的。在本发明某些实施方案中所述亲水性连接臂是-γ-Glu-γ-Glu-，在另一些实施方案中则 为-γ-Glu-Ado-Ado-γ-Glu；或在另一些实施方案中则是-Ado-Ado-γ-Glu。所述脂肪酰基优选选自C16-20脂肪酸或脂肪二酸。在某些实施方案中，所述脂肪酰基是C16或C18酰基；在某些特定实施方案中是C18、C20二酸单酯酰基。
本发明提供GLP-1/胰高血糖素双重激动作用的肽，通过本发明前述的结构设计，具有以下活性特点：本发明提供的多肽与GLP-1/GCGR各受体的天然配体相比至少具有1％的受体激动活性。通过一系列的结构优化设计，对GLP-1受体的激动作用强度与内源性天然配体GLP-1(7-36/37)相当或相当于天然配体的150％，200％，300％，500％，1000％或更多。对胰高血糖素受体的激动活性与内源性配体(GC)相当或相当于内源性激动剂作用强度的10～1000％。在某些实施方案中，本发明提供的多肽具有与内源性配体相等或更强的受体激动活性。在另一些实施方案中，对GLP-1受体的激动作用强于对胰高血糖素受体，或对两个受体的激动作用强度相等。对GLP-1和胰高血糖素受体的相对活性强度可以用效价比表示，即本发明提供的包含通式Ⅰ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10:1,9:1，8:1，7:1，6:1，3:1,1:1至1:10。
本发明提供的具有通式Ⅰ结构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，该技术为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，Fmo-8-氨基-3,6二氧杂辛酸，和Fmoc-γ-Glu-OtBu。脂肪链部分的引入，尤其是脂肪二酸单酯酰基，可以使用但不限于C18、C20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高脂饮食肥胖型糖尿病小鼠(Dio)模型评价了本发明提供多肽的对体重和血糖的影响，结果表明本发明提供的多肽衍生物具有显著的减轻体重和降血糖作用，体重减轻作用显著优于阳性对照药，提示在制备肥胖等代谢性疾病的控制和糖尿病的治疗药物方面具有潜在优势。
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:苯丙氨酸(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(Trt)的α-氨基用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％)。
以上述方法合成了表1所列化合物。
表1合成得到的化合物
化合物编号 序列号 结构
AKP 01 SEQ ID NO.4 HAibQGTFTSDK *SKYLEERAAKEFVEWLLEGRG-NH 2
AKP 02 SEQ ID NO.4 HAibQGTFTSDK *SKYLEERAAKEFVEWLLEGRG
AKP 03 SEQ ID NO.4 HAibQGTFTSDK **SKYLEERAAKEFVEWLLEGRG
AKP 04 SEQ ID NO.5 HAibQGTFTSDK *SKYLEERAAQEFVEWLLEGRG-NH 2
AKP 05 SEQ ID NO.5 HAibQGTFTSDK **SKYLEERAAQEFVEWLLEGRG
AKP 06 SEQ ID NO.7 HAibQGTFTSDK *SKYLEERAAKEFIEWLLEGRG-NH 2
AKP 07 SEQ ID NO.9 HAibQGTFTSDK *SKYLEERAAQEFIAWLLEGRG-NH 2
AKP 08 SEQ ID NO.9 HAibQGTFTSDK **SKYLEERAAQEFIAWLLEGRG-NH 2
AKP 09 SEQ ID NO.25 HAibQGTFTSDK **SKYLEERAAKEFIAWLLEGRG
AKP10 SEQ ID NO.22 HAibQGTFTSDYKSKYLEERAAK **EFIAWLLEGRG
AKP11 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK *EFVEWLLEGRG-NH 2
AKP12 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK *EFVEWLLEGRG
AKP13 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK **EFVEWLLEGRG
AKP14 SEQ ID NO.19 HAibQGTFTSDYSKYLEERAAK ***EFVEWLLEGRG
AKP15 SEQ ID NO.25 HAibQGTFTSDK *SKYLEERAAKEFVEWLLEGG-NH 2
AKP16 SEQ ID NO.27 HAibQGTFTSDK *SKYLEERAAQEFVAWLLEGG-NH 2
AKP17 SEQ ID NO.30 HAibQGTFTSDK *SKYLEERAAQEFIAWLLEGG-NH 2
AKP18 SEQ ID NO.31 HAibQGTFTSDYSKYLEERAAK *EFVEWLLEGG-NH 2
AKP19 SEQ ID NO.31 HAibQGTFTSDYSKYLEERAAK **EFVEWLLEGG-NH 2
AKP20 SEQ ID NO.28 HAibQGTFTSDK **SKYLEERAAQEFVEWLLEGG-NH 2
AKP21 SEQ ID NO.27 HAibQGTFTSDK **SKYLEERAAKEFVEWLLEGG
AKP22 SEQ ID NO.27 HAibQGTFTSDK ***SKYLEERAAKEFVEWLLEGG-NH 2
AKP23 SEQ ID NO.27 HAibQGTFTSDK ***SKYLEERAAKEFVEWLLEGRG
AKP24 SEQ ID NO.33 HAibQGTFTSDYSKYLEERAAK ***EFVEWLLEGRG
AKP25 SEQ ID NO.28 HAibQGTFTSDK ***SKYLEERAAQEFVEWLLEGG-NH 2
AKP26 SEQ ID NO.10 HAibQGTFTSDK *SKYLEERAAKEFVEWLLEGR-NH 2
AKP27 SEQ ID NO.11 HAibQGTFTSDK *SKYLEERAAQEFVEWLLEGR-NH 2
AKP28 SEQ ID NO.16 HAibQGTFTSDYSKYLEERAAK **EFVEWLLEGR-NH 2
AKP29 SEQ ID NO.24 HAibQGTFTSDYKSKYLEERAAK **EFIAWLLEGR-NH 2
AKP30 SEQ ID NO.5 HAibQGTFTSDK ***SKYLEERAAQEFVEWLLEGR
AKP31 SEQ ID NO.6 HAibQGTFTSD K **SKYLEERAAQEFVAWLLEGRG
AKP32 SEQ ID NO.12 HAibQGTFTSDK **SKYLEERAAQEFVAWLLEGR-NH 2
AKP33 SEQ ID NO.13 HAibQGTFTSDK ***SKYLEERAAKEFIEWLLEGR-NH 2
AKP34 SEQ ID NO.14 HAibQGTFTSDK **SKYLEERAAQEFIEWLLEGR-NH 2
AKP35 SEQ ID NO.15 HAibQGTFTSDK **SKYLEERAAQEFIAWLLEGR-NH 2
AKP36 SEQ ID NO.17 HAibQGTFTSDYSKYLEERAAK **EFVAWLLEGR-NH 2
AKP37 SEQ ID NO.18 HAibQGTFTSDYSKYLEERAAK **EFVEWLLEGR-NH 2
AKP38 SEQ ID NO.18 HAibQGTFTSDYSKYLEERAAK *EFVEWLLEGR-NH 2
AKP39 SEQ ID NO.20 HAibQGTFTSDYSKYLEERAAK **EFVAWLLEGRG
AKP40 SEQ ID NO.21 HAibQGTFTSDYSKYLEERAAK **EFIEWLLEGRG
AKP41 SEQ ID NO.23 HAibQGTFTSDYSKYLEERAAK **EFIEWLLEGR-NH 2
AKP42 SEQ ID NO.25 HAibQGTFTSDK **SKYLEERAAKEFIAWLLEGRG
AKP43 SEQ ID NO.25 HAibQGTFTSDK *SKYLEERAAKEFIAWLLEGRG
AKP44 SEQ ID NO.26 HAibQGTFTSDK *SKYLEERAAKEFIAWLLEGR-NH 2
AKP45 SEQ ID NO.26 HAibQGTFTSDK **SKYLEERAAKEFIAWLLEGR-NH 2
AKP46 SEQ ID NO.29 HAibQGTFTSDK **SKYLEERAAQEFVAWLLEGG-NH 2
AKP47 SEQ ID NO.29 HAibQGTFTSDK **SKYLEERAAQEFVAWLLEGG
AKP48 SEQ ID NO.30 HAibQGTFTSDK ***SKYLEERAAKEFIEWLLEGG
AKP49 SEQ ID NO.31 HAibQGTFTSDK **SKYLEERAAQEFIEWLLEGG-NH 2
AKP50 SEQ ID NO.32 HAibQGTFTSDK *SKYLEERAAQEFIAWLLEGG
AKP51 SEQ ID NO.34 HAibQGTFTSDYSKYLEERAAK **EFVAWLLEGG
AKP52 SEQ ID NO.35 HAibQGTFTSDYSKYLEERAAK **EFIEWLLEGG-NH 2
AKP53 SEQ ID NO.36 HAibQGTFTSDYSKYLEERAAK *EFIAWLLEGG
*：-γE-γE-OC 16H 31
**:-Ado-Ado-γE-OC 17H 32COOH
***:-Ado-Ado-γE-OC 19H 36COOH
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。
将转染有人GLP-1受体的中国豚鼠非细胞和转染GC受体的HEK293细胞接种到96孔培养板，(200000个/孔)，以Hanks’平衡盐缓冲液洗涤后，与不同浓度的受试多肽样品(10 -5～10 -12mol/L)，在200mol/L 3-异丁基-1-甲基茜草黄素存在下于37℃共孵20min。去除介质，溶解细胞，测定cAMP值,测定方法参照分析试剂盒说明。用Origin软件计算50％有效浓度(EC50)。结果见表2。
表2 AKP系列化合物对GLP-1/GC受体的激动作用
Figure PCTCN2019117614-appb-000001
Figure PCTCN2019117614-appb-000002
结论：如表2所示，受试化合物与内源性GC相比，对GLP-1受体的激动活性均不同程度的提高，其中，除AKP24、16以外的化合物活性强度相当或显著强于内源性GLP-1的活性。
实施例3
AKP系列化合物对DPP-IV酶降解的抗性
受试样品于37℃在50mmol/L TEA-HCl(pH7.8)中与纯化的猪DPP-Ⅳ(5毫单位)共孵0，2，4，8小时。HPLC法(色谱柱：Aeris widepore XB-C18 3.6μm,4.6×150mm；流动相：A:0.05％TFA,B:95％乙腈；检测波长：214nm)测定各时间点溶液中的残留样品峰面积，计算样品消除率。结果见表3。
表3 AKP系列化合物在DPP-IV酶体系中的半衰期
肽 半衰期(h)
GLp-1 2.9±0.30
GC 2.6±0.12
AKP01 ＞8
AKP02 ＞8
AKP03 ＞8
AKP15 ＞8
AKP18 ＞8
AKP23 ＞8
AKP27 ＞8
试验结果表明，本发明的受试化合物在含DPP-IV酶溶液体系中的半衰期均超过8个小时，说明采取的结构修饰策略可有效避免化合物被DPP-IV酶水解。
实施例4
AKP系列化合物的溶解性考察
称取2mg化合物，分别以1ml不同浓度(10、20mM)、不同pH值(7.5、8.0)的磷酸盐缓冲液溶解，离心，取上清液，HPLC法(色谱柱：Aeris widepore XB-C18 3.6μm,4.6×150mm；流动相：A:0.05％TFA,B:95％乙腈；检测波长：214nm)测定峰面积，与相应样品标准溶液比对，计算得到受试样品溶液的相对浓度。结果见表4。
表4 AKP系列化合物的溶解性
Figure PCTCN2019117614-appb-000003
Figure PCTCN2019117614-appb-000004
结论：如表4结果显示，本发明的受试化合物与内源性胰高血糖素相比，在机体可接受的注射液pH条件下的溶解性大幅改善，具备了有利于制剂的特性。
实施例5
AKP系列化合物对DIO鼠血糖和体重的影响
1)对体重的影响
C57BL/6J小鼠，35只，模型组(n＝30)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34周。首次给药前一天，模型组按照体重随机分组(平均体重45.2g)，分为6组，每组5只，分别为模型对照组、阳性对照组(索玛鲁肽)、受试样品组。空白对照组、模型对照组每天皮下给予生理盐水，阳性对照组、受试样品组每天皮下注射给药，共给药14天。每天称量记录动物体重，末次给药日与起始体重比较，计算体重变化率(％)。结果见表5。
计算公式：(起始体重-末次体重)/起始体重＝体重变化率(％)
计算结果正值表示降低，负值表示升高。
表5各组动物与起始体重相比末次未禁食体重变化率(％，n＝5)
空白组 模型组 AKP26 AKP02 AKP03 AKP05 阳性药组
-1.72 -1.11 19.14 ** 29.30 ** 25.72 ** 22.36 ** 13.22 **
**：与模型对照组相比P＜0.01； *：与模型对照组相比P＜0.05
2)对糖负荷模型动物的降血糖作用：
DIO鼠禁食16h，皮下注射给药1小时后，腹腔给予葡萄糖溶液(1g/kg)进行糖耐量试验，于给糖后0.5、1、1.5h分别测定血糖值，计算血糖浓度曲线下面积(AUC)与模型对照组比较，计算血糖抑制率(％)。结果见表6。
表6样品对动物糖负荷血糖的影响(n＝5)
Figure PCTCN2019117614-appb-000005
**：与模型对照组相比P＜0.01； *：与模型对照组相比P＜0.05
结论：如表5所示模型动物连续给药2周，与模型组相比，受试化合物和阳性对照药均显示了显著的减轻体重作用，其中，本发明化合物的作用强于阳性对照药。如表6所示，在单次给药糖负荷试验中，受试化合物显示了与阳性药相当的降血糖活性。结合对体重的影响和降血糖作用结果，说明本发明的化合物具备GC/GLP-1双重激动剂的特性，与体外受体活性评价结果相符，与单纯的GLP-1受体激动剂相比，更具应用潜力。

Claims (12)

  1. 一种多肽衍生物、其修饰衍生物或其盐，其特征在于，包含具有以下通式Ⅰ序列的多肽：
    通式Ⅰ：HX 2QGTFTSDX 10SX 12YLX 15EX 17X 18AX 20EFX 23X 24WLX 27X 28X 29X 30X 31，
    其中：
    X 2为Ser、D-Ser或Aib；
    X 10为Lys或Tyr；
    X 12为Lys或Arg；
    X 15为Asp或Glu；
    X 17为Arg或Lys；
    X 18为Lys、Ala或Arg；
    X 20为Gln或Lys；
    X 23为Val或Ile；
    X 24为Ala或Glu；
    X 27为Leu或Val；
    X 28为Ala或Glu；
    X 29为Gly或Glu；
    X 30为Glu、Arg或Gly；
    X 31为Gly、-NH 2或不存在；
    C-末端羧基游离或酰胺化；
    其中，X 10、X 17和X 20中有且只有一个侧链被修饰的Lys。
  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中，
    X 2为Aib；
    X 17为Arg；
    X 18为Ala；
    其中，X 10和X 20中有且只有一个侧链被修饰的Lys；并且，当X 20为侧链被修饰的Lys时，X 10是Tyr。
  3. 如权利要求2所述的多肽衍生物、其修饰衍生物或其盐，其特征在于，
    X 27为Leu，
    X 28为Glu，
    X 29为Gly，
    X 30为Arg。
  4. 如权利要求1所述的多肽衍生物、其修饰衍生物或其盐，所述多肽的序列选自SEQ ID NOs.4-26中的任一个。
  5. 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，
    X 27为Leu；
    X 28为Glu；
    X 29为Gly；
    X 30为Gly；
    X 31为-NH 2或不存在。
  6. 如权利要求5所述的多肽衍生物、其修饰衍生物或其盐，所述多肽的序列选自SEQ ID NOs.27-36中的任一个。
  7. 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，所述通式Ⅰ序列中X 10、X 17、X 20之一为侧链ε-氨基被修饰的Lys，所述Lys的侧链ε-氨基是通过亲水性连接片段酰化偶联脂肪酸而被修饰。
  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)中的一中或多种组成的片段；所述的亲水性连接片段优选为γGlu-γGlu-、Glu-γGlu-、Glu-γGlu-、γGlu-Gly-Gly、γGlu-Gly-γGlu-、γGlu-Ado-Ado-；Ado-Ado-γGlu-、或γGlu-Ado-Ado-γGlu-。
  9. 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，所述脂肪酸为C 14-20的脂肪酸；更优选为C 16-20脂肪二酸。
  10. 一种药物组合物，其含有如权利要求1-9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药学上可接受的辅料。
  11. 如权利要求1-9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。
  12. 一种治疗代谢性疾病的方法，包括给予需要的患者权利要求1-9中任一项所述的多肽衍生物、其修饰衍生物或其盐，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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