CN107236034A - A kind of glucagon-like peptide-1 analogs and its production and use - Google Patents

A kind of glucagon-like peptide-1 analogs and its production and use Download PDF

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CN107236034A
CN107236034A CN201610193152.7A CN201610193152A CN107236034A CN 107236034 A CN107236034 A CN 107236034A CN 201610193152 A CN201610193152 A CN 201610193152A CN 107236034 A CN107236034 A CN 107236034A
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peptide
glp
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CN107236034B (en
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赵娜夏
韩英梅
王玉丽
夏广萍
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Tianjin Institute of Pharmaceutical Research Co Ltd
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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
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    • A61K38/00—Medicinal preparations containing peptides
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    • 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

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Abstract

The invention provides a kind of glucagon-like peptide 1 analog monomeric peptide, the monomeric peptide has formula 1：Z1HAX1GTFTSDVSSYLE X2QAAKEFICWLVKGRX3；Wherein, Z1For hydrogen, acetyl group or trifluoroacetyl group；X1For Met, Leu, Pro, Phe or Tyr；X2For Gly, Glu or Aib (2 aminoisobutyric acid).Present invention also offers the preparation method and the purposes in medicine is prepared of the dimer formed by the monomeric peptide, and the dimer.The dimer for the analog monomers of GLP 1 that the present invention is provided has significant hypoglycemic effect, and Half-life in vivo can reach that the problem of overcoming natural 1 half-life shorts of GLP can greatly improve clinical practice compliance more than 12 72 hours.

Description

A kind of glucagon-like peptide-1 analogs and its production and use
Technical field
The present invention relates to pharmaceutical technology field, and in particular to a kind of glucagon with long-acting (GLP-1) analog of peptide -1 and its dimer.The invention further relates to the GLP-1 analogs dimer in system It is ready for use on the application in treatment and/or prevention diabetes, the medicine of fat and alzheimer disease.
Background technology
Glucagon-like-peptide-1 (GLP-1) is a kind of Entero hormone, mainly end jejunum, return Synthesized in the L cells of intestines and colon, circulation is discharged into reaction of having meal.GLP-1 (7-36,7-37) is GLP-1 chief active form in body circulation, by complicated mechanism control blood glucose, including insulin and The secretion of hyperglycemic factor, the emptying of stomach and the regulation of periphery insulin.GLP-1(7-36,7-37) Hypoglycemic effect is glucose dependency, can avoid hypoglycemia, and with suppress pancreas islet β- The apoptosis of cell, promotes the effect of the hyperplasia of pancreaticβ-cell, and disease development can be reversed.But natural GLP-1 Plasma half-life be only 1-2 minutes, metabolism instability limit it as the application of medicine.Research Show, acceptor knot in internal dipeptides kininase (DPPIV) specific recognition and GLP-1 structures of degrading Close active site N-terminal His-Ala fragments and make its rapid deactivation, while for example interior peptide of other proteolytic enzymes Enzyme etc. also assists in kidney filtration reset procedure.
Metabolic stability is improved, extends plasma half-life, so that it is to be based on to improve clinical application compliance The technical goal of GLP-1 course of drug development.Publication No. CN00806548.9, Record following in the Chinese patent application of CN99814187.9, Application No. 200410017667.9 etc. Some technologies：1) for the structure of modification of enzyme degraded critical sites；2) fat is introduced in parent peptide chain structure Fat acyl group group, improves with plasma protein adhesion to avoid polypeptide from quickly removing (such as Patent No. in vivo The Chinese patents such as CN201210513145.2, CN200810124641.2, CN20118000352.1 are remembered Carry)；3) GLP-1 analogs protein fusion technology；4) PEG modifications etc..Although carrying out for many years Many trials, but up to the present the parent peptide chain based on GLP-1 (7-36,7-37) is developed The medicine of listing only has Liraglutide, its by natural GLP-1 molecular structures by 34 lysines Replaced with arginine, and in adding a 16 carbon palms by glutamate-induced on the 26th lysine Fatty acid side chain.Although and Liraglutide significantly extends GLP-1 Half-life in vivo, stilling need daily Once, compliance is still to be improved for injection.
The present inventor is being authorized described in Patent No. CN 201110076380.3 Chinese patent before Single locus 10,15,22,23,30,33 in GLP-1 (7-37) sequence is respectively by half Guang Propylhomoserin replaces the dimer to be formed.
The content of the invention
Enzyme degraded sensitivity site during present inventor has found to sequence in follow-up optimizing research Replace, modify and contribute to the transformation to form secondary structure can further improve dimer activity and Extend Half-life in vivo.Therefore, the present inventor is by further investigation, there is provided a kind of pancreas with repetition test The analog monomeric peptide of blood glucose element sample peptide -1 and its dimer.
The invention provides a kind of glucagon-like peptide-1 analogs monomeric peptide, the monomeric peptide has Formula 1：Z1HAX1GTFTSDVSSYLEX2QAAKEFICWLVKGRX3；
Wherein, Z1For hydrogen, acetyl group or trifluoroacetyl group；
X1For Met, Leu, Pro, Phe or Tyr；
X2For Gly, Glu or Aib (2- aminoisobutyric acids).
X3To be connected to the NH of arginine residues C-terminal2Or 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。
Invention further provides a kind of dimer of glucagon-like peptide-1 analogs monomeric peptide, The dimer is connected by intermolecular disulfide bond by one or both of above-mentioned monomeric peptide and formed；It is excellent Selection of land, the dimer be by above-mentioned monomeric peptide with another have mutually homotactic monomeric peptide by divide Disulfide bond is formed between son.
Present invention also offers a kind of method for preparing above-mentioned dimer, methods described includes：
1) GLP-1 analog monomeric peptide crude products are synthesized by Fmoc methods；
2) by step 1) in obtain GLP-1 analog monomeric peptides purifying crude, concentration and lyophilized, Obtain freeze-dried powder；Preferably, the purifying is by step 1) obtained monomeric peptide dissolving crude product is in water Or 10~15% obtain in acetonitrile after the solution that concentration is 10~15mg/ml, using preparation HPLC method, C18 chromatographic columns, acetonitrile-water-trifluoroacetic acid system isolate and purify realization；
3) by step 2) in obtained freeze-dried powder be dissolved in deionized water, with ammonium bicarbonate method or DMSO methods formation GLP-1 analog dimers, purifying obtains GLP-1 analog dimers pure Product；Preferably, the monomeric peptide is dissolved in the dense of monomeric peptide described in the solution obtained in deionized water Spend for 1.5~2mmol/L.
It is logical when the N- ends of the monomeric peptide are acylated according in one embodiment of the invention Cross what the method comprised the steps was realized：
The peptide obtained in monomeric peptide building-up process-resin conjugate is removed into N-terminal Fmoc protection groups, so The peptide resin conjugate of the removal N-terminal Fmoc protection groups is suspended in pyridine afterwards, it is appropriate to add The acetic anhydride or TFAA of mol ratio, after mixing, placement, obtain the peptide that N- ends histidine is acylated - resin conjugate；N- ends group ammonia is obtained after the peptide that N- ends histidine is acylated-resin conjugate cracking The monomeric peptide crude product of acylating acid.
Invention further provides a kind of pharmaceutical composition, described pharmaceutical composition includes above-mentioned pancreas The analog of glucagon-like peptide -1 dimer or its salt.
According in one embodiment of the invention, described pharmaceutical composition is also comprising one or more medicines Auxiliary material is subjected on.
According in one embodiment of the invention, the auxiliary material is selected from water-soluble filler, pH and adjusted Save the one or more in agent, stabilizer, water for injection or osmotic pressure regulator；The water solubility is filled out Fill agent include but is not limited to mannitol, D-40, sorbierite, polyethylene glycol, glucose, Lactose, galactolipin etc.；The pH adjusting agent includes but is not limited to citric acid, phosphoric acid, lactic acid, wine The organic or inorganic acids such as stone acid, hydrochloric acid, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, carbonic acid Sodium, potassium carbonate, ammonium carbonate, saleratus, sodium acid carbonate, ammonium hydrogen carbonate etc. are physiologically acceptable Inorganic base or salt；The stabilizer includes but is not limited to EDTA-Na2, sodium thiosulfate, burnt sulfurous Sour sodium, sodium sulfite, dipotassium hydrogen phosphate, sodium acid carbonate, sodium carbonate, arginine, lysine, paddy Propylhomoserin, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxyl fiber Element or derivatives thereof, such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, dodecyl Sodium sulphate or trishydroxymethylaminomethane etc..The osmotic pressure regulator include but is not limited to sodium chloride or Potassium chloride.
Prepared present invention also offers above-mentioned glucagon-like peptide-1 analogs dimer or its salt For treating and/or preventing the application in diabetes, obesity, the medicine of alzheimer disease.
Further it is used to treat and/or prevents preparing the invention provides aforementioned pharmaceutical compositions Application in diabetes, obesity, the medicine of alzheimer disease.
The invention has the advantages that：
Tested in embodiment of the present invention using glucose tolerance in mice, using Liraglutide as positive control drug, It has rated hypoglycemic activity and the long-term effect of GLP-1 analog dimers.As a result show that the present invention provides GLP-1 analog dimers have significant hypoglycemic effect, and Half-life in vivo can reach that 12-72 is small When more than, the problem of overcoming natural GLP-1 half-life shorts can greatly improve clinical practice compliance, With potential application value.Further, the present invention is provided GLP-1 analogs dimer and endogenous GLP-1 very high homologies, can avoid security risks.
Brief description of the drawings
Fig. 1 is the column diagram of the hypoglycemic experiment of GLP-1 analog dimers in embodiment 5；
Fig. 2 is the column diagram of the hypoglycemic experiment of GLP-1 analog dimers in embodiment 6.
Embodiment
The present invention is further illustrated with reference to embodiment, it will be appreciated that embodiment is only used for further Illustrate and explain the present invention, be not intended to limit the present invention.
Embodiment 1The preparation of GLP-1 analogs monomer and dimer
The preparation of A.GLP-1 analog monomers：
1) synthesize：Using Fmoc methods, progressively implement synthetic method in accordance with the following steps：
A) it is coupled in the presence of activator systems by amino resins solid phase carrier and the Fmoc arginine protected Obtain Fmoc-Arg- resins；
B) amino acid is connected according to peptide sequence amino acid sequence by solid-phase synthesis, obtains N- ends Fmoc- Protection and peptide-resin conjugate of side chain protected；Band side chain amino acid takes following safeguard measure：Color ammonia Sour tertbutyloxycarbonyl (Boc), glutamic acid the oxygen tert-butyl group (OtBu), lysine tertiary butyloxycarbonyl Base (Boc), glutamine trityl (Trt), the tyrosine tert-butyl group (tBu), serine With trityl (Trt) or the tert-butyl group (tBu), aspartic acid is with the oxygen tert-butyl group (OtBu), ammonia of reviving The sour tert-butyl group (tBu), histidine is protected with trityl (Trt) or tertbutyloxycarbonyl (Boc).
C) crack, while deprotection base and resin, obtain the crude product of GLP-1 analog monomers；
2) purify：By the dissolving crude product in step c) in water or 10-15% acetonitriles (10-50mg/ml), adopt Preparation HPLC method is used, C18 chromatographic columns, acetonitrile-water-trifluoroacetic acid system is isolated and purified, concentrated, It is lyophilized, obtain GLP-1 analog monomers.
The preparation of B.GLP-1 analog dimers：
GLP-1 analogs monomer is dissolved in deionized water with debita spissitudo (1.5-2mmol/L), according to Ammonium bicarbonate method or DMSO methods formation dimer, purifying obtain GLP-1 analog dimer sterlings.
The dimer for obtaining being formed by following sequence monomers by the above method：
The GLP-1 analog dimer 1-1 of SEQ ID NO 1 and SEQ ID NO 1 formation,
The GLP-1 analog dimer 2-2 of SEQ ID NO 2 and SEQ ID NO 2 formation,
The GLP-1 analog dimer 9-9 of SEQ ID NO 9 and SEQ ID NO 9 formation,
The GLP-1 analog dimer 11-11 of SEQ ID NO 11 and SEQ ID NO 11 formation,
The GLP-1 analog dimer 18-18 of SEQ ID NO 18 and SEQ ID NO 18 formation,
The GLP-1 analog dimers 21-21 of SEQ ID NO 21 and SEQ ID NO 21 formation.
Embodiment 2The preparation for the GLP-1 analog dimers that N- ends are acylated
1) peptide for obtaining N- ends Fmoc- protections and side chain protected is synthesized according to the monomer synthesis of embodiment 1 - resin conjugate；
2) conventional method removes N- ends Fmoc protection groups, and resin-peptide conjugate is suspended in appropriate pyridine, The acetic anhydride or TFAA of appropriate mol ratio are added, is mixed, is placed, the histidine acylation of N- ends is obtained Peptide-resin conjugate；
3) crude product peptide is obtained by the cracking of the method for embodiment 1, purified, it is lyophilized to obtain objective monomer peptide；
4) dimer is prepared by the method for embodiment 1.
The GLP-1 analog dimers of following sequence monomers formation are prepared as stated above：
The GLP-1 analog dimer 3-3 of SEQ ID NO 3 and SEQ ID NO 3 formation,
The GLP-1 analog dimer 12-12 of SEQ ID NO 12 and SEQ ID NO 12 formation,
The GLP-1 analog dimer 14-14 of SEQ ID NO 14 and SEQ ID NO 14 formation,
The GLP-1 analog dimer 16-16 of SEQ ID NO 16 and SEQ ID NO 16 formation,
The GLP-1 analog dimer 20-20 of SEQ ID NO 20 and SEQ ID NO 20 formation,
The GLP-1 analog dimer 22-22 of SEQ ID NO 22 and SEQ ID NO 22 formation,
The GLP-1 analog dimers 23-23 of SEQ ID NO 15 and SEQ ID NO 15 formation.
Embodiment 3The preparation of GLP-1 analogs monomer and dimer
The preparation of A.GLP-1 analog monomers：
1) synthesize：Using Fmoc methods, progressively implement synthetic method in accordance with the following steps：
A) it is coupled in the presence of activator systems by amino resins solid phase carrier and the Fmoc glycine protected Obtain Fmoc-Gly- resins；
B) amino acid is connected according to peptide sequence amino acid sequence by solid-phase synthesis, obtains N- ends Fmoc- Protection and peptide-resin conjugate of side chain protected；Band side chain amino acid takes following safeguard measure：Color ammonia Sour tertbutyloxycarbonyl (Boc), glutamic acid the oxygen tert-butyl group (OtBu), lysine tertiary butyloxycarbonyl Base (Boc), glutamine trityl (Trt), the tyrosine tert-butyl group (tBu), serine With trityl (Trt) or the tert-butyl group (tBu), aspartic acid is with the oxygen tert-butyl group (OtBu), ammonia of reviving The sour tert-butyl group (tBu), histidine is protected with trityl (Trt) or tertbutyloxycarbonyl (Boc)
C) crack, while deprotection base and resin, obtain the crude product of GLP-1 analog monomers；
2) purify：By the dissolving crude product in step c) in water or 10-15% acetonitriles (10-50mg/ml), adopt Preparation HPLC method is used, C18 chromatographic columns, acetonitrile-water-trifluoroacetic acid system is isolated and purified, concentrated, It is lyophilized, obtain GLP-1 analog monomers.
The preparation of B.GLP-1 analog dimers：
GLP-1 analogs monomer is dissolved in deionized water with debita spissitudo (1.5-2mmol/L), according to Ammonium bicarbonate method or DMSO methods formation dimer, purifying obtain GLP-1 analog dimer sterlings.
The dimer for obtaining being formed by following sequence monomers by the above method：
The GLP-1 analog dimer 5-5 of SEQ ID NO 5 and SEQ ID NO 5 formation,
The GLP-1 analog dimer 6-6 of SEQ ID NO 6 and SEQ ID NO 6 formation,
The GLP-1 analog dimer 8-8 of SEQ ID NO 8 and SEQ ID NO 8 formation,
The GLP-1 analog dimers 17-17 of SEQ ID NO 17 and SEQ ID NO 17 formation.
Embodiment 4The preparation for the GLP-1 analog dimers that N- ends are acylated
1) peptide for obtaining N- ends Fmoc- protections and side chain protected is synthesized according to the monomer synthesis of embodiment 3 - resin conjugate；
2) conventional method removes N- ends Fmoc protection groups, and resin-peptide conjugate is suspended in appropriate pyridine, The acetic anhydride or TFAA of appropriate mol ratio are added, is mixed, is placed, the histidine acylation of N- ends is obtained Peptide-resin conjugate；
3) crude product peptide is obtained by the cracking of the method for embodiment 3, purified, it is lyophilized to obtain objective monomer peptide；
4) dimer is prepared by the method for embodiment 3.
The GLP-1 analog dimers of following sequence monomers formation are prepared as stated above：
The GLP-1 analog dimer 4-4 of SEQ ID NO 4 and SEQ ID NO 4 formation,
The GLP-1 analog dimer 7-7 of SEQ ID NO 7 and SEQ ID NO 7 formation,
The GLP-1 analog dimer 10-10 of SEQ ID NO 10 and SEQ ID NO 10 formation,
The GLP-1 analog dimer 13-13 of SEQ ID NO 13 and SEQ ID NO 13 formation,
The GLP-1 analog dimer 15-15 of SEQ ID NO 15 and SEQ ID NO 15 formation,
The GLP-1 analog dimer 19-19 of SEQ ID NO 19 and SEQ ID NO 19 formation,
The GLP-1 analog dimers 24-24 of SEQ ID NO 24 and SEQ ID NO 24 formation.
Embodiment 5The evaluation of GLP-1 analog dimers 5-5,8-8,11-11,18-18 hypoglycemic effect
The hypoglycemic work of GLP-1 analogs dimer of the present invention is evaluated using normal mouse carbohydrate tolerance test With.Method：Normal mouse 30 (is purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center), is randomly divided into 6 Group (blank control group, positive control, test group), every group 5；Weigh appropriate GLP-1 analogs Dimer sterling (>=98%) is dissolved in physiological saline, is configured to 0.1mg/ml sample solution.Test group Mouse, every 200 μ l sample solutions of hypodermic injection；Positive controls mouse, every 20 μ g of hypodermic injection Liraglutide；Blank control group mouse, every 200 μ l physiological saline of hypodermic injection.Injection is determined respectively Afterwards 4,24,48, the 72, sugar tolerance of 120 hours.Carbohydrate tolerance test：Oral administration of glucose 2g/kg, Measure 15,30,60min blood glucose value, calculate blood glucose value AUC (mg/dL.min).As a result figure is seen 1。
Result of the test shows that given the test agent is in administration display in 4 hours and the drop of positive control drug equality strength Sugar effect, positive control drug is invalid after 24 hours, but by reagent upon administration 96 hours still effectively, say Its bright Half-life in vivo significantly extends.
Embodiment 6
Using method same as Example 5 assess GLP-1 analog dimers 3-3,9-9,17-17, 21-21 hypoglycemic effect.The polypeptide that positive drug is selected in Chinese patent CN 201110076380.3 (HAEGTFTSDVSSYLEGCAAKEFIAW) dimer (the patent dimer 5/5 in Fig. 2), As a result Fig. 2 is seen.
Result of the test shows that given the test agent shows the hypoglycemic with positive control drug equality strength in administration 4hr Effect, positive control drug is invalid after 48 hours, by reagent upon administration 96 hours still effectively, wherein two Aggressiveness 17-17 and 21-21 effect are especially pronounced.
Although present invention has been a certain degree of description, it will be apparent that, do not departing from the spirit of the present invention Under conditions of scope, the appropriate change of each condition can be carried out.It is appreciated that the invention is not restricted to institute Embodiment is stated, and is attributed to the scope of claim, it includes the equivalent substitution of each factor.

Claims (10)

1. a kind of glucagon-like peptide-1 analogs monomeric peptide, it is characterised in that the monomeric peptide tool There is formula 1：Z1HAX1GTFTSDVSSYLEX2QAAKEFICWLVKGRX3；
Wherein, Z1For hydrogen, acetyl group or trifluoroacetyl group；
X1For Met, Leu, Pro, Phe or Tyr；
X2For Gly, Glu or Aib (2- aminoisobutyric acids)；
X3To be connected to the NH of arginine residues C-terminal2Or Gly-NH2。
2. monomeric peptide as claimed in claim 1, it is characterised in that the amino acid sequence of the monomeric peptide It is classified as：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、 SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23 Or SEQ ID NO:24.
3. a kind of dimer of glucagon-like peptide-1 analogs monomeric peptide, it is characterised in that described Dimer passes through intermolecular two sulphur by one or both of monomeric peptide as claimed in claim 1 or 2 Key connects to be formed；Preferably, the dimer be by monomeric peptide as claimed in claim 1 or 2 with Another there is mutually homotactic monomeric peptide to connect to be formed by intermolecular disulfide bond.
4. a kind of method for preparing dimer as claimed in claim 3, it is characterised in that the side Method includes：
1) GLP-1 analog monomeric peptide crude products are synthesized by Fmoc methods；
2) by step 1) in obtain GLP-1 analog monomeric peptides purifying crude, concentration and lyophilized, Obtain freeze-dried powder；Preferably, the purifying is by step 1) obtained monomeric peptide dissolving crude product is in water Or 10~15% obtain in acetonitrile after the solution that concentration is 10~15mg/ml, using preparation HPLC method, C18 chromatographic columns, acetonitrile-water-trifluoroacetic acid system isolate and purify realization；
3) by step 2) in obtained freeze-dried powder be dissolved in deionized water, with ammonium bicarbonate method or DMSO methods formation GLP-1 analog dimers, purifying obtains GLP-1 analog dimers pure Product；Preferably, the monomeric peptide is dissolved in the dense of monomeric peptide described in the solution obtained in deionized water Spend for 1.5~2mmol/L.
5. method as claimed in claim 4, is to pass through when the N- ends of the monomeric peptide are acylated What the method comprised the steps was realized：
The peptide obtained in monomeric peptide building-up process-resin conjugate is removed into N-terminal Fmoc protection groups, so The peptide resin conjugate of the removal N-terminal Fmoc protection groups is suspended in pyridine afterwards, it is appropriate to add The acetic anhydride or TFAA of mol ratio, after mixing, placement, obtain the peptide that N- ends histidine is acylated - resin conjugate；N- ends group ammonia is obtained after the peptide that N- ends histidine is acylated-resin conjugate cracking The monomeric peptide crude product of acylating acid.
6. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition includes such as claim 3 Described glucagon-like peptide-1 analogs dimer or its salt.
7. pharmaceutical composition as claimed in claim 6, it is characterised in that described pharmaceutical composition is also Include one or more pharmaceutically acceptable auxiliary materials.
8. pharmaceutical composition as claimed in claim 7, it is characterised in that the auxiliary material is selected from water-soluble One kind or many in property filler, pH adjusting agent, stabilizer, water for injection or osmotic pressure regulator Kind；Preferably, the water-soluble filler be selected from mannitol, D-40, sorbierite, One or more in polyethylene glycol, glucose, lactose and galactolipin；The pH adjusting agent is selected from Citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, ammonium hydroxide, Sodium carbonate, potassium carbonate, ammonium carbonate, saleratus, sodium acid carbonate, one kind in bicarbonate ammonium salt or It is a variety of；It is highly preferred that the stabilizer is selected from EDTA-Na2, sodium thiosulfate, sodium pyrosulfite, Sodium sulfite, dipotassium hydrogen phosphate, sodium acid carbonate, sodium carbonate, arginine, lysine, glutamic acid, Aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or its One or more in derivative, especially preferably, described carboxyl/hydroxylated cellulose or derivatives thereof is HPC, HPC-SL, HPC-L, HPMC, cyclodextrin, lauryl sodium sulfate or trihydroxy methyl ammonia Methylmethane；It is further preferred that the osmotic pressure regulator is sodium chloride or potassium chloride.
9. glucagon-like peptide-1 analogs dimer as claimed in claim 3 or its salt are in system It is ready for use on the application in treatment and/or prevention diabetes, obesity, the medicine of alzheimer disease.
10. pharmaceutical composition as any one of claim 6~8 prepare be used to treat and/ Or the application in prevention diabetes, obesity, the medicine of alzheimer disease.
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