CN102552883A - Polypeptide compound, pharmaceutical composition, its preparation method and application thereof - Google Patents
Polypeptide compound, pharmaceutical composition, its preparation method and application thereof Download PDFInfo
- Publication number
- CN102552883A CN102552883A CN201010581326XA CN201010581326A CN102552883A CN 102552883 A CN102552883 A CN 102552883A CN 201010581326X A CN201010581326X A CN 201010581326XA CN 201010581326 A CN201010581326 A CN 201010581326A CN 102552883 A CN102552883 A CN 102552883A
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- Prior art keywords
- exendin
- polypeptide
- complex
- pharmaceutical composition
- carrier polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
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- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
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- MGXWVYUBJRZYPE-YUGYIWNOSA-N incretin Chemical class C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=C(O)C=C1 MGXWVYUBJRZYPE-YUGYIWNOSA-N 0.000 description 1
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- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a polypeptide compound, which contains Exendin-4 and a carrier polypeptide. The carrier polypeptide can effectively prolong blood half-life time of Exendin-4, overcomes the present situation that Exendin-4 cannot be used in clinical practice due to its short half-life time, and has an application prospect in the field of drugs for treating diabetes and obesity. The invention also discloses a preparation method of the polypeptide compound and an application thereof. In addition, the invention further discloses a pharmaceutical composition containing the polypeptide compound.
Description
Technical field
The present invention relates to the relevant drug world of diabetes, particularly, the present invention relates to a kind of polypeptide complex, this complex has the interior half-life of body of the Exendin-4 of prolongation.The invention still further relates to the method for preparing and the application thereof of this polypeptide complex.
Background technology
What diabetes were that a kind of inherited genetic factors and multiple environmental factors be associated is the metabolism disturbance syndrome of characteristic with the chronic hyperglycemia.Because diabetes also are accompanied by many complication, have become at present and be only second to malignant tumor and cardiovascular disease the third-largest healthy killer afterwards.1984, glucagon peptide-1 (GLP-1) type medicine came to light, and it is a kind of 30 amino acid whose incretins that have.The main cause that GLP-1 is restricted as hypoglycemic drug be in the body of GLP-1 the half-life be merely 3-5 minute.
Exendin-4 is the straight-chain polypeptide that 39 aminoacid is formed, and the saliva separation and Extraction by the huge lizard in South America obtains at first.Its amino acid residue and mammal GLP-1 sequence have 52% homology; The maximum difference of itself and GLP-1 is that N holds the enzyme action effect of dipeptidyl peptidase (DPP-IV) in the 2nd the anti-blood of glycine; The same position of GLP-1 then is the alanine that is very easily cut off by DPP-IV, so the half-life that Exendin-4 is absorbed into behind the blood is grown (t
1/2=2.4 hours).Exendin-4 is a kind of strong effect GLP-1 receptor stimulating agent, can simulate the sugared regulating and controlling effect of this endogenous polypeptide of GLP-1, reduces on an empty stomach and post-prandial glycemia.The activity of Exendin-4 is mainly through mediating with human body pancreas GLP-1 receptors bind, by the synthetic and secretion of insulin that cyclic adenosine monophosphate (cAMP) relies on and β mechanism of cell differentiation initiation glucose relies on.Because the amino acid structure of Exendin-4 and biological activity and GLP-1 all have dependency, so it also is regarded as a kind of of incretin usually.
Because blood sugar reducing function time of Exendin-4 is long than GLP-1, delay gastric emptying and the inhibition impulsion of ingesting, the beneficial effect of adiposis patient body weight is different from traditional hypoglycemic medicine again, so its potentiality to be exploited as the type 2 diabetes mellitus medicine is huge.Along with Exendin-4 be activated monomer medicine (English general Exenatide by name, Exenatide, Lily) in 2005 in U.S.'s listing, Exendin-4 pays close attention to causing widely aspect the treatment diabetes as a member in the GLP-1 family.
But now the Exendin-4 medicine of listing still exists short problem of half-life, and medicinal Exenatide (Exenatide) is for injecting twice every day, greatly the patient's of increase misery.Therefore, exist at present solving the demand of the method for half-life weak point in the Exendin-4 medicine body.
Summary of the invention
An object of the present invention is that RT is shorter in vivo to Exendin-4 analog clinically, need the scarce limit of drug administration by injection every day, the long polypeptide complex that comprises carrier polypeptide and Exendin-4 of a kind of half-life is provided.
Another object of the present invention provides the method for preparing of aforementioned polypeptides complex, in addition, the application of aforementioned polypeptides complex in the medicine of preparation treatment diabetes and obesity is provided also.
Another purpose of the present invention provides the Pharmaceutical composition of a kind of polypeptide complex with Exendin-4 and carrier polypeptide as effective ingredient.
Be used to realize that the technical scheme of above-mentioned purpose is following:
On the one hand, the present invention provides a kind of polypeptide complex, and it comprises Exendin-4 and carrier polypeptide.
Wherein, said carrier polypeptide is preferably carrier polypeptide A or the carrier polypeptide B shown in sequence SEQ ID NO 2 shown in sequence SEQ ID NO 1.
In said polypeptide complex, the mol ratio of Exendin-4 and carrier polypeptide is 10: 1-1: 50, preferred 1: 2.5-1: 25, more preferably 1: 10-1: 25.
On the other hand, the present invention provides the method for preparing of aforementioned polypeptides complex, and this method comprises: according to molar ratio; Take by weighing Exendin-4 and carrier polypeptide A or B respectively; Be dissolved in an amount of normal saline, pure water or the PBS buffer, under 0-5 ℃ of temperature, ultrasonic mixing 1-15 minute; Perhaps stirred 1-3 hour, and perhaps placed and spend the night.
Again on the one hand, the present invention also provides the application of aforementioned polypeptides complex in the medicine of preparation treatment diabetes, and this polypeptide complex treats and/or prevents the application in the medicine of obesity in preparation.
Another aspect, the present invention provides a kind of pharmaceutical composition, and it comprises the aforementioned polypeptides complex.
Wherein, described pharmaceutical composition also comprises one or more acceptable accessories, and the weight ratio of polypeptide complex and adjuvant is preferably 5 in the wherein said pharmaceutical composition: 1-1: 50, further preferred 1: 1-1: 25.
And said pharmaceutical composition is preferably injection, further is preferably freeze-dried powder or injection of solution agent.
Combine the object of the invention that the present invention is described one by one at present.
(1) carrier polypeptide
Following sequence A of the sequence of carrier polypeptide of the present invention or B:
Sequence A (SEQ ID NO 1): GLWWKAWWKAWWKSLWWRKRKRKA
Sequence B (SEQ ID NO 2): GLWWKVWWKLWWKSLWWRKRLRKA
(2)Exendin-4
Exendin-4 is a natural product, and sequence is SEQ ID NO 9:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
(3) complex of Exendin-4 and carrier polypeptide
The complex of the different proportion that the ratio of Exendin-4 and carrier polypeptide: Exendin-4 (MW:4188) can form with carrier polypeptide A (MW:3329) or carrier polypeptide B (MW:3384); Calculate with mole; From 10: 1 to 1: 100; The complex that different ratios obtains has different release characteristics, and the complex that the ratio from 1: 1 to 1: 50 forms has the medicinal practicality of stronger conduct, and scope more preferably is 1: 2.5 to 1: 25.
The method for preparing of complex of the present invention: according to molar ratio, take by weighing an amount of Exendin-4 lyophilized powder, be dissolved in an amount of normal saline, pure water or the PBS buffer; Take by weighing an amount of carrier polypeptide A or B; Be dissolved in an amount of normal saline (or PBS, water), under 0-5 ℃ of temperature, ultrasonic mixing 1-15 minute; Perhaps stirred 1-3 hour, and perhaps placed and spend the night.The prepared solution that obtains can directly add adjuvant and make preparation, also can process preparation with other adjuvants again after the lyophilization.Annotate: PBS is a phosphate.
(4) Pharmaceutical composition of the present invention
The complex of Exendin-4 of containing of the present invention and carrier polypeptide can be processed Pharmaceutical composition jointly with one or more acceptable accessories, and these adjuvants comprise: water-soluble filler, PH regulator, stabilizing agent, water for injection, osmotic pressure regulator or the like.
Water-soluble filler adjuvant of the present invention is to be selected from following one or more: mannitol, low molecular dextran, sorbitol, Polyethylene Glycol, glucose, lactose, galactose etc.
The PH regulator is to be selected from following one or more: acceptable organic or inorganic bronsted lowry acids and bases bronsted lowry of nonvolatile acid such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid and potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide, sodium carbonate or potassium carbonate or physiology such as ammonium carbonate salts, sodium bicarbonate or potassium bicarbonate or bicarbonate ammonium salt and salt etc.
Stabilizing agent is to be selected from following one or more: EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, Macrogol 4000, sodium lauryl sulphate or Tris etc.Preferred sodium pyrosulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, Tris.
Osmotic pressure regulator is one or both combination of sodium chloride, potassium chloride.
(5) method for preparing of injection
Pharmaceutical composition of the present invention can be through muscle, intravenous, subcutaneous injection by way of carrying out administration, and preferred dosage form is lyophilized powder or injection of solution agent.
The method for preparing of freeze drying injection: get Exendin-4 and carrier polypeptide solution is an amount of, add water-soluble filler, stabilizing agent, osmotic pressure regulator etc., it is an amount of to add water for injection; Regulate pH value and make its dissolving to 4-8, thin up adds the 0.1-0.5% active carbon to debita spissitudo; Stirred 10-20 minute down at 0-10 ℃, the filtering with microporous membrane degerming is adopted in decarburization; Filtrating is carried out packing, adopts freeze-drying, makes the white loose block; Seal promptly and get, each specification contain Exendin-4 at 5 μ g between the 1mg.
The method for preparing of injection: it is an amount of to get Exendin-4 and carrier polypeptide solution or lyophilized powder, adds water-soluble filler, stabilizing agent, osmotic pressure regulator etc., and adding water for injection is an amount of; Regulate pH value and make its dissolving to 4-8, thin up adds the 0.1-0.5% active carbon to debita spissitudo; Stirred 10-20 minute down at 0-10 ℃, the filtering with microporous membrane degerming is adopted in decarburization; Filtrating is carried out packing, seal promptly and get, each specification contain Exendin-4 at 5 μ g between the 1mg.
(6) purposes of Pharmaceutical composition
Pharmaceutical composition of the present invention can be used in preparation Remedies for diabetes aspect.Particularly, compositions of the present invention can vein, muscle or subcutaneous injection agent form administration.Though dosage changes according to treatment target, administering mode, symptom and other factor, compositions 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 situation comprise by the condition of therapist, route of administration, age, body weight, the patient individual reaction to medicine; Order of severity of patient's symptom or the like, therefore above-mentioned dosage range is not to limit scope of the present invention by any way.
The present invention adopts the polypeptides in combination drug technique to overcome short problem of Exendin-4 half-life; In the polypeptide complex that is provided; Because the existence of carrier polypeptide makes the Exendin-4 half-life in vivo can reach more than 24 hours; The half-life of more individually dosed Exendin-4 obviously prolongs, and greatly facilitates the clinical expansion and the application of Exendin-4 (half-life is merely 2.4 hours).
Description of drawings
Below, specify embodiments of the invention in conjunction with accompanying drawing, wherein:
Fig. 1 is several kinds of blood sugar lowering experiments that contain the polypeptide complex of Exendin-4 and carrier polypeptide among the embodiment 18, wherein:
Exendin-4: natural Exendin-4;
Exendin-4+2.5A:Exendin-4 and carrier polypeptide are with 1: 2.5 complex of molar ratio;
Exendin-4+5A:Exendin-4 and carrier polypeptide are with 1: 5 complex of molar ratio;
Exendin-4+10A:Exendin-4 and carrier polypeptide are with 1: 10 complex of molar ratio;
Exendin-4+25A:Exendin-4 and carrier polypeptide are with 1: 25 complex of molar ratio;
Exendin-4+2.5B:Exendin-4 and carrier polypeptide are with 1: 2.5 complex of molar ratio;
Exendin-4+5B:Exendin-4 and carrier polypeptide are with 1: 5 complex of molar ratio;
Exendin-4+10B:Exendin-4 and carrier polypeptide are with 1: 10 complex of molar ratio;
Exendin-4+25B:Exendin-4 and carrier polypeptide are with 1: 25 complex of molar ratio.
Fig. 2 is the liquid phase separation result of the carrier polypeptide complex of Exendin-4 and different proportion, wherein
Fig. 2 A is Exendin-4, and Fig. 2 B is carrier polypeptide A, and Fig. 2 C is carrier polypeptide B, and Fig. 2 D is Exendin-4+ carrier polypeptide A, and Fig. 2 E is Exendin-4+ carrier polypeptide B.
The specific embodiment
Below in conjunction with the specific embodiment the present invention is further described in detail, the embodiment that provides has been merely and has illustrated the present invention, rather than in order to limit scope of the present invention.
In following embodiment, various processes and the method do not described in detail are conventional methods as known in the art.
Embodiment 1: the preparation of carrier polypeptide A
According to colibacillary preferred codon (but being not limited to preferred codon), utilize the synthetic base sequence that contains polypeptide A of the synthetic method of full gene, DNA sequence is following:
SEQ?ID?NO?3：
5TATG
ATTGAAGGCCGTGGCCTGTGGTGGAAAGCGTGGTGGAAAGCGTGGTGGAAATCCCTGTGGTGGCGTAAACGTAAACGTAAAGCGTAATAAG
3
SEQ?ID?NO?4：
5GATCCTTATTACGCTTTACGTTTACGTTTACGCCACCACAGGGATTTCCACCACGCTTTCCACCACGCTTTCCACCACAGGCC
ACGGCCTTCAAT
DNA sequence 3 and sequence 4 are carried out bonding, add respectively with SEQ ID NO3 and 4,95 ℃ of water-baths of SEQ ID NO of equimolar amounts and hatched 5 minutes, slowly cool to room temperature then.This double-stranded DNA can be connected by the coli expression carrier pET15b behind the NdeI/BamHI double digestion.As follows, the synthetic double-stranded DNA of said method contains the point property end of 5 ' NdeI and 3 ' BamHI, and the restriction enzyme site that other contains Factor Xa is used to remove the amino acid residue that N holds.
Enzyme incision technology among the present invention and interconnection technique are the technology of known in this area.
To make up the gained plasmid and transform B121 (DE3) competent cell, plasmid transforms and the preparation of competent cell is technology well-known in the art.Adopt general e. coli protein expression (IPTG guiding) that the polypeptide that relates among the present invention is expressed, bacterium liquid is collected precipitum after centrifugal.Precipitum is carried out fragmentation with the ultrasonic cell-break method, and centrifugal back is collected supernatant and is used the nickel post that the polypeptide among the present invention is carried out purification.Polypeptide behind the said method purification carries out Factor Xa enzyme action and is used to remove 6 * HisTag and other amino acid residues.Polypeptide behind the purification is removed the technology of knowing in these fields through ultraviolet sterilization, DNA and is carried out once more purification with the fulfilling medicinal demand.Protein purification described in the present invention, enzyme action and cryodesiccated method are knowledge well-known in the art and technology.This patent is equally applicable to use other polypeptide method for preparing, and for example chemiluminescent polypeptide is synthetic, yeast expression system and mammal expression system.
Embodiment 2: the preparation of carrier polypeptide B
According to colibacillary preferred codon (but being not limited to preferred codon), utilize the DNA sequence of the synthetic polypeptide B of the synthetic method of full gene following:
SEQ?ID?NO?5：
5TATG
ATTGAAGGCCGTGGCCTGTGGTGGAAAGTGTGGTGGAAACTGTGGTGGAAAAGCCTGTGGTGGCGCAAACGCCTGCGCAAAGCGTAATAAG
3
SEQ?ID?NO?6：
5GATCCTTATTACGCTTTGCGCAGGCGTTTGCGCCACCACAGGCTTTTCCACCACAGTTTCCACCACACTTTCCACCACAGGCC
ACGGCCTTCAAT
To make up the gained plasmid and transform B121 (DE3) competent cell, plasmid transforms and the preparation of competent cell is technology well-known in the art.Adopt general e. coli protein expression (IPTG guiding) that the polypeptide that relates among the present invention is expressed, bacterium liquid is collected precipitum after centrifugal.Precipitum is carried out fragmentation with the ultrasonic cell-break method, and centrifugal back is collected supernatant and is used Ni-NTA nickel post that the polypeptide among the present invention is carried out purification.Polypeptide behind the said method purification carries out Factor Xa enzyme action and is used to remove 6 * His Tag and other amino acid residues.Polypeptide behind the purification is removed the technology of knowing in these fields through ultraviolet sterilization, DNA and is carried out once more purification with the fulfilling medicinal demand.Protein purification described in the present invention, enzyme action and cryodesiccated method are knowledge well-known in the art and technology.This patent is equally applicable to use other polypeptide method for preparing, and for example chemiluminescent polypeptide is synthetic, yeast expression system and mammal expression system.
Embodiment 3: preparation and the purification technique of reorganization Exendin-4
Exendin-4 is a natural product, does not have patent limitation.Adopt biological method to carry out the preparation of Exendin-4 among the present invention, this patent is equally applicable to use other polypeptide method for preparing, and for example chemiluminescent polypeptide is synthetic.
SEQ?ID?NO?9：
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPS?SGAPPPS
Carry out the synthetic and preparation of coding DNA according to the aminoacid sequence (SEQ ID NO 9) of Exendin-4, two complementary dna single chains are given birth to the worker by Shanghai and are synthesized, and see sequence 7, sequence 8.And said with reference to embodiment 1, the double-stranded DNA of preparation coding Exendin-4, this double-stranded DNA has the restriction enzyme site (NdeI/BamHI) that is connected with carrier (pET15b).With reference to the content of embodiment 1, make up the expression plasmid of Exendin-4.
Reorganization Exendin-4 prepares with reference to protein expression, purification, the drying means described among the embodiment 1.Polypeptide behind the purification carries out once more purification with the fulfilling medicinal demand through the technology of knowing in ultraviolet sterilization, these fields of removal DNA.
SEQ?ID?NO?7：
5TATGATTGAAGGCCGTCATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTGCGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA?TCGTAATAAG
3
SEQ?ID?NO?8：
5GATCCTTATTACGATGGCGGAGGTGCCCCGCTACTTGGTCCTCCGTTCTTAAGCCACTCAATAAATAACCGCACTGCCTCCTCTTCCATCTGTTTTGACAAGTCACTGGTAAATGTTCCTTCACCATG
ACGGCCTTCAAT
1: 10 complex of embodiment 4:Exendin-4 and carrier polypeptide A
According to Exendin-4 and carrier polypeptide A molar ratio (following ratio is not indicated and is all mol ratio) 1: 10, take by weighing Exendin-4 (MW:4188) lyophilized powder of 1mg, be dissolved in the 1ml normal saline; Fully behind the mixing, take by weighing 7.94mg carrier polypeptide A (MW:3329) and be dissolved in the normal saline of the above-mentioned Exendin-4 of containing, fully behind the mixing; Under 0-10 ℃ of temperature, ultrasonic mixing 5 minutes obtains complex solution; If prepare preparation immediately, can directly down operate does not need lyophilizing.
10: 1 complex of embodiment 5:Exendin-4 and carrier polypeptide A
Take by weighing the Exendin-4 lyophilized powder of 4.2mg, be dissolved in the 1ml normal saline, fully behind the mixing; Take by weighing 0.33mg carrier polypeptide A lyophilized powder and be dissolved in the above-mentioned Exendin-4 normal saline, fully behind the mixing, under 0-10 ℃ of temperature; Stirred 3 hours, lyophilization obtains the complex pressed powder.
1: 2.5 complex of embodiment 6:Exendin-4 and carrier polypeptide A
Take by weighing the Exendin-4 lyophilized powder of 2.1mg, be dissolved in the 1ml pure water, fully behind the mixing; Take by weighing 4.1mg carrier polypeptide A lyophilized powder and be dissolved in the above-mentioned Exendin-4 aqueous solution, fully behind the mixing, under 0-5 ℃ of temperature; Stirred 3 hours, lyophilization obtains the complex pressed powder.
1: 25 complex of embodiment 7:Exendin-4 and carrier polypeptide A
Take by weighing the Exendin-4 lyophilized powder of 0.21mg, be dissolved in the 1ml pure water, fully behind the mixing; Take by weighing 4.1mg carrier polypeptide A lyophilized powder and be dissolved in the above-mentioned Exendin-4 aqueous solution, fully behind the mixing, under 0-5 ℃ of temperature; Stirred 3 hours, lyophilization obtains the complex pressed powder.
1: 1 complex of embodiment 8:Exendin-4 and carrier polypeptide B
Take by weighing the Exendin-4 lyophilized powder of 0.42mg; Be dissolved in 1ml PBS buffer, fully behind the mixing, take by weighing 0.34mg carrier polypeptide B (MW:3384) lyophilized powder and be dissolved in the PBS buffer of the above-mentioned Exendin-4 of containing; Fully behind the mixing; Under 0-5 ℃ of temperature, placement is spent the night, and lyophilization obtains the complex pressed powder.
1: 10 complex of embodiment 9:Exendin-4 and carrier polypeptide B
Take by weighing the Exendin-4 lyophilized powder of 0.42mg, be dissolved in the 1ml pure water, fully behind the mixing; Take by weighing 3.4mg carrier polypeptide B lyophilized powder and be dissolved in the solution of the above-mentioned Exendin-4 of containing, fully behind the mixing, under 0-5 ℃ of temperature; Placement is spent the night, and lyophilization obtains the complex pressed powder.
1: 25 complex of embodiment 10:Exendin-4 and carrier polypeptide B
Take by weighing the Exendin-4 lyophilized powder of 0.21mg, be dissolved in 1ml PBS buffer, fully behind the mixing; Take by weighing 4.25mg carrier polypeptide B lyophilized powder and be dissolved in the PBS buffer of the above-mentioned Exendin-4 of containing, fully behind the mixing, under 0-10 ℃ of temperature; Stirred 1 hour, lyophilization obtains the complex pressed powder.
1: 50 complex of embodiment 11:Exendin-4 and carrier polypeptide B
Take by weighing the Exendin-4 lyophilized powder of 0.21mg, be dissolved in 1ml PBS buffer, fully behind the mixing; Taking by weighing 8.5mg carrier polypeptide B lyophilized powder is dissolved in the PBS buffer of the above-mentioned Exendin-4 of containing; Fully behind the mixing,, place about 0 ℃ 0 ℃ of ultrasonic mixing in the left and right sides 5 minutes; Subsequent use, be used to prepare injection in 1-3 hour.
1: 100 complex of embodiment 12:Exendin-4 and carrier polypeptide B
Take by weighing the Exendin-4 lyophilized powder of 0.21mg, be dissolved in the 2ml normal saline, fully behind the mixing; Take by weighing 17mg carrier polypeptide B lyophilized powder and be dissolved in the normal saline of the above-mentioned Exendin-4 of containing, fully behind the mixing, under 0-10 ℃ of temperature; Placement is spent the night, and lyophilization obtains the complex pressed powder.
Embodiment 13: lyophilized powder type preparation of drug combination
Get an amount of container and add poloxamer 0.05g, mannitol 0.2g, lactose 0.1g, water for injection 3ml; Stirring makes dissolving; The citric acid or the sodium hydroxide that add 1mol/L are regulated PH to 6.0, are cooled to 5 ℃, and the complex solution 5ml (containing Exendin-45mg+39.7mg carrier polypeptide A) that gets the preparation of embodiment 4 methods adds wherein; Continue to transfer and mend PH to 6.0, add water to 10ml.Add the 20mg activated carbon, stirred 20 minutes down at 5 ℃, the filtering with microporous membrane degerming is adopted in decarburization; Filtrating is carried out packing by every 0.2ml, and pre-freeze is after 2 hours, and freezing drying under reduced pressure down 12 hours is to sample temperature to 5 ℃; Dry 2 hours again, make the white loose block, seal the pharmaceutical composition that promptly gets the Exendin-4 polypeptide complex, place pre-filled syringe; Specification is that 100 μ g/ prop up, and preserves below 4 ℃, dissolves the back administration with 200 μ l waters for injection before the injection.
Embodiment 14: injection of solution drug form preparation of compositions
Get an amount of container and add sorbitol 0.1g, lactose 0.1g, NaCl 20mg, citric acid 10mg, water for injection 7ml stir and make dissolving; The citric acid or the sodium hydroxide that add 1mol/L are regulated PH to 6.5; Be cooled to 0 ℃, the composite powder an amount of (containing Exendin-45mg+9.8mg carrier polypeptide A) that the method with embodiment 6 of getting makes adds wherein, continues stirring and dissolving; Transfer and mend PH to 6.5, add water to 10ml.Add the 10mg activated carbon, stirred 20 minutes down at 0-4 ℃, the filtering with microporous membrane degerming is adopted in decarburization, and filtrating is divided the precharging type syringe of packing into by every 100 μ l, preserves below the sample temperature to 5 ℃, and specification is that 50 μ g/ prop up.
Embodiment 15: lyophilized powder type preparation of drug combination
Get an amount of container and add sorbitol 0.05g, lactose 0.06g and water for injection 5ml; Stirring makes dissolving; The hydrochloric acid or the sodium hydroxide that add 1mol/L are regulated PH to 7.5, are cooled to 5 ℃, and the complex an amount of (containing Exendin-45mg+97.5mg carrier polypeptide A) of getting the preparation of embodiment 7 methods adds wherein; Continue to transfer and mend PH to 6.0, add water to 10ml.Add the 10mg activated carbon, stirred decarburization 20 minutes down at 5 ℃; Adopt the filtering with microporous membrane degerming, filtrating is carried out packing by every 1ml, and pre-freeze is after 2 hours; Freezing down drying under reduced pressure 12 hours, to sample temperature to 5 ℃, dry 5 hours again; Make the white loose block, seal and promptly get Exendin-4 polypeptide drugs complex freeze-dried powder, specification is that 500 μ g/ prop up.
Embodiment 16: injection of solution drug form preparation of compositions
Get an amount of container and add NaCl 40mg, water for injection 7ml stirs and makes dissolving; The citric acid or the sodium hydroxide that add 1mol/L are regulated PH to 6.5; Be cooled to 0 ℃, the composite powder an amount of (containing Exendin-45mg+40.5mg carrier polypeptide B) that the method with embodiment 9 of getting makes adds wherein, continues stirring and dissolving; Transfer and mend PH to 6.5, add water to 10ml.Add the 10mg activated carbon, stirred 20 minutes down at 0-4 ℃, the filtering with microporous membrane degerming is adopted in decarburization, and filtrating is divided the precharging type syringe of packing into by every 200 μ l, preserves below the sample temperature to 5 ℃, and specification is that 100 μ g/ prop up.
Embodiment 17: lyophilized powder type preparation of drug combination
Get an amount of container and add NaCl 20mg and water for injection 5ml; Stirring makes dissolving; The hydrochloric acid or the sodium hydroxide that add 1mol/L are regulated PH to 7.5, are cooled to 5 ℃, and the complex of getting the preparation of embodiment 10 methods adds wherein an amount of (being calculated as 5mg with Exendin-4 weight) (containing Exendin-4 5mg+101.2mg carrier polypeptide B); Continue to transfer and mend PH to 6.0, add water to 10ml.Add the 10mg activated carbon, stirred decarburization 20 minutes down at 5 ℃; Adopt the filtering with microporous membrane degerming, filtrating is carried out packing by every 0.5ml, and pre-freeze is after 2 hours; Freezing down drying under reduced pressure 12 hours, to sample temperature to 5 ℃, dry 5 hours again; Make the white loose block, seal and promptly get Exendin-4 polypeptide drugs complex freeze-dried powder, specification is that 250 μ g/ prop up.
Embodiment 18: polypeptide complex is in the intravital function of blood sugar reduction experiment of mice
(calculating dosage with Exendin-4 is 100 μ g/kg to give the complex of mouse mainline different proportion; Mixed proportion was respectively 1: 2.5, and 1: 5,1: 10; 1: 25; Above mixed proportion is the molar ratio of Exendin-4 and carrier polypeptide), respectively at 0.5,4,6,8,12,24 and 48 hour injectable dextrose monohydrate (3g/kg) after the administration, measure the halfhour blood sugar content in injection back; The result shows that Exendin-4 completely lost its function of blood sugar reduction after 4 hours; But but still there is function of blood sugar reduction in complex in administration after 48 hours, and the result shows that Exendin-4 and carrier polypeptide form the function of blood sugar reduction that has not only effectively kept natural Exendin-4 behind the complex, and the result sees Fig. 1.Fig. 2 has shown the blended liquid chromatograph result of the carrier polypeptide of Exendin-4 and different proportion, and the result has shown that Exendin-4 and carrier polypeptide complex are confirmed as complex (20 minutes peaks) in liquid phase.
Embodiment 19: polypeptide complex is in the intravital half-life experiment of rat
Rat injection Exendin-4 and (amounting to Exendin-4 is 1000 μ g/kg with the complex of polypeptide A, polypeptide B respectively; Mixed proportion was respectively 1: 2.5, and 1: 5,1: 10; 1: 25; Above mixed proportion is the molar ratio of Exendin-4 and carrier polypeptide), get blood about 0.2ml in eye clump vein respectively at before the injection and injection back after 0.5,1,3,6,9,12,24,36 and 48 hour, preparation serum is subsequent use.
Exendin-4 method for measurement of concentration: adopt enzyme linked immunological adsorption method (ELISA) to detect the concentration of polypeptide complex in the rat blood serum, operate as follows: 4 ℃ of centrifugal separation plasmas.(Phoenix Pharmaceuticals INC) measures the concentration of the Exendin-4 in the Mus blood plasma with the Exendin-4EIA test kit.Mus plasma sample after the dilution is added 96 orifice plates (50 μ l), add the sheep anti mouse one of 25 μ l Exendin-4 anti-after, hatched 2 hours for 37 ℃.Use washing liquid to clean 96 orifice plates 4 times, hatched 1 hour after adding 100 μ lSA-HRP.Same method is cleaned 96 orifice plates, adds 100 μ lTMB then, hatches 1 hour for 37 ℃.With measuring the OD450 value in after the 2N hydrochloric acid stopped reaction 20 minutes, and compare with the Exendin-4 of normal concentration and calculate pharmacokinetic parameter according to ELISA result.
The body giving drugs into nose of Exendin-4 and complex thereof is seen table 1 for kinetic results, and the result shows that more independent Exendin-4 of Exendin-4 complex half-life in vivo of the present invention obviously prolongs, and has long-acting characteristic.
Table 1Exendin-4 and complex thereof are in the intravital half-life of rat
| Sample source | Half-life (hour) |
| Exendin-4 | 2.4 |
| Exendin-4+ polypeptide A (1: 2.5) | 5.0 |
| Exendin-4+ polypeptide A (1: 5) | 12.2 |
| Exendin-4+ polypeptide A (1: 10) | 16.5 |
| Exendin-4+ polypeptide A (1: 25) | 24.0 |
| Exendin-4+ polypeptide B (1: 2.5) | 3.5 |
| Exendin-4+ polypeptide B (1: 5) | 9.8 |
| Exendin-4+ polypeptide B (1: 10) | 15.8 |
| Exendin-4+ polypeptide B (1: 25) | 24.5 |
Claims (10)
1. polypeptide complex, wherein, said polypeptide complex comprises Exendin-4 and carrier polypeptide.
2. polypeptide complex as claimed in claim 1, wherein, said carrier polypeptide is carrier polypeptide A or the carrier polypeptide B shown in sequence SEQ ID NO 2 shown in sequence SEQID NO 1.
3. according to claim 1 or claim 2 polypeptide complex, wherein, the mol ratio of said Exendin-4 and carrier polypeptide is 10: 1-1: 50, further preferred 1: 2.5-1: 25, more preferably 1: 10-1: 25.
4. like the method for preparing of each described polypeptide complex of claim 1-3, wherein, said method comprises: according to molar ratio; Take by weighing Exendin-4 and carrier polypeptide A or B respectively; Be dissolved in an amount of normal saline, pure water or the PBS buffer, under 0-5 ℃ of temperature, ultrasonic mixing 1-15 minute; Perhaps stirred 1-3 hour, and perhaps placed and spend the night.
5. like the application of each described polypeptide complex of claim 1-3 in the medicine of preparation treatment diabetes.
6. treat and/or prevent the application in the medicine of obesity like each described polypeptide complex of claim 1-3 in preparation.
7. pharmaceutical composition, it comprises each described polypeptide complex like claim 1-3.
8. pharmaceutical composition as claimed in claim 7, wherein, said pharmaceutical composition also comprises one or more acceptable accessories.
9. like claim 7 or 8 described pharmaceutical compositions, wherein, the weight ratio of polypeptide complex and adjuvant is 5 in the said pharmaceutical composition: 1-1: 50, preferred 1: 1-1: 25.
10. like each described pharmaceutical composition among the claim 7-9, wherein, said pharmaceutical composition is an injection, is preferably freeze-dried powder or injection of solution agent.
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Cited By (6)
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| CN102558308A (en) * | 2010-12-31 | 2012-07-11 | 天津药物研究院 | Carrier polypeptide for forming medical compositions and preparation method and application of carrier polypeptide |
| CN103833833A (en) * | 2012-11-20 | 2014-06-04 | 天津药物研究院 | Self-assembled polypeptide, preparation method and application thereof |
| CN106459170A (en) * | 2014-04-07 | 2017-02-22 | 赛诺菲 | GLP-1/glucagon receptor dual agonists derived from exendin-4 |
| CN108187060A (en) * | 2018-01-11 | 2018-06-22 | 王�琦 | Pharmaceutical carrier, pharmaceutical preparation and preparation method |
| CN109395065A (en) * | 2017-08-18 | 2019-03-01 | 中国药科大学 | A kind of polypeptide is used to prepare the purposes of slimming medicine |
| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PE20151239A1 (en) | 2012-12-21 | 2015-09-08 | Sanofi Sa | DERIVATIVES OF EXENDIN-4 FUNCTIONALIZED |
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| CN101525386A (en) * | 2008-03-05 | 2009-09-09 | 浙江华阳药业有限公司 | Fusion protein of Exendin-4 tandem polypeptide and human serum albumin, preparation and application thereof |
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| CN102558308A (en) * | 2010-12-31 | 2012-07-11 | 天津药物研究院 | Carrier polypeptide for forming medical compositions and preparation method and application of carrier polypeptide |
| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
| CN103833833A (en) * | 2012-11-20 | 2014-06-04 | 天津药物研究院 | Self-assembled polypeptide, preparation method and application thereof |
| CN106459170A (en) * | 2014-04-07 | 2017-02-22 | 赛诺菲 | GLP-1/glucagon receptor dual agonists derived from exendin-4 |
| CN106459170B (en) * | 2014-04-07 | 2020-08-04 | 赛诺菲 | G L P-1/glucagon receptor dual agonists derived from exendin-4 |
| CN109395065A (en) * | 2017-08-18 | 2019-03-01 | 中国药科大学 | A kind of polypeptide is used to prepare the purposes of slimming medicine |
| CN108187060A (en) * | 2018-01-11 | 2018-06-22 | 王�琦 | Pharmaceutical carrier, pharmaceutical preparation and preparation method |
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