CN109384839B - Glucagon-like peptide-1 analogs and uses thereof - Google Patents
Glucagon-like peptide-1 analogs and uses thereof Download PDFInfo
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- CN109384839B CN109384839B CN201710660343.4A CN201710660343A CN109384839B CN 109384839 B CN109384839 B CN 109384839B CN 201710660343 A CN201710660343 A CN 201710660343A CN 109384839 B CN109384839 B CN 109384839B
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- Prior art keywords
- peptide
- glucagon
- pharmaceutically acceptable
- acceptable salt
- analog
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Images
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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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a precursor polypeptide of a glucagon-like peptide-1 analogue or pharmaceutically acceptable salt thereof, wherein the precursor polypeptide has an amino acid sequence shown in the following general formula I; general formula I HX1X2GTFTSDVSSYLEEX3AAX4EFIX5WLVKX6X7X8X9(ii) a Wherein, X is1、X2、X3、X4、X5、X6、X7、X8And X9Represents an arbitrary amino acid. The invention also provides a glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof. The glucagon-like peptide-1 analog is obtained by conjugating polypeptide optimized based on endogenous GLP-1(7-36/37) sequence and polyethylene glycol with specific structure, has stronger hypoglycemic activity, can reduce the dosage, obviously prolongs the half life in vivo, and is expected to improve the clinical compliance, and the precursor polypeptide sequence provided by the invention is highly homologous with endogenous GLP-1(7-36/37) and can reduce the immunogenicity, thereby having better potential for developing and applying as a medicament.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a glucagon-like peptide-1 analogue and medical application thereof.
Background
Glucagon-like peptide 1(GLP-1) is an enterogenic hormone that is released into the circulation primarily in the L-cell synthesis of the terminal jejunum, ileum and colon, in a meal response. GLP-1(7-36, 7-37) is the major active form of GLP-1 in the systemic circulation, controlling blood glucose by complex mechanisms including secretion of insulin and glucagon, gastric emptying and regulation of peripheral insulin. The hypoglycemic effect of GLP-1(7-36, 7-37) is glucose-dependent, can avoid hypoglycemia, inhibit apoptosis of islet beta-cells, promote proliferation of islet beta-cells, and reverse disease development. However, the plasma half-life of native GLP-1 is only 1-2 minutes, and the metabolic instability limits the application of the native GLP-1 as a medicine.
Enzymatic degradation and renal clearance are the major pathways for in vivo metabolism of polypeptides. Research shows that in vivo dipeptidyl kininase (DPPIV) specifically recognizes and degrades the N-terminal His-Ala segment of the receptor binding active site in the GLP-1 structure to quickly inactivate the receptor binding active site, and other proteolytic enzymes such as endopeptidase and the like are also involved in the in vivo degradation process of the polypeptide. The kidney plays an important role in eliminating peptide, protein and other substances, and the molecular weight in blood plasma is less than 5KD and the effective radius is less than
The free part of the molecule is easily filtered by glomeruli, and in the renal circulation, peptide hormones (such as calcitonin, GLP-1) are degraded by metabolic enzymes in the renal cortex and further excreted into urine. The study reported that the kidney was responsible for the clearance of at least 80% of Exendin-4(CN 1372570).
The technical goals of the GLP-1-based drug development field are to improve the metabolic stability and prolong the half-life period of blood plasma so as to improve the clinical drug compliance. In the prior patent technology, only one elimination factor of enzyme degradation is considered in the structural modification (CN00806548.9, CN99814187.9, CN200410017667.9 and the like) of the enzyme degradation key site of the human GLP-1 sequence, and the ideal long-acting effect cannot be achieved; the technology of introducing fatty acyl groups into a parent peptide chain structure to improve the binding force with plasma protein so as to avoid the polypeptide from being rapidly eliminated in vivo (CN201210513145.2, CN200810124641.2, CN20118000352.1 and the like) can prolong the half-life to a certain extent (for example, the liraglutide on the market is administered once a day), the drug compliance of the technology still needs to be improved, the drug effect is delayed due to high binding rate of the plasma protein, the solubility of the technology is reduced due to the introduction of the fatty acyl groups into the peptide chain, and an organic solvent is needed to be used in the preparation.
Polyethylene glycol (PEG) technology is a more applicable long-acting technology in the field of current protein/polypeptide drug administration. The protein/polypeptide is modified by linear chain or branched polyethylene glycol, so that the physical and chemical property stability of the protein/polypeptide can be improved, the immunogenicity is reduced, the protease degradation resistance is improved, the metabolism of the kidney clearing effect on the medicament is reduced, the in vivo half-life period of the medicament is obviously prolonged, and the medicament solubility and the penetrating power of cell membranes are improved. Generally speaking, high molecular weight (20 KD or more) PEG modification is more beneficial to prolonging the in vivo half-life of polypeptide or protein drugs, but generally, each PEG molecule only has 1 active end group coupled with precursor active molecules, the drug-loading rate of the molecule is limited, and the binding effect of the polypeptide modified by the high molecular weight and a receptor is generally weakened to influence the drug effect, so that the activity intensity of the precursor polypeptide is an important factor for determining the drug property of a final modified product. In addition, the physicochemical properties of the precursor polypeptide have a large influence on the efficiency of the modification reaction, the yield and quality of the final product, and therefore, selection of an appropriate GLP-1 analogue molecule as a high-molecular modified precursor polypeptide is an important factor for achieving a long-lasting drug effect of the target molecule.
Disclosure of Invention
In view of the limitations of the prior art, it is an object of the present invention to provide a glucagon-like peptide-1 analog which is a high molecular weight polymer of a polypeptide precursor having an optimized sequence of endogenous GLP-1(7-36/37), and more specifically a polyethylene glycol conjugate of the analog.
The invention unexpectedly discovers that the GLP-1 analogue obtained by structure transformation has obviously enhanced agonistic activity to a GLP-1 receptor and obviously improved solubility and stability, and the GLP-1 analogue is used as a precursor polypeptide modified by macromolecules, thereby not only improving the bioactivity of a target product, but also improving the reaction efficiency of the modification of the macromolecules, and the yield and the quality of a final product.
The glucagon-like peptide-1 analogue has stronger hypoglycemic activity, can reduce the dosage of the medicament, obviously prolongs the half life in vivo, is expected to improve clinical compliance, has highly homologous precursor polypeptide sequence with endogenous GLP-1(7-36/37) and can reduce immunogenicity, thereby having better potential for developing and applying as the medicament.
In one aspect, the invention provides a precursor polypeptide of a glucagon-like peptide-1 analog, or a pharmaceutically acceptable salt thereof, wherein the precursor polypeptide has an amino acid sequence shown in the following general formula I;
general formula I HX1X2GTFTSDVSSYLEEX3AAX4EFIX5WLVKX6X7X8X9；
Wherein, X is1、X2、X3、X4、X5、X6、X7、X8And X9Represents an arbitrary amino acid.
Preferably, the sequence of the precursor polypeptide of the glucagon-like peptide-1 analogue is shown in any one of SEQ ID NO 1-56;
in another aspect, the present invention provides a glucagon-like peptide-1 analog, or a pharmaceutically acceptable salt thereof, which is a polyethylene glycol conjugate of the above-described precursor polypeptide, or a pharmaceutically acceptable salt thereof;
preferably, the polyethylene glycol has an average molecular weight in the range of 5 to 50KDa, more preferably 20 to 50 KDa; further preferably 40-50 KDa;
preferably, the polyethylene glycol is a linear, branched polyethylene glycol;
preferably, the polyethylene glycol is selected from branched polyethylene glycols having an average molecular weight in the range of 40-50kDa, more preferably a unbranched polyethylene glycol having an average molecular weight in the range of 40-50 kDa.
Preferably, the polyethylene glycol is conjugated to the glucagon-like peptide-1 analog or the pharmaceutically acceptable salt thereof through an activating group on a molecular terminal group, wherein the activating group is selected from active functional groups such as maleimide group, sulfydryl group, succinimidyl group, aldehyde group, halogen and the like, and is preferably maleimide group;
preferably, the polyethylene glycol is conjugated to the lysine side chain or the cysteine side chain of any one of SEQ ID NOs 1-56 via an activating group; more preferably, the polyethylene glycol is conjugated to any one of SEQ ID NOs 9-12, 17-20 via an activating group.
It is another object of the present invention to provide a pharmaceutical composition comprising a precursor polypeptide of any of the above, or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog, or a pharmaceutically acceptable salt thereof, preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant; more preferably, the carrier and/or the auxiliary material is selected from one or more of water-soluble filler, pH regulator, stabilizer, water for injection or osmotic pressure regulator;
preferably, the water-soluble filler is selected from one or more of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose; the pH regulator includes but is not limited to organic or inorganic acids such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid and the like, and one or more of physiologically acceptable inorganic bases or salts such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium bicarbonate salts; the stabilizer is selected from one or more of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxy cellulose or derivatives thereof such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, sodium dodecyl sulfate or tris (hydroxymethyl) aminomethane; the osmotic pressure regulator is sodium chloride and/or potassium chloride.
The invention also aims to provide a precursor polypeptide of the glucagon-like peptide-1 analogue or a pharmaceutically acceptable salt thereof, application of the glucagon-like peptide-1 analogue or the pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for treating diabetes, obesity and metabolic syndrome, and application of the pharmaceutical composition in preparing a medicament for treating the diabetes, the obesity and the metabolic syndrome.
Detailed description of the invention
Polypeptide sequence
The precursor polypeptide of the glucagon-like peptide-1 analog is an artificial modified form of GLP-1 (7-36/37). The natural sequence of GLP-1(7-36/37) is:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRNH2and/G. The N-terminal His-Ala dipeptide fragment is cleaved hydrolytically by dipeptidyl kininase (DPPIV) in blood, and the remaining GLP-1(9-36/37) sequence loses or diminishes biological activity, in an embodiment of the invention, to X in the endogenous sequence8Or X9The experimental results of example 3 of the present invention show that the measures taken in the present invention can effectively prevent the enzymatic degradation inactivation of polypeptides by carrying out appropriate amino acid substitutions.
Generally, the polypeptide ligand acting on the B-type GCGR receptor is combined with the receptor by an alpha-helix secondary structure, so that the structural transformation which is favorable for forming the alpha-helix is beneficial to improving the binding force of the polypeptide and the receptor and enhancing the biological activity of the polypeptide.
In an embodiment of the invention, amino acid substitutions are made at appropriate sites in the human GLP-1(7-36/37) sequence, such as G22/E、G35Aib to enhance the biological activity of the precursor polypeptide. In the embodiment of the invention, the precursor polypeptide provided by the invention is mixed with Gly2、Cys37GLP-1(7-37) (SEQ ID NO:57 sequence)
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRC) and Gly2、Aib35、Ala38、Cys39GLP-1(7-37) (SEQ ID NO: 58)
HGEGTFTSDVSSYLEGQAAKEFIWLVKAIbRGAC), and the results show that the polypeptide sequence obtained by the transformation has obviously enhanced in-vitro agonistic activity on GLP-1 receptors, and shows stronger hypoglycemic activity and better in-vivo stability in-vivo animal tests.
Meanwhile, in order to facilitate site-directed conjugation modification of polyethylene glycol groups, cysteine residues are introduced or substituted into appropriate sites of the sequence (A)30/C、C37、C38) Or by replacing it appropriatelyAlternatively, only one lysine residue (K) is retained in the sequence34/R、K26R) is added. The results of the studies conducted in the examples of the present invention show that these structural changes have no influence on the activity of the polypeptide. The experimental result shown in the embodiment 5 of the invention shows that the agonistic activity of the polyethylene glycol modified body of the preferred polypeptide provided by the invention to the GLP-1 receptor is obviously stronger than that of other precursor polypeptide modified bodies with relatively weak activity, and the research result of the hypoglycemic drug effect in the embodiment 8 also indicates that the polyethylene glycol conjugate of the preferred polypeptide has better drug effect and long-acting property, and the dosage is reduced, so that the polyethylene glycol conjugate has more drug potential.
In general, the physicochemical properties of the precursor polypeptide used for modification of a polymer have a large influence on the efficiency of the modification reaction, the yield and the quality of the final product. The precursor polypeptide has poor solubility in a modification reaction system (generally, a buffer solution with a proper pH value), polypeptide aggregates are precipitated, the reaction efficiency is reduced, steps for removing residual polypeptide, such as centrifugation, molecular sieve chromatographic separation and the like, are required to be added in the post-treatment, even an organic solvent, such as DMSO and the like, is directly used as a reaction solvent, the post-treatment difficulty is increased, and the preparation period is prolonged. In the embodiment of the invention, the polypeptide provided by the structural transformation is unexpectedly found to have obviously improved solubility and stability, and is more suitable to be used as a precursor polypeptide for macromolecular modification. As example 4, the precursor polypeptides are well dissolved in the reaction system, the reaction efficiency is high, the final product has no residual polypeptide or other impurities, the subsequent purification treatment is simple, and the yield (average 75-80%) and quality (purity is more than or equal to 98%) of the target product reach the expected level.
Pegylation of polyethylene
Polypeptide hormones are mainly metabolized in the body through enzymatic degradation and renal clearance pathways, wherein renal clearance is dominant and is a main factor influencing the half-life of polypeptide drugs in the body. Structural modification on side chains of some amino acid residues in the polypeptide sequence, particularly the conjugation of alkylated or polyethylene glycol and other macromolecular groups can delay renal clearance and effectively prolong the biological half-life.
In a particular embodiment of the invention, the polypeptide sequence provided comprises at least oneThe site being Cys, or Lys, so that the thiol group in the side chain of Cys or the epsilon-NH in the side chain of Lys2The upper site is covalently conjugated with a polyethylene glycol (PEG) group. The average molecular weight of the polyethylene glycol is 5-50 KD; more preferably, the polyethylene glycol has an average molecular weight of 20-50 KDa; further preferably, the polyethylene glycol has an average molecular weight of 40-45 KDa;
polyethylene glycols used for modification are generally linear or branched structures comprising one activating group, wherein branched polyethylene glycols include di-and tetra-branched types, and the activating group refers to a group capable of covalently binding to a free thiol or amino group in the polypeptide structure, such as an aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine group.
The di-branched polyethylene glycol is preferred in embodiments of the present invention and preferably contains 1 or 2 activated functional groups, preferably maleimide groups.
The polyethylene glycols of the present invention are available from a variety of sources, including commercially available or self-prepared according to methods known in the art. The polyethylene glycol used for modification in the embodiments of the present invention is preferably, but not limited to, selected from the following structures:
the PEG modification described herein can be achieved by any method known in the art, including via acylation, reductive alkylation, Michael addition, thiolation, or other chemoselective conjugation methods via the reactive group of the PEG moiety (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine group). In a particular embodiment of the invention, the pegylated polypeptides are linked by thioether bonds via a Michael addition reaction, i.e. modification of the sulfhydryl side chain of Cys with maleimide activated PEG.
Use of
In response to the limitations of current drug therapies for the treatment of diabetes, obesity, metabolic syndrome, and the like, the present invention provides a novel therapeutic approach involving the administration of a pharmaceutical composition comprising a precursor polypeptide of the present invention, or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition containing the precursor polypeptide or the pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 analogue or the pharmaceutically acceptable salt thereof has long-acting property while effectively reducing blood sugar, can improve the medication compliance and has more clinical application potential.
Pharmaceutical composition
In yet another aspect, the present invention also provides a pharmaceutical composition comprising the above precursor polypeptide or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
More preferably, the carrier and/or adjuvant is selected from one or more of water-soluble filler, pH regulator, stabilizer, water for injection or osmotic pressure regulator.
Preferably, the water-soluble filler is selected from one or more of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose; the pH regulator includes but is not limited to organic or inorganic acids such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid and the like, and one or more of physiologically acceptable inorganic bases or salts such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium bicarbonate salts; the stabilizer is selected from one or more of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxy cellulose or derivatives thereof such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, sodium dodecyl sulfate or tris (hydroxymethyl) aminomethane; the osmotic pressure regulator is sodium chloride and/or potassium chloride.
In still another aspect, the present invention provides the use of the above precursor polypeptide or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment of diabetes, obesity, metabolic syndrome.
Preferably, the composition of the present invention can be administered in the form of intravenous, intramuscular or subcutaneous injections or orally, rectally or nasally. The dosage may range from 5 μ g to 10mg per dose, depending on the subject being treated, the mode of administration, the indication, and other factors.
Synthesis of
The basic peptide chain of the precursor polypeptide having the structure of formula I provided in the present invention is prepared by methods known in the art:
1) synthesis by conventional solid or liquid phase methods, stepwise or by fragment assembly;
2) expressing a nucleic acid construct encoding the polypeptide in a host cell and recovering the expression product from the host cell culture;
3) effecting cell-free in vitro expression of a nucleic acid construct encoding the polypeptide and recovering the expression product;
or by any combination of methods 1), 2) or 3) to obtain peptide fragments, followed by ligation of the fragments to obtain the target peptide.
Preferably, the target peptide is prepared using Fmoc solid phase synthesis.
Preferably, the pegylation modification of the target polypeptide is accomplished by: reacting the activated PEG with the polypeptide of the invention at pH5.0-7.0, wherein the molar ratio of the PEG to the peptide is 1-10, the reaction time is 0.5-12 hours, and the reaction temperature is 4-37 ℃.
Following the conjugation reaction, the product of interest may be isolated by suitable methods known in the art. Suitable methods include, but are not limited to, ultrafiltration, dialysis, or chromatography.
Activity evaluation
According to the embodiment of the invention, a normal mouse glucose load test is adopted, and the liraglutide is used as a positive control drug to evaluate the hypoglycemic activity and long-acting property of the precursor polypeptide provided by the invention.
In another embodiment of the invention, the ob/ob diabetes model mouse is adopted to evaluate the hypoglycemic effect and the influence on the body weight of the glucagon-like peptide-1 analogue, and the result shows that the glucagon-like peptide-1 analogue provided by the invention has a remarkable hypoglycemic effect, the administration frequency is reduced, and the application advantage is obvious.
Drawings
FIG. 1 is an HPLC chromatogram of a precursor polypeptide of a glucagon-like peptide-1 analog of the present invention, a glucagon-like peptide-1 analog, and a modification group of the analog, wherein FIG. A is a precursor polypeptide of a glucagon-like peptide-1 analog of the present invention SEQ ID NO:9, FIG. B is a glucagon-like peptide-1 analog SEQ ID NO: 9-II, and FIG. C is a structure II (m PEG)2-HPLC chromatogram of Mal (40 KDa);
FIG. 2 is a MALDI-TOF plot of the glucagon-like peptide-1 analog of the present invention SEQ ID NO 9-II;
FIG. 3 is a comparison of the hypoglycemic effects and long-term effects of the glucagon-like peptide-1 analogs of the present invention, SEQ ID NO 9-II, and SEQ ID NO 58-II (see example 2).
Detailed Description
The present invention will be further described with reference to the following examples. The present examples are merely illustrative of the present invention and are not meant to limit the inventive content in any way.
Description 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: alpha-aminoisobutyric acid
Description of the abbreviations of reagents
Boc tert-butoxycarbonyl
Tert-butyl group as Tert-Bu
DCM dichloromethane
DIC: diisopropylcarbodiimide
Fmoc 9-fluorenylmethoxycarbonyl
HoBt 1-hydroxybenzotriazole
HBTU 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyl-uronium hexafluorophosphate
HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyl-uronium hexafluorophosphate
Mtt: 4-Methyltriphenylmethyl
NMP N-methylpyrrolidone
DMF dimethylformamide
Trt triphenylmethyl
EDT ethanedithiol
TFA trifluoroacetic acid
TIS triisopropylsilane
FBS fetal bovine serum
EXAMPLE 1 preparation of glucagon-like peptide-1 analog precursor Polypeptides
A precursor polypeptide of formula I is prepared by the following steps
1) Synthesizing: stepwise synthesis using Fmoc strategy with a CS 336 polypeptide synthesizer (CS Bio) according to the following procedure:
a) coupling a resin solid phase carrier and Fmoc protected C-terminal amino acid in the presence of an activator system to obtain Fmoc-amino acid-resin; wherein, amino resin such as Rink Amide AM, Rink Amide and Rink MBHA is adopted for synthesizing the C-terminal amidated polypeptide.
b) Elongation of peptide chain: connecting amino acids according to the sequence of peptide sequence amino acids by a solid phase synthesis method to obtain a peptide-resin conjugate with protected N-terminal and side chain; the amino acid with side chain adopts the following protective measures: tryptophan with Boc, glutamic acid with OtBu, lysine with Boc, glutamine with Trt, tyrosine with tBu, serine with Trt or tBu, aspartic acid with OtBu, threonine with tBu, cysteine with Trt, histidine with Trt or Boc, arginine with Pbf. The coupling activating agents used are HOBT/HBTU/DIEA and HOBT/HATU/DIEA, and the reaction efficiency is detected by an indetrione method.
c) Cleavage of the polypeptide on the resin: TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5v/v) solution, left to react at room temperature for 90min, deprotected and deresinated. Filtering to obtain filtrate, precipitating crude polypeptide with excessive diethyl ether, centrifuging, collecting precipitate, washing precipitate with small amount of diethyl ether, and vacuum drying to obtain crude polypeptide. Simultaneously removing protecting groups and resin to obtain crude glucagon-like peptide-1 and glucagon fragment analog chimeric polypeptide;
2) and (3) purification: dissolving the crude polypeptide in water or 10-15% acetonitrile (10-50mg/ml), adding 50-100mM dithiothreitol DTT or beta-mercaptoethanol for denaturation, separating and purifying by preparative HPLC, C18 chromatographic column and acetonitrile-water-trifluoroacetic acid system, concentrating, and lyophilizing to obtain pure polypeptide with free sulfhydryl.
The precursor polypeptides shown in the following SEQ ID NO 1-56 are prepared by the method.
SEQ ID NO:1HAPGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:2HAPGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:3HAPGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:4HAPGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:5HAPGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:6HAPGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:7HAPGTFTSDVSSYLEERAAKEFIAWLVKGC-NH2
SEQ ID NO:8HAPGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:9HGEGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:10 HGEGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:11 HGEGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:12 HGEGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:13HGEGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:14HGEGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:15 HGEGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:16 HGEGTFTSDVSSYLEERAAKEFICWLVKAibR-NH2
SEQ ID NO:17 H(d-A)EGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:18 H(d-A)EGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:19 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:20 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:21 H(d-A)EGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:22 H(d-A)EGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:23 H(d-A)EGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:24 H(d-A)EGTFTSDVSSYLEERAAKEFICWLVKAibR-NH2
SEQ ID NO:25 HAPGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:26 HAPGTFTSDVSSYLEEQAAKEFIAWLVRAibR-NH2
SEQ ID NO:27 HAPGTFTSDVSSYLEERAAKEFIAWLVRAibRG-NH2
SEQ ID NO:28 HAPGTFTSDVSSYLEERAAKEFIAWLVRAibR-NH2
SEQ ID NO:29 HAPGTFTSDVSSYLEERAAKEFIAWLVRG-NH2
SEQ ID NO:30 HGEGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:31 HGEGTFTSDVSSYLEEQAAKEFIAWLVRAibG-NH2
SEQ ID NO:32 HGEGTFTSDVSSYLEERAAKEFIAWLVRAibRG-NH2
SEQ ID NO:33 HGEGTFTSDVSSYLEERAAKEFIAWLVRAibG-NH2
SEQ ID NO:34 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:35 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVRAibG-NH2
SEQ ID NO:36 HAPGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:37 HAPGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:38 HAPGTFTSDVSSYLEERAAREFIAWLVKAibRG-NH2
SEQ ID NO:39 HAPGTFTSDVSSYLEERAAREFIAWLVKAibR-NH2
SEQ ID NO:40 HAPGTFTSDVSSYLEERAAREFIAWLVKG-NH2
SEQ ID NO:41 HGEGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:42 HGEGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:43 H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:44 H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:45HAPGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:46HAPGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:47HAPGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:48HAPGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
SEQ ID NO:49HGEGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:50HGEGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:51HGEGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:52HGEGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
SEQ ID NO:53H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:54H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:55H(d-A)EGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:56H(d-A)EGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
EXAMPLE 2 solubility and stability assays of glucagon-like peptide-1 analog precursor Polypeptides
The preferred polypeptides provided by the invention were tested for solubility and solution stability evaluation, and simultaneously compared to the polypeptide sequence which is simply a function of the original sequence of GLP-1 (Gly2, Cys37 GLP-1(7-37): SEQ ID NO:57HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRC) and the sequences mentioned in the patent (CN201610211144.0) (Gly2, Aib35, Ala38, Cys39GLP-1(7-37): SEQ ID NO:58
HGEGTFTSDVSSYLEGQAAKEFIWLVKAIbRGAC), and the structural transformation in the technical scheme of the invention effectively solves the solubility and the solution stability of the precursor polypeptide.
Sample preparation:
SEQ ID NO:57、SEQ ID NO:58、
1, 9, 11, 15, 17 and 24 of SEQ ID NO, and the purity is more than or equal to 98 percent.
Solubility:
preparing the sample solution with the target concentration of 2mg/ml, dissolving a proper amount of sample in 20mMPBS with the pH of 6.0 and 7.4 at room temperature, centrifuging at 4000r, and taking supernatant for detection. A solution of appropriate concentration of the polypeptide sample in acetonitrile-water (30%) was prepared as a control solution for testing.
HPLC-UV determination:
a chromatographic column: aeriswidcore XB-C183.6 μm, 4.6 × 150 mm;
mobile phase: a0.05% TFA/H2O; b0.05% TFA/acetonitrile
Detection wavelength: 214nm
Solution stability:
weighing appropriate amount of above sample, dissolving with 20mM sodium acetate buffer solution of pH4.2 and 20mM PBS of pH 6.0 and 7.4 at room temperature to obtain 2mg/ml, standing at 25 deg.C for 8hr, measuring polypeptide amount in solution according to the above method, and comparing with initial amount to calculate residual amount percentage. The results are shown in Table 1.
TABLE 1 results of the solubility and stability test of the polypeptide samples
Example 3 evaluation of hypoglycemic Effect of precursor polypeptide of glucagon-like peptide-1 analog
The hypoglycemic effect of part of the polypeptides provided by the invention is evaluated by adopting a normal mouse glucose load model.
And (3) testing a sample:
57, 58, 4, 9, 11, 15, 17 and 19 of SEQ ID NO, and the purity is more than or equal to 98 percent
The method comprises the following steps:
animals (n-8) were fasted overnight before the experiment and injected subcutaneously with normal saline (10mL/kg) as a control group; a single subcutaneous injection of liraglutide (200 μ g/kg) as a positive control group; blood glucose was measured before administration, the positive drug and test sample (200. mu.g/kg) were administered, glucose (4.5g/kg) was injected into the abdominal cavity 2 to 4 hours after administration, blood was taken from the tail tip, and the blood glucose level 30min after administration was measured, and the blood glucose inhibition rate (%) relative to the positive control drug was calculated. The results are shown in Table 2.
TABLE 2 hypoglycemic Effect of Polypeptides on Normal mouse glucose Loading model
Note that the blood glucose inhibition in the table is the percentage relative to the positive control.
And (4) conclusion: as can be seen from the data of blood sugar inhibition rate relative to the positive drug in the above Table 2, the precursor naked peptide designed by the invention still shows stronger blood sugar reducing effect after being administrated for 2-4 hours, and the sequence Gly which is not optimized2，Cys37GLP-1(7-37) aloneShows activity immediately upon administration. The in vivo activity and stability of the precursor polypeptide optimized by the invention are obviously superior to those of the unoptimized sequence.
EXAMPLE 4 preparation of glucagon-like peptide-1 analogs
1) Connecting:
maleic acyl functionalization: the polypeptide shown as SEQ ID NO. 3 was dissolved in 50mM sodium phosphate buffer solution containing 5mM EDTA, pH6, at a concentration of 2 mg/mL. Adding solid PEG-maleimide 1.2-1.5 times of the molar weight, stirring to dissolve, and reacting at room temperature for 0.5-2 hr. The reaction was monitored by HPLC, quenched with 5mM β -mercaptoethanol, and purified after 30min at room temperature.
Iodine acetyl functionalization: polyethylene glycol modification was accomplished by reaction of peptide precursors and iodoacetyl functionalized mPEG (1:1) in 7M urea/50 mM Tris buffer (pH7.5-8.5) with stirring at room temperature for 45 minutes to form covalent thioether bonds between PEG and Cys in the peptide chain.
2) Purifying by preparative ion exchange column chromatography, eluting with SP Sepharose HP filler, and linear gradient eluting with 0-500mM sodium chloride pH6 phosphate buffer solution. Detecting the effluent by HPLC and SDS-electrophoresis, collecting PEG-polypeptide fraction, ultrafiltering, concentrating, and freeze drying. The results are shown in FIG. 1, wherein FIG. A is a precursor polypeptide of the glucagon-like peptide-1 analog of the present invention SEQ ID NO:9, FIG. B is a glucagon-like peptide-1 analog SEQ ID NO: 9-II, and FIG. C is Structure II (mPEG)2-HPLC chromatogram of Mal (40 kDa).
3) And carrying out full molecular weight scanning on the pure polypeptide by MALDI-TOF to determine the average molecular weight. The results are shown in FIG. 2, and FIG. 2 is a MALDI-TOF diagram of the glucagon-like peptide-1 analog of the present invention of SEQ ID NO 9-II.
The polypeptide polyethylene glycol modifications shown in the following table are prepared by the method.
TABLE 3 polypeptide polyethylene glycol modifications
Example 5 pancreatic hypertensionPrecursor polypeptide of glucagons-like peptide-1 analogue and glucagons-like peptide-1 analogue pair
Agonism of GLP-1 receptor
And (3) testing a sample:
GLP-1, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 4, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 9-II, SEQ ID NO 11-II, SEQ ID NO 57-II, SEQ ID NO 58-II, and purity is more than or equal to 98%
The method comprises the following steps: the test compound was made 2-fold higher than the working concentration with serum-free medium (containing 0.1% BSA,0.5mM IBMX). The cells (HEK293) were digested, suspended in serum-free medium (containing 0.1% BSA,0.5mM IBMX) and counted, and cells were added to a 384 well plate at 2000 c/5. mu.l/well, followed by 5. mu.l of test compound and reacted at room temperature in the dark for 30 min. After the reaction is finished, adding cAMP detection substrate, and reacting for 60min at room temperature in a dark place. After the reaction is finished, the reaction product is detected by an Envision2104 multifunctional microplate reader. The activation rate (% Response) was calculated for each sample at each concentration. EC50 values were calculated by nonlinear fitting of the log X of% Response versus sample concentration.
The results are shown in Table 4 below.
TABLE 4 agonistic activity of GLP-1 analog polypeptides and PEG modifications on GLP-1 receptor
As can be seen from the above table, the polypeptide with the improved structure by the technical scheme of the invention has obviously higher agonistic activity to a receptor than endogenous GLP-1 and other GLP-1 analogues which are simply changed; the activity intensity of PEG modified receptor is reduced to about 1/10 of that of precursor polypeptide, so that the stronger the activity of precursor polypeptide, the more beneficial the activity of target modified product is exerted.
Example 6 evaluation of hypoglycemic Effect and Long-term Effect of Linear PEG-modified Polypeptides
The hypoglycemic effect and the long-acting property of the polypeptide are evaluated by adopting a normal mouse glucose load test.
Animals (n-8) were fasted overnight before the experiment and injected subcutaneously with normal saline (10mL/kg) as a control group; injecting liraglutide (100nmol/kg, 1 time per day) subcutaneously as a positive control drug group; the other test groups were SEQ ID NO 1(mPEG20, 40KD), SEQ ID NO 9-mal-mPEG20KD, and SEQ ID NO 11-mal-mPEG20KD, respectively, at a dose of 100 nmol/kg. The dose was 100 nmol/kg.
Blood glucose was measured before administration, test samples were administered, glucose (4.5g/kg) was intraperitoneally injected at different time periods after administration, blood was taken from the tail tip to measure the blood glucose level 30min after administration, and the blood glucose inhibition rate was calculated.
The results are shown in Table 5.
And (4) conclusion: the 4 tested samples all show different degrees of hypoglycemic activity and long-acting performance, are superior to the positive control drug, and simultaneously show that the increase of the molecular weight is helpful for prolonging the drug effect. The modifier of SEQ ID NO 9 and 11 has NO obvious difference in hypoglycemic effect, and shows that the modification of 30 th site and C-terminal in the sequence can realize the activity maintaining and long-acting effect.
TABLE 5 hypoglycemic action of straight-chain PEG-modified Polypeptides (n ═ 8)
Example 7 hypoglycemic Effect and Long-acting Properties of branched PEG-modified Polypeptides
And (3) testing a sample: 1-I, 9-II, 11-II, 15-II, 17-II of SEQ ID NOs prepared in example 4, evaluation method: the same as in example 6. The results are shown in Table 6 below.
TABLE 6 branched PEG-modified polypeptides for hypoglycemic action and long-lasting effect
And (4) conclusion: from the results in table 6, it can be seen that the drug effect of the positive drug liraglutide only lasts for 28 hours, while the drug effect of the test group samples lasts for 148 hours, and the expected long-lasting effect can be determined by considering the difference between the species of metabolism of the mouse and the human body.
EXAMPLE 8 Effect of different polypeptide precursors on the drug efficacy of PEG-modified products
Comparing the hypoglycemic effect and the long-acting effect of the SEQ ID NO 9-II and the SEQ ID NO 58-II.
The tested samples are all self-made, and the purity is more than or equal to 98 percent.
The positive control drug is liraglutide.
9-II of the test sample set 250, 25, 2.5nmol/kg (indicated as samples b-1, b-2, b-3 in the figure), Positive drug and Gly2，Aib35，Ala38，Cys39GLP-1(7-37) -II (indicated as sample a in the figure) are all set at a dose of only 25 nmol/kg.
The procedure is as in example 6, and the results are shown in FIG. 3. It can be seen from the figure that the drug effect of the tested sample SEQ ID NO 9-II can stably last for more than 120hr under the high dose set in the experiment, and the drug effect and the long-acting property are both in dose correlation. The positive drug liraglutide showed efficacy at 25nmol/kg at 12hr, while efficacy declined at 24 hours. The drug effect of SEQ ID NO 9-II can still be maintained to 76hr under the dosage of 25nmol/kg, while the blood sugar inhibition rate of SEQ ID NO 58-II is greatly weakened at 52hr, which shows that the optimization of the precursor polypeptide sequence is helpful to improve the drug effect of the final product.
Example 9 blood glucose regulating Effect of PEG-modified products on ob/ob mice
ob/ob model mouse, male, 6 weeks old, purchased by Beijing Hua Pookang Biotech GmbH, license number: SCXK (Jing) 2014-.
After animal quarantine and feeding for two weeks, fasting is used for measuring fasting blood glucose, the animals are randomly divided into 5 groups according to the fasting blood glucose, each group comprises 8 animals, namely, SEQ ID NO 9-II, SEQ ID NO 11-II and SEQ ID NO 17-II, a positive control drug liraglutide group and a model control group, subcutaneous administration is carried out, the drug dose is 100nmol/kg, the positive control drug liraglutide is administered for 2 times every day, other three samples are administered for 1 time every 4 days and are continuously administered for 21 days, the fasting blood glucose is monitored every week, glucose is administered for 2.5g/kg to the abdominal cavity after 21 days, the blood glucose values are measured before and after 0.5h, 3 h and 6h after the glucose administration, and the area under the blood glucose concentration-time curve (AUC) is calculated. The results are shown in tables 7 and 8.
TABLE 7 continuous administration of PEG-modified Polypeptides for 21 days and weekly determination of fasting blood glucose values
＊(
n＝8，100nmol/kg)
Table 8 blood glucose concentration-time profile after 21 consecutive days of PEG-modified polypeptide administration (
n＝8)
Comparison with model control group:*P<0.05，**P<0.01.
as can be seen from the above table, the test sample, i.e., the long-acting polypeptide conjugate provided by the present invention, shows comparable blood glucose regulation effect to that of the positive drug liraglutide in the diabetes animal model, but the administration frequency is reduced (2 times per day vs 4 times per day 1 time), and has obvious application advantages. In consideration of the metabolic difference between mice and humans, the experimental data of the invention supposes that the administration frequency of 1 time per week can be satisfied clinically, and the medication compliance can be obviously improved.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Sequence listing
<110> Tianjin research institute of pharmaceuticals, Inc
<120> glucagon-like peptide-1 analogs and uses thereof
<130> DIC17110002
<160> 58
<170> PatentIn version 3.3
<210> 1
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage of Cys to NH2
<400> 1
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 2
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 2
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 3
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 3
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 4
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 4
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 5
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage of Cys to NH2
<400> 5
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 6
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> C-terminal linkage of Cys to NH2
<400> 6
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 7
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (30)..(30)
<223> C-terminal linkage of Cys to NH2
<400> 7
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 8
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 8
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 9
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage of Cys to NH2
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 10
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> C-terminal linkage of Cys to NH2
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 11
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 12
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 12
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 13
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage NH of Cys
<400> 13
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 14
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> C-terminal linkage of Cys to NH2
<400> 14
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 15
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 16
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 16
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 17
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 17
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 18
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 18
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 19
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage of Cys to NH2
<400> 19
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 20
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> C-terminal linkage of Cys to NH2
<400> 20
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 21
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (32)..(32)
<223> C-terminal linkage of Cys to NH2
<400> 21
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 22
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> C-terminal linkage of Cys to NH2
<400> 22
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 23
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 23
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 24
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 24
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 25
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 25
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 26
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 26
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg
20 25 30
<210> 27
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 27
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 28
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 28
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg
20 25 30
<210> 29
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Gly C terminal connected NH2
<400> 29
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
20 25
<210> 30
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 30
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 31
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Gly C terminal connected NH2
<400> 31
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 32
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 32
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 33
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Gly C terminal connected NH2
<400> 33
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 34
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 34
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 35
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Gly C terminal connected NH2
<400> 35
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 36
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 36
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 37
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 37
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 38
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 38
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 39
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 39
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 40
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Gly C terminal connected NH2
<400> 40
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly
20 25
<210> 41
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 41
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 42
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 42
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 43
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 43
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 44
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Arg at C-terminus to NH2
<400> 44
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 45
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 45
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 46
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 46
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 47
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 47
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 48
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 48
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 49
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 49
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 50
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 50
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 51
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 51
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 52
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 52
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 53
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 53
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 54
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 54
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 55
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (31)..(31)
<223> Gly C terminal connected NH2
<400> 55
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 56
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> precursor polypeptide
<220>
<221> misc_feature
<222> (2)..(2)
<223> D form amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa represents Aib, α -aminoisobutyric acid
<220>
<221> misc_feature
<222> (30)..(30)
<223> Lys C-terminal attachment NH2
<400> 56
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 57
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> Polypeptides
<400> 57
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 58
<211> 33
<212> PRT
<213> Artificial sequence
<220>
<223> Polypeptides
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa can be any naturally occurring amino acid
<400> 58
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Ala
20 25 30
Cys
Claims (19)
1. A precursor polypeptide of a glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof, wherein the sequence of the precursor polypeptide is shown as any one of SEQ ID NO 1, 4, 9, 11, 15, 17 and 19.
2. A glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof, wherein the glucagon-like peptide-1 analog or pharmaceutically acceptable salt thereof is a polyethylene glycol conjugate of the precursor polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.
3. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol has an average molecular weight in the range of 5-50 KDa.
4. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol has an average molecular weight in the range of 20-50 KDa.
5. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol has an average molecular weight in the range of 40-50 Kda.
6. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol is a linear, branched polyethylene glycol.
7. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol is selected from the group consisting of branched polyethylene glycols having an average molecular weight ranging from 40-50 KDa.
8. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol is selected from the group consisting of a bifurcated polyethylene glycol having an average molecular weight ranging from 40-50 KDa.
9. The glucagon-like peptide-1 analog of claim 2, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol is conjugated to the glucagon-like peptide-1 analog or a pharmaceutically acceptable salt thereof through an activation group on a molecular terminal group.
10. The glucagon-like peptide-1 analog of claim 9, wherein the activating group is selected from the group consisting of a maleimide group, a thiol group, a succinimidyl group, an aldehyde group, a halogen.
11. The glucagon-like peptide-1 analog of claim 9, or a pharmaceutically acceptable salt thereof, wherein the activating group is a maleimide group.
12. The glucagon-like peptide-1 analog of claim 9, or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol is conjugated to the cysteine side chain of any of SEQ ID NOs 1, 4, 9, 11, 15, 17, and 19 through an activating group.
13. A pharmaceutical composition comprising a precursor polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog of any one of claims 2-12, or a pharmaceutically acceptable salt thereof.
14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier and/or adjuvant.
15. The pharmaceutical composition of claim 14, wherein the carrier and/or excipient is selected from one or more of a water-soluble filler, a pH adjuster, a stabilizer, water for injection, or an osmotic pressure regulator.
16. The pharmaceutical composition of claim 15, wherein the water soluble filler is selected from one or more of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose; the pH regulator is selected from citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, and one or more of potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium bicarbonate salt; the stabilizer is selected from one or more of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxy cellulose or derivatives thereof, cyclodextrin, sodium dodecyl sulfate or tris (hydroxymethyl) aminomethane; the osmotic pressure regulator is sodium chloride and/or potassium chloride.
17. The pharmaceutical composition of claim 16, wherein the carboxy/hydroxycellulose or derivative thereof is HPC, HPC-SL, HPC-L or HPMC.
18. Use of a precursor polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof, or a glucagon-like peptide-1 analog of any of claims 2-12, or a pharmaceutically acceptable salt thereof, in the preparation of a pharmaceutical composition for the treatment of diabetes.
19. Use of a pharmaceutical composition according to any one of claims 13-17 in the manufacture of a medicament for the treatment of diabetes.
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| CN201710660343.4A CN109384839B (en) | 2017-08-04 | 2017-08-04 | Glucagon-like peptide-1 analogs and uses thereof |
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| CN201710660343.4A CN109384839B (en) | 2017-08-04 | 2017-08-04 | Glucagon-like peptide-1 analogs and uses thereof |
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| CN109384839B true CN109384839B (en) | 2021-03-09 |
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| CN112898404B (en) * | 2019-12-03 | 2024-11-12 | 天津药物研究院有限公司 | A long-acting modified glucagon peptide analog or its salt and its use |
| CN113336840B (en) * | 2020-03-02 | 2022-09-23 | 武汉帕肽生物医药有限责任公司 | Stapled peptides, methods of making and uses thereof |
| CN111574597B (en) * | 2020-05-07 | 2023-03-31 | 中国科学院微生物研究所 | anti-HIV polypeptide modified by high molecular weight PEG (polyethylene glycol), preparation method and application thereof |
| CN115634285A (en) * | 2021-07-20 | 2023-01-24 | 派格生物医药(苏州)股份有限公司 | Application of polypeptide conjugate in preparation of medicines for treating diseases related to glycometabolism |
| CN115947821B (en) * | 2021-10-09 | 2023-06-30 | 合肥天汇生物科技有限公司 | GLP-1 analogues |
| CN115850438B (en) * | 2021-12-28 | 2023-05-12 | 北京惠之衡生物科技有限公司 | Long-acting GLP-1 derivative |
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| WO2007124461A2 (en) * | 2006-04-20 | 2007-11-01 | Amgen Inc. | Glp-1 compounds |
| CN106554408A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | - 1 analog dimer of long-acting glucagon peptide and its application |
| CN106554409A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | A kind of -1 analog of long-acting glucagon peptide and its application |
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2017
- 2017-08-04 CN CN201710660343.4A patent/CN109384839B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007124461A2 (en) * | 2006-04-20 | 2007-11-01 | Amgen Inc. | Glp-1 compounds |
| CN106554408A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | - 1 analog dimer of long-acting glucagon peptide and its application |
| CN106554409A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | A kind of -1 analog of long-acting glucagon peptide and its application |
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