CN102766204B - Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof - Google Patents

Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof Download PDF

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CN102766204B
CN102766204B CN201110115152.2A CN201110115152A CN102766204B CN 102766204 B CN102766204 B CN 102766204B CN 201110115152 A CN201110115152 A CN 201110115152A CN 102766204 B CN102766204 B CN 102766204B
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polypeptide
mutant
peptide
glucagon
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CN102766204A (en
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付刚
龚珉
徐为人
王玉丽
汤立达
任晓文
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Tianjin Institute of Pharmaceutical Research Co Ltd
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Tianjin Institute of Pharmaceutical Research Co Ltd
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Abstract

The invention provides a glucagon-like peptide-1 mutant polypeptide, its preparation method and an application thereof. The mutant series polypeptide is formed by adding Cys series Exendin-4 (31-39) to the C terminal of the glucagon-like peptide-1 mutant. And the mutant series polypeptide folds itself to form a disulfide bond. The glucagon-like peptide-1 mutant polypeptide has an amino acid sequence as shown in SEQ ID NO 1, which is generated by point mutation to Cys occurring at the 9th Asp, 16th Gly or 27th Val of amino acid residues and the addition of Cys to the C terminal. The Exendin-4(31-39) has an amino acid sequence as shown in SEQ ID NO 5. The glucagon-like peptide-1 mutant series polypeptide is used for preparation of pharmaceutical compositions for treating diabetes and treating and/or preventing obesity.

Description

Glucagon-like-peptide-1 mutant polypeptide and preparation method thereof and its application
Technical field
The present invention relates to the pharmaceutical field that diabetes are relevant, particularly, the present invention relates to a kind of novel glucagon-like-peptide-1 (GLP-1) mutant polypeptide, this polypeptide has the Half-life in vivo of the GLP-1 of prolongation.The invention still further relates to preparation method and the application thereof of this polypeptide.
Background technology
Diabetes are metabolism disturbance syndromes taking chronic hyperglycemia as feature that a kind of inherited genetic factors and multiple environmental factors are associated.Because diabetes are also accompanied by many complication, now become and be only second to malignant tumour and cardiovascular disorder the third-largest Health Killer afterwards.1984, glucagon-like-peptide-1 (Glucagon-like peptide-1, GLP-1) be found, it is that one has 30 amino acid whose incretins, have the insulin secretion of promotion and biosynthesizing, the secretion of glucagon suppression, promotes insulinoma cell proliferation, suppress Intra-islet Apoptosis, preserve the different physiological roles such as the susceptibility of cell to blood sugar.After GLP-1 and its receptors bind, can increase cAMP concentration activated protein kinase A(PKA in cell), transcribing and translating by insulin gene in cAMP/PKA kinase pathway enhancing cell, and improve the susceptibility to glucose stimulus signal, thereby increase amount of insulin secretion.GLP-1 also by with cell on the secretion of glucagon-like peptide-1 receptor (GLP-1R) effect glucagon suppression, or indirectly by promoting the secretion of Regular Insulin and Somatostatin to carry out the secretion of glucagon suppression.The major cause that GLP-1 is restricted as hypoglycemic drug is that GLP-1 can be degraded and lose activity by dipeptidyl peptidase-IV (DPP-IV) rapidly in vivo.
Exendin-4 (Exendin-4) is the straight-chain polypeptide of a 39 amino acid composition, is obtained at first by the saliva separation of the huge lizard in South America.It and people source GLP-1 sequence have 53% homology.Exendin-4 equally can with GLP-1 receptors bind, produce physiological actions such as promoting insulin secretion, and second amino acid of the N of Exendin-4 end is glycine, can tolerate the decomposition of DPP-IV in blood plasma, therefore the transformation period that Exendin-4 is absorbed into after blood is grown (t1/2=2.4 hour); C holds than many 9 amino acid of GLP-1, has formed " tryptophane cage " (" Trp-cage ") in structure, can with the combination more closely of GLP-1 acceptor.
Because GLP-1 is people source polypeptide, there is no the immunogenicity problem of Exendin-4, comparatively slight according to the side effect in clinical trial of the GLP-1 analogue of its design, become the ideal prototype that is designed for the GLP-1 receptor stimulant for the treatment of diabetes B, potentiality to be exploited is huge.2010, the Liraglutide(Chinese name Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of Novo Nordisk Co.,Ltd taking GLP-1 as prototype, trade(brand)name in U.S.'s listing, because its good clinical effectiveness, lower side effect and better patient compliance degree (injection in a day once) have won extensive concern, thereby started the exploitation upsurge of GLP-1 receptor stimulant.
Although the GLP-1 receptor stimulants such as the Liraglutide going on the market have now extended the transformation period, but still need patient's injectable drug every day, still have room for improvement in drug use comfort level.Therefore be necessary further to improve the Half-life in vivo of GLP-1 class polypeptide drugs, improve patient compliance degree.
Summary of the invention
Therefore, the object of the invention is that retention time is shorter in vivo for GLP-1 analogue clinically, need the scarce limit of drug administration by injection every day, the GLP-1 mutant polypeptide that a kind of transformation period grows is provided, this GLP-1 mutant polypeptide transformation period is longer, do not need every day to patient infusion, can effectively improve patient's the attitude of complying with.
Another object of the present invention is to provide the preparation method of aforementioned polypeptides, in addition, also provides the application of aforementioned polypeptides in the medicine of preparation treatment diabetes and obesity.
Another object of the present invention is to provide a kind of pharmaceutical composition using GLP-1 mutant polypeptide as effective constituent.
As follows for realizing the technical scheme of above-mentioned purpose:
On the one hand, the invention provides a kind of glucagon-like-peptide-1 mutant polypeptide, described mutant polypeptide is that sudden change produces in SEQ ID NO1 sequence basis as follows:
7HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 37
Wherein, the 9th Asp of above-mentioned sequence amino-acid residue, 16 Gly and/or 27 Val origination points sport Cys, and add Cys at C-terminal.
Glucagon-like-peptide-1 mutant polypeptide as above, in the 16th Gly and/or the 27th Val origination point sudden change; Preferably, the 27th Val origination point sudden change.
Glucagon-like-peptide-1 mutant polypeptide as above, is preferably selected from:
SEQ?ID?NO2： 7HAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGC 38；
SEQ ID NO3: 7hAEGTFTSDVSSYLECQAAKEFIAWLCKGRGC 38; With
SEQ?ID?NO4： 7HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC 38。
On the other hand, the invention provides a kind of glucagon-like-peptide-1 mutant tandem polypeptide, the Cys series connection Exendin-4 being added by glucagon-like-peptide-1 mutant polypeptide C-terminal described above 31-39form, and described glucagon-like-peptide-1 mutant tandem polypeptide self is folded into disulfide linkage, described Exendin-4 31-39there is the aminoacid sequence shown in SEQ ID NO5.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, the Cys that its C-terminal adds and SEQ ID NO1 sequence are in the 9th Asp of amino-acid residue, 16 Gly or 27 Cys formation that Val origination point is mutated into.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, the Cys that the Cys that described disulfide linkage is added by glucagon-like-peptide-1 mutant polypeptide C-terminal and SEQ ID NO1 sequence are mutated at the 16th Gly of amino-acid residue or 27 Val origination points forms.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, Cys and SEQ ID NO1 sequence that described disulfide linkage is added by glucagon-like-peptide-1 mutant polypeptide C-terminal form at the 27th Cys that Val origination point is mutated into of amino-acid residue.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, 1,2 or 3 Exendin-4 of Cys series connection that described glucagon-like-peptide-1 mutant polypeptide adds by C-terminal 31-39.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, 2 Exendin-4 of Cys series connection that described glucagon-like-peptide-1 mutant polypeptide adds by C-terminal 31-39.
Glucagon-like-peptide-1 mutant tandem polypeptide as above, it is preferably selected from: sequence is the polypeptide of SEQ ID NO6-14.
Again on the one hand, the invention provides glucagon-like-peptide-1 mutant polypeptide as above or glucagon-like-peptide-1 mutant tandem polypeptide as above in the application for the preparation for the treatment of and/or preventing in the pharmaceutical composition of diabetes and/or obesity; Preferably, described pharmaceutical composition is injection; More preferably, described injection is freeze-dried powder or injection of solution agent.
Another aspect, the invention provides a kind of pharmaceutical composition, and described composition contains glucagon-like-peptide-1 mutant polypeptide as above or glucagon-like-peptide-1 mutant tandem polypeptide as above.
Pharmaceutical composition as above, it also contains and also comprises one or more pharmaceutically acceptable carriers;
Preferably, described pharmaceutically acceptable carrier is selected from: water-soluble filler, pH adjusting agent, stablizer, water for injection and osmotic pressure regulator;
More preferably, described water-soluble filler is selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose and semi-lactosi;
More preferably, described pH adjusting agent is the acceptable acid of physiology, alkali and/or salt, is preferably selected from following one or more:
Non-volatile acid is as Citric Acid, phosphoric acid, lactic acid, tartrate or hydrochloric acid,
Alkali is as potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide,
Salt is as sodium carbonate or salt of wormwood or ammonium carbonate salts, sodium bicarbonate or saleratus or hydrogen-carbonate ammonium salt;
More preferably, described stablizer is to be selected from following one or more: EDTA-2Na, Sulfothiorine, Sodium Pyrosulfite, S-WAT, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, L-glutamic acid, polyethylene glycol 6000, Macrogol 4000, sodium lauryl sulphate or Tutofusin tris etc., further preferably from one or more in Sodium Pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, Tutofusin tris;
More preferably, described osmotic pressure regulator is selected from sodium-chlor and/or Repone K.
Technical scheme of the present invention also can further be expressed as follows:
One aspect of the present invention provides a kind of GLP-1 mutant polypeptide, described GLP-1 mutant polypeptide has the sequence shown in SEQ ID NO1 and sports Cys at the 9th Asp of amino-acid residue, 16 Gly or 27 Val origination points, and adds at C-terminal the aminoacid sequence that Cys produces.
Preferably, in the 16th Gly or the 27th Val origination point sudden change.
Preferably, the 27th Val origination point sudden change.
The present invention also provides above-mentioned GLP-1 mutant tandem polypeptide on the other hand, the Cys series connection Exendin-4 that described mutant tandem polypeptide is added by GLP-1 mutant polypeptide C-terminal described above 31-39form, and described mutant tandem polypeptide self is folded into disulfide linkage, described Exendin-4 31-39there is the aminoacid sequence shown in SEQ ID NO5.
Preferably, the Cys that the sequence shown in described disulfide linkage is added by GLP-1 mutant polypeptide C-terminal Cys and SEQ IDNO1 is mutated at the 9th Asp of amino-acid residue, 16 Gly or 27 Val origination points forms.
Preferably, the Cys that the sequence shown in described disulfide linkage is added by GLP-1 mutant polypeptide C-terminal Cys and SEQ IDNO1 is mutated at the 16th Gly of amino-acid residue or 27 Val origination points forms.
Preferably, the sequence shown in described disulfide linkage is added by GLP-1 mutant polypeptide C-terminal Cys and SEQ IDNO1 forms at the 27th Cys that Val origination point is mutated into of amino-acid residue.
Preferably, 1,2 or 3 Exendin-4 of Cys series connection that described GLP-1 mutant polypeptide adds by C-terminal 31-39.
Preferably, 2 Exendin-4 of Cys series connection that described GLP-1 mutant polypeptide adds by C-terminal 31-39.
Another aspect of the invention also provides the preparation method of above-mentioned GLP-1 mutant tandem polypeptide, the method comprises: GLP-1 mutant tandem polypeptide is after the preparation of Peptide synthesizer solid phase, add the DTT(disulfide group threitol of 50-100mM) or mercaptoethanol carry out sex change, then in 4 DEG C of slow oxidationes or pass into oxygen form disulfide linkage, to obtain final product.
Further aspect of the present invention also provides the application of above-mentioned GLP-1 mutant tandem polypeptide in the medicine of preparation treatment diabetes, and this polypeptide complex treats and/or prevents the application in the medicine of obesity in preparation.
And described pharmaceutical composition is preferably injection, be more preferably freeze-dried powder or injection of solution agent.
Another aspect of the present invention also provides a kind of pharmaceutical composition, comprises GLP-1 mutant tandem polypeptide described above.
Preferably, also comprise acceptable adjunct ingredient.
Now in conjunction with object of the present invention, the present invention is described one by one.
(1) GLP-1 polypeptide
GLP-1 peptide sequence of the present invention is as follows:
SEQ?ID?NO1： 7HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 37
(2) GLP-1 mutant polypeptide
The following sequence of peptide sequence of GLP-1 mutant of the present invention:
SEQ?ID?NO2： 7HAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGC 38
SEQ?ID?NO3： 7HAEGTFTSDVSSYLECQAAKEFIAWLCKGRGC 38
SEQ?ID?NO4： 7HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC 38
（3）Exendin-4 31-39
Exendin-4 31-39for rear 9 amino acid of natural product, SEQ ID NO5: 31pSSGAPPPS 39
(4) GLP-1 mutant and Exendin-4 31-39tandem polypeptide
The C of three kinds of GLP-1 mutant polypeptides holds connect respectively 1,2 and 3 Exendin-4 31-39sequence, shown in the rear following SEQ ID of the peptide sequence NO6-14 of series connection:
SEQ?ID?NO6：
HAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGCPSSGAPPPS
SEQ?ID?NO7：
HAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGCPSSGAPPPSPSSGAPPPS
SEQ?ID?NO8：
HAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGCPSSGAPPPSPSSGAPPPSPSSGAPPPS
SEQ?ID?NO9：
HAEGTFTSDVSSYLECQAAKEFIAWLCKGRGCPSSGAPPPS
SEQ?ID?NO10：
HAEGTFTSDVSSYLECQAAKEFIAWLCKGRGCPSSGAPPPSPSSGAPPPS
SEQ?ID?NO11：
HAEGTFTSDVSSYLECQAAKEFIAWLCKGRGCPSSGAPPPSPSSGAPPPSPSSGAPPPS
SEQ?ID?NO12：
HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGCPSSGAPPPS
SEQ?ID?NO13：
HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGCPSSGAPPPSPSSGAPPPS
SEQ?ID?NO14：
HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGCPSSGAPPPSPSSGAPPPSPSSGAPPPS
(5) pharmaceutical composition of the present invention
The GLP-1 mutant tandem polypeptide of tool disulfide linkage of the present invention can be made pharmaceutical composition jointly with one or more pharmaceutically acceptable auxiliary materials, and these auxiliary materials comprise: water-soluble filler, pH adjusting agent, stablizer, water for injection, osmotic pressure regulator etc.
Water-soluble filler auxiliary material of the present invention is to be selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose, semi-lactosi etc.
Described pH adjusting agent is selected from following one or more: the nonvolatile acid such as Citric Acid, phosphoric acid, lactic acid, tartrate, hydrochloric acid, and the acceptable organic or inorganic acid of physiology, alkali or the salt etc. such as potassium hydroxide, sodium hydroxide, potassium hydroxide or ammonium hydroxide, sodium carbonate, salt of wormwood, ammonium carbonate salts, sodium bicarbonate, saleratus or hydrogen-carbonate ammonium salt.
Described stablizer is selected from following one or more: EDTA-2Na, Sulfothiorine, Sodium Pyrosulfite, S-WAT, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, L-glutamic acid, polyethylene glycol 6000, Macrogol 4000, sodium lauryl sulphate or Tutofusin tris etc.Be preferably Sodium Pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, Tutofusin tris.
Described osmotic pressure regulator is sodium-chlor and/or Repone K.
(5) preparation method of injection
Pharmaceutical composition of the present invention can be by muscle, intravenously, subcutaneous injection by way of carrying out administration, and preferred formulation is lyophilized powder or injection of solution agent.
The preparation method of freeze drying injection: get tandem polypeptide solution appropriate, add water-soluble filler, stablizer, osmotic pressure regulator etc., add water for injection appropriate, regulate pH value to make its dissolving to 4-8, be diluted with water to proper concn, add 0.1-0.5% gac, at 0-10 DEG C, stir 10-20 minute, decarburization, adopts filtering with microporous membrane degerming, and filtrate is carried out packing, adopt freeze-drying, make white loose block, seal and get final product, each specification contains GLP-1 mutant tandem polypeptide at 5 μ g-1mg.
The preparation method of injection liquid: get tandem polypeptide solution or lyophilized powder appropriate, add water-soluble filler, stablizer, osmotic pressure regulator etc., add water for injection appropriate, regulate pH value to make its dissolving to 4-8, be diluted with water to proper concn, add 0.1-0.5% gac, at 0-10 DEG C, stir 10-20 minute, decarburization, adopt filtering with microporous membrane degerming, filtrate is carried out packing, seals and get final product, and each specification contains GLP-1 mutant tandem polypeptide at 5 μ g-1mg.
Pharmaceutical composition of the present invention can be by muscle, intravenously, subcutaneous injection by way of carrying out administration, and preferred formulation is lyophilized powder or injection of solution agent.Although dosage changes according to treatment target, administering mode, symptom and other factors, composition of the present invention is effective in quite wide dosage range.In adult's treatment, dosage range is 5 μ g/ people-1mg/ people, once a day or every several days single administrations.Actual dose should be decided according to relevant situation by doctor, these situations comprise the person's of being treated physical state, route of administration, age, body weight, the individual reaction of patient to medicine, severity of patient's symptom etc., therefore above-mentioned dosage range is not to limit the scope of the invention by any way.
The present invention utilizes tandem polypeptide technology, at peptide C end several Exendin-4 that connected 31-39sequence; increased the binding ability of polypeptide and GLP-1 acceptor, and designed cysteine mutation site on wild GLP-1 basis, therefore mutant polypeptide can self be folded to form disulfide linkage; protect the polypeptide N end that is subject to DPP IV degraded, overcome short problem of transformation period.In the series connection mutant polypeptide providing, due to C end Exendin-4 31-39existence; the binding ability of itself and GLP-1 acceptor is increased to; and the provide protection of the polypeptide secondary structure forming because of the existence of disulfide linkage; mutant polypeptide transformation period in vivo that makes to connect can reach more than 24 hours; the transformation period of more individually dosed GLP-1 obviously extends, and greatly facilitating the GLP-1(transformation period is only 2 minutes) clinical expansion and application.
Brief description of the drawings
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
The result schematic diagram of the function of blood sugar reduction experiment of Fig. 1 GLP-1 mutant of the present invention tandem polypeptide in Mice Body.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.
synthesizing of embodiment 1GLP-1 mutant tandem polypeptide
1.1:GLP-1 the determining of peptide sequence, preparation and sequence verification.Be subject to the amino acid of Fmoc protection as raw material taking aminoterminal; adopt solid-phase synthesis to synthesize in the full-automatic microwave polypeptide of Liberty1 single passage synthesis system (being purchased from Pei An scientific & technical corporation of U.S. Beijing office), synthetic method is carried out with reference to manufacturer's instrument specification sheets.Through deprotection, coupling and the last cleavage reaction of Fmoc amino acid (being purchased from Shanghai gill biochemistry), finally form desired polypeptides.Gained polypeptide is confirmed through the order-checking of Shanghai Sheng Gong biotech company.
1.2:Exendin-4 31-39determine, preparation and sequence verification.Be subject to the amino acid of Fmoc protection as raw material taking aminoterminal, adopt solid-phase synthesis to synthesize in the full-automatic microwave polypeptide of Liberty1 single passage synthesis system, synthetic method is carried out with reference to manufacturer's instrument specification sheets.Gained polypeptide is confirmed through the order-checking of Shanghai Sheng Gong biotech company.
1.3: the determining of mutant tandem polypeptide, preparation and sequence verification are subject to the amino acid of Fmoc protection as raw material taking aminoterminal; adopt solid-phase synthesis to synthesize in the full-automatic microwave polypeptide of Liberty1 single passage synthesis system, synthetic method is carried out with reference to manufacturer's instrument specification sheets.Gained polypeptide is confirmed through the order-checking of Shanghai Sheng Gong biotech company.
GLP-1 mutant tandem polypeptide, after Peptide synthesizer solid phase preparation, adds the DTT(disulfide group threitol of 50-100mM) or-mercaptoethanol carries out sex change, then in 4 DEG C of slow oxidationes or pass into oxygen and form disulfide linkage, obtains GLP-1 mutant tandem polypeptide.
Described GLP-1 mutant tandem polypeptide is passed through 1,2 or 3 Exendin-4 of Cys series connection of C-terminal by GLP-1 mutant polypeptide 31-39form, preferably, described mutant tandem polypeptide is passed through 2 Exendin-4 of Cys series connection of C-terminal by GLP-1 mutant 31-39form, and described GLP-1 mutant tandem polypeptide passes through the sequence shown in Cys and the SEQ ID NO1 of C-terminal at the 9th Asp, the Cys that 16 Gly or 27 Val origination points are mutated into is folded into disulfide linkage, preferably, the Cys that described GLP-1 mutant tandem polypeptide is mutated at the 16th Gly or 27 Val origination points by the sequence shown in Cys and the SEQ ID NO1 of C-terminal is folded into disulfide linkage, more preferably, described GLP-1 mutant tandem polypeptide is folded into disulfide linkage by the sequence shown in Cys and the SEQ ID NO1 of C-terminal at the 27th Cys that Val origination point is mutated into, described Exendin-4 31-39there is the aminoacid sequence shown in SEQID NO5, described GLP-1 mutant polypeptide has the sequence shown in SEQ ID NO1 and sports Cys at the 9th Asp of amino-acid residue, 16 Gly or 27 Val origination points, and add at C-terminal the aminoacid sequence (SEQ ID NO2-4) that Cys produces, preferably, in the 16th Gly origination point sudden change (SEQ ID NO3) or the 27th Val origination point sudden change (SEQ IDNO3), more preferably, in the 27th Val origination point sudden change (SEQ ID NO3).
the external GLP-1 receptors bind experiment of embodiment 2GLP-1 mutant tandem polypeptide
It is that INS-1 (rat β insulinoma cell, ATCC, Manassas, VA) carries out that receptors bind experiment utilizes GLP-1 acceptor high expressing cell.First INS-1 cell is inoculated on 12 orifice plates, and within first 2 hours, rinses cell with PBS damping fluid in experiment, then use under 4% paraformaldehyde room temperature fixed cell 10 minutes.After cell counting, 10 5individual cells/well is hatched 2 hours jointly respectively at GLP-1 mutant tandem polypeptide (SEQ ID NO6-14) and natural GLP-1.PBS, by after not rinsing with the polypeptide of Cell binding, uses GLP-1ELISA test kit to detect.As can be seen from Table 1, GLP-1 mutant tandem polypeptide is larger has improved the binding ability of itself and GLP-1 acceptor, compares GLP-1.
The binding constant of table 1, GLP-1 mutant tandem polypeptide and GLP-1 polypeptide and external GLP-1 receptors bind
Polypeptide (sequence) Binding constant (nM)
SEQ?ID?NO1 16.64±1.75
SEQ?ID?NO6 12.46±2.21
SEQ?ID?NO7 16.88±1.86
SEQ?ID?NO8 8.75±1.65
SEQ?ID?NO9 9.26±2.53
SEQ?ID?NO10 12.64±2.86
SEQ?ID?NO11 9.15±1.75
SEQ?ID?NO12 13.24±2.65
SEQ?ID?NO13 10.25±1.74
SEQ?ID?NO14 9.26±1.22
the function of blood sugar reduction experiment of embodiment 3GLP-1 mutant tandem polypeptide in Mice Body
Give mouse mainline 9 kinds of different GLP-1 mutant tandem polypeptides of the present invention (injected dose is 100 μ g/kg), with the positive contrast of Exendin-4, respectively at 0.5,4,6,8,12,24 and 48 hour injectable dextrose monohydrate (2g/kg) after administration, measure the rear halfhour blood-sugar content of injection, as can be seen from Figure 1, Exendin-4 completely lost its function of blood sugar reduction after 4 hours, but 9 kinds of GLP-1 mutant tandem polypeptides but still exist function of blood sugar reduction after 48 hours in administration.
the transformation period experiment of embodiment 4GLP-1 mutant tandem polypeptide in rat body
Rat 9 kinds of GLP-1 mutant tandem polypeptides of injection (injected dose is 100 μ g/kg), respectively at injecting front and injecting after latter 0.5,1,3,6,9,12,24,36,48,72,96 and 120 hour, in the about 0.2mL of eye clump venous blood sampling, prepare serum for subsequent use.
GLP-1 and analogue method for measurement of concentration: adopt Enzyme-linked Immunosorbent Assay method (ELISA) to detect the concentration of polypeptide complex in rat blood serum, operate as follows: 4 DEG C of centrifugal separation plasmas.The concentration of GLP-1 in mouse blood plasma is measured with GLP-1EIA test kit (Phoenix Pharmaceuticals, INC).Mouse plasma sample after dilution is added to 96 orifice plates (50 μ L), add after the sheep anti mouse primary antibodie of 25 μ L Exendin-4, hatch 2 hours for 37 DEG C.Use washing lotion to clean 96 orifice plate 4 times, hatch 1 hour after adding 100 μ LSA-HRP.Same method is cleaned 96 orifice plates, then adds 100 μ LTMB, hatches 1 hour for 37 DEG C.In after 2N hydrochloric acid stopped reaction 20 minutes, measure OD450 value, and compare with the GLP-1 of normal concentration and calculate pharmacokinetic parameter according to ELISA result.
In the body of 9 kinds of GLP-1 mutant tandem polypeptides and positive control, pharmacokinetic results is in table 2, and result shows that sudden change tandem polypeptide of the present invention more independent GLP-1 of transformation period in vivo obviously extends, and has long-acting characteristic.
table 2, GLP-1 mutant tandem polypeptide and the transformation period of Exendin-4 in rat body
Polypeptide (sequence) Transformation period (hour)
SEQ?ID?NO5 <4.0
SEQ?ID?NO6 24
SEQ?ID?NO7 60
SEQ?ID?NO8 60
SEQ?ID?NO9 75
SEQ?ID?NO10 70
SEQ?ID?NO11 75
SEQ?ID?NO12 65
SEQ?ID?NO13 72
SEQ?ID?NO14 78
the preparation of embodiment 5 lyophilized powder type pharmaceutical compositions
Get appropriate container and add poloxamer 0.05g, N.F,USP MANNITOL 0.2g, lactose 0.1g, water for injection 3mL, be stirred to dissolve, the Citric Acid or the sodium hydroxide that add 1mol/L regulate pH to 6.0, be cooled to 5 DEG C, the tandem polypeptide solution 5mL that gets tool disulfide linkage adds wherein, continue to adjust and mend pH to 6.0, add water to 10mL.Add 20mg activated carbon, at 5 DEG C, stir decarburization 20 minutes, adopt filtering with microporous membrane degerming, filtrate is carried out packing by every 0.2mL, and pre-freeze is after 2 hours, freezing lower drying under reduced pressure 12 hours, to sample temperature to 5 DEG C, drier 2 hours, make white loose block, seal and obtain the pharmaceutical composition of GLP-1 mutant tandem polypeptide, be placed in pre-filled syringe, specification is that 100 μ g/ prop up, 4 DEG C of following preservations, administration after dissolving with 200 μ L waters for injection before injection.
the preparation of embodiment 6 injection of solution drug form compositions
Get appropriate container and add sorbyl alcohol 0.1g, lactose 0.1g, NaCl20mg, Citric Acid 10mg, water for injection 7mL, be stirred to dissolve, the Citric Acid or the sodium hydroxide that add 1mol/L regulate pH to 6.5, be cooled to 0 DEG C, the tandem polypeptide powder 5mg that gets the tool disulfide linkage of preparing with embodiment 5 methods adds wherein, continues stirring and dissolving, adjust and mend pH to 6.5, add water to 10mL.Add 10mg activated carbon, stir 20 minutes at 0-4 DEG C, decarburization, adopts filtering with microporous membrane degerming, and filtrate is distributed into precharging type syringe by every 100 μ L, sample temperature to 5 DEG C following preservation, and specification is that 50 μ g/ prop up.

Claims (13)

1. a glucagon-like-peptide-1 mutant tandem polypeptide, the Cys being added by glucagon-like-peptide-1 mutant polypeptide C-terminal series connection Exendin-4 31-39form, and described glucagon-like-peptide-1 mutant tandem polypeptide self is folded into disulfide linkage, described Exendin-4 31-39for the aminoacid sequence shown in SEQ ID NO:5;
Wherein, described glucagon-like-peptide-1 mutant polypeptide is that sudden change produces in SEQ ID NO:1 sequence basis as follows:
7HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 37；
Wherein, described tandem polypeptide is selected from: sequence is the polypeptide of SEQ ID NO:6-14.
2. glucagon-like-peptide-1 mutant tandem polypeptide claimed in claim 1 is in the application for the preparation for the treatment of and/or preventing in the pharmaceutical composition of diabetes and/or obesity.
3. application according to claim 2, wherein, described pharmaceutical composition is injection.
4. application according to claim 3, wherein, described injection is freeze-dried powder or injection of solution agent.
5. a pharmaceutical composition, is characterized in that, described composition contains glucagon-like-peptide-1 mutant tandem polypeptide claimed in claim 1.
6. pharmaceutical composition according to claim 5, is characterized in that, also comprises one or more pharmaceutically acceptable carriers.
7. pharmaceutical composition according to claim 6, wherein, described pharmaceutically acceptable carrier is selected from: water-soluble filler, pH adjusting agent, stablizer, water for injection and osmotic pressure regulator.
8. pharmaceutical composition according to claim 7, wherein, described water-soluble filler is selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose and semi-lactosi.
9. pharmaceutical composition according to claim 7, wherein, described pH adjusting agent is the acceptable acid of physiology, alkali and/or salt.
10. pharmaceutical composition according to claim 9, wherein, the acceptable acid of described physiology, alkali and/or salt are selected from following one or more:
Non-volatile acid, is selected from Citric Acid, phosphoric acid, lactic acid or tartrate,
Alkali, is selected from potassium hydroxide, sodium hydroxide or ammonium hydroxide,
Salt, is selected from sodium carbonate, salt of wormwood, ammonium carbonate salts, sodium bicarbonate, saleratus or hydrogen-carbonate ammonium salt.
11. pharmaceutical compositions according to claim 7, wherein, described stablizer is to be selected from following one or more: EDTA-2Na, Sulfothiorine, Sodium Pyrosulfite, S-WAT, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, L-glutamic acid, polyethylene glycol 6000, Macrogol 4000, sodium lauryl sulphate or Tutofusin tris.
12. pharmaceutical compositions according to claim 11, wherein, described stablizer is selected from: one or more in Sodium Pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, Tutofusin tris.
13. pharmaceutical compositions according to claim 7, wherein, described osmotic pressure regulator is selected from sodium-chlor and/or Repone K.
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