CN111234000A

CN111234000A - Exenatide analogs

Info

Publication number: CN111234000A
Application number: CN201811432450.2A
Authority: CN
Inventors: 张贵民; 李铁健; 刘�东
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2020-06-05
Anticipated expiration: 2038-11-28
Also published as: WO2020108228A1; CN111234000B

Abstract

The invention belongs to the field of biological medicines, and particularly discloses an exenatide peptide analogue. The invention designs a plurality of amino acid structures at the C end of the amino acid sequence of the Exendin-4 of the exenatide, including the addition or deletion of amino acids, the mutation of the amino acid sequence and the addition of sites which can be used for fixed point modification, so as to obtain an exenatide analogue. The activity of the analogue for stimulating the islet tumor cells of the mouse to secrete insulin is obviously higher than that of exenatide Ex-4, the in-vivo hypoglycemic activity of the diabetes model mouse C57BL/KsJ-db/db is also obviously better than that of exenatide Ex-4, and the analogue can be used for preventing, treating or relieving other diseases such as diabetes, obesity and/or related complications thereof.

Description

Exenatide analogs

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an exenatide peptide analog.

Background

Mammalian GLP-1 is derived from a raw glucagon-like peptide secreted by L cells of small intestinal mucosa, and a natural analogue of GLP-1, namely Exenatide developed by America and Amylin company, is found in saliva secretion of exendin, and an amino acid sequence of the natural analogue has sequence similarity with a plurality of members in a GLP family and has 53 percent of homology with human GLP-1 (7-36).

The expression and activity of human GLP-1 (7-36) in vivo are strictly regulated, and after the second bit Ala at the N end of the human GLP-1 is hydrolyzed by dipeptidyl peptidase (DPP), inactive GLP-1 (9-36) is formed, and the metabolite is also an in vivo natural antagonist of GLP-1R. Therefore, the natural human active GLP-1 has short half-life in vivo and the metabolism rate of 2 min; in addition, under physiological conditions, GLP-1 is mainly excreted through the kidney, and the clinical application of the human GLP-1 is limited. The second amino acid Gly of non-human-derived artificially synthesized Exenatide is different from Ala of human GLP-1, and can effectively resist the degradation of dipeptide acyl peptidase; the C-terminal rigid (PSSGAPPPS) amino acid sequence of Exenatide can increase the stability of polypeptide, and the blood sugar reducing capability of Exenatide in vivo is about 1000 times stronger than that of GLP-1.

Exenatide (also known as Exenatide or Exenatide, Exenatide or Ex-4, trade name Byetta) is a polypeptide consisting of 39 amino acids, the molecular weight of which is 4186.6, and the molecular formula is C₁₈₄H₂₈₂N₅₀O₆₀S, CAS number 141758-74-9, amino acid sequence as shown below.

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-NH₂The exenatide is approved by the FDA to be marketed in 4 months of 2005, belongs to a medicinal subcutaneous injection preparation, has the effects of promoting glucose-dependent insulin secretion, restoring first-phase insulin secretion, inhibiting glucagon secretion, slowing emptying stomach contents, improving functions of pancreatic β cells and the like, and is very suitable for treating type II diabetes, for example, for improving and controlling blood sugar of type II diabetes patients who are not ideal for treatment by metformin and sulfonylurea drugs.

Exenatide peptide secondary structure: the N end of Exenatide is irregularly curled, amino acid residues with opposite charge side chains are alternately arranged on the same side surface of the middle part of Exenatide, a helix is formed through a salt bridge or a polar hydrogen bond, and the C end of Exenatide is hydrophilic Trp-Cage. The interaction mechanism of Exenatide with the GLP-1 receptor has been studied clearly.

A series of different structural designs have been developed to extend the structural half-life of GLP-1 analogs and enhance biological activity. CN1384755 discloses a novel Exendin agonist preparation and an administration method thereof, and discloses a compound structure and a preparation method of Exenatide. CN102532303 discloses the modification of amino group of lysine or amino group of N-terminal histidine residue in Exenatide with methoxypolyethylene glycol. CN200980111088 discloses fatty acid-PEG-Exenatide. Patent CN101125207 reports the structural modification of Exenatide into short peptide. CN105753963A discloses Exenatide analogs with single-point or multi-point amino acid mutations. CN102397558 discloses the substitution of certain amino acids in Exenatide with cysteine and the modification with PEG or PEG terminally substituted with methyl.

Although many efforts are made in the development of exenatide analogue long-acting drugs, the exenatide analogue on the market at present has poor stability and low drug effect, and innovative modification and research of the exenatide analogue are still a very important topic as the structure and activity basis of long-acting drug development.

Based on the above analysis and research, we select exenatide as the modified polypeptide sequence of the invention, combine computer model calculation and binding test, and focus on modifying the amino acid sequence of the C-terminal, and add modified active sites for long-acting drug development.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides an exenatide peptide analogue which has long half-life period, good stability and high activity, can simultaneously keep better blood sugar reducing effect and low toxicity, and simultaneously provides the application of the exenatide peptide analogue in preparing medicines for treating diabetes, obesity and/or complications.

The invention specifically comprises the following contents:

the invention provides an exenatide analog which is characterized in that amino acids from 37 th site to 45 th site of an amino acid sequence of exenatide Ex-4 are replaced to obtain the exenatide analog; the amino acid sequence is shown in SEQ.ID NO. 1, and the amino acid sequence comprises:

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，

wherein 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.

Further preferably, the amino acid sequence of the preferred epothilone sodium peptide analogue is shown in SEQ.ID NO. 2-7:

wherein, Xaa37 is Gly, Xaa38 is Ser, Xaa39 is Gly, Xaa40 is Gly, Xaa41 is Thr, Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide analogue Ex-4i is obtained, and the amino acid sequence of the exenatide analogue is shown in SEQ.ID NO. 2.

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.IDNO:2)。

Wherein Xaa37 is Pro, Xaa38 is Gly, Xaa39 is Gly, Xaa40 is Gly, Xaa41 is Thr, Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide analogue Ex-4ii is obtained, and the amino acid sequence of the exenatide analogue is shown in SEQ.ID NO. 3.

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.IDNO:3)。

Wherein Xaa37 is Pro, Xaa38 is Ser, Xaa39 is Gly, Xaa40 is Gly, Xaa41 is Thr Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide peptide analogue Ex-4iii is obtained, and the amino acid sequence of the exenatide peptide analogue is shown in SEQ.ID NO. 4.

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.IDNO:4)。

Wherein Xaa37 is Pro, Xaa38 is Ser, Xaa39 is Ser, Xaa40 is Gly, Xaa41 is Thr, Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide analogue Ex-4iv is obtained, and the amino acid sequence of the exenatide analogue is shown in SEQ.ID NO. 5.

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)。

Wherein Xaa37 is Pro, Xaa38 is Ser, Xaa39 is Ser, Xaa40 is Ala, Xaa41 is Thr, Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide analogue Ex-4v is obtained, and the amino acid sequence of the exenatide analogue is shown in SEQ.ID NO. 6.

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)。

Wherein Xaa37 is Pro, Xaa38 is Ser, Xaa39 is Ser, Xaa40 is Gly, Xaa41 is Ala, Xaa42 is Gly, Xaa43 is Gly, Xaa44 is Ser, and Xaa45 is Cys; the exenatide peptide analogue Ex-4vi is obtained, and the amino acid sequence of the exenatide peptide analogue is shown in SEQ ID NO. 7.

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.IDNO:7)。

The C-terminal of the exenatide analog is amidated, and the amidation is the same as that of natural GLP-1 and exenatide.

Another object of the present invention is to provide the use of the exenatide analog of the present invention in the preparation of a medicament for the treatment of diabetes, obesity and/or related complications.

The application is that the medicine contains the exenatide analogue and a pharmaceutically acceptable carrier.

The exenatide analogue of the invention may be administered as a single drug or may be administered in combination with other drugs.

The exenatide peptide analogue of the invention may form a salt, including various inorganic or organic salts, such as hydrochloride, hydrobromide, phosphate, sulphate, maleate, oxalate, citrate, tartrate, fumarate, mandelate and lactate; salifying with various inorganic and organic bases, such as sodium hydroxide, tris (hydroxymethyl) aminomethane and N-methyl-glucosamine.

The exenatide analogue of the invention may be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition comprises the exenatide analogue or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" does not destroy the pharmaceutical activity of the compounds of the present invention and their useful salts, while the effective amount thereof is non-toxic to humans. The "pharmaceutically acceptable carrier" can be used without limitation: ion exchange materials, aluminum stearate, lecithin, serum proteins for pharmaceutical preparations, saturated vegetable fatty acids, cellulosic substances, ethylene-polyoxyethylene-block polymers, cyclodextrins or chemically modified derivatives or other soluble derivatives thereof, and the like.

Other pharmaceutically acceptable excipients, fillers such as anhydrous lactose, starch, lactose beads and glucose, binders such as microcrystalline cellulose, disintegrants such as croscarmellose sodium, croscarmellose starch, low-substituted hydroxypropylcellulose, lubricants such as magnesium stearate, absorption enhancers, excipients, solubilizers, colorants and the like may also be added to the pharmaceutical composition of the present invention.

The exenatide analogue or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of the invention as described above may be administered by the enteral or parenteral route. Parenteral routes include subcutaneous, intradermal, intramuscular, nasal, mucosal administration or inhalation. The preparation can be developed into injection, cream, ointment, patch, aerosol, etc.

The exenatide analog or the pharmaceutically acceptable salt and the pharmaceutical composition thereof can be used for single-drug or combined-drug treatment of related diseases, and are within the scope of understanding of the skilled person.

The invention does not limit the preparation method of the exenatide Ex-4 and the exenatide analogue, and can adopt a chemical solid phase synthesis method or a gene expression method. One embodiment of the present invention employs Fmoc solid phase synthesis.

The results of pharmaceutical tests show that the activity of the 32 exenatide analogs in vitro stimulating the mouse islet tumor cell RIN-m5F to release cAMP is obviously higher than that of non-mutated exenatide; and the blood sugar reducing effect of subcutaneous injection administration on db/db model mice is also obviously superior to that of exenatide. Therefore, the exenatide peptide analogue can be used for better preventing, preventing or alleviating diabetes, obesity and/or complications.

The differences between the 32 exenatide analogs of the present invention and unmutated exenatide were analyzed by using MOE software (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada), and the results showed that the interaction and number of bonds between the 32 exenatide analogs of the present invention and the receptor were changed, preferably, the C-terminal amino acid sequence of the 6 exenatide analogs Ex-4(i-vi) formed a new binding effect, which stabilized the helical structure of the polypeptide α, promoted its binding to the active core structure field, and increased the biological activity.

The exenatide peptide analogue designed by the invention shows enhanced activity. Under different drug concentrations, the exenatide Ex-4 and exenatide analogues stimulate the cAMP releasing activity of mouse islet tumor cells RIN-m 5F. The in vitro bioactivity is increased in a dose-dependent manner. Preferably, exenatide peptide analog Ex-4(i-vi) stimulates murine insulinoma cells RIN-5mF to release cAMP with greater activity than other analogs. More preferably, exenatide peptide analogs Ex-4ii, Ex-4iii and Ex-4iv stimulate mouse islet tumor cells RIN-m5F to release cAMP with higher activity than exenatide peptide analogs Ex-4i, Ex-4v and Ex-4 vi.

Ex-4 and Ex-4(i-vi) groups can obviously reduce the blood sugar level (P <0.01) after being injected subcutaneously for 0.5 h; the blood sugar reducing effect of the Ex-4 single administration can be maintained for 8 hours, and the blood sugar concentration is obviously increased after 12 hours of administration. The blood glucose reducing effect of the Ex-4(i-vi) can be maintained for 12 hours by single administration, and the blood glucose level of the Ex-4(i-vi) group is still maintained to be lower than that of the model group (P <0.05) by single administration for 12 hours. Therefore, six analogues of Ex-4(i-vi) have the hypoglycemic effect, and have longer hypoglycemic drug effect compared with Ex-4, and particularly have more remarkable hypoglycemic effects of Ex-4ii, Ex-4iii and Ex-4 iv. Ex-4(i-vi) has stronger activity than Ex-4, long efficacy maintaining time and longer half-life.

Compared with Ex-4, the exenatide analogue provided by the invention can be obtained through an in-vivo blood sugar reducing effect test of a type II diabetes model db/db mouse, and the blood sugar level of the exenatide analogue can be obviously reduced after the exenatide analogue is subcutaneously administrated for 0.5 h; the hypoglycemic effect of the exenatide in a single administration can be maintained for 8 hours, the blood sugar concentration is obviously increased after the administration for 12 hours, the blood sugar-reducing maintaining time of the exenatide analogue designed by the invention is longer than that of the exenatide Ex-4, and the blood sugar-reducing maintaining time reaches more than 10 hours. Preferably, the hypoglycemic effect of the Ex-4(i-vi) peptide analogue in a single administration can be maintained for 12 hours, and the blood sugar level of the Ex-4(i-vi) group is maintained to be lower than that of the model group (P <0.05) in a single administration for 12 hours. Therefore, the exenatide analogs have the hypoglycemic effect, and have longer hypoglycemic effect compared with exenatide Ex-4. The exenatide analogue has stronger activity than the exenatide, long efficacy maintaining time and longer half-life.

The exenatide analogue designed by the invention has the advantages of long half-life period, good stability and high activity, and can simultaneously keep good blood sugar reducing effect and low toxicity.

Term(s) for

In this specification, the term "exenatide" is a functional analogue of glucagon-like peptide-1 isolated from the salivary glands of exendin inhabiting the southwest united states. Exenatide, academic name "exenatide-4", as a physiologically active peptide consisting of 39 amino acids, has 53% amino acid similarity compared to glucagon-like peptide-1 present in human living bodies. Denoted Ex-4 in the following examples is exenatide, the amino acid sequence of which is shown in SEQ. ID NO: 1.

In the present specification, the term exenatide analog means that the rigid amino acid sequence at the C-terminus of exenatide-4 is deleted and replaced by another flexible amino acid sequence, still being a physiologically active peptide consisting of 39 amino acids. The amino acid sequence of the preferred Ex-4(i-vi) peptide analogues of Ex-4 is shown in SEQ ID NO: 2-7. +

Drawings

FIG. 1 is a schematic representation of the interaction of exenatide Ex-4 with a G protein-coupled receptor;

FIG. 2 is a diagram showing the binding pattern of Ex-4C-terminal exenatide to a G protein-coupled receptor;

FIG. 3 is a schematic representation of the interaction of exenatide peptide analogue Ex4iii with a G protein-coupled receptor;

FIG. 4 is a graph showing the binding pattern of the Ex4 iiiiiiiC terminus of an exenatide analog to a G protein-coupled receptor;

FIG. 5 shows the activity of exenatide Ex-4 and its analogue Ex-4(i-vi) on the release of cAMP by murine insulinoma cells RIN-m 5F.

Detailed Description

The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art from this disclosure that these examples are merely for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1 preparation of Ex-4 and Ex-A peptide analogues

This example used Fmoc solid phase synthesis to synthesize one of the exenatide analogs of the invention, and the other analogs were synthesized similarly.

Synthesis process

Fmoc synthesis method is adopted, and Fmoc-Rink resin is selected for synthesis. The synthesis steps are as follows:

19 Fmoc-amino acid raw material with side chain protecting groups → solid phase synthesis → removal of side chain protecting groups → HPLC purification → freeze drying → exenatide peptide analogues.

Specifically, the method comprises the following steps:

1. amino acid monomer

2. Solid phase synthesis and crude polypeptide preparation process

Activating amino acid by HOBt method, connecting to amino resin according to sequence, and synthesizing in 39 steps.

(1) The instrument equipment comprises:

CS536XT vertical polypeptide synthesizer and magnetic stirring apparatus.

(2) Reagent:

(3) solid phase synthesis operation: weighing 5.5g of resin (the resin loading rate is 0.45mmol/g), pouring into a reactor of a polypeptide synthesizer, weighing 10mmol of corresponding amino acid with a protecting group from the C-end to the N-end according to the amino acid sequence of the exenatide analog, and arranging in the synthesizer. Under the condition of room temperature, the 39-step synthesis reaction is automatically completed according to a computer program.

And after the synthesis is finished, obtaining the polypeptide resin with side chain protecting groups. The polypeptide resin is taken out and put into a vacuum drier, the drying temperature is 30 ℃, and the polypeptide resin is dried to the constant weight (+/-0.2 g).

(4) Deprotection and precipitation

Putting the exenatide peptide analogue polypeptide resin with the protecting group into a triangular flask with a plug, adding a cleavage reagent TFA/water/TIS/EDT (TFA/water/TIS/EDT) ═ 94:2:2:2(V/V) according to 13 ml/g of the peptide resin, and stirring and reacting for 4 hours at the constant temperature of 25 ℃; the filtrate was filtered and collected, the resin was washed with a small amount of trifluoroacetic acid, and the collected solutions were combined by filtration. 500mL of glacial ethyl ether (-10 ℃) are added dropwise with stirring to give a white precipitate, which is filtered, the crude product is washed with a small amount of glacial ethyl ether and dried overnight in a vacuum drier to give crude peptide.

3. HPLC purification

(1) Instrumentation and reagents:

(2) purification of

The crude product of the exenatide peptide analogue, namely the trifluoroacetate solution is prepared by HPLC reversed phase purification, and the purity is more than 98%.

① chromatographic column, 50mm × 300mm RP-1810 μm

② mobile phase:

a: 0.1% aqueous trifluoroacetic acid solution

B: 0.1% trifluoroacetic acid acetonitrile solution

③ sample solution crude polypeptide is prepared into 10.0mg/ml solution with 0.1% trifluoroacetic acid solution in water and passed through 0.22 μm filter.

④ the elution condition is that gradient elution is adopted, the flow rate is 50ml/min, the ultraviolet light is 215nm for detection, and the elution gradient is as follows:

⑤ collecting the eluate with main peak purity higher than 98% to obtain the eluent.

⑥ removal of acetonitrile:

pouring the collected preparation eluent into a rotary evaporation bottle, carrying out rotary evaporation at 25 ℃ and-0.099 Mpa, removing all acetonitrile, and filtering the residual liquid through a 0.22-micron filter membrane for freeze-drying.

4. Freeze drying

(1) The instrument equipment comprises: GOLD-SIM vertical freeze dryer

(2) The operation is as follows:

and pouring the aqueous solution of the exenatide analogue trifluoroacetic acid into a sample tray of a vacuum freeze dryer, and carrying out freeze-drying according to a computer program to obtain the required compound.

Exenatide Ex-4 and other exenatide analogs are prepared according to the above methods.

Example 2 computer simulation-assisted polypeptide screening

The result shows that compared with the Exenatide Ex-4, the 32 Exenatide analogs have enhanced interaction with a receptor binding domain, the number of the interaction between the Exenatide analogs and the receptor is increased, and preferably 6 Exenatide analogs Ex-4(i-vi) form new interaction between the C terminal of the polypeptide and the receptor, including new hydrogen bonds, new hydrophobic interaction and new ionic bonds.

The analysis of MOE software shows that a new interaction is formed between the C terminal and a receptor by introducing a flexible amino acid sequence cross-linking region or replacing an exenatide peptide analogue by a flexible Linker, and the interaction schematic diagrams of Ex-4 and Ex-4iii and the receptor are shown in figures 1 to 4, the C terminal of Ex-4iii and the receptor form a new hydrogen bond and hydrophobic interaction, meanwhile, the spiral structure of active α is more stable, and the binding force of the active domain of the receptor is improved.

Example 3 Activity of Exenatide analogs in vitro stimulation of cAMP Release from murine insulinoma cells RIN-m5F

(1) Test material and related reagent formulations

PBS: 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, adding water to dissolve and dilute the mixture to 1000ml, adjusting the pH value to 7.2, autoclaving at 121 ℃ for 15min, and storing at 4 ℃.

Preparation of exenatide Ex-4 and exenatide analogue Ex-4(i-vi) polypeptide samples: accurately weighing 10mg of each polypeptide sample, accurately preparing a sample solution of 0.1mg/ml by using sterile water for injection, determining the content of the polypeptide by a nitrogen determination method, diluting the polypeptide sample solution to 100ng/ml according to the content of the polypeptide, and then diluting the polypeptide sample solution by a 2-fold ratio to obtain 8 dilution sample solutions with polypeptide concentrations of 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.12ng/ml, 1.56ng/ml and 0.78ng/ml respectively.

(2) Test method

After digestion of the RIN-m5F cells in good growth status, the cells were counted at 5X 10⁵～8×10⁵Culturing the strain per mL at 37 ℃ for 18-36 h; digesting with 0.25% pancreatin at a ratio of 3.5X 10⁵one/mL, inoculated into 24-well cell plates at 0.5 mL/well, incubated at 37 ℃ with 5% CO₂And continuously culturing for 24-36 h. Discarding cell sap, adding cell maintenance solution (1.0 mL/well of RPMI1640 culture solution containing 5mg/mL bovine serum albumin), at 37 deg.C and 5% CO₂Culturing for 15 min. The supernatant was discarded, and a cell maintenance medium containing 1mM IBMX (BioRad) was added thereto at 0.9 mL/well at 37 ℃ with 5% CO₂Culturing for 15 min. Adding 0.1 mL/hole sample to be tested as soon as possible, shaking gently and mixing uniformly, then, at 37 ℃, 5% CO₂Culturing for 15 min. The cell culture plate was removed and placed on ice, the supernatant discarded, washed twice with 1.0 mL/well of pre-chilled PBS, and the PBS discarded. Add 300. mu.L of cell lysate to each well and freeze-thaw twice at-80 ℃ and 37 ℃ for 30min each time. Taking out cell lysate, centrifuging at 12000r/min for 10min, taking supernatant for OD value determination according to the detection instruction of cAMP-ELISA kit, and making a curve of OD value and sample concentration, wherein the result is shown in figure 5.

As can be seen from FIG. 5, Ex-4(i-vi) which is the peptide analogue of exenatide designed by the invention shows enhanced activity. Ex-4 and Ex-4(i-vi) stimulate cAMP releasing activity of mouse islet tumor cells RIN-m5F at different drug concentrations. The in vitro bioactivity is increased in a dose-dependent manner. Ex-4ii, Ex-4iii and Ex-4iv stimulate mouse islet tumor cells RIN-m5F to release cAMP with higher activity than Ex-4i, Ex-4v and Ex-4 vi. At the same concentration, ED50 for Ex-4 and Ex-4(i-vi) to stimulate cAMP release from mouse islet tumor cells RIN-m5F were determined to be 0.080nmol/L, 0.072nmol/L, 0.067nmol/L, 0.061nmol/L, 0.063nmol/L, 0.073nmol/L, 0.075nmol/L, respectively. Wherein, Ex-4ii, Ex-4iii and Ex-4iv are respectively increased by 16.2 percent, 23.8 percent and 21.2 percent compared with Ex-4.

Example 4 in vivo hypoglycemic Effect in type II diabetes model db/db mice

SPF grade BKS. Cg-m +/+ Leprdb/J (db/db) spontaneously diabetic mice: a total of 128 animals, 6-8 weeks old, half male and half female, were purchased from Beijing Hua Fuyang Biotechnology GmbH. The production license number is SYXK (Jing) 2009-: 0172750.

SPF-grade bks. cg-m +/+ Leprdb/J negative control mice (db/dm), used for control, 16 animals total, 6-8 weeks old, hermaphrodite, purchased from beijing caruncle biotechnology gmbh. The production license number is SYXK (Jing) 2009-: 0172750.

mouse SPF animal house breeding environmental conditions: 10000 levels of air cleanliness, 0.1-0.2 m/s of air flow velocity, 20-50 Pa of environmental pressure difference, 20-25 ℃ of temperature, 40-70% of humidity and ammonia concentration less than or equal to 14mg/m³Ventilation of 10 to 20The animal illumination is 15-20 lux, the working illumination is 150-300 lux, the noise is less than or equal to 60dB, the light and shade alternating time is 12h/12h day and night, male and female animals are separately fed, 4 animals are fed in each cage, and the test is carried out after the animals are fed adaptively for one week.

Dividing 128 db/db spontaneous diabetic mice into sexes, and randomly dividing the mice into 8 groups according to the blood glucose value measured in advance, namely (1) a model group; (2) ex-4 group, 5. mu.g/kg; (3) ex-4i group, 5. mu.g/kg; (4) ex-4 group, 5. mu.g/kg; (5) ex-4iii group, 5. mu.g/kg; (6) ex-4iv group, 5. mu.g/kg; (7) ex-4v group, 5. mu.g/kg; and (8) Ex-4vi group, 5. mu.g/kg. Each group had 16 animals, each half of a female. (9)16 db/dm mice served as normal control mice.

9 groups of mice were administered 1 time by subcutaneous injection, 10mL/kg of physiological saline was injected to the control group and the model group, 5. mu.g/kg of Ex-4 sample was injected to the Ex-4 group, and 5. mu.g/kg of Ex-4(i-vi) sample was injected to the sample group.

After administration, all mice are sampled with blood, and fasting blood sugar is detected for 0.5-12 h. The intensity and duration of the hypoglycemic effect (M + -SD, N-16) of the Ex-4 and Ex-4(i-vi) samples after a single administration were determined. The results are shown in Table 2.

TABLE 2 Effect of Exenatide Ex-4 and its analogues Ex-4(i-vi) on blood glucose levels in mice following fasting administration

*P<0.01vs negative control group;

^#P<0.01vs model set

The experimental results show that compared with the model group, the Ex-4 and Ex-4(i-vi) groups can obviously reduce the blood sugar level (P <0.01) after being subcutaneously injected for 0.5 h; the blood sugar reducing effect of the Ex-4 single administration can be maintained for 8 hours, and the blood sugar concentration is obviously increased after 12 hours of administration. The blood glucose reducing effect of the Ex-4(i-vi) can be maintained for 12 hours by single administration, and the blood glucose level of the Ex-4(i-vi) group is still maintained to be lower than that of the model group (P <0.05) by single administration for 12 hours. Therefore, six analogues of Ex-4(i-vi) have the hypoglycemic effect, and have longer hypoglycemic drug effect compared with Ex-4, and particularly have more remarkable hypoglycemic effects of Ex-4ii, Ex-4iii and Ex-4 iv. Ex-4(i-vi) has stronger activity than Ex-4, long efficacy maintaining time and longer half-life.

Sequence listing

<110> Lunan pharmaceutical group, Inc

<120> exenatide peptide analog

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>39

<212>PRT

<213> Artificial Sequence (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> Artificial Sequence (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> Artificial Sequence (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> Artificial Sequence (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> Artificial Sequence (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> Artificial Sequence (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

2025 30

Ser Ala Thr Gly Gly Ser Cys

35

<210>7

<211>39

<212>PRT

<213> Artificial Sequence (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

1. An exenatide analog, wherein the amino acid sequence is shown as SEQ ID NO. 1, and the exenatide analog comprises:

wherein 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.

2. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4i peptide analogue, the amino acid sequence of which is shown in SEQ.ID NO. 2.

3. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4ii peptide analogue, the amino acid sequence of which is shown in SEQ ID NO. 3.

4. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4iii analogue of the exenatide peptide, and the amino acid sequence is shown in SEQ.ID NO. 4.

5. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4iv analogue of the exenatide peptide, and the amino acid sequence of the Ex-4iv analogue of the exenatide peptide is shown in SEQ ID NO. 5.

6. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4v peptide analogue of the Exenatide, and the amino acid sequence of the Exenatide analogue is shown as SEQ ID NO. 6.

7. The exenatide peptide analog according to claim 1, wherein 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; namely the Ex-4vi peptide analogue of the Ex-4vi, and the amino acid sequence of the Ex-4vi peptide analogue is shown in SEQ ID NO. 7.

8. The exenatide peptide analog according to claim 1, wherein the C-terminus of amino acids of exenatide peptide analog Ex-4(i-vi) is amidated.

9. Use of an exenatide peptide analogue according to any of claims 1-8 for the preparation of a medicament for the treatment of diabetes, obesity and/or related complications.

10. The use according to claim 9, wherein the medicament comprises an exenatide peptide analogue according to any one of claims 1-8 and a pharmaceutically acceptable carrier.