CN107236034B - Glucagon-like peptide-1 analogue, preparation method and application thereof - Google Patents
Glucagon-like peptide-1 analogue, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a glucagon-like peptide-1 analogue monomer peptide, which has a general formula 1: z1HAX1GTFTSDVSSYLE X2QAAKEFICWLVKGRX3(ii) a Wherein Z is1Is hydrogen, acetyl or trifluoroacetyl; x1Met, Leu, Pro, Phe, or Tyr; x2Is Gly, Glu or Aib (2-aminoisobutyric acid). The invention also provides a dimer formed by the monomer peptide, a preparation method of the dimer and application of the dimer in preparing medicines. The dimer of the GLP-1 analogue monomer provided by the invention has a remarkable hypoglycemic effect, the half-life period in vivo can reach more than 12-72 hours, the problem of short half-life period of natural GLP-1 is solved, and the compliance of clinical application can be greatly improved.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a glucagon-like peptide-1 (GLP-1) analogue with a long-acting effect and a dimer thereof. The invention also relates to application of the GLP-1 analogue dimer in preparing a medicament for treating and/or preventing diabetes, obesity and Alzheimer's disease.
Background
Glucagon-like peptide-1 (GLP-1) is an enterogenic hormone that is synthesized primarily in L cells of the terminal jejunum, ileum, and colon, and is released into the circulation in a meal response. GLP-1 (7-36, 7-37) is the major active form of GLP-1 in the systemic circulation, controlling blood glucose by complex mechanisms including secretion of insulin and glucagon, gastric emptying and regulation of peripheral insulin. GLP-1 (7-36, 7-37) has glucose-dependent hypoglycemic effect, can prevent hypoglycemia, inhibit apoptosis of pancreatic islet beta-cells, promote proliferation of pancreatic islet beta-cells, and reverse disease development. However, the plasma half-life of natural GLP-1 is only 1-2 minutes, and the metabolic instability limits the application of the natural GLP-1 as a medicine. Research shows that in vivo dipeptidyl kininase (DPPIV) specifically recognizes and degrades the N-terminal His-Ala segment of the receptor binding active site in the GLP-1 structure to quickly inactivate the receptor binding active site, and other proteolytic enzymes such as endopeptidase and the like are also involved in the process of renal filtration and clearance.
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. Chinese patent applications with publication numbers CN00806548.9, CN99814187.9, application number 200410017667.9, etc. describe the following technologies: 1) structural modification aiming at enzyme degradation key sites; 2) fatty acyl group is introduced into parent peptide chain structure to improve the binding force with plasma protein so as to avoid the polypeptide from being rapidly cleared in vivo (as described in Chinese patents such as CN201210513145.2, CN200810124641.2 and CN 20118000352.1); 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 parent peptide chain of GLP-1 (7-36, 7-37) are liraglutide, which is obtained by substituting arginine for lysine at position 34 in the molecular structure of native GLP-1 and adding a glutamic acid-mediated 16-carbon palmitic fatty acid side chain to lysine at position 26. While the liraglutide greatly prolongs the in vivo half-life of GLP-1, the liraglutide still needs to be injected once a day, and the medication compliance still needs to be improved.
The present inventors describe dimers in which single sites 10, 15, 22, 23, 30, 33 in the GLP-1 (7-37) sequence are each replaced by cysteine in a previously granted Chinese patent No. CN 201110076380.3.
Disclosure of Invention
In subsequent optimization studies, the inventors of the present application found that the substitution, modification and modification of the site in the sequence that is susceptible to enzymatic degradation, which contributes to the formation of secondary structure, can further improve the activity of the dimer and prolong the half-life in vivo. Therefore, the inventor provides a monomeric peptide of a glucagon-like peptide-1 analogue and a dimer thereof through intensive research and repeated experiments.
The invention provides a glucagon-like peptide-1 analogue monomer peptide, which has a general formula 1: z1HAX1GTFTSDVSSYLEX2QAAKEFICWLVKGRX3；
Wherein Z is1Is hydrogen, acetyl or trifluoroacetyl;
X1met, Leu, Pro, Phe, or Tyr;
X2is Gly, Glu or Aib (2-aminoisobutyric acid).
X3To NH C-terminal of an arginine residue2Or Gly-NH2。
Preferably, the amino acid sequence of the monomeric peptide is:
SEQ ID NO 1：HAMGTFTSDVSSYLEGQAAKEFICWLVKGR-NH2；
SEQ ID NO 2：HALGTFTSDVSSYLEGQAAKEFICWLVKGR-NH2；
SEQ ID NO 3：Ac-HAMGTFTSDVSSYLEEQAAKEFICWLVKGRNH2；
SEQ ID NO 4：Ac-HAFGTFTSDVSSYLEGQAAKEFICWLVKGRG -NH2；
SEQ ID NO 5：HAYGTFTSDVSSYLEEQAAKEFICWLVKGRG-NH2；
SEQ ID NO 6：HAMGTFTSDVSSYLEAibQAAKEFICWLVKGRG -NH2；
SEQ ID NO 7：Ac-HALGTFTSDVSSYLEEQAAKEFICWLVKGRG -NH2；
SEQ ID NO 8：HAPGTFTSDVSSYLEAibQAAKEFICWLVKGRG -NH2；
SEQ ID NO 9：HAFGTFTSDVSSYLEAibQAAKEFICWLVKGR-NH2；
SEQ ID NO 10：CF3CO-HAYGTFTSDVSSYLEGQAAKEFICWLVKG RG-NH2；
SEQ ID NO 11：HAPGTFTSDVSSYLEGQAAKEFICWLVKGR-NH2；
SEQ ID NO 12：Ac-HALGTFTSDVSSYLEEQAAKEFICWLVKG R-NH2；
SEQ ID NO 13：CF3CO-HAPGTFTSDVSSYLEEQAAKEFICWLVKGR G-NH2；
SEQ ID NO 14：CF3CO-HAFGTFTSDVSSYLEEQAAKEFICWLVKGR -NH2；
SEQ ID NO 15：Ac-HAYGTFTSDVSSYLEGQAAKEFICWLVKGRG -NH2；
SEQ ID NO 16：CF3CO-HAFGTFTSDVSSYLEAibQAAKEFICWLVK GR-NH2；
SEQ ID NO 17：HAFGTFTSDVSSYLEEQAAKEFICWLVKGRG-NH2；
SEQ ID NO 18：HALGTFTSDVSSYLEAibQAAKEFICWLVKGR-NH2；
SEQ ID NO 19：CF3CO-HALGTFTSDVSSYLEAibQAAKEFICWLVKG RG-NH2；
SEQ ID NO 20：CF3CO-HAMGTFTSDVSSYLEGQAAKEFICWLVK GR-NH2；
SEQ ID NO 21：HAYGTFTSDVSSYLEGQAAKEFICWLVKGR-NH2；
SEQ ID NO 22：CF3CO-HAPGTFTSDVSSYLEGQAAKEFICWLVKGR -NH2；
SEQ ID NO 23：Ac-HAYGTFTSDVSSYLEGQAAKEFICWLVKGR -NH2；
SEQ ID NO 24：Ac-HAPGTFTSDVSSYLEEQAAKEFICWLVKGRG -NH2。
the invention further provides a dimer of the glucagon-like peptide-1 analogue monomer peptide, wherein the dimer is formed by connecting one or two of the monomer peptides through an intermolecular disulfide bond; preferably, the dimer is formed by linking the above monomer peptide with another monomer peptide having the same sequence through an intermolecular disulfide bond.
The invention also provides a method for preparing the dimer, which comprises the following steps:
1) synthesizing a crude product of GLP-1 analogue monomer peptide by an Fmoc method;
2) purifying, concentrating and freeze-drying the crude GLP-1 analogue monomer peptide obtained in the step 1) to obtain freeze-dried powder, preferably, the purification is realized by dissolving the crude GLP-1 analogue monomer peptide obtained in the step 1) in water or 10 ~ 15% acetonitrile to obtain a solution with the concentration of 10 ~ 15mg/ml and then separating and purifying by adopting a preparative HPLC method, a C18 chromatographic column and an acetonitrile-water-trifluoroacetic acid system;
3) dissolving the freeze-dried powder obtained in the step 2) in deionized water, forming GLP-1 analogue dimer by an ammonium bicarbonate method or a DMSO method, purifying to obtain a pure GLP-1 analogue dimer product, and preferably, dissolving the monomer peptide in the deionized water to obtain a solution with the concentration of 1.5 ~ 2 mmol/L.
In one embodiment according to the present invention, when the N-terminal acylation of the monomeric peptide is carried out by a method comprising the steps of:
removing an N-terminal Fmoc protecting group from a peptide-resin conjugate obtained in the monomer peptide synthesis process, then suspending the peptide-resin conjugate from which the N-terminal Fmoc protecting group is removed in pyridine, adding acetic anhydride or trifluoroacetic anhydride in a proper molar ratio, uniformly mixing, and placing to obtain an N-terminal histidine acylated peptide-resin conjugate; and cracking the N-terminal histidine acylated peptide-resin conjugate to obtain a crude N-terminal histidine acylated monomer peptide product.
The present invention further provides a pharmaceutical composition comprising the glucagon-like peptide-1 analog dimer or salt thereof described above.
According to one embodiment of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
According to one embodiment of the invention, the adjuvant is selected from one or more of water-soluble fillers, pH regulators, stabilizers, water for injection or osmotic pressure regulators; 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, etc.; the stabilizer includes but is not limited to EDTA-Na2Sodium 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, cyclodextrin, sodium lauryl sulfate or tris (hydroxymethyl) aminomethane and the like. The tonicity modifier includes, but is not limited to, sodium chloride or potassium chloride.
The invention also provides application of the glucagon-like peptide-1 analog dimer or salt thereof in preparing medicaments for treating and/or preventing diabetes, obesity and Alzheimer's disease.
The invention further provides application of the pharmaceutical composition in preparing medicines for treating and/or preventing diabetes, obesity and Alzheimer's disease.
The invention has the following beneficial effects:
according to the embodiment of the invention, a mouse glucose tolerance test is adopted, and liraglutide is taken as a positive control drug to evaluate the hypoglycemic activity and long-acting property of the GLP-1 analogue dimer. The result shows that the GLP-1 analog dimer provided by the invention has a remarkable hypoglycemic effect, the half-life period in vivo can reach more than 12-72 hours, the problem of short half-life period of natural GLP-1 is solved, the clinical application compliance can be greatly improved, and the potential application value is realized. Furthermore, the GLP-1 analogue dimer provided by the invention is highly homologous with endogenous GLP-1, so that the safety risk can be avoided.
Drawings
FIG. 1 is a bar graph of a hypoglycemic assay of the dimer GLP-1 analog of example 5;
FIG. 2 is a bar graph of the hypoglycemic experiment of the dimer GLP-1 analog of example 6.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.
Example 1Preparation of GLP-1 analogue monomer and dimer
A. Preparation of GLP-1 analogue monomer:
1) synthesizing: the Fmoc method is adopted, and the synthesis method is implemented step by step according to the following steps:
a) coupling amino resin solid phase carrier and Fmoc protected arginine in the presence of an activator system to obtain Fmoc-Arg-resin;
b) connecting amino acids according to the sequence of peptide sequence amino acids by a solid phase synthesis method to obtain peptide-resin conjugates with N-terminal Fmoc-protection and side chain protection; the amino acid with side chain adopts the following protective measures: tryptophan was protected with tert-butyloxycarbonyl (Boc), glutamic acid with tert-butyl (OtBu), lysine with tert-butyloxycarbonyl (Boc), glutamine with trityl (Trt), tyrosine with tert-butyl (tBu), serine with trityl (Trt) or tert-butyl (tBu), aspartic acid with tert-butyl (OtBu), threonine with tert-butyl (tBu), histidine with trityl (Trt) or tert-butyloxycarbonyl (Boc).
c) Cracking, and removing the protecting group and the resin at the same time to obtain a crude product of the GLP-1 analogue monomer;
2) and (3) purification: dissolving the crude product in the step C) in water or 10-15% acetonitrile (10-50mg/ml), separating and purifying by using a preparative HPLC method, a C18 chromatographic column and an acetonitrile-water-trifluoroacetic acid system, concentrating, and freeze-drying to obtain the GLP-1 analogue monomer.
B. Preparation of GLP-1 analogue dimer:
dissolving GLP-1 analogue monomer in deionized water at a proper concentration (1.5-2mmol/L), forming a dimer according to an ammonium bicarbonate method or a DMSO method, and purifying to obtain a pure GLP-1 analogue dimer.
Dimers formed from the following sequence monomers were obtained by the above method:
GLP-1 analogue dimer 1-1 formed by SEQ ID NO1 and SEQ ID NO1,
GLP-1 analogue dimer 2-2 formed by SEQ ID NO2 and SEQ ID NO2,
GLP-1 analog dimer 9-9 formed by SEQ ID NO 9 and SEQ ID NO 9,
GLP-1 analog dimer 11-11 formed by SEQ ID NO 11 and SEQ ID NO 11,
GLP-1 analog dimer 18-18 formed by SEQ ID NO 18 and SEQ ID NO 18,
GLP-1 analog dimers 21-21 formed by SEQ ID NO21 and SEQ ID NO 21.
Example 2Preparation of N-terminally acylated GLP-1 analog dimers
1) Peptide-resin conjugates with Fmoc-protection at the N-terminus and side chain protection were synthesized according to the monomer synthesis method of example 1;
2) removing the Fmoc protecting group at the N-terminal by a conventional method, suspending the resin-peptide conjugate in a proper amount of pyridine, adding acetic anhydride or trifluoroacetic anhydride with a proper molar ratio, uniformly mixing, and standing to obtain an N-terminal histidine acylated peptide-resin conjugate;
3) cracking according to the method of example 1 to obtain crude peptide, purifying and freeze-drying to obtain monomer target peptide;
4) the dimer was prepared as in example 1.
GLP-1 analogue dimers formed by the following sequence monomers are prepared according to the method:
GLP-1 analog dimer 3-3 formed by SEQ ID NO3 and SEQ ID NO3,
GLP-1 analog dimer 12-12 formed by SEQ ID NO12 and SEQ ID NO12,
GLP-1 analog dimer 14-14 formed by SEQ ID NO 14 and SEQ ID NO 14,
GLP-1 analog dimer 16-16 formed by SEQ ID NO16 and SEQ ID NO16,
GLP-1 analog dimer formed from SEQ ID NO20 and SEQ ID NO20 20-20,
GLP-1 analog dimer 22-22 formed by SEQ ID NO 22 and SEQ ID NO 22,
GLP-1 analog dimer 23-23 formed by SEQ ID NO 15 and SEQ ID NO 15.
Example 3Preparation of GLP-1 analogue monomer and dimer
A. Preparation of GLP-1 analogue monomer:
1) synthesizing: the Fmoc method is adopted, and the synthesis method is implemented step by step according to the following steps:
a) coupling amino resin solid phase carrier and Fmoc protected glycine in the presence of an activator system to obtain Fmoc-Gly-resin;
b) connecting amino acids according to the sequence of peptide sequence amino acids by a solid phase synthesis method to obtain peptide-resin conjugates with N-terminal Fmoc-protection and side chain protection; the amino acid with side chain adopts the following protective measures: t-butyloxycarbonyl (Boc) for tryptophan, t-butyl (OtBu) for glutamic acid, t-butyloxycarbonyl (Boc) for lysine, trityl (Trt) for glutamine, t-butyl (tBu) for tyrosine, trityl (Trt) or t-butyl (tBu) for serine, t-butyl (OtBu) for aspartic acid, t-butyl (tBu) for threonine, trityl (Trt) or t-butyloxycarbonyl (Boc) for histidine
c) Cracking, and removing the protecting group and the resin at the same time to obtain a crude product of the GLP-1 analogue monomer;
2) and (3) purification: dissolving the crude product in the step C) in water or 10-15% acetonitrile (10-50mg/ml), separating and purifying by using a preparative HPLC method, a C18 chromatographic column and an acetonitrile-water-trifluoroacetic acid system, concentrating, and freeze-drying to obtain the GLP-1 analogue monomer.
B. Preparation of GLP-1 analogue dimer:
dissolving GLP-1 analogue monomer in deionized water at a proper concentration (1.5-2mmol/L), forming a dimer according to an ammonium bicarbonate method or a DMSO method, and purifying to obtain a pure GLP-1 analogue dimer.
Dimers formed from the following sequence monomers were obtained by the above method:
GLP-1 analog dimer 5-5 formed by SEQ ID NO 5 and SEQ ID NO 5,
GLP-1 analog dimer 6-6 formed by SEQ ID NO 6 and SEQ ID NO 6,
GLP-1 analog dimer 8-8 formed by SEQ ID NO8 and SEQ ID NO8,
GLP-1 analog dimer 17-17 formed by SEQ ID NO 17 and SEQ ID NO 17.
Example 4Preparation of N-terminally acylated GLP-1 analog dimers
1) Peptide-resin conjugates with Fmoc-protection at the N-terminus and side chain protection were synthesized according to the monomer synthesis method of example 3;
2) removing the Fmoc protecting group at the N-terminal by a conventional method, suspending the resin-peptide conjugate in a proper amount of pyridine, adding acetic anhydride or trifluoroacetic anhydride with a proper molar ratio, uniformly mixing, and standing to obtain an N-terminal histidine acylated peptide-resin conjugate;
3) cracking according to the method of the embodiment 3 to obtain crude peptide, purifying and freeze-drying to obtain monomer target peptide;
4) the dimer was prepared as in example 3.
GLP-1 analogue dimers formed by the following sequence monomers are prepared according to the method:
GLP-1 analogue dimer 4-4 formed by SEQ ID NO 4 and SEQ ID NO 4,
GLP-1 analog dimer 7-7 formed by SEQ ID NO7 and SEQ ID NO7,
GLP-1 analog dimer 10-10 formed by SEQ ID NO 10 and SEQ ID NO 10,
GLP-1 analog dimer 13-13 formed by SEQ ID NO 13 and SEQ ID NO 13,
GLP-1 analog dimer 15-15 formed by SEQ ID NO 15 and SEQ ID NO 15,
GLP-1 analog dimer 19-19 formed by SEQ ID NO 19 and SEQ ID NO 19,
GLP-1 analog dimer 24-24 formed by SEQ ID NO 24 and SEQ ID NO 24.
Example 5Evaluation of hypoglycemic Effect of GLP-1 analog dimer 5-5, 8-8, 11-11, 18-18
The hypoglycemic effect of the GLP-1 analogue dimer is evaluated by adopting a normal mouse glucose tolerance test. The method comprises the following steps: 30 normal mice (purchased from Shanghai laboratory animal center of Chinese academy of sciences) were randomly divided into 6 groups (blank control group, positive control group, test group), and 5 mice in each group; weighing a proper amount of pure GLP-1 analog dimer (more than or equal to 98 percent) and dissolving the pure GLP-1 analog dimer in normal saline to prepare a sample solution of 0.1 mg/ml. Mice in the test group were injected subcutaneously with 200. mu.l of each sample solution; positive control mice were injected subcutaneously with 20 μ g of liraglutide per mouse; mice in the blank control group were injected subcutaneously with 200. mu.l of physiological saline each. Glucose tolerance was measured 4, 24, 48, 72, 96 hours after injection, respectively. Sugar tolerance test: glucose was administered orally at 2g/kg, and blood glucose levels at 15, 30, and 60min were measured to calculate blood glucose level AUC (mg/dl.min). The results are shown in FIG. 1.
Test results show that the tested sample shows the same blood sugar reducing effect as the positive control drug in 4 hours after administration, the positive control drug is ineffective after 24 hours, but the tested drug is still effective after 96 hours after administration, which indicates that the half-life period in vivo of the tested drug is obviously prolonged.
Example 6
The hypoglycemic effects of GLP-1 analog dimers 3-3, 9-9, 17-17, 21-21 were evaluated in the same manner as in example 5. The positive drug is polypeptide (HAEGTFTSDVSSYLEGCAAKEFIAW) dimer (patent dimer 5/5 in figure 2) in chinese patent CN 201110076380.3, and the result is shown in figure 2.
Test results show that the tested sample shows the same blood sugar reducing effect as the positive control drug in 4hr after administration, the positive control drug is ineffective after 48 hours, the tested drug is still effective after 96 hours after administration, and the dimer 17-17 and dimer 21-21 have particularly obvious effects.
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 (16)
1. A glucagon-like peptide-1 analog monomeric peptide having the general formula 1: z1HAX1GTFTSDVSSYLEX2QAAKEFICWLVKGRX3；
Wherein Z is1Is hydrogen, acetyl or trifluoroacetyl;
X1met, Leu, Pro, Phe, or Tyr;
X2is Gly, Glu or Aib (2-aminoisobutyric acid);
X3to NH C-terminal of an arginine residue2Or Gly-NH2。
2. The monomeric peptide of claim 1, wherein the amino acid sequence of the monomeric peptide is: SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24.
3. A dimer of a glucagon-like peptide-1 analog monomer peptide formed by linking the monomer peptide of claim 1 or 2 to another monomer peptide having the same sequence via an intermolecular disulfide bond.
4. A method of preparing the dimer of claim 3, comprising:
1) synthesizing a crude product of GLP-1 analogue monomer peptide by an Fmoc method;
2) purifying, concentrating and freeze-drying the crude GLP-1 analogue monomer peptide product obtained in the step 1) to obtain freeze-dried powder;
3) dissolving the freeze-dried powder obtained in the step 2) in deionized water, forming GLP-1 analog dimer by an ammonium bicarbonate method or a DMSO method, and purifying to obtain pure GLP-1 analog dimer.
5. The method of claim 4, wherein in the step 2), the purification is achieved by dissolving the crude monomeric peptide obtained in the step 1) in water or 10 ~ 15% acetonitrile to obtain a solution with a concentration of 10 ~ 15mg/ml, and then separating and purifying the solution by using a preparative HPLC method, a C18 chromatographic column and an acetonitrile-water-trifluoroacetic acid system.
6. The method of claim 4, wherein in step 3), the concentration of the monomeric peptide in the solution of the monomeric peptide dissolved in deionized water is 1.5 ~ 2 mmol/L.
7. The method of claim 4, when the N-terminal acylation of the monomeric peptide is achieved by a method comprising the steps of:
removing an N-terminal Fmoc protecting group from a peptide-resin conjugate obtained in the monomer peptide synthesis process, then suspending the peptide-resin conjugate from which the N-terminal Fmoc protecting group is removed in pyridine, adding acetic anhydride or trifluoroacetic anhydride in a proper molar ratio, uniformly mixing, and placing to obtain an N-terminal histidine acylated peptide-resin conjugate; and cracking the N-terminal histidine acylated peptide-resin conjugate to obtain a crude N-terminal histidine acylated monomer peptide product.
8. A pharmaceutical composition comprising the glucagon-like peptide-1 analog dimer of claim 3.
9. The pharmaceutical composition of claim 8, further comprising one or more pharmaceutically acceptable excipients.
10. The pharmaceutical composition of claim 9, wherein the excipient is selected from one or more of a water-soluble filler, a pH adjuster, a stabilizer, water for injection, or an osmotic pressure adjuster.
11. The pharmaceutical composition of claim 10, wherein the water soluble filler is selected from one or more of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose and galactose; the pH regulator is one or more selected from citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate and ammonium bicarbonate.
12. The pharmaceutical composition of claim 10, wherein the stabilizer is selected from the group consisting of EDTA-Na2One or more of sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxy cellulose or derivatives thereof.
13. The pharmaceutical composition of claim 12, wherein the carboxy/hydroxycellulose or derivative thereof is HPC, HPC-SL, HPC-L, HPMC, cyclodextrin, sodium lauryl sulfate, or tris.
14. The pharmaceutical composition of claim 10, wherein the tonicity modifier is sodium chloride or potassium chloride.
15. Use of the glucagon-like peptide-1 analog dimer of claim 3 in the manufacture of a medicament for the treatment and/or prevention of diabetes, obesity.
16. Use of a pharmaceutical composition comprising a glucagon-like peptide-1 analog dimer according to any of claims 8-14 for the manufacture of a medicament for the treatment and/or prevention of diabetes, obesity.
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102718858A (en) * | 2011-03-29 | 2012-10-10 | 天津药物研究院 | Glucagon-like peptide-1 (GLP-1) analogue monomer and dimer, preparation method therefor and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Glucagon-like peptide-1 analogues: An overview;Vishal Gupta;《Indian Journal of Endocrinology and Metabolism》;20130630;第17卷(第3期);第413-421页 * |
| 糖尿病治疗新药—长效化胰高血糖素样肽-1(GLP-1)及其类似物的研究进展;沙向阳等;《海峡药学》;20111231;第23卷(第10期);第1-6页 * |
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