WO2020108228A1 - 艾塞纳肽类似物 - Google Patents

艾塞纳肽类似物 Download PDF

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WO2020108228A1
WO2020108228A1 PCT/CN2019/115200 CN2019115200W WO2020108228A1 WO 2020108228 A1 WO2020108228 A1 WO 2020108228A1 CN 2019115200 W CN2019115200 W CN 2019115200W WO 2020108228 A1 WO2020108228 A1 WO 2020108228A1
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gly
ser
exenatide
glu
thr
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PCT/CN2019/115200
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English (en)
French (fr)
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张贵民
李铁健
刘�东
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鲁南贝特制药有限公司
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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
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a class of exenatide analogs.
  • the mammalian GLP-1 is derived from the proglucagon-like peptide secreted by the small intestinal mucosa L cells, and the natural analogue of GLP-1, Exenatide, which was jointly developed by Eli Lilly and Amylin, was found in the saliva secretion of the poison lizard
  • the amino acid sequence has sequence similarity with several members of the GLP family, and 53% homology with human GLP-1 (7 ⁇ 36).
  • human GLP-1 (7-36) in vivo are strictly regulated.
  • DPP dipeptidyl peptidase
  • inactive GLP-1 9 ⁇ 36
  • this metabolite is also a natural antagonist of GLP-1R in vivo. Therefore, the natural human active GLP-1 has a short half-life in the body, and its metabolic rate is 2 min.
  • GLP-1 is mainly excreted through the kidneys, which limits the clinical application of human-derived GLP-1.
  • the non-human synthetic Exenatide second amino acid Gly is different from human GLP-1 Ala, which can effectively resist the degradation of dipeptide acyl peptidase; Exenatide C-terminal rigid (PSSGAPPPS) amino acid sequence can increase the stability of the peptide , Exenatide's hypoglycemic ability in the body is about 1000 times stronger than GLP-1.
  • PSSGAPPPS Exenatide C-terminal rigid
  • Exenatide (also known as Exenatide or Exenatide, Exenatide, or Ex-4, trade name Byetta) is a 39-amino acid peptide with a molecular weight of 4186.6 and a molecular formula of C 184 H 282 N 50 O 60 S, CAS No. 141758-74-9, the amino acid sequence is shown below.
  • pancreatic ⁇ -cells It is a medicinal subcutaneous injection preparation that promotes glucose-dependent insulin secretion, restores first-phase insulin secretion, inhibits glucagon secretion, and slows gastric content
  • the emptying, improving the function of pancreatic ⁇ -cells, etc. is very suitable for the treatment of type II diabetes; for example, for improving and controlling the blood sugar of patients with type II diabetes who are not ideal for the treatment of metformin and sulfonylurea drugs.
  • Exenatide peptide secondary structure the N-terminus of Exenatide is irregularly coiled, and the middle part alternately arranges amino acid residues with oppositely charged side chains on the same side, forming a helix through a salt bridge or polar hydrogen bond, and the C-terminus is a pro "Trp-Cage" of water.
  • the mechanism of interaction between Exenatide and GLP-1 receptor has been studied clearly.
  • CN1384755 discloses a new Exendin agonist preparation and its administration method, and discloses the compound structure and preparation method of Exenatide.
  • CN102532303 discloses the use of methoxypolyethylene glycol to modify the amino group of lysine or N-terminal histidine residue in Exenatide.
  • CN200980111088 discloses fatty acid-PEG-Exenatide.
  • Patent CN101125207 reported that the structure of Exenatide was modified into short peptides.
  • CN105753963A discloses Exenatide analogues with single-point or multi-point amino acid mutations.
  • CN102397558 discloses that certain amino acids in Exenatide are substituted with cysteine, and modified with PEG or PEG with methyl terminal substitution.
  • exenatide analog long-acting drugs Although many efforts have been made in the development of exenatide analog long-acting drugs, the exenatide analogs currently on the market have poor stability and low efficacy. As the structure and activity basis of long-acting drug development, exenatide The innovative transformation and research of analogues is still a very important subject.
  • exenatide as the modified polypeptide sequence of the present invention, combined with computer model calculation and binding test, focusing on the modification of the C-terminal amino acid sequence, and adding modified active sites for long-acting drug development.
  • the purpose of the present invention is to overcome the shortcomings of the prior art, to provide exenatide analogs with long half-life, good stability and high activity, which can simultaneously maintain a good hypoglycemic effect and low toxicity, and at the same time, the present invention also provides exenatide analogs Use of analogues in the preparation of medicaments for the treatment of diabetes, obesity and/or complications.
  • the present invention provides a class of exenatide analogs, which is characterized by replacing the amino acids 37 to 45 of the amino acid sequence of Exenatide Ex-4 to obtain a class of exenatide analogs; the amino acid sequence is as SEQ .ID NO:1, including:
  • Xaa37 is Pro or Gly
  • Xaa38 is Ser or Gly
  • Xaa39 is Ser or Gly
  • Xaa40 is Gly or Ala
  • Xaa41 is Ala or Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys.
  • amino acid sequence of the sodium exenatide analog is shown in SEQ.ID NO: 2 ⁇ 7:
  • Xaa37 is Gly
  • Xaa38 is Ser
  • Xaa39 is Gly
  • Xaa40 is Gly
  • Xaa41 is Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys
  • Ex-4 peptide analogue Ex-4i is obtained, its amino acid
  • the sequence is as SEQ.ID NO: 2 shows.
  • Xaa37 is Pro
  • Xaa38 is Gly
  • Xaa39 is Gly
  • Xaa40 is Gly
  • Xaa41 is Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys
  • Ex-4 peptide analogue Ex-4ii is obtained, its amino acid
  • the sequence is as SEQ.ID NO:3 shown.
  • Xaa37 is Pro
  • Xaa38 is Ser
  • Xaa39 is Gly
  • Xaa40 is Gly
  • Xaa41 is Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys; the Exenide peptide analog Ex-4iii is obtained, and its amino acid sequence As shown in SEQ.ID NO:4.
  • Xaa37 is Pro
  • Xaa38 is Ser
  • Xaa39 is Ser
  • Xaa40 is Gly
  • Xaa41 is Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys
  • Ex-4 peptide analogue Ex-4iv is obtained, its amino acid
  • the sequence is as SEQ.ID NO:5 shown.
  • Xaa37 is Pro
  • Xaa38 is Ser
  • Xaa39 is Ser
  • Xaa40 is Ala
  • Xaa41 is Thr
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys
  • Ex-4 peptide analogue Ex-4v is obtained, its amino acid
  • the sequence is as SEQ.ID NO:6 shown.
  • Xaa37 is Pro
  • Xaa38 is Ser
  • Xaa39 is Ser
  • Xaa40 is Gly
  • Xaa41 is Ala
  • Xaa42 is Gly
  • Xaa43 is Gly
  • Xaa44 is Ser
  • Xaa45 is Cys
  • Ex-4 peptide analog Ex-4vi is obtained The sequence is as SEQ.ID NO:7 shown.
  • the C-terminus of the exenatide analogue according to the present invention is amidated, which is the same as the C-terminus amidation of natural GLP-1 and exenatide.
  • Another object of the present invention is to provide the use of the Exenatide analogue of the present invention in the preparation of a medicament for treating diabetes, obesity and/or related complications.
  • the medicine contains the exenatide analogue of the present invention and a pharmaceutically acceptable carrier.
  • exenatide analog of the present invention may be administered in the form of a single drug or may be administered in combination with other drugs.
  • exenatide analogs of the present invention can form salts, including various inorganic or organic salts, such as hydrochloride, hydrobromide, phosphate, sulfate, maleate, oxalate, citrate, tartaric acid Salt, fumarate, mandelate and lactate; salt with various inorganic and organic bases, such as sodium hydroxide, trishydroxymethylaminomethane and N-methyl-glucosamine.
  • various inorganic or organic salts such as hydrochloride, hydrobromide, phosphate, sulfate, maleate, oxalate, citrate, tartaric acid Salt, fumarate, mandelate and lactate
  • salt with various inorganic and organic bases such as sodium hydroxide, trishydroxymethylaminomethane and N-methyl-glucosamine.
  • the exenatide analog of the present invention can be used alone or in the form of a pharmaceutical composition.
  • the pharmaceutical composition includes as an active ingredient the exenatide analog of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” will not destroy the pharmacological activity of the compound of the present invention and its available salts, and its effective dosage is non-toxic to the human body.
  • “Pharmaceutically acceptable carrier” may be used but not limited to: ion exchange materials, aluminum stearate, lecithin, serum proteins for pharmaceutical preparations, saturated vegetable fatty acids, cellulose materials, ethylene-polyoxyethylene-block polymers , Cyclodextrin or its chemically modified derivatives or other soluble derivatives.
  • excipients such as anhydrous lactose, starch, lactose beads and glucose, binders such as microcrystalline cellulose, disintegrating agents such as croscarmellose sodium, croscarmellose, Low-substituted hydroxypropyl cellulose, lubricants such as magnesium stearate, absorption enhancers, excipients, solubilizers, colorants, etc. can also be added to the pharmaceutical composition of the present invention.
  • binders such as microcrystalline cellulose
  • disintegrating agents such as croscarmellose sodium, croscarmellose, Low-substituted hydroxypropyl cellulose
  • lubricants such as magnesium stearate, absorption enhancers, excipients, solubilizers, colorants, etc.
  • exenatide analogue of the present invention or a pharmaceutically acceptable salt and pharmaceutical composition thereof can be administered by enteral or parenteral routes.
  • Parenteral routes include subcutaneous, intradermal, intramuscular, transnasal, and mucosal administration or inhalation.
  • the preparation can be developed as injections, creams, ointments, patches and aerosols.
  • exenatide analogue or the pharmaceutically acceptable salt and the pharmaceutical composition of the present invention can be used for the treatment of related diseases as single drugs or combined drugs, which is within the scope of those skilled in the art.
  • the present invention does not limit the preparation methods of Exenatide Ex-4 and Exenatide analogs, and can adopt a chemical solid phase synthesis method or a gene expression method.
  • One embodiment of the present invention uses the Fmoc solid-phase synthesis method.
  • the 6 exenatide analogs Ex-4(i-vi) C-terminal amino acid sequence forms a new binding effect, which can stabilize the polypeptide alpha helix structure and promote its interaction with the active core structure field. The combination improves the biological activity.
  • exenatide analogs designed by the present invention all show enhanced activity.
  • Exenatide Ex-4 and Exenatide analogues stimulated the activity of cAMP release from mouse islet tumor cells RIN-m5F.
  • In vitro biological activity showed a dose-dependent increase.
  • the Exenatide analog Ex-4 (i-vi) stimulates mouse islet tumor cells RIN-5mF to release cAMP with higher activity than other analogs.
  • exenatide analogs Ex-4ii, Ex-4iii and Ex-4iv stimulate mouse pancreatic islet tumor cells RIN-m5F to release cAMP more than exenatide analogs Ex-4i, Ex-4v and Ex-4vi Is more active.
  • Ex-4 and Ex-4( - ) Group 0.5 h after subcutaneous injection can significantly reduce blood glucose levels (P ⁇ 0.01); the single hypoglycemic effect of Ex-4 can be maintained for 8 hours, and the blood glucose concentration increases significantly after 12 hours of administration.
  • Ex-4( - ) The hypoglycemic effect of a single administration can be maintained for 12h. When the single administration is 12h, Ex-4( - ) The blood glucose level of the group remained lower than that of the model group (P ⁇ 0.05). Therefore, Ex-4( - ) The six analogs all have hypoglycemic effect, which has a longer hypoglycemic effect than Ex-4, especially Ex-4ii, Ex-4iii and Ex-4iv hypoglycemic effect.
  • Ex-4( - ) Stronger activity than Ex-4, long duration of drug effect, and longer half-life.
  • Exenatide analogs provided by the present invention can be obtained through the in vivo hypoglycemic effect test of type II diabetes model db/db mice.
  • Exenatide and its analogs are administered subcutaneously for 0.5 h It can significantly reduce the blood sugar level; the hypoglycemic effect of single administration of exenatide can be maintained for 8 hours, and the blood glucose concentration is significantly increased after 12 hours of administration.
  • the blood glucose lowering maintenance time reaches more than 10h.
  • the Exenatide analog Ex-4( - ) The hypoglycemic effect of a single administration can be maintained for 12h.
  • the exenatide analogue designed by the present invention has a long half-life, good stability and high activity, and can simultaneously maintain a good hypoglycemic effect and low toxicity.
  • the term "exenatide” is a functional analog of glucagon-like peptide-1 isolated from the salivary gland of a lizard inhabiting the southwestern United States.
  • Exenatide As a physiologically active peptide composed of 39 amino acids, Exenatide, whose academic name is “Exenatide-4”, has a 53% amino acid similarity to glucagon-like peptide-1 present in humans. Sex.
  • Ex-4 expressed as Ex-4 is Exenatide, the amino acid sequence is as SEQ.ID NO:1 shown.
  • exenatide analog means that the rigid amino acid sequence at the C-terminus of exenatide-4 is deleted and replaced by other flexible amino acid sequences, which is still a physiologically active peptide composed of 39 amino acids.
  • the amino acid sequence of the preferred Exenatide analog Ex-4 (i-vi) is shown in SEQ. ID NO: 2 ⁇ 7.
  • Figure 1 is a schematic diagram of the interaction of Exenatide Ex-4 with G protein-coupled receptors
  • Fig. 2 is a schematic diagram of the binding mode of Ex-4 peptide of Exenatide to G protein-coupled receptor;
  • Figure 3 is Exenatide analog Ex4 Schematic diagram of interaction with G protein-coupled receptors
  • Figure 4 is Exenatide analog Ex4 Binding mode diagram of C-terminal and G protein-coupled receptor
  • Figure 5 shows the activity of Exenatide Ex-4 and its analog Ex-4 (i-vi) on the release of cAMP from mouse islet tumor cells RIN-m5F.
  • the Fmoc solid phase synthesis method is used to synthesize one of the exenatide analogs of the present invention, and other analogs are synthesized similarly.
  • Adopt Fmoc synthesis method select Fmoc-Rink resin for synthesis.
  • the synthesis steps are as follows:
  • the HOBt method was used to activate the amino acids, which were connected to the amino resin according to the sequence, and a total of 39 steps of synthesis were performed.
  • a polypeptide resin with a side chain protecting group is obtained. Take out the peptide resin and put it in a vacuum desiccator at a drying temperature of 30°C and dry to a constant weight ( ⁇ 0.2g).
  • the initial trifluoroacetate solution of exenatide analogue was prepared by reverse phase purification by HPLC, and the purity was greater than 98%.
  • Sample loading solution The crude peptide was prepared with 0.1% trifluoroacetic acid aqueous solution to a concentration of 10.0 mg/ml solution, and passed through a 0.22 ⁇ m filter membrane.
  • Sample collection collect the effluent with the main peak purity greater than 98% to prepare the eluent.
  • Exenatide Ex-4 and other exenatide analogs were prepared according to the above method.
  • Exenatide Ex-4 and Exenatide analogs were analyzed using MOE software. The results show that, compared with Exenatide Ex-4, the interaction between 32 Exenatide analogs and the receptor binding domain is enhanced; the number of interactions between Exenatide analogs and receptors is increased; preferably The six exendin analogues Ex-4 (i-vi) formed new interactions between the C-terminal of the polypeptide and the receptor, including new hydrogen bonds, new hydrophobic interactions, and new ionic bonds. The presence of these new bonds can make the active ⁇ -helical structure more stable, and the binding force of the active domain of the receptor is improved, thereby improving the activity.
  • FIG. 1-4 shows the schematic diagram of the interaction between Ex-4 and Ex-4iii and the receptor.
  • the C-terminus of Ex-4iii forms a new hydrogen bond and hydrophobic interaction with the receptor.
  • the active alpha helix structure is more stable and the receptor active domain Increased binding power.
  • Example 3 The activity of exenatide analogues to stimulate mouse islet tumor cells RIN-m5F to release cAMP
  • PBS sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, dissolved in water and diluted to 1000 ml, pH adjusted to 7.2, autoclaved at 121°C for 15min, stored at 4°C .
  • Exenatide Ex-4 and Exenatide analog Ex-4 (i-vi) peptide sample preparation: accurately weigh 10mg each of the peptide samples, accurately prepare 0.1mg/ml sample solution with sterile water for injection, and determine nitrogen Method to determine the content of peptides, according to the content of peptides, dilute the peptide concentration to 100ng/ml, and then dilute by a factor of 2 to the polypeptide concentration of 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng /ml, 3.12ng/ml, 1.56ng/ml, 0.78ng/ml a total of 8 dilution sample solutions.
  • the cell count is 5 ⁇ 10 5 ⁇ 8 ⁇ 10 5 cells/mL, cultured at 37°C for 18 ⁇ 36h; digest with 0.25% trypsin, and press 3.5 ⁇ 10 5 cells/mL, Inoculate into a 24-well cell plate, 0.5mL per well, and keep culturing at 37°C, 5% CO 2 for 24 ⁇ 36h.
  • Discard the cell solution add the cell maintenance solution, which is RPMI1640 culture medium containing 5mg/mL bovine serum albumin, 1.0mL/well, and incubate at 37°C, 5% CO 2 for 15min.
  • the supernatant was discarded, and the cell maintenance solution containing 1 mM IBMX was added, 0.9 mL/well, and incubated at 37° C. and 5% CO 2 for 15 min.
  • Ex-4(i-vi) designed by the present invention all exhibit enhanced activity.
  • Ex-4 and Ex-4 (i-vi) stimulated mouse islet tumor cells RIN-m5F to release cAMP activity.
  • In vitro biological activity showed a dose-dependent increase.
  • Ex-4ii, Ex-4iii and Ex-4iv stimulate mouse islet tumor cells RIN-m5F to release cAMP more than Ex-4i, Ex-4v and Ex-4vi.
  • Ex-4ii, Ex-4iii and Ex-4iv increased by 16.2%, 23.8% and 21.2% respectively compared to Ex-4.
  • SPF grade BKS.Cg-m+/+Leprdb/J spontaneously diabetic mice: a total of 128 animals, 6-8 weeks old, half male and female, purchased from Beijing Huafu Biotechnology Co., Ltd.
  • the production license number is SYXK (Beijing) 2009-0004, and the quality certificate number is 0172750.
  • SPF grade BKS.Cg-m+/+Leprdb/J negative control mice used for control, a total of 16 animals, 6-8 weeks old, half male and female, purchased from Beijing Huafu Biotechnology Co., Ltd. the company.
  • the production license number is SYXK (Beijing) 2009-0004, and the quality certificate number is 0172750.
  • Environmental conditions for mouse SPF animal house rearing air cleanliness 10000, airflow speed 0.1 ⁇ 0.2m/s, ambient pressure difference 20 ⁇ 50Pa, temperature 20-25°C, humidity 40 ⁇ 70%, ammonia concentration ⁇ 14mg/m 3 , Ventilation 10 ⁇ 20 times/h, animal illumination 15 ⁇ 20lux, working illumination 150 ⁇ 300lux, noise ⁇ 60dB, day and night light and dark alternate time 12h/12h, male and female rearing separately, 4 per cage, test after one week of adaptive rearing .
  • 128 db/db spontaneously diabetic mice were divided into genders and randomly divided into 8 groups according to the pre-measured blood glucose value, namely (1) model group; (2) Ex-4 group, 5 ⁇ g/kg; (3) Ex-4i group, 5 ⁇ g/kg; (4) Ex-4 group, 5 ⁇ g/kg; (5) Ex-4iii group, 5 ⁇ g/kg; (6) Ex-4iv group, 5 ⁇ g/kg; (7) Ex -4v group, 5 ⁇ g/kg; and (8) Ex-4vi group, 5 ⁇ g/kg.
  • mice Nine groups of mice were administered by subcutaneous injection once, the control group and model group were injected with normal saline 10mL/kg, Ex-4 group was injected with Ex-4 sample at 5 ⁇ g/kg, and sample group was injected with Ex-4 at 5 ⁇ g/kg (i-vi) Sample.
  • Ex-4 and Ex-4( - ) Group 0.5 h after subcutaneous injection can significantly reduce blood glucose levels (P ⁇ 0.01); the single hypoglycemic effect of Ex-4 can be maintained for 8 hours, and the blood glucose concentration increases significantly after 12 hours of administration.
  • Ex-4( - ) The hypoglycemic effect of a single administration can be maintained for 12h. When the single administration is 12h, Ex-4( - ) The blood glucose level of the group remained lower than that of the model group (P ⁇ 0.05). Therefore, Ex-4( - ) The six analogs all have hypoglycemic effect, which has a longer hypoglycemic effect than Ex-4, especially Ex-4ii, Ex-4iii and Ex-4iv hypoglycemic effect.
  • Ex-4( - ) Stronger activity than Ex-4, long duration of drug effect, and longer half-life.

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Abstract

本发明属于生物医药领域，具体公开了一类艾塞纳肽类似物。本发明对艾塞纳肽Exendin-4的氨基酸序列C端的几个氨基酸结构设计，包括氨基酸的添加或缺失、氨基酸序列突变和增加可用于定点修饰的位点得到一类艾塞纳肽类似物。该类似物刺激小鼠胰岛瘤细胞分泌胰岛素的活性明显高于艾塞纳肽Ex-4，且糖尿病模型小鼠C57BL/KsJ-db/db体内降糖活性也显著优于艾塞纳肽Ex-4，可用于预防、治疗或减轻糖尿病和肥胖症和/或其相关并发症等其他疾病。

Description

艾塞纳肽类似物 技术领域
本发明属于生物医药领域，具体涉及一类艾塞纳肽类似物。
 背景技术
哺乳动物GLP-1来自于小肠粘膜L细胞分泌的原胰高血糖素样肽，而GLP-1的天然类似物即由美国礼来与Amylin公司共同研制的Exenatide发现于毒蜥的唾液分泌物中，其氨基酸序列与GLP家族中的数个成员有序列的相似性，与人源GLP-1（7~36）有53%的同源性。
人GLP-1（7~36）在体内的表达和活性受到严格的调控，当其N端的第二位Ala被二肽基酶（dipeptidylpeptidase，DPP）水解后，形成无活性的GLP-1（9~36），该代谢产物还是GLP-1R的体内天然拮抗剂。因此，天然人活性型GLP-1在体内的半衰期很短，其新陈代谢的速率为2min；加上在生理状态下，GLP-1主要通过肾脏排泄，限制了人源GLP-1的临床应用。非人源的人工合成Exenatide的第二位氨基酸Gly不同于人GLP-1的Ala，能有效抵抗二肽酰基肽酶的降解；Exenatide的C端的刚性（PSSGAPPPS）氨基酸序列，可增加多肽的稳定性，Exenatide在体内的降血糖能力比GLP-1强1000倍左右。
艾塞纳肽（又称依西纳肽或依克那肽，Exenatide，或Ex-4，商品名Byetta）是由39个氨基酸组成的多肽，其分子量为4186.6，分子式为C 184H 282N 50O 60S，CAS号为141758-74-9， 氨基酸序列如下所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2；由Amylin Pharmaceuticals和Eli Lilly公司生产并销售。艾塞纳肽已于2005年4月由FDA批准上市，属于药用皮下注射制剂，具有促进葡萄糖依赖的胰岛素分泌、恢复第一时相胰岛素分泌、抑制胰高血糖素的分泌，减慢胃内容物的排空，改善胰腺β细胞的功能等作用，非常适用于Ⅱ型糖尿病的治疗；例如，用于改善和控制二甲双胍和磺酰脲类药物治疗不理想的Ⅱ型糖尿病患者的血糖。
Exenatide肽二级结构：Exenatide的N端为不规则卷曲，中间部分在同一侧面上相互交替排列带相反电荷侧链的氨基酸残基，通过盐桥或极性氢键形成螺旋，C端则为亲水的“Trp-Cage”。Exenatide与GLP-1受体的相互作用机理已研究的比较清楚。
一系列不同结构设计已经开发用于延长GLP-1类似物结构半衰期，提高生物活性。CN1384755公开了新型Exendin激动剂制剂及其给药方法，公开了Exenatide的化合物结构和制备方法。CN102532303公开了使用甲氧基聚乙二醇修饰Exenatide中赖氨酸的氨基或N末端组氨酸残基的氨基。CN200980111088公开了脂肪酸-PEG-Exenatide。专利CN101125207报道了对Exenatide的结构改造成短肽。CN105753963A公开了单点或多点位氨基酸突变的Exenatide类似物。CN102397558公开了将Exenatide中某些氨基酸用半胱氨酸取代，并使用PEG或末端被甲基取代的PEG修饰。
虽然在艾塞纳肽类似物长效药物开发做了诸多努力，但是目前市场上的艾塞纳肽类似物稳定性较差和药效较低，作为长效药物研发的结构和活性基础，艾塞纳肽类似物的创新性改造和研究仍是一个非常重要的课题。
基于以上分析研究，我们选择艾塞纳肽作为本发明改造的多肽序列，结合计算机模型计算和结合测试，重点针对其C端的氨基酸序列进行改造，同时添加修饰活性位点用于长效药物开发。
 技术问题
本发明的目的在于克服现有技术的缺点，提供半衰期长，稳定性好，活性高，能够同时保持较好降糖效果和低毒性的艾塞纳肽类似物，同时，本发明还提供艾塞纳肽类似物在制备治疗糖尿病、肥胖症和/或并发症的药物中的用途。
 技术解决方案
本发明具体内容如下：
本发明提供一类艾塞纳肽类似物，其特征在于，对艾塞纳肽Ex-4的氨基酸序列第37位至45位的氨基酸进行替换，得到一类艾塞纳肽类似物；其氨基酸序列如SEQ.ID NO:1，包含：
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45，
其中，Xaa37为Pro或Gly；
Xaa38为Ser或Gly；
Xaa39为Ser或Gly；
Xaa40为Gly或Ala；
Xaa41为Ala或Thr;
Xaa42为Gly；
Xaa43为Gly；
Xaa44为Ser；
Xaa45为Cys。
进一步优选地，优选埃塞钠肽类似物的氨基酸序列如SEQ.ID NO:2~7所示：
其中，Xaa37为Gly，Xaa38为Ser，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4i，其氨基酸序列如SEQ.ID NO:2所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Gly-Ser-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:2)。
其中，Xaa37为Pro，Xaa38为Gly，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr ，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4ii，其氨基酸序列如SEQ.ID NO:3所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Gly-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:3)。
其中，Xaa37为Pro，Xaa38为Ser，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4iii，其氨基酸序列如SEQ.ID NO:4所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:4)。
其中，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Gly，Xaa41为Thr， Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4iv，其氨基酸序列如SEQ.ID NO:5所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:5)。
其中，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Ala，Xaa41为Thr， Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4v，其氨基酸序列如SEQ.ID NO:6所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Ala-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:6)。
其中，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Gly，Xaa41为Ala，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，Xaa45为Cys；得到艾塞纳肽类似物Ex-4vi，其氨基酸序列如SEQ.ID NO:7所示。
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Gly-Gly-Ser-Cys(SEQ.ID NO:7)。
本发明所述的艾塞纳肽类似物其C末端是酰胺化的，与天然GLP-1和艾塞纳肽的C端酰胺化相同。
本发明的另一目的在于提供本发明所述艾塞纳肽类似物在制备治疗糖尿病、肥胖症和/或相关并发症药物中的应用。
上述应用，所述的药物含由本发明所述艾塞纳肽类似物以及药学上可接受的载体。
本发明所述艾塞纳肽类似物可以以单一药物的形式给药或可以与其他药物联合给药。
本发明的艾塞纳肽类似物可以成盐，包括多种无机或有机盐，如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、马来酸盐、草酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、扁桃酸盐和乳酸盐；与各种无机、有机碱成盐，如氢氧化钠、三羟甲基氨基甲烷和N-甲基-葡萄糖胺成盐。
本发明的艾塞纳肽类似物可以单独使用或以药物组合物的形式使用。药物组合物包括作为活性成分的本发明的艾塞纳肽类似物或其可药用盐及药学上可接受的载体。“药学上可接受的载体”不会破坏本发明的化合物和其可用盐的药学活性，同时其有效用量对人体无毒。“药学上可接受的载体”可使用但不限于：离子交换材料、硬脂酸铝、卵磷脂、药物制剂用血清蛋白、饱和植物脂肪酸、纤维素物质、乙烯-聚氧乙烯-嵌段聚合物、环糊精或其经化学修饰的衍生物或其他可溶性衍生物等。
其他可药用辅料，填充剂如无水乳糖、淀粉、乳糖珠粒和葡萄糖，粘合剂如微晶纤维素，崩解剂如交联羧甲基纤维素钠、交联羧甲基淀粉、低取代羟丙基纤维素，润滑剂如硬脂酸镁，吸收促进剂、赋形剂、增溶剂和着色剂等也可加入本发明的药物组合物中。
上述本发明的艾塞纳肽类似物或其可药用盐以及药物组合物可通过肠道或者非肠道途径给药。非肠道途径包括皮下、皮内、肌内、经鼻、粘膜给药或吸入。制剂可开发注射剂、霜剂、软膏剂、贴剂和气雾剂等。
本发明艾塞纳肽类似物或其可药用盐以及药物组合物可用于相关疾病的单一用药或联合用药治疗，为本领域技术人员能够理解的范围。
本发明不限制艾塞纳肽Ex-4及艾塞纳肽类似物的制备方法，可以采用化学固相合成法，也可以采用基因表达法。本发明的一个实施例采用Fmoc固相合成法。
 有益效果
药学试验结果表明，本发明32种艾塞纳肽类似物体外刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性均明显高于未突变艾塞纳肽；且皮下注射给药对db/db模型小鼠的降血糖作用也均明显优于艾塞纳肽。因此，所述的艾塞纳肽类似物可用于更好地预防、防治或减轻糖尿病、肥胖症和/或并发症。
使用MOE软件（Molecular Operating Environment, Chemical Computing Group, Montreal, Canada）分析本发明32种艾塞纳肽类似物与未突变艾塞纳肽之间的差异，结果表明本发明32种艾塞纳肽类似物与受体之间的相互作用和成键数目发生变化，优选地，6种艾塞纳肽类似物Ex-4(i-vi)C末端氨基酸序列形成新的结合作用，可稳定多肽α螺旋结构，促进其与活性核心结构领域的结合，提高了生物活性。
本发明设计的艾塞纳肽类似物均表现出活性加强。在不同药物浓度下，艾塞纳肽Ex-4与艾塞纳肽类似物刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性。体外生物活性呈剂量依赖性增加。优选地，艾塞纳肽类似物Ex-4(i-vi)刺激小鼠胰岛瘤细胞RIN-5mF释放cAMP的活性较其他类似物高。更加优选地，艾塞纳肽类似物Ex-4ii、Ex-4iii和Ex-4iv刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性较艾塞纳肽类似物Ex-4i、Ex-4v和Ex-4vi的活性更高。
Ex-4及Ex-4(
Figure 614672dest_path_image001
-
Figure 325795dest_path_image002
)组皮下注射给药0.5h后即可显著降低血糖水平（P<0.01）；Ex-4单次给药的降糖作用可维持8h，给药12h后血糖浓度明显增高。Ex-4(
Figure 217528dest_path_image001
-
Figure 11171dest_path_image002
)单次给药的降糖作用可维持12h，单次给药12h时，Ex-4(
Figure 646552dest_path_image001
-
Figure 209251dest_path_image002
)组的血糖水平仍维持低于模型组（P<0.05）。因此，Ex-4(
Figure 271885dest_path_image001
-
Figure 411880dest_path_image002
)六种类似物均有降糖作用，相比于Ex-4具有更长的降糖药效，尤其Ex-4ii、Ex-4iii及Ex-4iv降糖作用更为显著。Ex-4(
Figure 991897dest_path_image001
-
Figure 533736dest_path_image002
)比Ex-4活性强，药效维持时间长，具有更长的半衰期。
本发明所提供的艾塞纳肽类似物相比于Ex-4，通过Ⅱ型糖尿病模型db/db小鼠的体内降血糖作用试验可得，艾塞纳肽及其类似物皮下给药0.5h后即可显著降低血糖水平；艾塞纳肽单次给药的降糖作用可维持8h，给药12h后血糖浓度明显增高，本发明所设计的艾塞纳肽类似物降血糖维持时间均高于艾塞纳肽Ex-4，降血糖维持时间达到10h以上。优选地，艾塞纳肽类似物Ex-4(
Figure 908217dest_path_image001
-
Figure 535508dest_path_image002
)单次给药的降糖作用可维持12h，单次给药12h时，Ex-4(
Figure 122478dest_path_image001
-
Figure 784403dest_path_image002
)组的血糖水平仍维持低于模型组（P<0.05）。因此，艾塞纳肽类似物均有降糖作用，相比于艾塞纳肽Ex-4具有更长的降糖药效。艾塞纳肽类似物比艾塞纳肽活性强，药效维持时间长，具有更长的半衰期。
本发明所设计的艾塞纳肽类似物其半衰期长，稳定性好，活性高，能够同时保持较好降糖效果和低毒性。
术语
在本说明书中，术语“艾塞纳肽”为从栖息于美国西南部的毒蜥的唾液腺分离的胰高血糖素样肽-1的功能性类似物。学术名为“艾塞纳肽-4”的艾塞纳肽作为由39个氨基酸组成的生理活性肽，与存在于人的活体内的胰高血糖素样肽-1相比，具有53%的氨基酸类似性。在以下实施例中被表示为Ex-4的为艾塞纳肽，其氨基酸序列如SEQ.ID NO:1所示。
在本说明书中，术语艾塞纳肽类似物意味着使艾塞纳肽-4的C末端的刚性氨基酸序列缺失，并由其他柔性氨基酸序列来代替，依然是39个氨基酸组成的生理活性肽。优选的艾塞纳肽类似物Ex-4(i-vi)的氨基酸序列如SEQ.ID NO:2~7所示。
 附图说明
图1是艾塞纳肽Ex-4与G蛋白偶联型受体相互作用示意图；
图2是艾塞纳肽Ex-4 C末端与G蛋白偶联型受体的结合模式图；
图3是艾塞纳肽类似物Ex4
Figure 329785dest_path_image003
与G蛋白偶联型受体相互作用示意图；
图4是艾塞纳肽类似物Ex4
Figure 178793dest_path_image003
C末端与G蛋白偶联型受体的结合模式图；
图5是艾塞纳肽Ex-4及其类似物Ex-4(i-vi)对小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性。
 本发明的实施方式
以下，通过实施例对本发明进行更加详细的说明。这些实施例仅用于更加具体地说明本发明，根据本发明的要旨，本发明的范围并不局限于这些实施例，这对于本发明所述技术领域的普通技术人员而言是显而易见的。
实施例1  艾塞纳肽Ex-4及艾塞纳肽类似物的制备
本实施例采用Fmoc固相合成法合成本发明艾塞纳肽类似物之一，其他类似物的合成与之类似。
合成工艺
采用Fmoc合成法，选择Fmoc-Rink树脂进行合成。合成步骤如下：
Figure 959667dest_path_image004
具体地，包括如下步骤：
Figure 351465dest_path_image005
2、固相合成与多肽粗品制备过程
采用HOBt法活化氨基酸，按照序列连接到氨基树脂上，共进行39步合成。
（1）仪器设备：
CS536XT立式多肽合成仪、磁力搅拌仪。
Figure 192382dest_path_image006
（3）固相合成操作：称取树脂5.5g（树脂装载率为0.45mmol/g），倒入多肽合成仪反应器内，按照艾塞纳肽类似物的氨基酸序列从C-端向N-端称取10mmol相应的带保护基氨基酸，并排列在合成仪中。在室温条件下，按照电脑程序自动进行完成39步合成反应。
合成结束后，得到带侧链保护基的多肽树脂。取出多肽树脂，放入真空干燥器中，干燥温度为30℃，干燥至恒重（±0.2g）。
（4）    脱保护基及沉淀
带保护基的艾塞纳肽类似物多肽树脂置入带塞的三角烧瓶中，按13ml/克肽树脂，加入裂解试剂TFA/水/TIS/EDT=94:2:2:2（V/V），恒温在25℃下，搅拌反应4小时；过滤、收集滤液，树脂用少量三氟乙酸洗涤，过滤合并收集液。在搅拌下，滴加500mL冰乙醚（-10℃），得到白色沉淀，过滤，用少量冰乙醚洗涤粗品，并将粗品放入真空干燥器中干燥过夜，得多肽粗品。
3、HPLC纯化
（1）仪器设备及试剂：
Figure 666701dest_path_image007
（2）纯化
通过HPLC反相纯化制备得到艾塞纳肽类似物初品三氟乙酸盐溶液，纯度大于98%。
色谱柱：50mm×300mm   RP-18 10μm  120Å
流动相：
A：0.1%三氟乙酸水溶液
B：0.1%三氟乙酸乙腈溶液
上样溶液：将多肽粗品用0.1%三氟乙酸水溶液配制成浓度为10.0mg/ml的溶液，并通过0.22μm滤膜通过。
洗脱条件：采用梯度洗脱，流速为50ml/min，紫外215nm检测，洗脱梯度如下表：
Figure 251266dest_path_image008
样品收集：收集主峰纯度大于98%的流出液，得制备洗脱液。
除去乙腈：
将收集的制备洗脱液倒入旋蒸瓶，25℃，-0.099Mpa条件下旋转蒸发，除去所有乙腈，剩余液体通过0.22μm滤膜过滤，留待冻干。
4、冷冻干燥
(1)仪器设备：GOLD-SIM立式冷冻干燥机
(2)操作：
将艾塞纳肽类似物三氟乙酸水溶液倒入真空冷冻干燥机样品盘中，按照电脑程序进行冻干，得到所需化合物。
按照以上方法制备艾塞纳肽Ex-4及其他艾塞纳肽类似物。
实施例2 计算机模拟辅助多肽筛选
使用MOE软件对 艾塞纳肽Ex-4及艾塞纳肽类似物与G蛋白偶联型受体相互作用进行分析。结果表明，与艾塞纳肽Ex-4相比，32种艾塞纳肽类似物与受体结合域之间作用有所增强；艾塞纳肽类似物与受体之间的相互作用数目增多；优选地6种艾塞纳肽类似物Ex-4(i-vi)在多肽C末端与受体之间形成了新的相互作用，包括新的氢键、新的疏水作用和新的离子键。这些新键的存在可使得活性α螺旋结构更加稳定，受体活性域结合力提高，进而提高了活性。
MOE软件分析显示，引入的柔性氨基酸序列交联区或柔性Linker替换艾塞纳肽类似物在C末端与受体之间形成了新的相互作用。图1-4显示Ex-4和Ex-4iii与受体的相互作用示意图，Ex-4iii的C末端与受体形成新的氢键和疏水作用，同时活性α螺旋结构更加稳定，受体活性域结合力提高。
 工业实用性
实施例3  艾塞纳肽类似物体外刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性
（1）试验材料及相关试剂配制
PBS：氯化钠8g、氯化钾0.2g、磷酸氢二钠1.44g、磷酸二氢钾0.24g，加水溶解并稀释至1000 ml，pH调至7.2，121℃高压灭菌15min，4℃保存。
艾塞纳肽Ex-4及艾塞纳肽类似物Ex-4(i-vi)多肽样品配制：精确称取多肽样品各10mg，用无菌注射用水精确配制成0.1mg/ml的样品溶液，定氮法测定多肽含量，根据多肽含量稀释至多肽浓度为100ng/ml，再按2倍倍比稀释，分别稀释成多肽浓度为100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、3.12ng/ml、1.56ng/ml、0.78ng/ml共8个稀释度样品溶液。
（2）试验方法
取生长状态良好的RIN-m5F细胞消化后，细胞计数5×10 5~8×10 5个/mL，37℃培养18~36h；用0.25%胰酶消化，按3.5×10 5个/mL，接种入24孔细胞板，每孔0.5mL，置37℃、5%CO 2继续培养24~36h。弃细胞液，加入细胞维持液即含5mg/mL牛血清白蛋白的RPMI1640培养液1.0mL/孔，37℃、5%CO 2培养15min。弃上清液，加入含1mM的IBMX的细胞维持液，0.9mL/孔，37℃、5%CO 2培养15min。尽可能快地加入0.1mL/孔待测样品，轻震荡混匀后，37℃、5%CO 2培养15min。取出细胞培养板置于冰上，弃上清液，加入1.0mL/孔预冷的PBS洗两次，弃PBS。每孔加入300µL细胞裂解液，-80℃和37℃反复冻溶两次，每次30min。取出细胞裂解液，12000r/min离心10min，参照cAMP-ELISA试剂盒检测说明书，取上清液用于OD值测定，做出OD值与样品浓度的曲线，结果见图5。
由图5可知，本发明设计的艾塞纳肽类似物Ex-4(i-vi)均表现出活性加强。在不同药物浓度下，Ex-4与Ex-4(i-vi)刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性。体外生物活性呈剂量依赖性增加。Ex-4ii、Ex-4iii和Ex-4iv刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的活性较Ex-4i、Ex-4v和Ex-4vi的活性更高。在相同浓度下，测定Ex-4与Ex-4(i-vi)刺激小鼠胰岛瘤细胞RIN-m5F释放cAMP的ED50分别为0.080nmol/L、0.072nmol/L、0.067nmol/L、0.061nmol/L、0.063nmol/L、0.073nmol/L、0.075nmol/L。其中，Ex-4ii、Ex-4iii和Ex-4iv较Ex-4分别提高了16.2%、23.8%和21.2%。
实施例4  Ⅱ型糖尿病模型db/db小鼠的体内降血糖作用
SPF级BKS.Cg-m+/+Leprdb/J(db/db)自发性糖尿病小鼠：共128只动物，6~8周龄，雌雄各半，购自北京华阜生物科技股份有限公司。生产许可证号为SYXK（京）2009-0004，质量合格证号：0172750。
SPF级BKS.Cg-m+/+Leprdb/J阴性对照小鼠（db/dm)，用于对照，共16只动物，6-8周龄，雌雄各半，购自北京华阜生物科技股份有限公司。生产许可证号为SYXK（京）2009-0004，质量合格证号：0172750。
小鼠SPF动物房饲养环境条件：空气洁净度10000级，气流速度0.1~0.2m/s，环境压差20~50Pa，温度20-25℃，湿度40~70%，氨浓度≤14mg/m 3，换气10~20次/h，动物照度15~20lux，工作照度150~300lux，噪音≤60dB，昼夜明暗交替时间12h/12h，雌雄分开饲养，每笼4只，适应性饲养一周后进行试验。
将128只db/db自发性糖尿病小鼠分性别，按预先测定的血糖值随机分层分为8组，即（1）模型组；（2）Ex-4组，5µg/kg；（3）Ex-4i组，5µg/kg；（4）Ex-4组，5µg/kg；（5）Ex-4iii组，5µg/kg；（6）Ex-4iv组，，5µg/kg；（7）Ex-4v组，5µg/kg；和（8）Ex-4vi组，5µg/kg。每组16只，雌雄各半。（9）16只db/dm小鼠作为正常对照组小鼠。
9组小鼠经皮下注射给药1次，对照组和模型组注射生理盐水10mL/kg，Ex-4组以5µg/kg量注射Ex-4样品，样品组以5µg/kg量注射Ex-4(i-vi)样品。
给药后，对所有小鼠采血，检测空腹0.5~12h血糖。测定Ex-4、Ex-4(i-vi)样品单次给药后的降糖作用强度和持续时间（M±SD，N=16）。测定结果见表2。
Figure 497571dest_path_image009
*P<0.01 vs阴性对照组； ‡P<0.05， #P<0.01 vs模型组
实验结果显示，与模型组相比，Ex-4及Ex-4(
Figure 509389dest_path_image001
-
Figure 208355dest_path_image002
)组皮下注射给药0.5h后即可显著降低血糖水平（P<0.01）；Ex-4单次给药的降糖作用可维持8h，给药12h后血糖浓度明显增高。Ex-4(
Figure 596611dest_path_image001
-
Figure 697422dest_path_image002
)单次给药的降糖作用可维持12h，单次给药12h时，Ex-4(
Figure 880142dest_path_image001
-
Figure 66404dest_path_image002
)组的血糖水平仍维持低于模型组（P<0.05）。因此，Ex-4(
Figure 992771dest_path_image001
-
Figure 948089dest_path_image002
)六种类似物均有降糖作用，相比于Ex-4具有更长的降糖药效，尤其Ex-4ii、Ex-4iii及Ex-4iv降糖作用更为显著。Ex-4(
Figure 567289dest_path_image001
-
Figure 99902dest_path_image002
)比Ex-4活性强，药效维持时间长，具有更长的半衰期。
序列表自由内容
                         序列表
 
<110>  鲁南贝特制药有限公司
 
<120>  艾塞纳肽类似物
 
<160>  7
 
<170>  SIPOSequenceListing 1.0
 
<210>  1
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  1
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15      
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Xaa
            20                  25                  30         
Xaa Xaa Xaa Pro Pro Pro Ser
        35                 
 
<210>  2
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  2
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Ser
            20                  25                  30         
Gly Gly Thr Gly Gly Ser Cys
        35                 
 
<210>  3
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  3
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Gly
            20                  25                  30         
Gly Gly Thr Gly Gly Ser Cys
        35                 
 
<210>  4
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  4
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
            20                  25                  30         
Gly Gly Thr Gly Gly Ser Cys
        35                 
 
<210>  5
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  5
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
            20                  25                  30         
Ser Gly Thr Gly Gly Ser Cys
        35                  
 
<210>  6
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  6
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
            20                  25                  30         
Ser Ala Thr Gly Gly Ser Cys
        35                 
 
<210>  7
<211>  39
<212>  PRT
<213>  人工序列(Artificial Sequence)
 
<400>  7
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1               5                   10                  15     
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
            20                  25                  30          
Ser Gly Ala Gly Gly Ser Cys
        35                 
。

Claims (10)

  1. 一类艾塞纳肽类似物，其特征在于，其氨基酸序列如SEQ.ID NO:1，包含：
    His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45，
    其中，Xaa37为Pro或Gly；
    Xaa38为Ser或Gly；
    Xaa39为Ser或Gly；
    Xaa40为Gly或Ala；
    Xaa41为Ala或Thr;
    Xaa42为Gly；
    Xaa43为Gly；
    Xaa44为Ser；
    Xaa45为Cys。
  2. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Gly，Xaa38为Ser，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4i，其氨基酸序列如SEQ.ID NO:2所示。
  3. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Pro，Xaa38为Gly，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4ii，其氨基酸序列如SEQ.ID NO:3所示。
  4. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Pro，Xaa38为Ser，Xaa39为Gly，Xaa40为Gly，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4iii，其氨基酸序列如SEQ.ID NO:4所示。
  5. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Gly，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4iv，其氨基酸序列如SEQ.ID NO:5所示。
  6. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Ala，Xaa41为Thr，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4v，其氨基酸序列如SEQ.ID NO:6所示。
  7. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，Xaa37为Pro，Xaa38为Ser，Xaa39为Ser，Xaa40为Gly，Xaa41为Ala，Xaa42为Gly，Xaa43为Gly，Xaa44为Ser，
    Xaa45为Cys；即艾塞纳肽类似物Ex-4vi，其氨基酸序列如SEQ.ID NO:7所示。
  8. 根据权利要求1所述的艾塞纳肽类似物，其特征在于，所述的艾塞纳肽类似物Ex-4（i-vi）氨基酸C末端是酰胺化的。
  9. 根据权利要求1-8任一项权利要求所述的艾塞纳肽类似物，在制备治疗糖尿病、肥胖症和/或相关并发症药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述的药物中含有权利要求1-8任一项所述艾塞纳肽类似物及药学上可接受的载体。
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EP1908778A1 (en) * 2005-06-29 2008-04-09 Changzhou Pharmaceutical Factory Co., Ltd. Exendin 4 polypeptide fragments and use thereof
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