CN107266557B - Glucagon-like peptide-1 analogue modified by polyethylene glycol - Google Patents

Glucagon-like peptide-1 analogue modified by polyethylene glycol Download PDF

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CN107266557B
CN107266557B CN201710221546.3A CN201710221546A CN107266557B CN 107266557 B CN107266557 B CN 107266557B CN 201710221546 A CN201710221546 A CN 201710221546A CN 107266557 B CN107266557 B CN 107266557B
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韩英梅
赵娜夏
王玉丽
夏广萍
孔维苓
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Tianjin Institute of Pharmaceutical Research Co Ltd
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Abstract

The invention provides a glucagon-like peptide-1 analogue modified by polyethylene glycol, wherein the glucagon-like peptide-1 analogue has an amino acid sequence shown in SEQ ID NO. 1-19, and a polyethylene glycol group is modified on a cysteine residue of the amino acid sequence. The invention also provides application of the analogue or the composition thereof in preparing medicaments for treating diabetes, obesity and Alzheimer's disease. The polypeptide has good metabolic stability, obviously prolongs the half-life period in vivo, overcomes the problem of short half-life period of natural GLP-1, can greatly improve the compliance of clinical application, and has good application value.

Description

Glucagon-like peptide-1 analogue modified by polyethylene glycol
The present application claims the priority of the invention patent application entitled "a polyethylene glycol modified glucagon-like peptide-1 analog" filed 2016, 4/6/2016 under the name of 20161021111440.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a glucagon-like peptide-1 analog modified by polyethylene glycol and medical application thereof.
Background
GLP-1(7-36,7-37) is the main active form of GLP-1 in systemic circulation, controls blood glucose through a complex mechanism including secretion of insulin and glucagon, gastric emptying and regulation of peripheral insulin, is glucose-dependent in hypoglycemic action, can avoid hypoglycemia, has the effects of inhibiting apoptosis of pancreatic β -cells, promoting proliferation of pancreatic β -cells and reversing disease development, but the plasma half-life of natural GLP-1 is only 1-2 minutes, and metabolic instability limits its application as a medicament.
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
Figure BDA0001263921280000011
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. The existing patent technology based on the human GLP-1 sequence only considers one elimination factor of enzyme degradation aiming at the structural modification (CN00806548.9, CN99814187.9, CN200410017667.9 and the like) of the key sites of the enzyme degradation, and does not achieve ideal long-acting effect; the half-life period (for example, the liraglutide on the market is administrated once a day) can be prolonged to a certain extent by 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), the drug compliance of the technology still needs to be improved, the solubility of the introduced fatty chain in the peptide chain is reduced, an organic solvent is required to be used in a preparation, and the preparation difficulty is increased.
Polyethylene glycol (PEG) technology is a more suitable technology in the field of protein/polypeptide administration at present. The protein/polypeptide is modified by linear direct-connected or branched polyethylene glycol, so that the physical and chemical property stability of the protein/polypeptide can be improved, the immunogenicity is remarkably reduced, the anti-protease degradation capability is remarkably improved, and the metabolism of the renal clearance effect on the medicament can be remarkably reduced along with the increase of the molecular weight of the polyethylene glycol, so that the in vivo half-life period of the medicament is remarkably prolonged, the solubility of the medicament is improved, and the penetrating power of a cell membrane is enhanced. The coupled PEG chain generates steric hindrance effect, reduces the hydrolysis of plasma proteolytic enzyme, thereby effectively prolonging the storage time of the PEG chain in a circulatory system, prolonging the half-life period of the PEG chain, increasing the exposure of the PEG chain to the systemic drug and improving the curative effect.
Although the prior patent technologies (CN1372570A, CN101125207A and the like) disclose polyethylene glycol modification technologies of Exendin-4, the prior technologies can prolong the half-life of Exendin-4 in vivo and achieve long-acting effect, the conjugates finally generate drug effect by releasing free Exendin-4 in vivo, and the drug resistance of generated antibodies (45 percent of people who take 30 weeks of drug) and the like caused by the immunogenicity of Exendin-4 cannot be solved.
Disclosure of Invention
Aiming at the limitations of the prior art, the invention provides a polyethylene glycol modified GLP-1 analogue which has longer in vivo half-life period and better hypoglycemic activity, is highly homologous with endogenous GLP-1(7-36/37), and can avoid safety risk.
The inventor finds in research that modifying the site easily degraded by enzyme in GLP-1(7-36,7-37) sequence, and simultaneously conjugating polyethylene glycol with proper molecular weight on proper active amino acid residue can maintain drug effect and effectively prolong half-life period in vivo, and the modified sequence is highly homologous (more than or equal to 90%) with human GLP-1, so that antibody generation can be avoided, the dissolubility is good, the preparation is convenient, and better application potential is achieved.
The glucagon-like peptide-1 analogs of the present invention are artificially modified forms of GLP-1 (7-36/37). The natural sequence of GLP-1(7-36/37) is: HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR-NH 2/G. In the invention, proper amino acid substitution is carried out on X8 or X9 and X35 in the sequence so as to avoid enzyme degradation inactivation, and in order to further improve the stability of the polypeptide, the C-terminal of a peptide chain is generally amidated in an embodiment of the invention unless special description is made; and simultaneously, introducing a cysteine residue at the X30 site or the C-terminal extension sequence X39 site, and modifying a polyethylene glycol group with proper molecular weight on the cysteine residue through maleimide activation to avoid the rapid elimination of the target polypeptide in vivo.
In one aspect, the invention provides a glucagon-like peptide-1 analog modified by polyethylene glycol, wherein the glucagon-like peptide-1 analog has a structure shown in a general formula I, and X in an amino acid sequence shown in the general formula I3Or X5A single site is modified with a polyethylene glycol group:
general formula I: HX1X2GTFTSDVSSYLEGQAAKEFIX3WLVKAibRX4X5X6
Preferably, the glucagon-like peptide-1 analogue modified by polyethylene glycol has the structure shown in the general formula I, and the site X in the amino acid sequence of the glucagon-like peptide-1 analogue with the structure shown in the general formula I3Or X5Is a cysteine residue;
preferably, the glucagon-like peptide-1 analog with the structure of the general formula I has the sequence shown in SEQ ID NO: 1-19;
SEQ ID NO:1 H(d-A)EGTFTSDVSSYLEGQAAKEFICWLVKAibRNH2
SEQ ID NO:2 H(d-A)EGTFTSDVSSYLEGQAAKEFICWLVKAibRG
SEQ ID NO:3 H(d-A)EGTFTSDVSSYLEGQAAKEFIAWLVKAibGCG
SEQ ID NO:4 H(d-A)EGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCA
SEQ ID NO:5 HGEGTFTSDVSSYLEGQAAKEFICWLVKAibRNH2
SEQ ID NO:6 HGEGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCG
SEQ ID NO:7 HGEGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCA
SEQ ID NO:8 HAPGTFTSDVSSYLEGQAAKEFICWLVKAibRNH2
SEQ ID NO:9 HAPGTFTSDVSSYLEGQAAKEFICWLVKAibRG
SEQ ID NO:10 HAPGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCG
SEQ ID NO:11 HAPGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCA
SEQ ID NO:12 HAFGTFTSDVSSYLEGQAAKEFICWLVKAibRNH2
SEQ ID NO:13 HAFGTFTSDVSSYLEGQAAKEFICWLVKAibRG
SEQ ID NO:14 HAFGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCG
SEQ ID NO:15 HAFGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCA
SEQ ID NO:16 HAYGTFTSDVSSYLEGQAAKEFICWLVKAibRNH2
SEQ ID NO:17 HAYGTFTSDVSSYLEGQAAKEFICWLVKAibRG
SEQ ID NO:18 HAYGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCG
SEQ ID NO:19 HAYGTFTSDVSSYLEGQAAKEFIAWLVKAibRGCA
preferably, the molecular weight of the polyethylene glycol in the polyethylene glycol modified glucagon-like peptide-1 analogue is 20000-50000 dalton;
preferably, the polyethylene glycol is selected from modified PEG;
more preferably, the polyethylene glycol is selected from monomethoxy PEG, branched PEG.
Preferably, the polyethylene glycol group is activated by a maleimido group;
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. PEG modification with different molecular weights has influence on polypeptide properties and biological activity. Preferred PEG molecular weights for the present invention range between 20000 and 50000 daltons, including 20000 and 500000 daltons.
The polyethylene glycol modification of the polypeptide is realized by connecting polyethylene glycol activated by maleimide with sulfydryl on a side chain of a cysteine residue. Maleimidopolyethylene glycols can be obtained commercially or prepared by themselves according to techniques well known in the art.
It is another object of the present invention to provide a composition comprising at least one PEG modification of a glucagon-like peptide-1 analog represented by formula I.
Preferably, the composition further comprises pharmaceutically acceptable carriers, diluents and the like.
Preferably, the composition of the present invention consists of the glucagon-like peptide-1 analog and one or more pharmaceutically acceptable excipients. The medicinal adjuvants comprise water-soluble filler, pH regulator, stabilizer, water for injection, osmotic pressure regulator, etc.
Preferably, the water-soluble filler includes, but is not limited to, mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, etc.; 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, etc., and physiologically acceptable inorganic bases or salts such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate, ammonium bicarbonate salts, etc.; such stabilizers include, but are not limited to, 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, carboxy/hydroxy cellulose or derivatives thereof such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrins, sodium lauryl sulfate or tris (hydroxymethyl) aminomethane and the like; the tonicity modifier includes, but is not limited to, sodium chloride or potassium chloride.
The invention also aims to provide application of the PEG modified glucagon-like peptide-1 analogue shown in the general formula I or the composition thereof in preparing medicaments for treating diabetes, obesity and Alzheimer's disease.
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.
The glucagon-like peptide-1 analog with the structure of the general formula I provided in the invention is prepared by the method 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.
In the embodiment provided by the invention, the target peptide is preferably prepared by using Fmoc solid phase synthesis method.
In a specific embodiment provided by the present invention, the pegylation modification of the target polypeptide is accomplished by: the activated PEG and the target polypeptide of SEQ ID NO 1-19 of the invention are at pH5.0-7.0, the molar ratio of PEG to 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.
According to the embodiment of the invention, a db/db diabetic mouse model is adopted to evaluate the hypoglycemic effect of the PEG-modified GLP-1 analogue, and the result shows that the PEG-modified glucagon-like peptide-1 analogue provided by the invention has a remarkable hypoglycemic effect and still shows activity after 144 hours of administration, so that the designed sequence polypeptide has good metabolic stability, the half-life period in vivo is remarkably prolonged, the problem of short half-life period of natural GLP-1 is solved, the clinical application compliance can be greatly improved, and the PEG-modified glucagon-like peptide-1 analogue has good application value. The in vitro agonistic activity test on GLP-1 receptor shows that the modified peptide provided by the invention is superior to endogenous receptor agonist GLP-1 (7-36-NH)2) The activity of (3) indicates that it is more suitable for long-acting through macromolecular modification.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the results of the evaluation of the hypoglycemic effect of linear monomethoxyPEG (40000 Dalton) modified SEQ ID NO:5 and linear monomethoxyPEG (42000 Dalton) modified SEQ ID NO:12 and PEG (45000 Dalton) modified SEQ ID NO:7 of example 4;
FIG. 2 shows the results of the evaluation of the hypoglycemic effect of linear monomethoxyPEG (40000 Dalton) modified SEQ ID NO:9, linear monomethoxyPEG (42000 Dalton) modified SEQ ID NO:11, PEG (45000 Dalton) modified SEQ ID NO:14 in example 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1
The present invention will be further described with reference to the following examples. The examples are merely illustrative of the invention and do not limit the scope of the invention in any way. For a better understanding of the invention, the abbreviations used in the reagents are detailed in Table 1 in comparison with the Chinese names.
TABLE 1 comparison of reagent abbreviations with Chinese names
Figure BDA0001263921280000061
Example 1
A. Preparation of glucagon-like peptide-1 analogues
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 (tert-butyloxycarbonyl), glutamic acid with OtBu (tert-butyloxy), lysine with Boc (tert-butyloxycarbonyl), glutamine with Trt (trityl), tyrosine with tBu (tert-butyl), serine with Trt (trityl) or tBu (tert-butyl), aspartic acid with OtBu (tert-butyloxy), threonine with tBu (tert-butyl), and histidine with Trt (trityl) or Boc (tert-butyloxycarbonyl). 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 a crude glucagon-like peptide-1 analogue;
2) purifying by dissolving the crude glucagon-like peptide-1 analog in water or 10-15% acetonitrile (10-50mg/ml), adding 50-100mM dithiothreitol DTT or β -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 glucagon-like peptide-1 analog represented by SEQ ID NO 1-19 is prepared by the method.
Example 2
Linear monomethyl PEG (21000 daltons) modified SEQ ID NO 1
1) Ligation the polypeptide of SEQ ID NO. 1 was dissolved in 50mM sodium phosphate buffer solution containing 5mM EDTA, pH6, and the solution was added at a concentration of 2mg/mL, 1.2-1.5 times the molar amount of solid PEG-maleimide was added, the solution was stirred and dissolved, the reaction was allowed to proceed at room temperature for 2hr, the reaction was monitored by HPLC, the reaction was stopped with 5mM β -mercaptoethanol, and the reaction was allowed to stand at room temperature for 30min and then purified.
2) And (3) purifying, namely performing preparative ion exchange column chromatography, using SP Sepharose HP as a filler, and eluting by a linear gradient of 0-500mM sodium chloride solution. Detecting the effluent by HPLC and SDS-electrophoresis, collecting PEG-polypeptide fraction, ultrafiltering and concentrating or desalting by Sephadex G-25 method, and lyophilizing.
The purity of the obtained PEG modified polypeptide is detected by RP-HPLC, and the molecular weight is determined by MALDI-TOF.
Example 3
Linear monomethyl PEG (30000 daltons) modified SEQ ID NO 8
PEG conjugates of linear monomethyl PEG (30000 daltons) modified SEQ ID NO:8 and the following glucagon-like peptide-1 analogs were prepared as in example 2.
Linear monomethoxy PEG (45000 Dalton) modified SEQ ID NO 7
Linear monomethoxy PEG (43000 dalton) modified SEQ ID NO 8
Linear monomethoxy PEG (45000 Dalton) modified SEQ ID NO 9
Linear monomethoxy PEG (41000 Dalton) modified SEQ ID NO 10
Linear monomethoxy PEG (42000 Dalton) modified SEQ ID NO 11
Linear monomethoxy PEG (35000 dalton) modified SEQ ID NO 13
Linear monomethoxy PEG (42000 Dalton) modified SEQ ID NO 14
Linear monomethoxy PEG (22000 dalton) modified SEQ ID NO 16
Linear monomethoxy PEG (45000 Dalton) modified SEQ ID NO 18
Linear monomethoxy PEG (46000 Dalton) modified SEQ ID NO 19
Linear monomethoxy PEG (20000 dalton) modified SEQ ID NO 7
Linear monomethoxy PEG (40000 dalton) modified SEQ ID NO 7
Y-type PEG (40000 dalton) modified SEQ ID NO 7
4-arm PEG (40000 dalton) modified SEQ ID NO 7
Linear monomethoxy PEG (20000 dalton) modified SEQ ID NO 3
Y-type PEG (40000 dalton) modified SEQ ID NO 3
Linear monomethoxy PEG (20000 dalton) modified SEQ ID NO 3
Linear monomethoxy PEG (40000 dalton) modified SEQ ID NO 4
Y-type PEG (40000 dalton) modified SEQ ID NO. 4
4-arm PEG (40000 dalton) modified SEQ ID NO 4
Y-type PEG (40000 dalton) modified SEQ ID NO:5
4-arm PEG (40000 dalton) modified SEQ ID NO:5
4-arm monomethoxy PEG (40000 dalton) modified SEQ ID NO 6
Y-type PEG (40000 dalton) modified SEQ ID NO 6
Example 4
Evaluation of the hypoglycemic Effect of the Linear monomethoxyPEG (30000 Dalton) modified SEQ ID NO:5 (sample 8) and PEG (45000 Dalton) modified SEQ ID NO:7 (sample 14) and 12 (sample 12)
A db/db model mouse is adopted to evaluate the blood sugar reducing effect of the GLP-1 analogue PEG modifier. The method comprises the following steps: 40 db/db mice (purchased from the Shanghai laboratory animal center of the Chinese academy of sciences, having blood glucose levels of at least 250mg/dL) were randomly divided into 4 groups (blank control group, 2 test groups), 10 mice per group; a sample solution of a suitable amount of PEG modification of GLP-1 analog 0.1mg/ml was weighed. Mice in the test group were injected subcutaneously with 200. mu.l of each sample solution; mice in the blank control group were injected subcutaneously with 200. mu.l of physiological saline each. Feeding was not restricted during the trial. Blood glucose levels were measured 2, 4, 8, 24, 72, 96, and 120 hours after injection, respectively. The results are shown in FIG. 1.
Example 5
Evaluation of the hypoglycemic Effect of Linear Monomethoxy PEG (45000 daltons) modified SEQ ID NO 9 (sample 17) and Linear Monomethoxy PEG (42000 daltons) modified SEQ ID NO 11 (sample 21) and PEG (42000 daltons) modified SEQ ID NO 14 (sample 26)
The evaluation method was the same as in example 4, and the results are shown in FIG. 2.
Example 6
Evaluation of GLP-1 receptor agonistic Activity
The agonistic activity of the polypeptides of the invention at the GLP-1 receptor is assessed by the effect on GLP-1 receptor mediated in vitro cAMP production.
Chinese guinea pig lung cells transfected with the human GLP-1 receptor were inoculated into 96-well plates, (200000 cells/well), washed with Hanks' balanced salt buffer, and incubated with test polypeptide samples (10-5-10-12mol/L) at different concentrations for 20min at 37 ℃ in the presence of 200. mu. mol/L of 3-isobutyl-1-methylrubixanthin. Medium was removed, cells were lysed and cAMP values were determined according to the assay kit instructions. The 50% effective concentration was calculated using Origin software. The results are shown in Table 2.
TABLE 2 induced cAMP production by Polypeptides
Peptide number GLP-1R(nM EC50)
GLP-1 0.17±0.21
SEQ ID NO:1 0.07±0.14
SEQ ID NO:3 0.10±0.26
SEQ ID NO:4 0.08±0.33
SEQ ID NO:5 0.09±0.19
SEQ ID NO:6 0.16±0.20
SEQ ID NO:7 0.06±0.09
SEQ ID NO:8 0.18±0.22
SEQ ID NO:11 0.08±0.13
SEQ ID NO:12 0.13±0.17
SEQ ID NO:14 0.16±0.14
SEQ ID NO:19 0.20±0.31
And (4) conclusion: SEQ ID NO 1, 4, 7 and 11 in the polypeptide sequence show stronger receptor agonistic activity than endogenous GLP-1.
Example 7
Evaluation of hypoglycemic effect and long-acting property of PEG modified SEQ ID NO. 7 with different molecular weights and different structures
The hypoglycemic effect and the long-acting property of different PEG modifiers of SEQ ID NO. 7 are evaluated by adopting a normal mouse glucose load model.
Sample preparation:
the samples of SEQ ID NO. 7 (C-terminal amidation) modified with mPEG (20KD), mPEG (40KD), Y-type PEG (40KD) and 4-arm type PEG (40KD) were prepared and characterized by the method of example 3, with a purity (HPLC) > 98.0%; (37C) mPEG (20KD) modifier of GLP-1(7-36), prepared and characterized by the method of example 3, with purity (HPLC) > 98.0%;
positive drugs: liraglutide (Nonhuode company)
The method comprises the following steps:
evaluation by a normal mouse sugar load model: normal mice (n is 6) are fasted for 12 hours, blood sugar values are measured before drugs are administered subcutaneously, glucose (4mg/kg) is injected into the abdominal cavity at different time points after drugs are administered (the mice are fasted for 4 hours before the drugs are administered), the blood sugar values of the mice are measured 0.5 hour after the drugs are administered, the inhibition rate of the samples on the increase of the blood sugar of the glucose-loaded normal mice is calculated, the calculation formula is that the inhibition rate is (1-the blood sugar average value of an administration group/the blood sugar average value of a model control group)%, and the liraglutide is a positive control drug.
Administration dose: the positive drug liraglutide and the sample are both 100 nmol/kg.
As a result: see Table 3 below
TABLE 3 hypoglycemic Effect of PEG-modified SEQ.7 series samples over different time periods
Figure BDA0001263921280000101
And (4) conclusion: positive drugs liraglutide and mPEG (20KD) modified37The hypoglycemic effect of CGLP-1 is basically disappeared after 24 hours of administration, the PEG-modified sample series of SEQ.7 shows the hypoglycemic effect superior to that of the positive drug in different time periods, the drug effect duration of 4 samples of mPEG (20KD), mPEG (40KD), YPEG (40KD) and 4Arm PEG (40KD) is 48, 120 and 144 hours respectively, the drug effect fluctuation of the branched PEG-modified sample is small, and the 4-Arm PEG (40KD) modified sample is significantly superior to other samples. mPEG (2)0KD)-37Compared with mPEG (20KD) SEQ.7, the CGLP-1 has faster activity attenuation, the activity of the CGLP-1 in 24 hours is only 33.1 percent, the activity is not active in 48 hours, and the drug effect of the CGLP-1 can be stably maintained for 48 hours, which is obviously superior to that of the mPEG. A series of replacement and modification of the sequence effectively avoids enzyme degradation, improves the stability and the activity of the naked peptide, and is more beneficial to the drug effect exertion of a modifier and the prolongation of the half-life period in vivo.
Example 8
The hypoglycemic effects and the long-lasting effect of the Y-type PEG (40KD) modified forms of SEQ ID NOS 4, 5 and 6 were evaluated in the same manner as in example 7. The results are shown in Table 4.
Figure BDA0001263921280000102
Figure BDA0001263921280000111
And (4) conclusion: the hypoglycemic effect and the duration of the drug effect of the Y-type PEG (40KD) modified bodies of SEQ ID NO. 4 and SEQ ID NO. 6 are similar, while the activity and the duration of the activity of SEQ ID NO. 5 are greatly different from those of the former two groups. Indicating that PEG modification at different sites has a great influence on the exertion and duration of activity.
Sequence listing
<110> Tianjin research institute of pharmaceuticals, Inc
<120> glucagon-like peptide-1 analogue modified by polyethylene glycol
<130>DIC17110020
<160>19
<170>PatentIn version 3.5
<210>1
<211>30
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(2)..(2)
<223> D form amino acid
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<220>
<221>MISC_FEATURE
<222>(30)..(30)
<223> Arg at C-terminus to NH2
<400>1
His Ala 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 Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210>2
<211>31
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(2)..(2)
<223> D form amino acid
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>2
His Ala 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 Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210>3
<211>32
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(2)..(2)
<223> D form amino acid
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>3
His Ala 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 Gly Cys Gly
20 25 30
<210>4
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(2)..(2)
<223> D form amino acid
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>4
His Ala 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 Cys
20 25 30
Ala
<210>5
<211>30
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<220>
<221>MISC_FEATURE
<222>(30)..(30)
<223> Arg at C-terminus to NH2
<400>5
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 Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210>6
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>6
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 Cys
20 25 30
Gly
<210>7
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>7
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 Cys
20 25 30
Ala
<210>8
<211>30
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<220>
<221>MISC_FEATURE
<222>(30)..(30)
<223> Arg at C-terminus to NH2
<400>8
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210>9
<211>31
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>9
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210>10
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>10
His Ala Pro 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 Cys
20 25 30
Gly
<210>11
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>11
His Ala Pro 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 Cys
20 25 30
Ala
<210>12
<211>30
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<220>
<221>MISC_FEATURE
<222>(30)..(30)
<223> Arg at C-terminus to NH2
<400>12
His Ala Phe Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210>13
<211>31
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>13
His Ala Phe Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210>14
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>14
His Ala Phe 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 Cys
20 25 30
Gly
<210>15
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>15
His Ala Phe 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 Cys
20 25 30
Ala
<210>16
<211>30
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<220>
<221>MISC_FEATURE
<222>(30)..(30)
<223> Arg at C-terminus to NH2
<400>16
His Ala Tyr Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln 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>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>17
His Ala Tyr Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
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>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>18
His Ala Tyr 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 Cys
20 25 30
Gly
<210>19
<211>33
<212>PRT
<213> Artificial sequence
<220>
<221>MISC_FEATURE
<222>(29)..(29)
<223> Xaa represents Aib, 2-aminoisobutyric acid
<400>19
His Ala Tyr 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 Cys
20 25 30
Ala

Claims (8)

1. The glucagon-like peptide-1 analogue modified by polyethylene glycol is characterized in that the amino acid sequence of the glucagon-like peptide-1 analogue is selected from one of the amino acid sequences shown in SEQ ID NO. 4, 6-7, 12 and 14, and a polyethylene glycol group is modified on a cysteine residue of the amino acid sequence;
wherein the molecular weight of the polyethylene glycol is 20000-50000 dalton.
2. The analog of claim 1, wherein the polyethylene glycol is a modified polyethylene glycol.
3. The analog of claim 2, wherein the polyethylene glycol is selected from the group consisting of monomethoxypolyethylene glycol, branched polyethylene glycol, and branched polyethylene glycol.
4. The analog of claim 3, wherein the polyethylene glycol is monomethoxypolyethylene glycol.
5. The analog of any of claims 1 to 4, wherein the polyethylene glycol group is activated by a maleimide group.
6. A composition comprising the analog of any one of claims 1-5, or a salt thereof.
7. The composition of claim 6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
8. The use of an analogue according to any one of claims 1 to 5, a composition according to claim 6 or 7 for the manufacture of a medicament for the treatment of diabetes, obesity.
CN201710221546.3A 2016-04-06 2017-04-06 Glucagon-like peptide-1 analogue modified by polyethylene glycol Active CN107266557B (en)

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Publication number Priority date Publication date Assignee Title
CN109021093B (en) * 2018-08-29 2021-09-07 上海生物制品研究所有限责任公司 Polyethylene glycol modified GLP-1 derivatives and medicinal salts thereof
CN109400695B (en) 2018-10-31 2020-06-30 中南大学湘雅医院 Polypeptide modification method and application
CN111378028A (en) * 2018-12-30 2020-07-07 万新医药科技(苏州)有限公司 Synthesis of acylated GLP-1 compounds and modified groups thereof
CN112898406B (en) * 2019-10-12 2023-11-10 深圳纳福生物医药有限公司 GLP-1 analogue peptide modified dimer with different configurations and application of preparation method thereof in treatment of type II diabetes
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
CN114591416B (en) * 2022-03-03 2022-11-18 河北科技大学 A kind of N-glycan modified glucagon-like peptide-1 analogue and its preparation method and application

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