CN108341880A - The chimeric polyeptides of fragment analogue and application thereof containing glucagon-like-peptide-1 and glucagon - Google Patents

The chimeric polyeptides of fragment analogue and application thereof containing glucagon-like-peptide-1 and glucagon Download PDF

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Publication number
CN108341880A
CN108341880A CN201810043273.2A CN201810043273A CN108341880A CN 108341880 A CN108341880 A CN 108341880A CN 201810043273 A CN201810043273 A CN 201810043273A CN 108341880 A CN108341880 A CN 108341880A
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xaa
arg
gln
chimeric polyeptides
glu
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CN108341880B (en
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韩英梅
赵娜夏
王玉丽
夏广萍
刘冰妮
孔维苓
石玉
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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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The present invention provides a kind of chimeric polyeptides of the fragment analogue comprising glucagon-like peptide 1 and glucagon, and the sequence of the polypeptide is as shown in following general formula I：General formula I：HX2EGTFTSDYSKYLX15X16X17AA X20X21FX23X24WLVKX29X30X31X32.The present invention also provides the conjugate of the chimeric polyeptides and polymer, wherein the polymer is to be formed by connecting by Cys residue covalents by above-mentioned chimeric polyeptides or be covalently attached by connector.The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition includes above-mentioned chimeric polyeptides, the conjugate of above-mentioned chimeric polyeptides, polymer.Chimeric polyeptides provided by the invention have significant hypoglycemic effect and the effect that loses weight, its drug effect and long-term effect are better than positive control drug, and dissolubility and stability are good, are more suitable for preparation, can be used for preparing the medicine of the metabolic diseases such as diabetes and obesity.

Description

Fragment analogue containing glucagon-like-peptide-1 and glucagon it is chimeric more Peptide and application thereof
It is entitled " containing glucagon-like-peptide-1 and glucagon this application claims submitting January 23 within 2017 Chimeric polyeptides of fragment analogue and application thereof ", application No. is the excellent of 201710050234.0 Chinese invention patent application First weigh.
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, and in particular to one kind containing glucagon-like-peptide-1 and pancreas hyperglycemia Chimeric polyeptides, its conjugate, polymer and the chimeric polyeptides, its conjugate, the polymer of the fragment analogue of element are being made Be ready for use on prevention and/or treat diabetes, obesity and/or metabolic syndrome drug in purposes.
Background technology
Fat and diabetes B (T2DM) is one of the major public health problem for perplexing modern society.Fat and its companion With insulin resistance be diabetes B morbidity key factor, according to investigations, the diabetes B patient of 80-90% is overweight or fertile Fat (Zou great Jin etc., Shanghai medicine, 2014,37 (9), 729~734).Therefore, effectively control blood glucose and weight are related grind always Study carefully the focus project in field.
In past more than ten year, enteron aisle hormone especially acts on controlling for glucagon-like-peptide-1 (GLP-1) access Drug is treated to be widely recognized as by industry.Glucagon-like-peptide-1 (GLP-1) is a kind of Entero hormone, mainly by being located at end The L cells synthesis for holding jejunum, ileum and colon, the circulatory system is discharged into reaction of having meal.GLP-1 (7-36,7-37) is body The chief active form of GLP-1 in cycle includes point of insulin and glucagon by complicated mechanism control blood glucose It secretes, the emptying of stomach and the adjusting of periphery insulin.GLP-1 (7-36,7-37) hypoglycemic effect is glucose dependency, can Hypoglycemia is avoided, and with the apoptosis for inhibiting pancreaticβ-cell, promotes the effect of hyperplasia of pancreatic islet beta cell, disease can be reversed Feelings develop.But the plasma half-life of natural GLP-1 is only 1-2 minutes, to limit its application as drug.Research shows that Receptor-binding activity position N-terminal His-Ala in internal dipeptides kininase (DPPIV) specific recognition and GLP-1 structures of degrading Dipeptide fragment, and make its rapid deactivation, while other proteolytic enzymes such as endopeptidase etc. also assists in kidney filtration reset procedure.
Substantially pass through structure of modification to improve metabolic stability based on the people source GLP-1 medicament research and developments carried out, extends blood For the purpose of starching half-life period, disclosed patented technology can be attributed to：1) it is directed to the structure of modification of enzyme degradation critical sites (specially such as China Disclosed in profit application CN00806548.9, CN99814187.9, CN200410017667.9 etc.)；2) parent peptide chain is covalently even Join fatty acyl group, improves and remove (such as Chinese patent with plasma protein binding force to avoid quick in vivo Disclosed in CN201210513145.2, CN200810124641.2, CN20118000352.1 etc.)；3) albumen coupling/fusion Technology；4) PEG modifications etc..The polypeptide that molecular weight is less than 5000 dalton is made from internal main eliminating rate for glomerular filtration With, therefore be to extend in GLP-1 receptor stimulating agent class polypeptide drugs extension bodies partly to decline to increase molecular weight and volume by modifying The technical way of phase.And to have more druggability excellent for the trim of high-activity mother body polypeptide when taking these structural modification measures Gesture.
The receptor of the enteron aisles hormone such as the glucagon (GC) in preceding glucagon source, GLP-1, GLP-2 is G-protein Coupled receptor, and these ligands are combined with receptor in the form of α-helixstructure.Studies have shown that the tendency of α-helixstructure It is proportionate (Biochemistry, 46,5830-5840) with the binding ability of hGLP-1R, therefore, in addition to receptor swashs in structure Except dynamic necessity segment, support that the α-helixstructure segment that it is combined with receptor is also to influence ligand polypeptide bioactivity intensity Key factor.People source GLP-1 structures include the 7-13 segments that the ends N- irregularly crimp and are not connected by what Gly22 flexible was connected Two sections of continuous α-helixstructures (13-20,24-36).Exenatide (Exendin-4) is the GLP-1 receptor agonisms listed earliest Agent is derived from the polypeptide of America gilamonster Yi salivas, because of the segment (11-27) of continuous α spirals in its structure and is conducive to Formed and stable alpha spiral the ends C-, compare people source GLP-1 have stronger receptor agonist activity (Biochemistry, 46, 5830-5840).Either short-acting product or the durative action preparation of weekly administration 1 time, dosage are both less than based on people source GLP- 1 sequence changes structure kind, such as Liraglutide (15 μ g Exenatide of 0.8-1.8mg vs).But Exenatide and people source GLP- 1 only 53% sequence homology and there is immunogenicity, so that 41% tested is resisted in patient body 30 Zhou Houhui are administered Body (Diabets Care, 2004,27,2628-2635).
Glucagon (GC) is 33 to 61 amino acid by corresponding to preceding glucagon (pro-glucagon) The derived peptide containing 29 amino acid of composition, amino acid sequence are：HSQGTFTSDYSKYLDSRRAQDFVQWLMNT.GC tri- The X-ray diffraction structure of aggressiveness crystal is studies have shown that 10-25 segments are in continuous alpha-helix secondary structure in its sequence (Nature 1975,257,751-757)。
However, the drug for being currently based on people source GLP-1 research and development needs in activity, stability and immunogenicity etc. In improvement.
Invention content
Based on the deficiencies of the prior art, present inventors have surprisingly found that, GLP-1 sequences are replaced with the specific fragment in GC structures The specific fragment of row, and the chimeric polyeptides for carrying out specific amino acid replacement acquisition have selectivity GLP-1 receptor agonist activities, Its activity is better than endogenous GLP-1, and also shows good hypoglycemic effect and enzyme metabolic stability, can be used as and be used for Effectively control blood glucose and the therapeutic agent with more potentiality to be exploited to lose weight.
Therefore, the purpose of the present invention is to provide the embedding of a kind of glucagon-like-peptide-1 and glucagon fragment analogue Close polypeptide, its conjugate, polymer.The chimeric polyeptides of the present invention are selective GLP-1 receptor stimulating agents, and activity is better than endogenous Property GLP-1, there is better metabolic stability, modified to be more suitable for long-actingization, and chimeric polyeptides provided by the invention Sequence is formed has high homology (＞ 80%) with endogenous GLP-1 and GC, it is possible to reduce immunogenicity, more application are latent Power.
On the one hand, the present invention provides a kind of the embedding of the fragment analogue comprising glucagon-like-peptide-1 and glucagon Close polypeptide, wherein the sequence of the chimeric polyeptides is as shown in following general formula I：
General formula I：
HX2EGTFTSDYSKYLX15X16X17AAX20X21FX23X24WLVKX29X30X31X32
Wherein：
X2For D-Ala, Gly or Aib (2- aminoisobutyric acids)；
X15For Asp or Glu；
X16For Glu or Aib；
X17For Arg or Gln；
X20For Gln or Lys；
X21For Glu or Asp；
X23For Ile or Val；
X24For Ala, Gln or Cys；
X29For Gly or Aib；
X30For Arg or Cys；
X31For Gly, Cys ,-NH2Or it is not present；
X32For Cys ,-NH2Or it is not present；
Also, in the sequence shown in general formula I, has and only there are one Cys residues；The sequence shown in the general formula I is residual containing Aib When base, X2、X16And X29In three sites only there are one site be Aib.
Preferably, in the sequence shown in general formula I, X2For D-Ala or Aib, and X16For Glu or Aib；It is highly preferred that In sequence shown in general formula I, X2For Aib, and X16For Glu；
Preferably, the sequence of the chimeric polyeptides such as SEQ ID NO:In 1-42 shown in any bar；
It is further preferred that the sequence of the chimeric polyeptides such as SEQ ID NO:In 16-36 shown in any bar；
On the other hand, the present invention provides the conjugates of the chimeric polyeptides, it is preferable that the conjugate is described chimeric The carbowax modifier of polypeptide is covalently attached polyethylene glycol on the side chain of the Cys residues of the chimeric polyeptides；More preferably The average molecular weight on ground, the polyethylene glycol is 5-50KD；More preferably 20-45KD；Preferably, the polyethylene glycol is straight Chain or branched chair polymacrogol；It is highly preferred that the polyethylene glycol is the branched chain type polyethylene glycol of molecular weight 40-45KDa.
On the other hand, the present invention also provides the polymer of the chimeric polyeptides, it is preferable that the polymer is described embedding The dimer of polypeptide is closed, the dimer is that the chimeric polyeptides are formed by connecting or covalent by connector by Cys residue covalents It is formed by connecting；
Preferably, the dimer reacts with the sulfydryl of polypeptide by being connected with the connector of bifunctional amine's crosslinked group to be formed；
Preferably, the connector is bismaleimide-polyethylene glycol (Mal-PEG-Mal)；
It is highly preferred that the polyethylene glycol average molecular weight range is 3-20KD；
Preferably, the dimer is homodimer；
In another aspect, the present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition include above-mentioned chimeric polyeptides, Its conjugate, polymer.
Preferably, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material.
It is highly preferred that the carrier and/or auxiliary material be selected from water-soluble filler, pH adjusting agent, stabilizer, water for injection or It is one or more in osmotic pressure regulator；
Preferably, the water-soluble filler is selected from mannitol, D-40, sorbierite, polyethylene glycol, grape It is one or more in sugar, lactose or galactolipin；The pH adjusting agent is selected from physiologically acceptable organic acid or inorganic acid, example Such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid or physiologically acceptable inorganic base, such as potassium hydroxide, hydroxide It is one or more in sodium, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, saleratus, sodium bicarbonate, bicarbonate ammonium salt； The stabilizer is selected from EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, carbon Sour sodium, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxyl Cellulose or derivatives thereof such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trihydroxy methyl amino It is one or more in methane；The osmotic pressure regulator is sodium chloride and/or potassium chloride.
Yet another aspect, the present invention provides include above-mentioned chimeric polyeptides, its conjugate, polymer or said medicine group Object is closed to prepare for treating the purposes in diabetes, obesity and/or the drug of metabolic syndrome.
Preferably, composition of the present invention can be vein, muscle or subcutaneous injection agent form, or oral, rectum, The form of nasal-cavity administration.Dosage range can be 5 μ g-10mg/ time, this depending on treatment object, administering mode, indication and Other factors etc..
For a better understanding of the present invention, detailed description are as follows：
The basic structure of glucagon-like-peptide-1 and glucagon segment chimeric polyeptides
Endogenous GLP-1 receptor stimulating agents are GLP-1 (7-36/37), and polypeptid acid sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (7-36) or HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (7-37)； And the polypeptide sequence of endogenous glucagon is：HSQGTFTSDYSKYLDSRRAQDFVQWLMNT(1-29).
In embodiments of the invention, chimeric polyeptides are the 12-26 segments by GLP-1 (7-36/37) sequence with GC sequences The long short-movie section of difference of the 6-24 of row is replaced and is obtained.The ends N- are easy by dipeptidyl peptidase in GLP-1 (7-36/37) original sequences (DPP-IV) it identifies, degradation removes the dipeptides of His-Ala and loses activity.The inventors discovered that by the X of chimeric polyeptides2It replaces position It is changed to D-Ala or Aib, greatly improves the resistance degraded to DPP-IV.The present inventors have additionally discovered that by from GC's (16-20) segment is suitably changed, such as by Ser16Replace with Glu, Arg18Replace with Ala, greatly increase GLP-1 by The combination activity of body.The present inventor further has found, the C- terminal carboxyl amidations in the application sequence are helped to improve Polypeptide stability.
By above structure design, polypeptide and the endogenous GLP-1 (7- of the sequence provided by the invention as shown in general formula I 36/37) it compares, has the following advantages that：
1) polypeptide of the invention is easier to form continuous α-helixstructure in interior environment, to further enhance it Activity in vivo；
2) polypeptide of the invention is not easy to be identified by DPP-IV, is inactivated so as to avoid being degraded by DPP-IV enzymes；
3) polypeptide of the invention not only has GLP-1 receptors the agonist activity of enhancing, but also is selective GLP-1 receptors Agonist, simultaneously internal stability be improved, be more advantageous to long-actingization modification.
4) 24 or the ends C- are replaced or are introduced Cys residues and do not influence the work of polypeptide in polypeptide sequence provided by the invention Property, it may select for decorating site.And it is known to those skilled in the art that C- terminal carboxyl amidations contribute to polypeptid system Row are stablized, and therefore, the C- terminal carboxyl groups that the present invention still further provides particular sequence form amide.
Pegylation
Peptide hormone mainly removes approach metabolism in body by enzyme degradation and kidney, and wherein kidney removing accounts for leading, is Influence the principal element of polypeptide drug Half-life in vivo.Structural modification in polypeptide sequence on some amino acid residue sides is special Not being the conjugated of the macro-radicals such as alkylation or polyethylene glycol can delay kidney to remove, and effectively extend biological half-life.
In the particular embodiment of the present invention, there is I sequences polypeptide of general formula, X24、X30、X31、X32A position in site Point may alternatively be Cys, and covalently conjugated polyethylene glycol (PEG) group is pinpointed on the thiol side chain of Cys.The PEG refers to The linear chain or branched chain polyethylene glycol of average molecular weight range 20-50KD, preferred straight chain mono methoxy in embodiment of the present invention PEG。
Polyethylene glycol of the present invention is can to obtain through a variety of ways, including commercial sources obtain or according to this Known method is voluntarily prepared in field.The general formula of branched chain type polyethylene glycol in embodiment of the present invention for modification is Mal-(mPEG)2, it is preferably but not limited to selected from such as lower structure：
PEG of the present invention, which is modified, to be realized by any method well known in the art, including via acylation, reproducibility Alkanisation, Michael additions, mercaptan alkanisation pass through the active group of peg moiety (such as aldehyde, amino, ester, mercaptan, alpha-halogenate second Acyl group, dimaleoyl imino or diazanyl) other chemo-selective conjugation methods.In the particular embodiment of the present invention, lead to Michael addition reactions are crossed, that is, uses the thiol side chain of the PEG modifications Cys of maleimide activation, generates the poly- of thioether key connection Glycation polypeptide.In certain embodiments, by nucleophilic substitution, i.e., sulphur is modified with the PEG that haloacetyl activates Alcohol obtains the PEGylated polypeptides of thioether key connection.
Dimer
The dimer or multimeric forms of polypeptide are also the effective means for extending polypeptide Half-life in vivo.The present invention also provides The dimeric forms of the polypeptide.Include by the thiol side chain in sequence on 24 Cys residues with intermolecular disulfide bond shape At homodimer or via the covalently bound dimeric forms of connector.These polypeptide dimers are by forming steric hindrance It effectively shields the restriction enzyme site in peptide chain and active peptides is delayed to degrade.
In specific embodiments, the side chain that the single chain polypeptide of I sequential structure of general formula passes through cysteine residues in structure Sulfhydryl oxidase forms intermolecular disulfide bond.Routine techniques well-known in the art, including sky can be used in disulfide bond formation method Gas oxidation, glutathione, K3Fe(CN)6, I2, DMSO oxidizing process etc..
The dimer is the connector and monomer by being connected with bifunctional amine's crosslinked group in some specific embodiments The sulfydryl of peptide chain reacts to be formed.The connector is but not limited to bismaleimide-polyethylene glycol (Mal-PEG-Mal), described Polyethylene glycol average molecular weight range is but not limited to 3000-20000 dalton.
Activity
In embodiments of the present invention, by confirming offer of the present invention to the external agonist activity experiment of GLP-1, GC receptor Glucagon-like-peptide-1 and glucagon fragment analogue chimeric polyeptides are the selective agonists of GLP-1 receptors, to GC Receptor does not influence, and is better than endogenous ligands GLP-1 (7-36) to the agonist activity of GLP-1 receptors.It is repaiied by PEG long-actingization The polypeptide of decorations in vitro receptor active evaluation in show with the comparable receptor agonist activity intensity of endogenic ligand, illustrate this hair The precursor polypeptide sequence of bright offer is more advantageous to the bioactivity for ensureing long-actingization modified body.
Purposes
For the limitation of the existing drug therapies such as current treatment diabetes, obesity, metabolic syndrome, the present invention provides one Kind novel method for the treatment of, the method are related to giving the drug of the chimeric peptide comprising the present invention, its conjugate, polymer or its salt Composition.Including the chimeric polyeptides of the present invention, its conjugate, polymer or its salt pharmaceutical composition be effectively reduced blood glucose While have and promote loss of weight and to prevent effect of gain, with unexpected beneficial effect with existing drug compared with.
Pharmaceutical composition
In another aspect, the present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition includes above-mentioned chimeric polyeptides Or its salt, its conjugate, polymer.
Preferably, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material.
It is highly preferred that the carrier and/or auxiliary material include water-soluble filler, pH adjusting agent, stabilizer, water for injection, It is one or more in osmotic pressure regulator.
Preferably, the water-soluble filler includes but not limited to mannitol, D-40, sorbierite, poly- second Glycol, glucose, lactose, galactolipin etc.；The pH adjusting agent includes but not limited to citric acid, phosphoric acid, lactic acid, tartaric acid, salt The organic or inorganic acids such as acid and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, bicarbonate Physiologically acceptable inorganic base or the salt such as potassium, sodium bicarbonate, bicarbonate ammonium salt；The stabilizer includes but not limited to EDTA- 2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, Glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or derivatives thereof as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane etc.；The osmotic pressure tune It includes but not limited to sodium chloride or potassium chloride to save agent.
Yet another aspect, the present invention provides above-mentioned chimeric polyeptides to prepare for treating diabetes, obesity and/or metabolism Purposes in the drug of syndrome.
Preferably, composition of the present invention can in the form of vein, muscle or subcutaneous injection agent or oral, rectum, Nasal-cavity administration.Dosage range can be 5 μ g-10mg/ time, this depending on treatment object, administering mode, indication and other because Element etc..
Synthesis
It is by means commonly known in the art in the basic peptide chain of the chimeric polyeptides provided by the invention with logical structure shown in formula I It is prepared：
1) synthesis gradually or by segment is assembled by conventional solid or liquid phase process；
2) expression encodes the nucleic acid construct of polypeptide in host cell, and recycles expression production from host cell cultures Object；
3) the cell free in vitro expression of the nucleic acid construct of coding polypeptide is influenced, and recycles expression product；
Or pass through method 1), 2) arbitrary combination or 3) obtain peptide fragment, then connect these segments to obtain target Peptide.
Preferably, target peptide is prepared using Fmoc solid phase synthesis process.
Preferably, the pegylation of target polypeptides is completed by the following method：By PEG and this hair through overactivation Bright polypeptide is reacted at pH5.0-7.0, and the molar ratio of wherein PEG and peptide is 1-10, and the reaction time is 0.5-12 hours, instead It is 4-37 DEG C to answer temperature.
After conjugation reaction, target product can be detached by appropriate method well known in the art.Applicable method packet Include but be not limited to ultrafiltration, dialysis or chromatography etc..
Preferably, the dimeric forms of polypeptide of the present invention are the sequences polypeptide such as SEQ ID NO indicated by general formula I: Any monomer in 1-42 connects in such a way that the side chain thiol of cysteine residues in structure aoxidizes and to form intermolecular disulfide bond It connects.Disulfide bond formation method can be used routine techniques well-known in the art, including air oxidation, glutathione, K3Fe(CN)6、I2, DMSO oxidizing process etc..
Activity rating
It is had rated by the receptor-mediated external cAMP of the GLP-1/GC influences generated in embodiment of the present invention described Effect of the polypeptide to GLP-1/GC receptors.
It is tested using glucose tolerance in mice in embodiment of the present invention, using Exenatide as positive control drug, has rated this Invention provides the hypoglycemic activity of polypeptide.
It is repaiied using high fat diet obesity mice (Dio) model evaluation PEG in another embodiment of the invention Adorn the hypoglycemic effect of polypeptide and the influence to weight, the results showed that polyethyleneglycol modified polypeptide provided by the invention has notable Hypoglycemic effect and losing weight effect, drug effect and long-term effect are better than positive control drug, and dissolubility and stability are good, It is more suitable for preparation, can be used for preparing the control of metabolic diseases and the medicines of diabetes such as obesity.
Description of the drawings
Hereinafter, illustrate the embodiment of the present invention in conjunction with attached drawing, wherein
Fig. 1 is in embodiment 8 such as SEQ ID NO:18, the PEG40KD conjugate for drug delivery of polypeptide sequence shown in 40 two weeks The evaluation result that Dio mouse weights are influenced.
Fig. 2 is in embodiment 9 such as SEQ ID NO:18, the PEG40KD conjugate for drug delivery of polypeptide sequence shown in 40 two weeks The evaluation result that Dio mouse blood sugars are influenced.
Fig. 3 is the evaluation that branched chain type (Y) PEG modified polypeptides pg016, pg019 influences Dio mouse weights in embodiment 11 As a result.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.The present embodiment is only to explain the present invention, unexpectedly Taste limit the invention in any way content.
The explanation of amino acid abbreviations：
Gly：Glycine (G)
Ala：Alanine (A)
Val：Valine (V)
Leu：Leucine (L)
Phe：Phenylalanine (F)
Trp：Tryptophan (W)
Ser：Serine (S)
Thr：Threonine (T)
Glu：Glutamic acid (E)
Gln：Glutamine (Q)
Asp：Aspartic acid (D)
Asn：Asparagine (N)
Tyr：Phenylalanine (Y)
Arg：Arginine (R)
Lys：Lysine (K)
His：Histidine (H)
Aib：2- aminoisobutyric acids
The explanation of reagent abbreviation
Boc:Tert-butoxycarbonyl
Tert-Bu:Tertiary butyl
DCM:Dichloromethane
DIC：Diisopropylcarbodiimide
Fmoc:9- fluorenes methoxycarbonyls
HoBt:I-hydroxybenzotriazole
HBTU:2- (1H- benzotriazole -1- bases) -1,1,3,3- tetramethyls-urea hexafluorophosphoric acid ester
HATU:O- (7- azepine benzos triazol-1-yl)-N, N, N ', N '-tetramethyls-urea hexafluorophosphoric acid ester
Mtt:4- methyltrityls
NMP:N-Methyl pyrrolidone
DMF:Dimethylformamide
Pbf:2,2,4,6,7- pentamethyl Dihydrobenzofuranes
Trt:Trityl group
EDT:Dithioglycol
TFA:Trifluoroacetic acid
TIS:Tri isopropyl silane
FBS:Fetal calf serum
Embodiment 1
Polypeptide shown in general formula I is prepared by following step
1) it synthesizes：Using Fmoc strategies, with 336 type Peptide synthesizers of CS (CS Bio), gradually close in accordance with the following steps At：
A) it is coupled to obtain Fmoc- by resin solid phase carrier and fmoc-protected C-terminal amino acid in the presence of activator systems Amino acid-resin；Wherein, synthesis C- end amidation polypeptides use amino resins, such as Rink Amide AM, Rink Amide, Rink MBHA etc..
B) extension of peptide chain：By solid-phase synthesis according to peptide sequence amino acid sequence connect amino acid, obtain the ends N- and Peptide-resin conjugate of side chain protection；Band side chain amino acid takes following safeguard measure：Tryptophan Boc, glutamic acid are used OtBu, lysine Boc, glutamine Trt, tyrosine tBu, serine Trt or tBu, aspartic acid OtBu, Soviet Union Propylhomoserin tBu, cysteine Trt, histidine Trt or Boc, arginine are protected with Pbf.The coupling activator used is HOBT/HBTU/DIEA and HOBT/HATU/DIEA, ninhydrin method detect reaction efficiency.
C) on resin polypeptide cracking：TFA/EDT/TIS/H2O(92.5:2.5:2.5:2.5v/v) solution is set at room temperature React 90min, deprotection and deresination.Filtrate is filtered to obtain, precipitates thick polypeptide with excess diethyl ether, precipitation is collected in centrifugation, then with less Ether washing is measured to precipitate, it is dry under vacuum, obtain crude product polypeptide.Deprotection base and resin simultaneously, obtain crude product glucagon Sample peptide -1 and glucagon fragment analogue chimeric polyeptides；
2) it purifies：By crude product glucagon-like-peptide-1 and glucagon fragment analogue chimeric polyeptides be dissolved in water or 10-15% acetonitriles (10-50mg/ml), the disulfide group threitol DTT or beta -mercaptoethanol that 50-100mM is added are denaturalized, are adopted With preparative HPLC method, C18 chromatographic columns, acetonitrile-water-trifluoroacetic acid system isolates and purifies, and concentrates, and it is free to obtain sulfydryl for freeze-drying Sterling polypeptide.
Following SEQ ID NO are prepared in method described above:Polypeptide shown in 1-42.
SEQ ID NO:1H(d-A)EGTFTSDYSKYLDERAAQEFICWLVKGRNH2
SEQ ID NO:2H(d-A)EGTFTSDYSKYLDERAAQDFVCWLVKGRG
SEQ ID NO:3H(d-A)EGTFTSDYSKYLEAibRAAQEFIAWLVKGRCNH2
SEQ ID NO:4H(d-A)EGTFTSDYSKYLEERAAQEFIAWLVKGRGC
SEQ ID NO:5H(d-A)EGTFTSDYSKYLEAibRAAQEFVCWLVKGRNH2
SEQ ID NO:6H(d-A)EGTFTSDYSKYLDAibRAAQEFVQWLVKGRGC
SEQ ID NO:7H(d-A)EGTFTSDYSKYLEAibRAAQEFIAWLVKGCNH2
SEQ ID NO:8H(d-A)EGTFTSDYSKYLDERAAQEFICWLVKAibRNH2
SEQ ID NO:9HGEGTFTSDYSKYLDERAAQEFICWLVKAibRG
SEQ ID NO:10HGEGTFTSDYSKYLEERAAQEFIAWLVKGRGC
SEQ ID NO:11HGEGTFTSDYSKYLDAibRAAQEFIAWLVKGRGCSEQ ID NO: 12HGEGTFTSDYSKYLEERAAQEFIAWLVKAibRCNH2
SEQ ID NO:13HGEGTFTSDYSKYLDAibRAAQEFIAWLVKGRCNH2
SEQ ID NO:14HGEGTFTSDYSKYLDAibRAAQEFICWLVKGR NH2
SEQ ID NO:15HGEGTFTSDYSKYLEAibRAAQEFICWLVKGRNH2
SEQ ID NO:16HAibEGTFTSDYSKYLDERAAQEFICWLVKGRNH2
SEQ ID NO:17HAibEGTFTSDYSKYLEERAAQEFIAWLVKGRCNH2
SEQ ID NO:18HAibEGTFTSDYSKYLDERAAQEFIAWLVKGRCNH2
SEQ ID NO:19HAibEGTFTSDYSKYLEERAAQEFICWLVKGRNH2
SEQ ID NO:20HAibEGTFTSDYSKYLDERAAQEFIAWLVKGCNH2
SEQ ID NO:21HAibEGTFTSDYSKYLEERAAQEFICWLVKGRG
SEQ ID NO:22HAibEGTFTSDYSKYLDERAAQEFIAWLVKGRGC
SEQ ID NO:23HAibEGTFTSDYSKYLEERAAQDFVQWLVKGRCNH2
SEQ ID NO:24HAibEGTFTSDYSKYLDERAAQEFVQWLVKGRCNH2
SEQ ID NO:25HAibEGTFTSDYSKYLDERAAQDFVQWLVKGCNH2
SEQ ID NO:26HAibEGTFTSDYSKYLEERAAQEFVCWLVKGRNH2
SEQ ID NO:27HAibEGTFTSDYSKYLDERAAQEFVQWLVKGRGC
SEQ ID NO:28HAibEGTFTSDYSKYLDERAAQEFVCWLVKGRNH2
SEQ ID NO:29HAibEGTFTSDYSKYLEERAAQDFVCWLVKGRNH2
SEQ ID NO:30HAibEGTFTSDYSKYLDERAAQDFVCWLVKGRNH2
SEQ ID NO:31HAibEGTFTSDYSKYLEERAAQEFVCWLVKGRG
SEQ ID NO:32HAibEGTFTSDYSKYLDERAAQEFVCWLVKGRG
SEQ ID NO:33HAibEGTFTSDYSKYLEERAAQDFVCWLVKGRG
SEQ ID NO:34HAibEGTFTSDYSKYLDERAAQDFVCWLVKGRG
SEQ ID NO:35HAibEGTFTSDYSKYLEERAAQDFVQWLVKGRGC
SEQ ID NO:36HAibEGTFTSDYSKYLEERAAQEFVQWLVKGRGC
SEQ ID NO:37HAibEGTFTSDYSKYLDEQAAKEFICWLVKGRNH2
SEQ ID NO:38HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRCNH2
SEQ ID NO:39HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGCNH2
SEQ ID NO:40HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRGC
SEQ ID NO:41HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRCNH2
SEQ ID NO:42HAibEGTFTSDYSKYLDEQAAKEFIAWLVKGRGCNH2
Embodiment 2
The preparation of the carbowax modifier of polypeptide
1) it connects：
Maleoyl functionalization：It will be such as SEQ ID NO:Polypeptide shown in 3 is dissolved in the 50mM phosphorus of the pH6 containing 5mM EDTA In sour sodium buffer solution, a concentration of 2mg/mL.The solid PEG- maleimides of 1.2-1.5 times of mole are added, stirring is molten Solution, in room temperature reaction 2hr.Reaction is monitored with HPLC, is terminated and is reacted with 5mM beta -mercaptoethanols, purified after setting room temperature 30min.
Iodoacetyl functionalization：Pass through the mPEG (1 of peptide precursor and iodoacetyl functionalization:1) in 7M ureas/50mM Tris It is polyethyleneglycol modified that reaction completion in 45 minutes is stirred at room temperature in buffer solution (pH7.5-8.5), between the Cys on PEG and peptide chain Form covalent thioether key.
2) it purifies:Using preparative ion-exchange chromatography, with SP SepharoseHP fillers, with 0-500mM sodium chloride PH6 phosphate buffer linear gradient elutions.Efflux collects PEG- polypeptide flow points with HPLC and SDS- electrophoresis detections, It is concentrated by ultrafiltration, is freeze-dried and obtains.
3) sterling polypeptide carries out full molecular weight scanning through MILD-Q-TOF, determines average molecular weight.
Polypeptide carbowax modifier shown in following table is prepared for using the above method.
1 polypeptide carbowax modifier of table
Embodiment 3
The preparation of dimer (disulfide bond connection)
GLP-1 analogs monomer is dissolved in debita spissitudo (1.5-2mmol/L) in deionized water, according to ammonium hydrogen carbonate Method or DMSO methods form dimer, are purified by purification process in embodiment 1, obtain GLP-1 analog dimer sterlings.
It is prepared for following dimer according to the method.
SEQ ID NO:1 homodimer
SEQ ID NO:3 homodimer
SEQ ID NO:13 homodimer
SEQ ID NO:15 homodimer
SEQ ID NO:17 homodimer
SEQ ID NO:18 homodimer
SEQ ID NO:19 homodimer
SEQ ID NO:22 homodimer
SEQ ID NO:26 homodimer
SEQ ID NO:28 homodimer
SEQ ID NO:32 homodimer
SEQ ID NO:36 homodimer
SEQ ID NO:38 homodimer
SEQ ID NO:40 homodimer
SEQ ID NO:41 homodimer
Embodiment 4
The preparation of Cys- bismaleimide dimers
By the SEQ ID NO of 0.05mmol:The span of the average molecular weight 3500 of 1 monomer polypeptide and 0.15-0.2mol comes Acid imide polyethylene glycol is dissolved in the phosphate buffer containing 0.01mM EDTA of pH6, after stirring 16 hours at room temperature, Continue the monomer polypeptide that 0.05mmol is added in the reaction system, continue to be stirred to react 16-20 hours, monitored and reacted with HPLC, It is terminated and is reacted with 5mM beta -mercaptoethanols, set room temperature 30min.Using preparative ion-exchange chromatography, with SP SepharoseHP Filler, with 0-500mM sodium chloride solution linear gradient elutions.Efflux collects PEG- polypeptides with HPLC and SDS- electrophoresis detections Flow point is concentrated by ultrafiltration, is further purified with preparative HPLC, and corresponding flow point concentration, freeze-drying obtains target dimer.Same method SEQ ID NO are prepared:15,18,24,28,32,40,42 homodimer connected by bismaleimide.
Embodiment 5
The polypeptide of the present invention is in vitro to the stability of DPP-IV degradation
Given the test agent is total in 50mmol/L TEA-HCl (pH7.8) with the pig DPP-IV of purifying (5 milliunit) in 37 DEG C It incubates 0,2,4,8 hour.
HPLC method (chromatographic columns：3.6 μm of Aeris widepore XB-C18,4.6 × 150mm；Mobile phase：A:0.05% TFA, B:95% acetonitrile；Detection wavelength：The residual sample peak area in each time point solution 214nm) is measured, sample is calculated and eliminates Rate.It the results are shown in Table 2.
Table 2 is in vitro to the stability of DPP-IV degradation
Peptide Half-life period (h)
GLP-1 3.8±0.21
GC 2.4±0.16
SEQ ID NO:1 ＞ 8
SEQ ID NO:8 ＞ 8
SEQ ID NO:9 ＞ 8
SEQ ID NO:18 ＞ 8
SEQ ID NO:24 ＞ 8
SEQ ID NO:41 ＞ 8
SEQ ID NO:42 ＞ 8
Embodiment 6
Effect to GLP-1/GC receptors
The polypeptide is evaluated to GLP-1/GC receptors by the influence generated to the receptor-mediated external cAMP of GLP-1/GC Effect.
By the HEK293 cell inoculations of the Chinese cavy pneumonocyte of transfection someone's GLP-1 receptors and transfection GC receptors to 96 holes Culture plate, (200000/hole), after being washed with Hanks ' balanced salt solutions, the subject polypeptide sample (10 with various concentration-5- 10-12Mol/L), 20min is incubated altogether in 37 DEG C in the presence of 200 μm of ol/L 3- isobutyl group -1- methyl madder flavine.Medium is removed, Cell is dissolved, measures cAMP values, assay method is with reference to assay kit explanation.50% effective concentration is calculated with Origin softwares. It the results are shown in Table 3.
3 polypeptide of table acts the induction of cAMP
Embodiment 7
Agonism of the carbowax modifier of polypeptide to GLP-1 receptors
Effect of the polypeptide to GLP-1 receptors is evaluated by the influence generated to the receptor-mediated external cAMP of GLP-1.
The Chinese Guinea pig lung cell inoculations of someone's GLP-1 receptors will be transfected to 96 well culture plates (200000/hole), with After the washing of Hanks ' balanced salt solutions, the subject polypeptide sample (10 with various concentration-5-10-12Mol/L), in 200 μm of ol/L 20min is incubated altogether in 37 DEG C in the presence of 3- isobutyl group -1- methyl madder flavine.Medium is removed, cell is dissolved, measures cAMP values, is surveyed Method is determined with reference to assay kit explanation.50% effective concentration is calculated with Origin softwares.It the results are shown in Table 4.
Agonism of the 4 branched chain type PEG modified polypeptides of table to GLP-1 receptors
Conclusion：The polyethyleneglycol modified polypeptide sequence sample of the present invention of branched chain type is equivalent to the agonist activity of receptor endogenous Property ligand (hGLP-1 (7-37)-NH2), people source GLP-1 sequences obviously weaken through PEG modification activities, illustrate the poly- second two of polypeptide The activity intensity of alcohol trim depends on Precursor Peptide.PEG modifications generally make polypeptide active weaken or disappear, but Precursor Peptide The activity of the stronger PEG modified bodies of activity retains more.
Embodiment 8
The hypoglycemic effect of Precursor Peptide is evaluated
Using the hypoglycemic effect of the polypeptide of the normal mouse glucose load experimental evaluation present invention.Animal (n=8) in Physiological saline (10mL/kg) is subcutaneously injected as a control group in overnight fasting before experiment；Be subcutaneously injected Exenatide (5 μ g, daily 2 It is secondary) it is used as positive control drug group；Remaining test component Wei not SEQ ID NO:9、18、24、40.
It weighs and is dissolved in physiological saline by test product (>=98%) in right amount, be configured to the sample solution of 50 μ g/ml.Test group is small Mouse, every is subcutaneously injected 200 μ l by test sample solution；
Blood glucose is measured before administration, gives given the test agent at once after glucose (4.5g/kg) intraperitoneal injection, tail point takes hematometry The blood glucose value of 30min after medicine.
It the results are shown in Table 5.
It tries 4 samples and shows different degrees of hypoglycemic activity, wherein SEQ ID NO:9,24 drug effects and the positive Medicine Exenatide is suitable, SEQ ID NO:18 and 40 are better than positive drug.
The hypoglycemic effect of the polypeptide of 5. present invention of table
Grouping Number of animals Blood glucose value
Physiological saline 8 15.7±5.81
Positive control 8 6.06±3.74
SEQ ID NO:9 8 6.53±2.62
SEQ ID NO:18 8 5.73±3.52
SEQ ID NO:24 8 6.21±2.69
SEQ ID NO:40 8 5.69±3.18
Embodiment 9
Influence of the PEG modified polypeptides to Dio mouse weights and blood glucose
The Dio mouse of average weight 52.7g, 4 groups, every group 10, blank group gives physiological saline, and positive controls give Exendin 25nmol/kg, 2 times a day, test component do not give SEQ ID NO shown in embodiment 2:18, the PEG of 40 polypeptides Trim (abbreviation dp-1, dp-2 respectively) 100nmol/kg is administered 2 weeks once a week.After injection 0,2,4,6,8,10, 12, changes of weight is measured within 14 days, detection random plasma glucose is horizontal.The result is shown in Figure 1, Fig. 2.
Test medicine group, i.e. dp-1, dp-2 weekly administration 1 time, successive administration 2 weeks, it is shown that it is similar to Exenatide or Slightly good hypoglycemic effect, and compare positive control drug and more significantly mitigate weight, 14 days mitigation amplitudes of administration can reach 7-8%.
Embodiment 10
Branched chain type (Y) PEG modified polypeptides hypoglycemic effect and long-term effect
The hypoglycemic effect and long-term effect of polypeptide of the present invention are had rated using normal mouse glucosieloading test.
Physiological saline (10mL/kg) is subcutaneously injected as a control group in animal (n=8) overnight fasting before experiment；Subcutaneous note It penetrates Liraglutide (100nmol/kg, one time a day) and is used as positive control drug group；Remaining test component does not give the PEG of embodiment 7 Modified polypeptide pg016, pg019, pg029, dosage 100nmol/kg.
Blood glucose is measured before administration, gives given the test agent, and glucose is injected intraperitoneally in different time sections after administration After (2.5g/kg), tail point takes hematometry to calculate blood glucose inhibiting rate to the blood glucose value of 30min after sugar.
It the results are shown in Table 6.
6. branched chain type of table (Y) PEG modified polypeptide sample hypoglycemic effect timeliness
Conclusion：Positive drug Liraglutide hypoglycemic effect continued to 28 hours, and given the test agent pg016, pg019, pg029 It can steadily continue to 100 hours, weaken since 124 hours.Speculated according to mouse and body metabolism difference, given the test agent exists Human body drug effect can continue to 1 week or more.
Embodiment 11
Influence of branched chain type (Y) the PEG modified polypeptides to animal pattern weight and sugar tolerance
The Dio mouse of average weight 45.9g, 4 groups, every group 10, blank group gives physiological saline, and positive controls give Liraglutide 100nmol/kg, one time a day, test component do not give PEG modified polypeptide pg016, pg019 of embodiment 7, dosage 100nmol/kg, 4 primary, is administered 4 weeks.Changes of weight is measured within 0,8,16,24,28 day upon administration, and it is resistance to measure sugar within the 28th day Amount.As a result see Fig. 3, table 7.
As a result：4 weeks weight loss 12.3%, the polypeptide sample of branched chain type PEG modifications is administered in positive control drug Liraglutide Tested group of weight of pg016, pg019 mitigates 20.1%, 17.4% respectively, is better than Liraglutide.
28th day carbohydrate tolerance test-blood glucose AUC value (n=5) after table 7 is administered
Group AUC(mmol/L)
Model group 19.8
Liraglutide 9.1
pg016 9.6
pg019 8.2
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right It is required that range comprising the equivalent replacement of each factor.
Sequence table
<110>Tianjin Inst. of Materia Medica Co., Ltd
<120>The chimeric polyeptides of fragment analogue and application thereof containing glucagon-like-peptide-1 and glucagon
<130> DIC16110047
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 1
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 2
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 2
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 3
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 3
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 4
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 4
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 5
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 5
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 6
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 6
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 7
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 7
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 8
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is D type alanine
<220>
<221> MUTAGEN
<222> (29)..(29)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 8
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 9
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (29)..(29)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 10
<211> 32
<212> PRT
<213> artificial sequence
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 11
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 12
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (29)..(29)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (29)..(29)
<223> The 'Xaa' at location 29 stands for Gln, Arg, Pro, or Leu.
<400> 12
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 13
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 13
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 14
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 14
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 15
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (16)..(16)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 16
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 16
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 17
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 17
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 18
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 18
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 19
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 19
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 20
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 20
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 21
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 21
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 22
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 22
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 23
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 23
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 24
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 24
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 25
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 25
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Cys
20 25 30
<210> 26
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 26
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 27
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 27
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 28
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 28
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 29
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 29
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 30
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 30
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 31
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 31
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 32
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 32
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 33
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 33
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 34
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 34
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Cys Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 35
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 35
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 36
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 36
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Gln Glu Phe Val Gln Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 37
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Arg connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 37
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Gly Arg
20 25 30
<210> 38
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 38
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 39
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (30)..(30)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 39
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 40
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 40
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 41
<211> 31
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (31)..(31)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 41
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 42
<211> 32
<212> PRT
<213> artificial sequence
<220>
<221> MUTAGEN
<222> (2)..(2)
<223>Xaa is Aib, 2- aminoisobutyric acids
<220>
<221> BINDING
<222> (32)..(32)
<223>The C-terminal of Cys connects NH2
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (2)..(2)
<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.
<400> 42
His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30

Claims (10)

1. a kind of chimeric polyeptides of the fragment analogue comprising glucagon-like-peptide-1 and glucagon, which is characterized in that The sequence of the chimeric polyeptides is as shown in following general formula I：
General formula I：
HX2EGTFTSDYSKYLX15X16X17AAX20X21FX23X24WLVKX29X30X31X32
Wherein：
X2For D-Ala, Gly or Aib (2- aminoisobutyric acids)；
X15For Asp or Glu；
X16For Glu or Aib；
X17For Arg or Gln；
X20For Gln or Lys；
X21For Glu or Asp；
X23For Ile or Val；
X24For Ala, Gln or Cys；
X29For Gly or Aib；
X30For Arg or Cys；
X31For Gly, Cys ,-NH2Or it is not present；
X32For Cys ,-NH2Or it is not present；
Also, in the sequence shown in general formula I, has and only there are one Cys residues；The sequence residue containing Aib shown in the general formula I When, X2、X16And X29In three sites only there are one site be Aib.
2. chimeric polyeptides as described in claim 1, which is characterized in that in the sequence shown in general formula I, X2For D-Ala or Aib, And X16For Glu or Aib；Preferably, in the sequence shown in general formula I, X2For Aib, and X16For Glu.
3. chimeric polyeptides as claimed in claim 1 or 2, which is characterized in that the sequence of the chimeric polyeptides such as SEQ ID NO: In 1-42 shown in any bar；Preferably, the sequence of the chimeric polyeptides such as SEQ ID NO:In 16-36 shown in any bar.
4. the conjugate of chimeric polyeptides as claimed in any one of claims 1-3, which is characterized in that the conjugate is described The carbowax modifier of chimeric polyeptides is covalently attached polyethylene glycol on the side chain of the Cys residues of the chimeric polyeptides.
5. conjugate as claimed in claim 4, which is characterized in that the average molecular weight of the polyethylene glycol is 5-50KD, excellent Selection of land is 20-45KD；More preferably 40-45KD；It is highly preferred that the polyethylene glycol is linear chain or branched chain polyethylene glycol；
It is further preferred that the polyethylene glycol is the branched chair polymacrogol of average molecular weight 40-45KD.
6. the polymer of chimeric polyeptides as claimed in any one of claims 1-3, which is characterized in that the polymer is described The dimer of chimeric polyeptides, the dimer are that the chimeric polyeptides are formed by connecting by Cys residue covalents or are total to by connector What valence was formed by connecting；
Preferably, the dimer is by being connected with the connector of bifunctional amine's crosslinked group and such as any one of claim 1-3 institutes The sulfydryl for the chimeric polyeptides stated reacts to be formed；
Preferably, the connector is bismaleimide-polyethylene glycol (Mal-PEG-Mal)；It is highly preferred that the polyethylene glycol Average molecular weight range is 3-20KD；
Preferably, the dimer is homodimer.
7. a kind of pharmaceutical composition, described pharmaceutical composition includes chimeric polyeptides as claimed in any one of claims 1-3, such as Conjugate described in claim 4 or 5 or polymer as claimed in claim 6；
Preferably, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material.
8. pharmaceutical composition as claimed in claim 7, which is characterized in that the carrier and/or auxiliary material are selected from water soluble bulk It is one or more in agent, pH adjusting agent, stabilizer, water for injection or osmotic pressure regulator；
Preferably, the water-soluble filler be selected from mannitol, D-40, sorbierite, polyethylene glycol, glucose, It is one or more in lactose or galactolipin；The pH adjusting agent is selected from physiologically acceptable organic acid or inorganic acid, such as Citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid or physiologically acceptable inorganic base, for example, potassium hydroxide, sodium hydroxide, It is one or more in ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, saleratus, sodium bicarbonate, bicarbonate ammonium salt；It is described Stabilizer be selected from EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, Arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxyl fiber Element or derivatives thereof such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane In it is one or more；The osmotic pressure regulator is sodium chloride and/or potassium chloride.
9. chimeric polyeptides as claimed in any one of claims 1-3, conjugate as described in claim 4 or 5 or such as right It is required that the polymer described in 6 is being prepared for treating the purposes in diabetes, obesity and/or the drug of metabolic syndrome.
10. pharmaceutical composition as claimed in claim 7 or 8 is being prepared for treating diabetes, obesity and/or metabolic syndrome Drug in purposes.
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