CN106554409B

CN106554409B - Long-acting glucagon-like peptide-1 analogue and application thereof

Info

Publication number: CN106554409B
Application number: CN201510639405.4A
Authority: CN
Inventors: 韩英梅; 赵娜夏; 王玉丽
Original assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Current assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Priority date: 2015-09-30
Filing date: 2015-09-30
Publication date: 2020-04-07
Anticipated expiration: 2035-09-30
Also published as: CN106554409A

Abstract

The invention provides a long-acting glucagon-like peptide-1 analogue, which is characterized in that the polypeptide has a structure represented by a general formula I: z₁‑HX₈EX₁₀T FTSDV SSYLE X₂₂QAAK EFIX₃₀W LVKX₃₅RG‑Z₂In the general formula I, X₈、X₁₀、X₂₂、X₃₀、X₃₅Any two residues in the amino acid residue are cysteine, Z₂is-H or-NH₂；X₈、X₁₀、X₂₂、X₃₀、X₃₅In which any one residue is cysteine, Z₂Is a sequence of-CG. The glucagon-like peptide-1 analogue represented by the general formula I always contains two cysteine residues to form an intramolecular disulfide bond, thereby avoiding the rapid degradation in vivo and obviously prolonging the half-life in vivo.

Description

Long-acting glucagon-like peptide-1 analogue and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a glucagon-like peptide-1 analogue with a long-acting effect and application thereof.

Background

GLP-1(7-36, 7-37) is a major active form of GLP-1 in systemic circulation, controlling blood glucose by complex mechanisms including secretion of insulin and glucagon, gastric emptying and regulation of peripheral insulin, the hypoglycemic action of GLP-1(7-36, 7-37) is glucose dependent, avoiding hypoglycemia, inhibiting apoptosis of pancreatic islet β -cells, promoting proliferation of pancreatic islet β -cells and reversing disease progression, but the plasma half-life of native GLP-1 is only 1-2 minutes, metabolic instability limits its use as a drug, and in vivo dipeptidyl peptidase (DPPIV) specifically recognizes and degrades the N-terminal His-Ala fragment of the receptor-binding active site in GLP-1 structure, inactivating it rapidly, while other proteolytic enzymes such as endopeptidase are involved in renal filtration.

The technical goals of the GLP-1-based drug development field are to improve the metabolic stability and prolong the half-life period of blood plasma so as to improve the clinical drug compliance. The patent technologies that have been disclosed can be summarized as: 1) structural modification aiming at key sites of enzyme degradation (CN00806548.9, CN99814187.9, CN200410017667.9 and the like); 2) fatty acyl groups are introduced into a parent peptide chain structure, so that the binding force with plasma protein is improved to avoid the polypeptide from being rapidly eliminated in vivo (CN201210513145.2, CN200810124641.2, CN20118000352.1 and the like); 3) GLP-1 analogue protein fusion technology; 4) PEG modification, and the like. Despite various attempts over the years, the only drugs on the market developed so far based on the GLP-1(7-36, 7-37) parent peptide chain were liraglutide. Although liraglutide prolongs GLP-1 half-life in vivo, it still needs to be injected once a day, and drug compliance still needs to be improved.

The invention patent CN201110361927.4 relates to a method for prolonging half-life by connecting cysteine-containing groups to the N-end and the C-end of GLP-1(7-37) respectively to form intramolecular disulfide bond; the invention patent 201410243272.4 relates to a method for prolonging the C-terminal of GLP-1(7-37) and introducing cysteine to make the peptide chain contain at least two cysteine residues and introducing amino fatty acid on the C-terminal cysteine residue of the peptide chain, which is a technical proposal for prolonging the half-life by combining intramolecular disulfide cyclization and introducing fatty acid group to enhance the combination of the polypeptide and plasma protein. However, the technical schemes involved in the two publications have the problems of excessive cysteine residues in molecules, high synthesis difficulty, excessive introduced hydrophobic groups, easy aggregation of final products, poor solubility and difficult quality control of the final products.

Disclosure of Invention

Aiming at the limitations of the prior art, the invention provides a long-acting glucagon-like peptide-1 analogue which has longer half-life period in vivo and better hypoglycemic activity, is highly homologous with endogenous GLP-1(7-37) and can avoid safety risk.

The glucagon-like peptide-1 analogs of the present invention are artificially modified forms of GLP-1 (7-37). The natural sequence of GLP-1(7-37) is: HAEGT FTSDV SSYLE GQAAK EFIAW LVKGRG are provided. The invention addresses X in the sequence₈、X₁₀、X₂₂、X₃₀、X₃₅The site is appropriately substituted by amino acid, especially two corresponding sites in the original sequence are substituted by cysteine, or the cysteine residue is introduced by extending the sequence, so that two cysteine residues are contained in the peptide chain, an intramolecular disulfide bond is formed, and the part which is easily recognized by a degrading enzyme in the peptide chain is contained in a ring structure, thereby avoiding the rapid elimination of the target polypeptide in vivo.

In one aspect of the present invention, there is provided a long-acting glucagon-like peptide-1 analog, characterized in that said polypeptide has a structure represented by general formula i:

Z₁-HX₈EX₁₀T FTSDV SSYLE X₂₂QAAK EFIX₃₀W LVKX₃₅RG-Z₂

general formula I

Wherein Z is₁is-H or CH₃CO- (hereinafter, Ac);

X₈is Ala, Gly, Cys, Ser or D-Ala;

X₁₀gly, Ser or Cys;

X₂₂is Gly, Glu, Cys or Ser;

X₃₀cys or Ala;

X₃₅is Gly, Cys or Aib (wherein Aib represents 2-aminoisobutyric acid);

Z₂is-H, -NH₂or-CG.

In another aspect of the present invention, there is provided a long-acting glucagon-like peptide-1 analog characterized by having the structure represented by the general formula i:

wherein, X₈、X₁₀、X₂₂、X₃₀、X₃₅Any two residues in the amino acid residue are cysteine, Z₂is-H or-NH₂；

X₈、X₁₀、X₂₂、X₃₀、X₃₅In which any one residue is cysteine, Z₂is-CG.

Preferably, the two cysteine residues form an intramolecular disulfide bond.

Preferably, when Z is₂is-H or-NH₂，X₈、X₂₂When it is a cysteine residue, X₁₀Is Gly or Ser, X₃₀Is Ala, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₁₀，X₂₂When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₃₀Is Ala, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₂₂，X₃₅When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly or Ser, X₃₀Is Ala, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₂₂、X₃₀When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly or Ser, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₃₀，X₃₅When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly, Ser, X₂₂Is Gly, Glu or Ser, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₈，X₃₅When it is a cysteine residue, X₁₀Is Gly or Ser, X₂₂Is Gly, Glu or Ser, X₃₀Is Ala, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₈，X₃₀When it is a cysteine residue, X₁₀Is Gly, Ser, X₂₂Is Gly, Glu or Ser, X₃₅Is Gly or Aib, Z₁is-H, CH₃CO-；

When Z is₂is-H or-NH₂，X₁₀，X₃₀When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₂₂Is Gly, Glu or Ser, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-H or-NH₂，X₁₀，X₃₅When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₂₂Is Gly, Glu or Ser, X₃₀Is Ala, Z₁is-H or CH₃CO-；

When Z is₂is-CG, X₈When it is a cysteine residue, X₁₀Is Gly or Ser, X₂₂Is Gly, Glu or Ser, X₃₀Is Ala, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-CG, X₁₀When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₂₂Is Gly, Glu or Ser, X₃₀Is Ala, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-CG, X₂₂When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly or Ser, X₃₀Is Ala, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-；

When Z is₂is-CG, X₃₀When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly or Ser, X₂₂Is Gly, Glu or Ser, X₃₅Is Gly or Aib, Z₁is-H or CH₃CO-; and

when Z is₂is-CG, X₃₅When it is a cysteine residue, X₈Is Ala, Gly, Ser or D-Ala, X₁₀Is Gly or Ser, X₂₂Is Gly, Glu or Ser, X₃₀Is Ala, Z₁is-H or CH₃CO-。

In another aspect of the invention, glucagon-like peptide-1 analogs represented by SEQ ID NOs 1-16 are provided:

SEQ ID NO1：HCEGT FTSDV SSYLE CQAAK EFIAW LVKAibRGNH2

SEQ ID NO2：HCEGT FTSDV SSYLE CQAAK EFIAW LVKAibRG

SEQ ID NO3：AcHAECT FTSDV SSYLE CQAAK EFIAW LVKAibRG

SEQ ID NO4：HGECT FTSDV SSYLE CQAAK EFIAW LVKAibRGNH2

SEQ ID NO5：AcHAEGT FTSDV SSYLE CQAAK EFIAW LVKCRG

SEQ ID NO6：HGEGT FTSDV SSYLE CQAAK EFIAW LVKCRGNH2

SEQ ID NO7：HGEGT FTSDV SSYLE SQAAK EFICW LVKCRGNH2

SEQ ID NO8：AcHAEGT FTSDV SSYLE GQAAK EFICW LVKAibRGCG

SEQ ID NO9：HGEGT FTSDV SSYLE CQAAK EFIAW LVKAibRGCG

SEQ ID NO10：AcHAEGT FTSDV SSYLE CQAAK EFIAW LVKGRGCG

SEQ ID NO11：HCEGT FTSDV SSYLE GQAAK EFIAW LVKGRGCG

SEQ ID NO12：HCEGT FTSDV SSYLE GQAAK EFICW LVKAibRGNH2

SEQ ID NO13：HCEGT FTSDV SSYLE GQAAK EFICW LVKAibRG

SEQ ID NO14：AcHAECT FTSDV SSYLE GQAAK EFICW LVKAibRG

SEQ ID NO15：HGECT FTSDV SSYLE GQAAK EFIAW LVKAibRGCG

SEQ ID NO16：HGEGT FTSDV SSYLE CQAAK EFICW LVKAibRG

in another aspect of the invention, a pharmaceutical composition is provided comprising at least one glucagon-like peptide-1 analog of formula i.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, diluents and other excipients.

Preferably, the pharmaceutical composition of the present invention is composed of the glucagon-like peptide-1 analog and one or more pharmaceutically acceptable excipients. The medicinal adjuvants comprise water-soluble filler, pH regulator, stabilizer, water for injection, osmotic pressure regulator, etc.

Preferably, the water-soluble filler includes, but is not limited to, mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, etc.; the pH regulator includes, but is not limited to, organic or inorganic acids such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, etc., and physiologically acceptable inorganic bases or salts such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate salts, etc.; such stabilizers include, but are not limited to, EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxy/hydroxy cellulose or derivatives thereof, such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrins, sodium lauryl sulfate or tris (hydroxymethyl) aminomethane, and the like; the tonicity modifier includes, but is not limited to, sodium chloride or potassium chloride.

In another aspect of the invention, the invention provides the use of the glucagon-like peptide-1 analog represented by the general formula I, the pharmaceutically acceptable salt thereof or the composition thereof in the preparation of medicaments for treating and/or preventing diabetes, obesity and Alzheimer's disease.

Preferably, the composition of the present invention can be administered in the form of intravenous, intramuscular or subcutaneous injections or orally, rectally or nasally. The dosage may range from 5 μ g to 10mg per dose, depending on the subject being treated, the mode of administration, the indication, and other factors.

In another aspect of the invention, there is provided a method of preparing a glucagon-like peptide-1 analog comprising:

1) synthesis by conventional solid or liquid phase methods, stepwise or by fragment assembly;

2) expressing a nucleic acid construct encoding the polypeptide in a host cell and recovering the expression product from the host cell culture;

3) effecting cell-free in vitro expression of a nucleic acid construct encoding the polypeptide and recovering the expression product;

or by any combination of methods 1), 2) or 3) to obtain peptide fragments, followed by ligation of the fragments to obtain the target peptide.

Preferably, the target peptide is prepared using Fmoc solid phase synthesis.

The side chain sulfydryl of two cysteine residues in the glucagon-like peptide-1 analogue is oxidized to form an intramolecular disulfide bond. The disulfide bond formation method can be performed by conventional techniques known in the art, including cleaving the polypeptide from the resin, diluting to a concentration of less than 1mmol/L, and subjecting to air oxidation, glutathione, K₃Fe(CN)₆、I₂And DMSO oxidation method, etc. to finally form intramolecular disulfide bonds.

In the embodiment of the invention, a mouse glucose tolerance test is adopted, and the liraglutide is taken as a positive control drug to evaluate the hypoglycemic activity and long-acting property of the glucagon-like peptide-1 analogue. The result shows that the glucagon-like peptide-1 analog provided by the invention has obvious hypoglycemic effect and still shows activity after 96 hours after administration, which indicates that the designed sequence polypeptide has better metabolic stability, the half-life period in vivo is obviously prolonged, the problem of short half-life period of natural GLP-1 is overcome, the clinical application compliance can be greatly improved, and the glucagon-like peptide-1 analog has potential application value.

Drawings

Embodiments of the present invention are described below with reference to the drawings, in which

FIG. 1 is a hypoglycemic test of glucagon-like peptide-1 analog of example 2;

FIG. 2 is a hypoglycemic test of glucagon-like peptide-1 analog of example 3;

figure 3 is a hypoglycemic assay of the glucagon-like peptide-1 analog of example 4.

Detailed Description

The present invention will be further described with reference to the following examples. The examples are merely illustrative of the invention and do not limit the scope of the invention in any way.

Example 1

A. Preparation of glucagon-like peptide-1 analogues

1) Synthesizing: using Fmoc strategy, a model CS 336 polypeptide synthesizer (CS Bio) was used according to

The synthesis is carried out step by step according to the following steps:

a) coupling a resin solid phase carrier and Fmoc protected glycine in the presence of an activator system to obtain Fmoc-Gly-resin; wherein, amino resin such as Rink Amide AM, Rink Amide and Rink MBHA is adopted for synthesizing the C-terminal amidated polypeptide.

b) Connecting amino acids according to the sequence of peptide sequence amino acids by a solid phase synthesis method to obtain a peptide-resin conjugate with protected N-terminal and side chain; the amino acid with side chain adopts the following protective measures: tryptophan with Boc, glutamic acid with OtBu, lysine with Boc, glutamine with Trt, tyrosine with tBu, serine with Trt or tBu, aspartic acid with OtBu, threonine with tBu, cysteine with Trt, histidine with Trt or Boc.

c) Cracking, and simultaneously removing a protecting group and resin to obtain a crude glucagon-like peptide-1 analogue; the N-terminal acetylation sequence is subjected to N-terminal acetylation by using an acylating agent, and then the operations of removing a protecting group and resin are performed.

2) Purifying by dissolving the crude glucagon-like peptide-1 analog in water or 10-15% acetonitrile (10-50mg/ml), adding 50-100mM dithiothreitol DTT or β -mercaptoethanol for denaturation, separating and purifying by preparative HPLC, C18 chromatographic column and acetonitrile-water-trifluoroacetic acid system, concentrating, and lyophilizing to obtain pure polypeptide with free sulfhydryl.

B. Intramolecular disulfide bond formation

Dissolving the purified glucagon-like peptide-1 analog in deionized water at a proper concentration (less than or equal to 0.5mg/ml), oxidizing by ammonium bicarbonate method, DMSO method or introducing oxygen at 4 deg.C, removing salt and oxidation medium by gel chromatography, and purifying by HPLC to obtain pure glucagon-like peptide-1 analog.

The glucagon-like peptide-1 analogue represented by SEQ NOs 1-16 is prepared by the method.

Example 2

Evaluation of hypoglycemic Effect of glucagon-like peptide-1 analog represented by SEQ NOs 1-4

The hypoglycemic effect of the glucagon-like peptide-1 analogue is evaluated by adopting a normal mouse glucose tolerance test. The method comprises the following steps: normal mice (purchased from shanghai laboratory animal center in chinese academy of sciences) were randomly divided into 6 groups (model, positive control, test group), each of 6 mice; weighing pure product (more than or equal to 98%), preparing into test solution of 0.1mg/ml with physiological saline, injecting 200 μ l per test group subcutaneously, measuring

sugar tolerance

4, 24, 48, 72, 96hr after administration, and administering liraglutide (20 μ g/body) to positive control group; blank control was injected with 200. mu.l of physiological saline. Sugar tolerance test: glucose was administered orally at 2g/kg, and blood glucose values at 15, 30, and 60min were measured to calculate blood glucose value AUC (mg/dl.min). The results are shown in FIG. 1.

Test results show that the test sample shows the same blood sugar reduction effect as the positive control drug in 4hr after administration, the positive control drug is ineffective after 24hr, but the test drug still has effect in 96hr after administration, which indicates that the half-life period in vivo is obviously prolonged.

Example 3

Evaluation of hypoglycemic Effect of glucagon-like peptide-1 analogs represented by SEQ ID NOS 5, 7, 8 and 9

The evaluation method was the same as in example 2, and the results are shown in FIG. 2.

The results show that the tested sample shows a remarkable effect of reducing the blood sugar of the mice with the sugar load at the time of administration for 4hr, and the intensity is equivalent to that of the positive control drug. However, the hypoglycemic effect of the positive control drug disappears after 24hr, and the drug effect is still shown by the tested sample 96hr after the drug, wherein the hypoglycemic effect of the glucagon-like peptide-1 analogues represented by SEQ ID NO7 and 9 is particularly remarkable.

Example 4

Evaluation of hypoglycemic Effect of glucagon-like peptide-1 analogs represented by SEQ ID NO 11, 12, 14, 15

The evaluation method was the same as in example 2, and the results are shown in FIG. 3.

The results show that the tested sample shows a remarkable effect of reducing the blood sugar of the mice with the sugar load at the time of administration for 4hr, and the intensity is equivalent to that of the positive control drug. However, the hypoglycemic effect of the positive control drug disappears after 24 hours, and the drug effect is still shown by the tested sample 96 hours after the drug, wherein the hypoglycemic effect of the glucagon-like peptide-1 analogues represented by SEQ ID NO12 and SEQ ID NO 15 is particularly obvious.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. A long-acting glucagon-like peptide-1 analog, wherein said polypeptide has a structure represented by formula i:

Z₁-HX₈EX₁₀T FTSDV SSYLE X₂₂QAAK EFIX₃₀W LVKX₃₅RG-Z₂

general formula I

Wherein Z is₁is-H or CH₃CO-；

X₈Is Ala, Gly, Cys, Ser or D-Ala;

X₁₀gly, Ser or Cys;

X₂₂is Gly, Glu, Cys or Ser;

X₃₀cys or Ala;

X₃₅is Gly, Cys or Aib;

Z₂is-H, -NH₂or-CG;

characterized in that when Z is₂is-H or-NH₂When, X₈、X₁₀、X₂₂、X₃₀、X₃₅Any two residues are cysteine; when Z is₂When it is-CG, X₈、X₁₀、X₂₂、X₃₀、X₃₅Any one residue is cysteine; the two cysteines form an intramolecular disulfide bond.

2. The long-acting glucagon-like peptide-1 analog of claim 1, wherein the analog is selected from the group consisting of the structures represented by seq id NOs 1-16.

3. A pharmaceutical composition, characterized in that it comprises at least one glucagon-like peptide-1 analog of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.

5. Use of a glucagon-like peptide-1 analog of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 3 or 4, for the manufacture of a medicament for the treatment and/or prevention of diabetes, obesity, alzheimer's disease.