CN102363633B - Glucagon like peptide-1 mutant polypeptide and preparation method, medicinal composition and use thereof - Google Patents
Glucagon like peptide-1 mutant polypeptide and preparation method, medicinal composition and use thereof Download PDFInfo
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Abstract
The invention discloses a glucagon like peptide(GLP)-1 mutant polypeptide and a preparation method, a medicinal composition and use thereof. The mutant polypeptide is formed by bonding a Cys-containing elongation peptide to the N terminal of the glucagon like peptide-1 mutant, the mutant polypeptide itself folds to form a disulfide bond. The glucagon like peptide-1 mutant polypeptide is used for preparing a medicinal composition for treating diabetes and treating and preventing obesity. For overcoming the drawbacks of short retention time in body and need of daily injection administration of clinic GLP-1 analogue, the invention provides the GLP-1 analogue which has a longer half-life period. With the longer half-life period, the GLP-1 mutant polypeptide is not required to be injected into a patient every day, and can increase the obedience of the patient effectively.
Description
Technical field
The present invention relates to the relevant pharmaceutical field of a kind of diabetes, particularly, the present invention relates to a kind of glucagon-like-peptide-1 mutant polypeptide and preparation method thereof, pharmaceutical composition and its application of Half-life in vivo of glucagon-like-peptide-1 of prolongation.
Background technology
Diabetes are metabolism disturbance syndromes take chronic hyperglycemia as feature that a kind of inherited genetic factors and multiple environmental factors are associated., because diabetes also are 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 a kind of 30 amino acid whose incretins that have, have the insulin secretion of promotion and biosynthesizing, the secretion of glucagon suppression, promote insulinoma cell proliferation, suppress Intra-islet Apoptosis, preserve the different physiological roles such as susceptibility of cell to blood sugar.Can increase cAMP concentration and activated protein kinase A(PKA in cell after GLP-1 and its receptors bind), transcribing and translating by insulin gene in cAMP/PKA kinase pathway enhancing cell, and improve susceptibility to the 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, perhaps indirectly by the secretion that promotes Regular Insulin and Somatostatin, 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 that 39 amino acid forms, and obtains the saliva separation by the huge lizard in South America at first.It and people source GLP-1 sequence have 53% homology.Exendin-4 equally can with the 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 end is Duoed 9 amino acid than GLP-1, has formed " tryptophane cage " (" Trp-cage ") on structure, can with the combination more closely of GLP-1 acceptor.
Because GLP-1 is people source polypeptide, the immunogenicity problem that there is no Exendin-4, comparatively slight according to GLP-1 analogue side effect in clinical trial 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 take 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 is once) have won extensive concern, thereby started the exploitation upsurge of GLP-1 receptor stimulant.
Although the GLP-1 receptor stimulants such as Liraglutide of listing have extended the transformation period now, but still need patient's injectable drug every day, still have room for improvement on the drug use comfort level.Therefore be necessary further to improve the Half-life in vivo of GLP-1 class polypeptide drugs, improve the patient compliance degree.
Summary of the invention
Technical problem to be solved by this invention is that a kind of long glucagon-like-peptide-1 mutant polypeptide of the phase of declining of carrying is provided.
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 purpose of the present invention is to provide the pharmaceutical composition of a kind of GLP-1 of using mutant polypeptide as effective constituent.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of glucagon-like-peptide-1 mutant polypeptide, described glucagon-like-peptide-1 mutant polypeptide is to add the mutant of the prolongation polypeptide formation that can form disulfide linkage at the N of SEQ ID NO1 sequences polypeptide end, and the general structure of described mutant is as follows:
X
1(n
1)-C-X
2(n
2)-C-X
3(n
3)SEQ?ID?NO1
Wherein:
Above-mentioned sequence amino-acid residue X
1Refer to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine), n
1Refer to above-mentioned X
1Amino acid whose number, n
1=1～5;
Above-mentioned sequence amino-acid residue X
2Refer to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine), n
2Refer to above-mentioned X
2Amino acid whose number, n
2=1～5;
Above-mentioned sequence amino-acid residue X
3Refer to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine), n
3Refer to above-mentioned X
3Amino acid whose number, n
2=1～5;
Two halfcystines in described glucagon-like-peptide-1 mutant, polypeptide form disulfide linkage.
Described glucagon-like-peptide-1 mutant polypeptide is selected from any in SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8 or SEQ ID NO9.
A kind of preparation method of above-mentioned glucagon-like-peptide-1 mutant polypeptide, the method comprises: the glucagon-like-peptide-1 mutant polypeptide is after the preparation of Peptide synthesizer solid phase, add disulfide group threitol DTT or the beta-mercaptoethanol of 50-100mM to carry out sex change, then in 4 ℃ of slow oxidationes or pass into oxygen and form disulfide linkage.
A kind of pharmaceutical composition, described composition contain above-mentioned glucagon-like-peptide-1 mutant polypeptide.
Also contain one or more pharmaceutically acceptable carriers, described pharmaceutically acceptable carrier is selected from: water-soluble filler, pH adjusting agent, stablizer, water for injection or osmotic pressure regulator.
Described water-soluble filler is selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose or semi-lactosi;
Described pH adjusting agent is the acceptable acid of physiology, alkali and/or salt;
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;
Described osmotic pressure regulator is selected from sodium-chlor and/or Repone K.
Described pH adjusting agent is selected from following one or more:
Non-volatile acid: Citric Acid, phosphoric acid, lactic acid, tartrate or hydrochloric acid;
Alkali: potassium hydroxide, sodium hydroxide, potassium hydroxide or ammonium hydroxide;
Salt: sodium carbonate, salt of wormwood, ammonium carbonate salts, sodium bicarbonate, saleratus or hydrogen-carbonate ammonium salt;
Described pharmaceutical composition is injection.
Described injection is freeze-dried powder or injection of solution agent.
Described glucagon-like-peptide-1 mutant polypeptide be used for the treatment of and/or the pharmaceutical composition of prevent diabetes and/or obesity in application.
The invention has the beneficial effects as follows: retention time is shorter in vivo for GLP-1 analogue clinically, the scarce limit that needs drug administration by injection every day, long GLP-1 mutant polypeptide of a kind of transformation period 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.
Description of drawings
The result schematic diagram of the function of blood sugar reduction experiment of Fig. 1 GLP-1 mutant polypeptide of the present invention in Mice Body.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail:
The invention provides a kind of glucagon-like-peptide-1 mutant polypeptide, the general structure of the mutant that relates in the present invention is as follows:
X
1(n
1)-C-X
2(n
2)-C-X
3(n
3)SEQ?ID?NO1
Be X
1(n
1)-C-X
2(n
2)-C-X
3(n
3)
7HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
36
Wherein, above-mentioned sequence amino-acid residue X1 refers to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine).n
1Refer to above-mentioned X
1Amino acid whose number, n
1=1～5.
Above-mentioned sequence amino-acid residue X
2Refer to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine).n
2Refer to above-mentioned X
2Amino acid whose number, n
2=1～5.
Above-mentioned sequence amino-acid residue X
3Refer to glycine (Glycine), L-Ala (Alanine), α-amino-isovaleric acid (Valine), leucine (Leucine) or Isoleucine (Isoleucine).n
3Refer to above-mentioned X
3Amino acid whose number, n
2=1～5
Two halfcystines in glucagon-like-peptide-1 mutant as above, polypeptide form disulfide linkage.
On the one hand, the invention provides the application of glucagon-like-peptide-1 mutant polypeptide as above in the pharmaceutical composition for the preparation for the treatment of and/or preventing diabetes and/or obesity again; 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.
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 or 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 such as Citric Acid, phosphoric acid, lactic acid, tartrate or hydrochloric acid,
Alkali such as potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide,
Salt such 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., and further preferred 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.
Another aspect of the invention also provides the preparation method of above-mentioned GLP-1 mutant polypeptide, the method comprises: the GLP-1 mutant polypeptide is after the preparation of Peptide synthesizer solid phase, the DTT(disulfide group threitol that adds 50-100mM) or beta-mercaptoethanol carry out sex change, then in 4 ℃ of slow oxidationes or pass into oxygen and form disulfide linkage, obtain.
Further aspect of the present invention also provides the application of above-mentioned GLP-1 mutant polypeptide in the medicine of preparation treatment diabetes, and this polypeptide complex treats and/or prevents application in the medicine of obesity in preparation.
And described pharmaceutical composition is preferably injection, is 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 polypeptide described above.
Preferably, also comprise acceptable adjunct ingredient.
Now in conjunction with purpose 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, namely
7HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
36
(2) GLP-1 mutant polypeptide
The following sequence of GLP-1 mutant polypeptide sequence of the present invention (SEQ ID NO2-9), that is:
SEQ?ID?NO2：GCGCGHAEGTFTSCVSSYLEGQAAKEFIAWLVKGRGC
SEQ?ID?NO3:GGCGGCGGHAEGTFTSDVSSYLECQAAKEFIAWLCKGRGC
SEQ?ID?NO4：GGGCGGGCGGGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
SEQ?ID?NO5:GCGGCGGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
SEQ?ID?NO6:GCGGGCGGGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
SEQ?ID?NO7:GGCGGCGGGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
SEQ?ID?NO8:GGGCGCGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
SEQ?ID?NO9:GGGCGGCGGHAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGC
(3) pharmaceutical composition of the present invention
The GLP-1 mutant 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 the physiology 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, alkali or salt etc.
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.
(4) 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 polypeptide solution appropriate, add water-soluble filler, stablizer, osmotic pressure regulator etc., add water for injection appropriate, regulate the pH value and make its dissolving to 4-8, be diluted with water to proper concn, add the 0.1-0.5% gac, stir 10-20 minute under 0-10 ℃, decarburization, adopt the filtering with microporous membrane degerming, and filtrate is carried out packing, adopt freeze-drying, make the white loose block, seal and get final product, each specification contains the GLP-1 mutant polypeptide at 5 μ g-1mg.
The preparation method of injection liquid: get string peptide solution or lyophilized powder appropriate, add water-soluble filler, stablizer, osmotic pressure regulator etc., add water for injection appropriate, regulate the pH value and make its dissolving to 4-8, be diluted with water to proper concn, add the 0.1-0.5% gac, stir 10-20 minute, decarburization under 0-10 ℃, adopt the filtering with microporous membrane degerming, filtrate is carried out packing, seals and get final product, and each specification contains the GLP-1 mutant 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 factor, 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 the doctor, these situations comprise the person's of being treated physical state, route of administration, age, body weight, the patient individual reaction 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 disulfide linkage,, at the N-terminal formation hairpin structure of GLP-1, has protected the polypeptide N end that is subject to the DPPIV degraded, has overcome short problem of transformation period.In the mutant polypeptide that provides, the transformation period in body all can reach more than 24 hours, and 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.
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment that provides is only in order to illustrate the present invention, rather than 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.
The embodiment 1 external GLP-1 receptors bind experiment of GLP-1 mutant polypeptide
It is INS-1(Islet cells-1 that the receptors bind experiment utilizes GLP-1 acceptor high expressing cell, available from Shanghai Chinese Academy of Sciences animal center) carry out.At first the INS-1 cell is inoculated on 12 orifice plates, and in experiment, rinsed cell with the PBS damping fluid in front 2 hours, then used under 4% paraformaldehyde room temperature fixed cell 10 minutes.After cell counting, 10
5Individual cells/well was hatched 2 hours jointly respectively at GLP-1 mutant polypeptide (SEQ ID2, SEQ ID3, SEQ ID4, SEQ ID5, SEQ ID6, SEQ ID7, SEQ ID8 and SEQ ID9) and natural GLP-1 (SEQ ID NO1).PBS does not use the GLP-1ELISA test kit to detect after will be not with the polypeptide of Cell binding, rinsing.As can be seen from Table 1, the GLP-1 mutant 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 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?NO2 | 12.46±2.21 |
| SEQ?ID?NO3 | 16.88±1.86 |
| SEQ?ID?NO4 | 8.75±1.65 |
| SEQ?ID?NO5 | 9.26±2.53 |
| SEQ?ID?NO6 | 12.64±2.86 |
| SEQ?ID?NO7 | 9.15±1.75 |
| SEQ?ID?NO8 | 13.24±2.65 |
| SEQ?ID?NO9 | 9.26±1.22 |
The function of blood sugar reduction experiment of embodiment 2 GLP-1 mutant polypeptides in Mice Body
Give mouse mainline 8 kinds of different GLP-1 mutant 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 but still there is function of blood sugar reduction in 8 kinds of GLP-1 mutant polypeptides after 48 hours in administration.
The transformation period experiment of embodiment 3 GLP-1 mutant polypeptides in the rat body
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 ℃ of centrifugal separation plasmas.(Phoenix Pharmaceuticals, INC) measures the concentration of the GLP-1 in mouse blood plasma with the GLP-1EIA test kit.Mouse plasma sample after dilution is added 96 orifice plates (50 μ L), after adding the sheep anti mouse primary antibodie of 25 μ L Exendin-4, hatched 2 hours for 37 ℃.Use washing lotion to clean 96 orifice plate 4 times, hatched 1 hour after adding 100 μ L SA-HRP.Same method is cleaned 96 orifice plates, then adds 100 μ LTMB, hatches 1 hour for 37 ℃.Measured the OD450 value in after 2N hydrochloric acid stopped reaction 20 minutes, and with the GLP-1 of normal concentration, compare and according to the ELISA result, calculate pharmacokinetic parameter.
In the body of 8 kinds of GLP-1 mutant polypeptides and positive control, pharmacokinetic results is in Table 2, and result shows that more independent GLP-1 of sudden change tandem polypeptide transformation period in vivo of the present invention obviously extends, and has long-acting characteristic.
Table 2, GLP-1 mutant polypeptide and the transformation period of Exendin-4 in the rat body
| Polypeptide (sequence) | Transformation period (hour) |
| Exendin-4 | <4.0 |
| SEQ?ID? |
24 |
| SEQ?ID?NO3 | 30 |
| SEQ?ID?NO4 | 33 |
| SEQ?ID?NO5 | 25 |
| SEQ?ID?NO6 | 30 |
| SEQ?ID?NO7 | 35 |
| SEQ?ID?NO8 | 35 |
| SEQ?ID?NO9 | 40 |
The preparation of embodiment 4 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 are regulated pH to 6.0, be cooled to 5 ℃, the polypeptide solution 5mL that gets the tool disulfide linkage adds wherein, continue to transfer and mend pH to 6.0, add water to 10mL.Add the 20mg activated carbon, stirred 20 minutes decarburization under 5 ℃, adopt the 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 ℃, drier 2 hours, make the white loose block, sealing namely obtains the pharmaceutical composition of GLP-1 mutant polypeptide, is placed in pre-filled syringe, and specification is that 100 μ g/ prop up, preserve administration after dissolving with 200 μ L waters for injection before injection below 4 ℃.
The preparation of embodiment 5 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 are regulated pH to 6.5, be cooled to 0 ℃, the polypeptide powder 5mg that gets the tool disulfide linkage for preparing with embodiment 5 methods adds wherein, continues stirring and dissolving, transfer and mend pH to 6.5, add water to 10mL.Add the 10mg activated carbon, stirred under 0-4 ℃ 20 minutes, decarburization, adopt the filtering with microporous membrane degerming, and filtrate is distributed into precharging type syringe by every 100 μ L, preserves below sample temperature to 5 ℃, and specification is that 50 μ g/ prop up.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (8)
1. glucagon-like-peptide-1 mutant polypeptide, it is characterized in that, described glucagon-like-peptide-1 mutant polypeptide is selected from any in SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8 or SEQ ID NO9.
2. the preparation method of a glucagon-like-peptide-1 mutant polypeptide claimed in claim 1, it is characterized in that, the method comprises: the glucagon-like-peptide-1 mutant polypeptide is after the preparation of Peptide synthesizer solid phase, add disulfide group threitol DTT or the beta-mercaptoethanol of 50-100mM to carry out sex change, then in 4 ℃ of slow oxidationes or pass into oxygen and form disulfide linkage.
3. a pharmaceutical composition, is characterized in that, described composition contains the described glucagon-like-peptide-1 mutant polypeptide of any one in claim 1-2.
4. pharmaceutical composition according to claim 3, it is characterized in that, also contain one or more pharmaceutically acceptable carriers, described pharmaceutically acceptable carrier is selected from: water-soluble filler, pH adjusting agent, stablizer, water for injection or osmotic pressure regulator.
5. pharmaceutical composition according to claim 4, is characterized in that, described water-soluble filler is selected from following one or more: N.F,USP MANNITOL, low molecular dextran, sorbyl alcohol, polyoxyethylene glycol, glucose, lactose or semi-lactosi;
Described pH adjusting agent is the acceptable acid of physiology, alkali and/or salt;
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;
Described osmotic pressure regulator is selected from sodium-chlor and/or Repone K.
6. pharmaceutical composition according to claim 5, is characterized in that, described pH adjusting agent is selected from following one or more:
Non-volatile acid: Citric Acid, phosphoric acid, lactic acid or tartrate;
Alkali: sodium hydroxide, potassium hydroxide or ammonium hydroxide;
Salt: sodium carbonate, salt of wormwood, ammonium carbonate salts, sodium bicarbonate, saleratus or hydrogen-carbonate ammonium salt.
7. pharmaceutical composition according to claim 4, is characterized in that, described pharmaceutical composition is injection.
8. pharmaceutical composition according to claim 7, is characterized in that, described injection is freeze-dried powder or injection of solution agent.
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| CN102718858B (en) * | 2011-03-29 | 2014-07-02 | 天津药物研究院 | Glucagon-like peptide-1 (GLP-1) analogue monomer and dimer, preparation method therefor and application thereof |
| UA116217C2 (en) | 2012-10-09 | 2018-02-26 | Санофі | Exendin-4 derivatives as dual glp1/glucagon agonists |
| ES2688367T3 (en) | 2012-12-21 | 2018-11-02 | Sanofi | Derivatives of exendin-4 as dual agonists of GLP1 / GIP or trigonal of GLP1 / GIP / glucagon |
| CN103344764B (en) * | 2013-06-19 | 2014-11-26 | 天津美德太平洋科技有限公司 | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) |
| CN103405753B (en) * | 2013-08-13 | 2016-05-11 | 上海仁会生物制药股份有限公司 | Stable insulin secretion accelerating peptide liquid drugs injection pharmaceutical composition |
| WO2015086728A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Exendin-4 peptide analogues as dual glp-1/gip receptor agonists |
| EP3080149A1 (en) | 2013-12-13 | 2016-10-19 | Sanofi | Dual glp-1/glucagon receptor agonists |
| EP3080154B1 (en) | 2013-12-13 | 2018-02-07 | Sanofi | Dual glp-1/gip receptor agonists |
| TW201609796A (en) | 2013-12-13 | 2016-03-16 | 賽諾菲公司 | Non-acylated EXENDIN-4 peptide analogues |
| TW201625668A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| TW201625669A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4 |
| TW201625670A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Dual GLP-1/glucagon receptor agonists derived from EXENDIN-4 |
| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
| AR105319A1 (en) | 2015-06-05 | 2017-09-27 | Sanofi Sa | PROPHARMS THAT INCLUDE A DUAL AGONIST GLU-1 / GLUCAGON CONJUGATE HIALURONIC ACID CONNECTOR |
| AR105284A1 (en) | 2015-07-10 | 2017-09-20 | Sanofi Sa | DERIVATIVES OF EXENDINA-4 AS SPECIFIC DUAL PEPTIDE AGONISTS OF GLP-1 / GLUCAGÓN RECEPTORS |
| CN107266555B (en) * | 2016-04-06 | 2021-05-04 | 天津药物研究院有限公司 | Long-acting glucagon-like peptide-1 analogue dimer and medical application thereof |
| CN114685643A (en) * | 2020-12-29 | 2022-07-01 | 苏州康宁杰瑞生物科技有限公司 | Human GLP-1 polypeptide variant and application thereof |
| MX2024007861A (en) * | 2022-01-10 | 2024-07-09 | Jiangsu Hansoh Pharmaceutical Group Co Ltd | Stable pharmaceutical composition of receptor agonist, and preparation method and application thereof. |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676163A (en) * | 2004-09-30 | 2005-10-05 | 华东师范大学 | Human glucagon-like peptide-1 compound and its preparing method |
| CN1786031A (en) * | 2005-12-16 | 2006-06-14 | 暨南大学 | Glucagon kind polypeptide-1 analogue, its preparation method and application |
| CN101041693A (en) * | 2007-02-06 | 2007-09-26 | 珠海联邦制药股份有限公司 | Novel blood sugar lowing polypeptide and uses thereof |
| CN101056650A (en) * | 2004-11-12 | 2007-10-17 | 诺和诺德公司 | Stable formulations of insulinoptropic peptides |
| CN101273058A (en) * | 2005-09-22 | 2008-09-24 | 生物相容英国公司 | GLP-1 ( glucagon-like peptide-1 ) fusion polypeptides with increased peptidase resistance |
| CN101337989A (en) * | 2008-08-28 | 2009-01-07 | 中国药科大学 | Novel glucagon-like peptide-1(GLP-1) analogues and use thereof |
| JP4282485B2 (en) * | 2002-01-08 | 2009-06-24 | イーライ リリー アンド カンパニー | Elongated glucagon-like peptide-1 analog |
| CN102180963A (en) * | 2011-04-22 | 2011-09-14 | 中国药科大学 | Glucagons like peptide-1 (GLP-1) analog and application thereof |
| CN102229668A (en) * | 2011-06-03 | 2011-11-02 | 浙江贝达药业有限公司 | GLP-1 (glucagon-like peptide-1) derivative and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7235627B2 (en) * | 1996-08-30 | 2007-06-26 | Novo Nordisk A/S | Derivatives of GLP-1 analogs |
| US7601691B2 (en) * | 1999-05-17 | 2009-10-13 | Conjuchem Biotechnologies Inc. | Anti-obesity agents |
| WO2007030519A2 (en) * | 2005-09-08 | 2007-03-15 | Trustees Of Tufts College | Stabilized glp-1 analogs |
| EP1976873A2 (en) * | 2006-01-11 | 2008-10-08 | Brystol-Myers Squibb Company | Human glucagon-like-peptide-1 modulators and their use in the treatment of diabetes and related conditions |
| EP2021014A1 (en) * | 2006-05-26 | 2009-02-11 | Brystol-Myers Squibb Company | Sustained release glp-1 receptor modulators |
| US20100317057A1 (en) * | 2007-12-28 | 2010-12-16 | Novo Nordisk A/S | Semi-recombinant preparation of glp-1 analogues |
-
2011
- 2011-11-16 CN CN2011103619274A patent/CN102363633B/en not_active Expired - Fee Related
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4282485B2 (en) * | 2002-01-08 | 2009-06-24 | イーライ リリー アンド カンパニー | Elongated glucagon-like peptide-1 analog |
| CN1676163A (en) * | 2004-09-30 | 2005-10-05 | 华东师范大学 | Human glucagon-like peptide-1 compound and its preparing method |
| CN101056650A (en) * | 2004-11-12 | 2007-10-17 | 诺和诺德公司 | Stable formulations of insulinoptropic peptides |
| CN101273058A (en) * | 2005-09-22 | 2008-09-24 | 生物相容英国公司 | GLP-1 ( glucagon-like peptide-1 ) fusion polypeptides with increased peptidase resistance |
| EP2261245A1 (en) * | 2005-09-22 | 2010-12-15 | Biocompatibles Uk Ltd. | GLP-1 (glucagon-like peptide-1) fusion polypeptides with increased peptidase resistance |
| CN1786031A (en) * | 2005-12-16 | 2006-06-14 | 暨南大学 | Glucagon kind polypeptide-1 analogue, its preparation method and application |
| CN101041693A (en) * | 2007-02-06 | 2007-09-26 | 珠海联邦制药股份有限公司 | Novel blood sugar lowing polypeptide and uses thereof |
| CN101337989A (en) * | 2008-08-28 | 2009-01-07 | 中国药科大学 | Novel glucagon-like peptide-1(GLP-1) analogues and use thereof |
| CN102180963A (en) * | 2011-04-22 | 2011-09-14 | 中国药科大学 | Glucagons like peptide-1 (GLP-1) analog and application thereof |
| CN102229668A (en) * | 2011-06-03 | 2011-11-02 | 浙江贝达药业有限公司 | GLP-1 (glucagon-like peptide-1) derivative and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| JP特表2009-523177A 2009.06.18 |
| JP特表2011-509077A 2011.03.24 |
| JP特许第4282485号B2 2009.06.24 |
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