CN107266557A

CN107266557A - A kind of polyethyleneglycol modified glucagon-like peptide 1 analog

Info

Publication number: CN107266557A
Application number: CN201710221546.3A
Authority: CN
Inventors: 韩英梅; 赵娜夏; 王玉丽; 夏广萍; 孔维苓
Original assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Current assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Priority date: 2016-04-06
Filing date: 2017-04-06
Publication date: 2017-10-20
Anticipated expiration: 2037-04-06
Also published as: CN107266557B

Abstract

The present invention provides a kind of polyethyleneglycol modified glucagon-like peptide 1 analog, and the glucagon-like peptide 1 analog has SEQ ID NO:Polyethylene group is modified on amino acid sequence shown in 1 19, and the cysteine residues of the amino acid sequence.Present invention also offers the application of the analog or its composition in treatment diabetes, obesity, the medicine of alzheimer disease is prepared.The polypeptide of the present invention has preferable metabolic stability, and Half-life in vivo significantly extends, the problem of overcoming natural 1 half-life shorts of GLP, can greatly improve clinical practice compliance, with preferable application value.

Description

A kind of polyethyleneglycol modified glucagon-like peptide-1 analogs

This application claims entitled " a kind of polyethyleneglycol modified glucagon-like-peptide-1 submitted on April 6th, 2016 Analog ", the priority of the application for a patent for invention of Application No. 20161021111440.

Technical field

The invention belongs to pharmaceutical technology field, and in particular to a kind of polyethyleneglycol modified glucagon-like-peptide-1 is similar Thing and its medical usage.

Background technology

Glucagon-like peptide 1 (GLP-1) is a kind of Entero hormone, mainly thin in the L of end barnyard, ileum and colon Born of the same parents are synthesized, and circulation is discharged into reaction of having meal.GLP-1 (7-36,7-37) is the chief active form of GLP-1 in body circulation, is led to The mechanism control blood glucose of complexity is crossed, includes the secretion of insulin and hyperglycemic factor, the emptying of stomach and the tune of periphery insulin Section.GLP-1 (7-36,7-37) hypoglycemic effect is glucose dependency, can avoid hypoglycemia, and with suppression pancreas The apoptosis of island beta cell, promotes the effect of hyperplasia of pancreatic islet beta cell, and disease development can be reversed.But natural GLP-1 blood plasma partly declines Phase is only 1-2 minutes, and metabolism unstability limits it as the application of medicine.

Enzyme is degraded and kidney removing is the main path of metabolism in polypeptides matter body.Research shows, internal dipeptides kassinin kinin Receptor-binding activity position N-terminal His-Ala fragments in enzyme (DPPIV) specific recognition and GLP-1 structures of degrading and make its quick Inactivation, while other proteolytic enzymes such as endopeptidase also assists in degradation process in polypeptide body.Kidney is eliminating the things such as peptide, albumen Played an important role in matter, blood plasma middle-molecular-weihydroxyethyl is less than 5KD and effective radius is less thanFree fraction molecule it is easily small by kidney Ball is filtered, and peptide hormone (such as calcitonin, GLP-1) is degraded by the metabolic enzyme in cortex renis in kidney circulation, further row Let out in urine.Research report kidney is responsible for removing at least 80% Exendin-4 (CN1372570).

Metabolic stability is improved, extends plasma half-life, so that it is the medicine based on GLP-1 to improve clinical application compliance The technical goal of development field.For the knot of enzyme degraded critical sites in the existing patented technology based on people source GLP-1 sequences Structure transformation (CN00806548.9, CN99814187.9, CN200410017667.9 etc.) only considered enzyme degrade one eliminate because Element, does not reach preferably long-acting；And by introducing fatty acyl group in parent peptide chain structure, improve and plasma protein adhesion To avoid polypeptide from quickly removing (CN201210513145.2, CN200810124641.2, CN20118000352.1 etc.) in vivo Though technology can extend half-life period () to a certain extent such as the Liraglutide that has listed by daily single, its compliance is still Having much room for improvement, and aliphatic chain dissolubility is introduced in peptide chain reduces, and needs to use organic solvent in preparation, ease of formulation is increased.

Polyethylene glycol (PEG) change technology is the technology that current one, field of protein/polypeptide class administration is relatively applicable.Generally adopt Protein/polypeptide is modified with linear direct-connected or branched polyethylene glycol, protein/polypeptide physics and chemistry can be made Property stability is improved, and immunogenicity is remarkably decreased, and proteolytic degradation ability is significantly improved, with peg molecule The increase of amount, can significantly reduce metabolism of the kidney scavenging action to medicine, so that the Half-life in vivo of medicine significantly extends, medicine is molten Xie Du is improved and the penetration power of cell membrane is strengthened.The PEG chains of coupling produce space steric effect, reduce plasma protein hydrolysis The hydrolysis of enzyme, so that effectively extending its storage in the circulatory system stays the time, makes drug half-life extension and system medicine Thing exposure increases and improves curative effect.

Existing patented technology (CN1372570A, CN101125207A etc.) discloses the polyethyleneglycol modified of Exendin-4 Technology, although these existing technologies can extend Exendin-4 Half-life in vivo, can reach long-actingization, and these are conjugated Thing is all finally to produce drug effect by discharging free Exendin-4 in vivo, and what the immunogenicities of Exendin-4 in itself were brought Producing the drug resistances such as antibody (45% generation antibody in the crowd of medication 30 weeks) can not still solve.

The content of the invention

For the limitation of prior art, the present invention provides a kind of polyethyleneglycol modified GLP-1 analogs, its have compared with Long Half-life in vivo and preferable hypoglycemic activity, and with endogenous GLP-1 (7-36/37) very high homology, can avoid peace Full property risk.

The present inventor etc. have found to change the site being easily degraded by enzymes in GLP-1 (7-36,7-37) sequence under study for action The polyethylene glycol of appropriate molecular weight is conjugated while structure on suitable active amino acid residue, drug effect, effective extension body can be kept Interior half-life period, and because transformation sequence and people source GLP-1 very high homologies (>=90%) can avoid producing antibody, dissolubility is good, It is easy to preparation, there is more preferable application potential.

Glucagon-like peptide-1 analogs of the present invention are GLP-1 (7-36/37) manually modified forms.GLP- 1 (7-36/37) native sequences are：HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR-NH2/G.The present invention is to the sequence X8 or X9 and X35 carries out appropriate amino acid substitution to avoid enzyme degraded inactivation in row, is the further stability for improving polypeptide, C- ends if do not done peptide chain in specified otherwise embodiment of the present invention are usually amidated；Extend simultaneously in X30 sites or C-terminal 39 introducing cysteine residues of sequence X, appropriate molecule is modified on the cysteine residues by Maleimido activation The polyethylene group of amount, it is to avoid the quick elimination of target polypeptides in vivo.

On the one hand, the invention provides a kind of polyethyleneglycol modified glucagon-like peptide-1 analogs, the pancreas is high The analog of blood glucose element sample peptide -1 has the structure of below general formula I, and the X in the amino acid sequence of formula I₃Or X₅Repaiied on Single locus It is decorated with polyethylene group：

Formula I：HX₁X₂GTFTSDVSSYLEGQAAKEFIX₃WLVKAibRX₄X₅X₆

Preferably, polyethyleneglycol modified glucagon-like peptide-1 analogs of the present invention, the described knot of formula I Site X in the glucagon-like peptide-1 analogs amino acid sequence of structure₃Or X₅For cysteine residues；

Preferably, the glucagon-like peptide-1 analogs with logical structure shown in formula I of the present invention have SEQ ID NO： Amino acid sequence shown in 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 polyethylene glycol exists in the polyethyleneglycol modified glucagon-like peptide-1 analogs Between 20000-50000 dalton；

Preferably, the polyethylene glycol is selected from the PEG through modification；

It is highly preferred that the polyethylene glycol is selected from mono methoxy PEG, branch PEG, bifurcated PEG.

Preferably, the polyethylene group is activated by Maleimido；

Polyethylene glycol of the present invention can be obtained by number of ways, including commercial sources are obtained or according to this Known method is voluntarily prepared in field.Different molecular weight size PEG modifications all have an impact to polypeptide nature and bioactivity.This Preferred PEG molecular weight ranges are invented between 20000-50000 dalton, including 20000 and 500000 dalton.

The polyethyleneglycol modified of polypeptide is by maleimido-activated polyethylene glycol and cystein residue On sulfydryl connection and realize.Dimaleoyl imino polyethylene glycol can be obtained or according to known in this field by commercially available approach Technology is voluntarily prepared.

Another object of the present invention is to provide a kind of composition, the composition includes I table of at least one formula The PEG trims of the glucagon-like peptide-1 analogs shown.

Preferably, the composition is further comprising auxiliary materials such as pharmaceutically acceptable carrier, diluents.

Preferably, composition of the present invention is by the glucagon-like peptide-1 analogs and one or more medicines Acceptable auxiliary material composition on.The pharmaceutic adjuvant includes water-soluble filler, pH adjusting agent, stabilizer, water for injection, oozed Conditioning agent etc. is pressed thoroughly.

Preferably, the water-soluble filler includes but is not limited to mannitol, D-40, sorbierite, poly- second Glycol, glucose, lactose, galactolipin etc.；The pH adjusting agent includes but is not limited to citric acid, phosphoric acid, lactic acid, tartaric acid, salt The organic or inorganic acids such as acid and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, saleratus, Physiologically acceptable inorganic base or the salt such as sodium acid carbonate, bicarbonate ammonium salt；The stabilizer include but is not limited to EDTA-2Na, Sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium acid carbonate, sodium carbonate, arginine, lysine, paddy ammonia Acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or derivatives thereof as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane etc.；The osmotic pressure regulator Including but not limited to sodium chloride or potassium chloride.

It is also another object of the present invention to provide PEG modification formula I shown in glucagon-like peptide-1 analogs or Its composition is preparing the application in being used to treat diabetes, obesity, the medicine of alzheimer disease.

Preferably, composition of the present invention can be with vein, muscle or subcutaneous injection agent form or oral, rectum, nose Chamber is administered.Dosage range can be g-10mg/ times for 5 μ, and this depends on treatment target, administering mode, indication and other factors Deng.

The glucagon-like peptide-1 analogs with logical structure shown in formula I provided in the present invention are by well known in the art Method is prepared：

1) synthesis progressively or by fragment is assembled by conventional solid or liquid phase process；

2) nucleic acid construct of coded polypeptide is expressed in host cell, and expression production is reclaimed from host cell cultures Thing；

3) the cell free in vitro expression of the nucleic acid construct of influence coded polypeptide, and reclaim expression product；

Or by method 1), any combination 2) or 3) obtain fragments of peptides, these fragments are then connected obtain target Peptide.

In the embodiment that the present invention is provided preferably, target peptide is prepared using Fmoc solid phase synthesis process.

The pegylation of target polypeptides is completed in the specific embodiment that the present invention is provided by the following method：Will PEG and the SEQ ID NO of the present invention through overactivation:1-19 target polypeptides are in pH5.0-7.0, and the molar ratio of PEG and peptide is 1-10, the reaction time is 0.5-12 hours, and reaction temperature is 4-37 DEG C.

After conjugation reaction, target product can be separated by appropriate method well known in the art.Applicable method bag Include but be not limited to ultrafiltration, dialysis or chromatography etc..

The PEG have rated using db/db diabetic mouse models in embodiment of the present invention and modify GLP-1 analogs Hypoglycemic effect, as a result show that the polyethyleneglycol modified glucagon-like peptide-1 analogs that provide of the present invention have notable Hypoglycemic effect and also administration after still show activity within 144 hours, illustrate that designed sequences polypeptide has preferably metabolism steady Qualitative, Half-life in vivo significantly extends, the problem of overcoming natural GLP-1 half-life shorts, can greatly improve clinical practice compliance Property, with preferable application value.The agonist activity experiment to GLP-1 acceptors shows that the process that the present invention is provided is repaiied in vitro The structure peptide that changes of decorations is shown better than endogenous receptor activator GLP-1 (7-36-NH₂) activity, illustrate to be more suitable for by dividing greatly Son modification long-actingization.

Brief description of the drawings

Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：

Fig. 1 is the cathetus type mono methoxy PEG (40000 dalton) of embodiment 4 modification SEQ ID NO:5 and linear pattern list Methoxyl group PEG (42000 dalton) modification SEQ ID NO:12nd, PEG (45000 dalton) modifies SEQ ID NO:7 drop blood Sugared evaluation of effect result；

Fig. 2 is the cathetus type mono methoxy PEG (40000 dalton) of embodiment 5 modification SEQ ID NO:9 and linear pattern list Methoxyl group PEG (42000 dalton) modification SEQ ID NO:11st, PEG (45000 dalton) modifies SEQ ID NO:14 drop blood Sugared evaluation of effect result.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.

Embodiment 1

With reference to specific embodiment, the present invention is further illustrated.The present embodiment be only explain the present invention, not with Any mode limits present invention.In order to be better understood from the present invention, the abbreviation of agents useful for same is compareed with Chinese name refers to table 1。

The abbreviation of the reagent of table 1 is compareed with Chinese name

Embodiment 1

A. the preparation of glucagon-like peptide-1 analogs

1) synthesize：Using Fmoc strategies, with the type Peptide synthesizers of CS 336 (CS Bio), progressively close in accordance with the following steps Into：

A) the C-terminal amino acid coupling protected in the presence of activator systems by resin solid phase carrier and Fmoc obtains Fmoc- Amino acid-resin；Wherein, synthesis C- end amidatioon polypeptides use amino resins, such as Rink Amide AM, Rink Amide, Rink MBHA etc..

B) extension of peptide chain：By solid-phase synthesis according to peptide sequence amino acid sequence connect amino acid, obtain N- ends and Peptide-resin conjugate of side chain protected；Band side chain amino acid takes following safeguard measure：Tryptophan Boc (tertiary butyloxycarbonyls Base), glutamic acid OtBu (the oxygen tert-butyl group), lysine Boc (tertbutyloxycarbonyl), glutamine Trt (trityl), junket Propylhomoserin tBu (tert-butyl group), serine Trt (trityl) or tBu (tert-butyl group), aspartic acid OtBu (the tertiary fourths of oxygen Base), threonine is protected with tBu (tert-butyl group), histidine with Trt (trityl) or Boc (tertbutyloxycarbonyl).The coupling used Activator is HOBT/HBTU/DIEA and HOBT/HATU/DIEA, ninhydrin method detection reaction efficiency.

C) on resin polypeptide cracking：TFA/EDT/TIS/H2O(92.5:2.5:2.5:2.5v/v) solution, puts at room temperature React 90min, deprotection and deresination.Suction filtration obtains filtrate, and thick polypeptide is precipitated with excess diethyl ether, and precipitation is collected in centrifugation, then with less Measure and dried under ether washing precipitation, vacuum, obtain crude product polypeptide.Deprotection base and resin, obtain crude product hyperglycemic factor simultaneously The analog of sample peptide -1；

2) purify：Crude product glucagon-like peptide-1 analogs are dissolved in water or 10-15% acetonitriles (10-50mg/ml), The disulfide group threitol DTT or beta -mercaptoethanol for adding 50-100mM are denatured, using preparation HPLC method, C18 chromatographic columns, Acetonitrile-water-trifluoroacetic acid system is isolated and purified, and is concentrated, and is freezed, and obtains the free sterling polypeptide of sulfydryl.

SEQ ID NO are prepared for using the above method:Glucagon-like peptide-1 analogs represented by 1-19.

Embodiment 2

Linear pattern monomethyl PEG (21000 dalton) modification SEQ ID NO:1

1) connect：SEQ ID NO:1 polypeptide is dissolved in the 50mM buffer solution of sodium phosphate of the pH6 containing 5mM EDTA, Concentration is 2mg/mL.Add the PEG- maleimides of the solid-state of 1.2-1.5 times of mole, stirring and dissolving, in room temperature reaction 2hr.Reaction is monitored with HPLC, with 5mM beta -mercaptoethanol terminating reactions, is put and is purified after room temperature 30min.

2) purify:Using preparative ion-exchange chromatography, with SP SepharoseHP fillers, with 0-500mM sodium chloride Linear gradient is eluted.Efflux collects PEG- polypeptide flow points with HPLC and SDS- electrophoresis detections, is concentrated by ultrafiltration or uses The method desalination such as Sephadex G-25, freezes and obtains.

Gained PEG modified polypeptides detect purity using RP-HPLC, and molecular weight is determined using MALDI-TOF.

Embodiment 3

Linear pattern monomethyl PEG (30000 dalton) modification SEQ ID NO:8

Linear pattern monomethyl PEG (30000 dalton) modification SEQ ID NO are prepared by the method for embodiment 2:8 and following The PEG conjugates of glucagon-like peptide-1 analogs.

Linear pattern mono methoxy PEG (45000 dalton) modification SEQ ID NO:7

Linear pattern mono methoxy PEG (43000 dalton) modification SEQ ID NO:8

Linear pattern mono methoxy PEG (45000 dalton) modification SEQ ID NO:9

Linear pattern mono methoxy PEG (41000 dalton) modification SEQ ID NO:10

Linear pattern mono methoxy PEG (42000 dalton) modification SEQ ID NO:11

Linear pattern mono methoxy PEG (35000 dalton) modification SEQ ID NO:13

Linear pattern mono methoxy PEG (42000 dalton) modification SEQ ID NO:14

Linear pattern mono methoxy PEG (22000 dalton) modification SEQ ID NO:16

Linear pattern mono methoxy PEG (45000 dalton) modification SEQ ID NO:18

Linear pattern mono methoxy PEG (46000 dalton) modification SEQ ID NO:19

Linear pattern mono methoxy PEG (20000 dalton) modification SEQ ID NO:7

Linear pattern mono methoxy PEG (40000 dalton) modification SEQ ID NO:7

Y types PEG (40000 dalton) modification SEQ ID NO:7

4 arm type PEG (40000 dalton) modification SEQ ID NO:7

Linear pattern mono methoxy PEG (20000 dalton) modification SEQ ID NO:3

Y types PEG (40000 dalton) modification SEQ ID NO:3

Linear pattern mono methoxy PEG (20000 dalton) modification SEQ ID NO:3

Linear pattern mono methoxy PEG (40000 dalton) modification SEQ ID NO:4

Y types PEG (40000 dalton) modification SEQ ID NO:4

4 arm type PEG (40000 dalton) modification SEQ ID NO:4

Y types PEG (40000 dalton) modification SEQ ID NO:5

4 arm type PEG (40000 dalton) modification SEQ ID NO:5

4 arm type mono methoxy PEG (40000 dalton) modification SEQ ID NO:6

Y types PEG (40000 dalton) modification SEQ ID NO:6

Embodiment 4

Linear pattern mono methoxy PEG (30000 dalton) modification SEQ ID NO:5 (samples 8) and PEG (45000 dongles ) modification SEQ ID NO:The hypoglycemic effect evaluation of 7 (samples 14) and 12 (samples 12)

The hypoglycemic effect of GLP-1 analogs PEG trims of the present invention is evaluated using db/db model mices.Method：db/ Db mouse 40 (being purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, blood sugar level is at least 250mg/dL) are randomly divided into 4 groups of (blank Control group, 2 test groups), every group 10；Weigh the PEG trims 0.1mg/ml of appropriate GLP-1 analogs sample solution. Test group mouse, every 200 μ l sample solutions of hypodermic injection；Blank control group mouse, every 200 μ l physiology salts of hypodermic injection Water.Do not limit and ingest during experiment.Determine respectively 2 after injection, 4,8,24,72,96, the blood glucose value of 120 hours.As a result such as Fig. 1 It is shown.

Embodiment 5

Linear pattern mono methoxy PEG (45000 dalton) modification SEQ ID NO:9 (samples 17) and linear pattern mono methoxy PEG (42000 dalton) modification SEQ ID NO:11 (samples 21), PEG (42000 dalton) modification SEQ ID NO:14 (samples Product 26) hypoglycemic effect evaluation

Evaluation method be the same as Example 4, is as a result shown in Fig. 2.

Embodiment 6

GLP-1 receptor agonist activities are evaluated

By evaluating polypeptide of the present invention to GLP-1 acceptors to the receptor-mediated external cAMP of the GLP-1 influences produced Agonist activity.

The Chinese cavy pneumonocyte for transfecting someone's GLP-1 acceptors is inoculated into 96 well culture plates, (200000/hole), with After the washing of Hanks ' balanced salt solutions, the subject polypeptide sample (10-5-10-12mol/L) with various concentrations, in 200 μm of ol/ 20min is incubated altogether in 37 DEG C in the presence of L 3- isobutyl group -1- methyl madder flavine.Medium is removed, cell is dissolved, cAMP values are determined, Assay method is with reference to assay kit explanation.50% valid density is calculated with Origin softwares.It the results are shown in Table 2.

Induction of the polypeptide of table 2 to cAMP is acted

Peptide sequence 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

Conclusion：SEQ ID NO in shown peptide sequence:1st, 4,7,11 the receptor agonism for being better than endogenous GLP-1 is shown Activity.

Embodiment 7

Different molecular weight and different structure PEG modification SEQ ID NO:7 hypoglycemic effect and long-term effect are evaluated

The SEQ ID NO using normal mouse glucose load model evaluation:The hypoglycemic effect of 7 different PEG trims and Long-term effect

Sample:

The SEQ ID modified respectively with mPEG (20KD), mPEG (40KD), Y types PEG (40KD), 4 arm type PEG (40KD) NO:.7 (C- is terminus amidated) sample is prepared by the method for embodiment 3, characterized, purity (HPLC) ＞ 98.0%；(³⁷C)GLP-1(7- 36) mPEG (20KD) trim, is prepared by the method for embodiment 3, characterized, purity (HPLC) ＞ 98.0%；

Positive drug：Liraglutide (Novo Nordisk Co., Ltd's production)

Method：

Using normal mouse glucose load model evaluation：Normal mouse (n=6), fasting 12 hours, determines blood glucose value before medicine, Surveyed within 0.5 hour after different time points intraperitoneal injection glucose (4mg/kg) (to fasting before sugar 4 hours), sugar after subcutaneous administration, medicine Determine mouse blood sugar value, calculate inhibiting rate of the sample to glucose load normal mouse blood glucose rise, calculation formula is inhibiting rate %=(1- Administration group blood glucose average value/model control group blood glucose average value) %, Liraglutide is positive control drug.

Dosage：Positive drug Liraglutide and sample are 100nmol/kg.

As a result：It see the table below 3

Table 3.PEG modifies the different time sections hypoglycemic effect of SEQ.7 series of samples

Conclusion：What positive drug Liraglutide and mPEG (20KD) were modified³⁷CGLP-1 hypoglycemic effect bases after administration 24hr This disappearance, and the SEQ.7 of PEG modifications sample series show the hypoglycemic effect better than positive drug in different time sections, MPEG (20KD), mPEG (40KD), YPEG (40KD), 4 sample duration of efficacy of 4Arm PEG (40KD) are respectively 48, 120th, 144 hours, branching type PEG modification sample drug effect fluctuations were small, wherein 4 arm type PEG (40KD) modification samples are significantly better than it His sample.mPEG(20KD)-³⁷CGLP-1 compared with mPEG (20KD) SEQ.7, decay of activity faster, the former activity of 24 hours Only 33.1%, 48hr is inactive, and the latter's drug effect can steadily continue to 48hr, is significantly better than the former.Illustrate one to sequence Serial innovation effectively prevent enzyme degraded, improve naked peptide stability and activity be more beneficial for trim drug effect play and Extend Half-life in vivo.

Embodiment 8

As described in Example 7, SEQ ID NO be have rated:4th, the hypoglycemic effect of 5,6 Y types PEG (40KD) modified body And long-term effect.It the results are shown in Table 4.

Conclusion：SEQ ID NO:4 and 6 Y types PEG (40KD) modified body hypoglycemic effects and duration of efficacy is close, And SEQ ID NO:5 compared with first two groups activity and its duration have larger difference.Illustrate the PEG modifications pair of different loci The performance and duration of activity have a significant impact.

Sequence table

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Gly

<210> 7

<211> 33

<212> PRT

<213>Artificial sequence

<220>

<221> MISC_FEATURE

<222> (29)..(29)

<223>Xaa represents Aib, 2- aminoisobutyric acids

<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 acids

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223>Arg C-terminal connection 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 acids

<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 acids

<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 acids

<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 acids

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223>Arg C-terminal connection 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 acids

<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 acids

<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 acids

<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 acids

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223>Arg C-terminal connection 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 acids

<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 acids

<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 acids

<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

1. a kind of polyethyleneglycol modified glucagon-like peptide-1 analogs, it is characterised in that the glucagon-like peptide- 1 analog has SEQ ID NO:On amino acid sequence shown in 1-19, and the cysteine residues of the amino acid sequence Modify polyethylene group.

2. analog as claimed in claim 1, it is characterised in that the molecular weight of the polyethylene glycol is in 20000-50000 roads Between you pause.

3. analog as claimed in claim 1 or 2, it is characterised in that the polyethylene glycol is the polyethylene glycol through modification.

4. analog as claimed in claim 3, it is characterised in that the polyethylene glycol is selected from mono methoxy polyethylene glycol, divided Branch polyethylene glycol, bifurcated polyethylene glycol.

5. analog as claimed in claim 4, it is characterised in that the polyethylene glycol is mono methoxy polyethylene glycol.

6. the analog as any one of claim 1-5, it is characterised in that the polyethylene group is through maleimide Amido is activated.

7. a kind of composition, it includes the analog or its salt as any one of claim 1-6.

8. composition as claimed in claim 7, it is characterised in that described pharmaceutical composition is also carried comprising pharmaceutically acceptable Body and/or auxiliary material.

9. the analog as any one of claim 1-6, composition as claimed in claim 7 or 8 is preparing treatment Application in diabetes, obesity, the medicine of alzheimer disease.