CN109021093A - Polyethyleneglycol modified GLP-1 derivative and its pharmaceutical salts - Google Patents
Polyethyleneglycol modified GLP-1 derivative and its pharmaceutical salts Download PDFInfo
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Abstract
The present invention provides polyethyleneglycol modified GLP-1 derivative and its pharmaceutical salts.The sequence of the GLP-1 derivative is HX1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3‑NH2, wherein X1Selected from Aib, Ala, Ser or D-Ala, X2It is Gly or nothing for Gly or Glu, X3.The advantages that long half time of modification type GLP-1 derivative of the invention with outstanding effect of lowering blood sugar and in vivo is not easy to be removed by kidney or enzyme is degraded, in addition, also having human body heterologous smaller, is not easy to cause immune response, and physiological-toxicity is small, and safety is higher.
Description
Technical field
The present invention relates to GLP-1 derivative and its pharmaceutical salts, and in particular to a kind of the GLP-1 derivative and its medicine of modification type
With salt and preparation method thereof.
Background technique
Glucagon-like-peptide-1 (glucagon-like peptide-1, GLP-1) is the secretion of people's small intestine distal end L cell
The intestines peptide hormone with effect of lowering blood sugar, amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.GLP-
1 major physiological mechanism of action includes enhancing the insulin secretion of blood glucose dependence, glucagon suppression secretion and slowing down stomach to arrange
Null-rate.The promoting insulin secretion of GLP-1 also has blood glucose dependence, avoids risk of hypoglycemia.Gastric emptying rate decline,
Glucose absorption rate also slows down therewith, then the fluctuating range of postprandial blood sugar reduces, therefore GLP-1 is in terms of controlling postprandial blood sugar
Even more important effect is played than insulin.In addition, GLP-1 can also increase satiety, reduce appetite, and then lose weight.And
And in the therapeutic process of type-II diabetes, insulin resistance in weight gain meeting making patients' body leads to a variety of metabolic disorders,
Therefore the unique advantage that GLP-1 can lose weight makes its great market competitiveness in terms of type-II diabetes treatment.Although however,
GLP-1 has hypoglycemic activity outstanding, but its Half-life in vivo is very short can not become good type-II diabetes drug.Except tune
It controls outside blood glucose, GLP-1 receptor stimulating agent also has stimulation β hyperplasia, inhibits β Apoptosis, mitigates insulin resistance, protection
The effects of cardiovascular system.
Have at present and GLP-1 sequence is transformed, though sequence is not transformed relatively has longer Half-life in vivo, this
A little GLP-1 derivatives are still susceptible to the diacyl peptase -4 removed and can be widely present in vivo from kidney, NEP-24.11 enzyme drop
Solution, thus Half-life in vivo is undesirable.In addition, the existing method for improving Half-life in vivo generally changes GLP-1 sequence, with
Improve Half-life in vivo.But the heterologous of sequence and human body is bigger, the immune response of human body is stronger, so that existing by changing
The derivative for improving Half-life in vivo in turn into GLP-1 sequence is not met by treatment needs.Therefore, existing at present
For GLP-1 derivative when being applied to type-II diabetes treatment, Half-life in vivo is still undesirable.For example, marketed drugs hundred are secreted and are reached
For chemical synthesis Exenatide, one day administered twice is needed.
In conclusion there is an urgent need in the art to develop with outstanding effect of lowering blood sugar and Half-life in vivo is ideal and heterologous
The lesser modification type GLP-1 derivative of property and its pharmaceutical salts.
Summary of the invention
It is an object of the invention to provide with outstanding effect of lowering blood sugar and Half-life in vivo is ideal and heterologous is smaller
Modification type GLP-1 derivative and its pharmaceutical salts.
In the first aspect of the present invention, a kind of modification type GLP-1 derivative and its pharmaceutical salts are provided, wherein described
The sequence of modification type GLP-1 derivative are as follows:
HX1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3-NH2,
Wherein, X1Selected from Aib, Ala, Ser or D-Ala, X2It is Gly or nothing for Gly or Glu, X3；
And one or more (1, the 2 or 3) amino acid residue in sequence is modified through polyethylene glycol (PEG).
In another preferred example, the polyethylene glycol is branched polyethylene glycol.
In another preferred example, the polyethylene glycol is maleimide polyethylene glycol.
In another preferred example, the molecular weight of the polyethylene glycol is 10KD~60KD.
In another preferred example, the molecular weight of the polyethylene glycol is 20KD~40KD.
In another preferred example, the molecular weight of the polyethylene glycol is 10KD~30KD.
In another preferred example, the molecular weight of the polyethylene glycol is 30KD~50KD.
In another preferred example, the polyethylene glycol is two branched polyethylene glycols.
In another preferred example, the polyethylene glycol by the modification of cysteine (Cys) residue of GLP-1 derivative in
On the GLP-1 derivative.
In another preferred example, the structure of the polyethylene glycol are as follows:
In another preferred example, X1For Aib, Ala or Ser.
In another preferred example, the modification type GLP-1 derivative is
His——Ala——Glu——Gly——Thr——Phe——Thr——Ser——Asp——
Val——Ser——
Ser——Tyr——Leu——Glu——Gly——Gln——Ala——Ala——Lys——
Glu——Phe——
Ile——Ala——Trp——Leu——Val——Lys——Gly——Arg——Gly——Cys-
NH2
The second aspect of the present invention provides a kind of a kind of method for preparing modification type GLP-1 derivative and its pharmaceutical salts,
Comprising steps of
Coupling reaction is carried out with GLP-1 derivative and polyethylene glycol, obtains modified GLP-1 derivative.
In another preferred example, the coupling reaction carries out in the buffer solution of pH=6~9, preferably buffers molten
Liquid pH=7~8.
In another preferred example, the reaction time of the coupling reaction is 0.5~6h；Preferably, the coupling is anti-
The reaction time answered is 3~5h.
In another preferred example, the reaction temperature of the coupling reaction is 0~25 DEG C；It preferably, is 0~15 DEG C；More
It preferably, is 1~6 DEG C.
In another preferred example, the molar ratio of the GLP-1 derivative and the polyethylene glycol is 1: 0.5~1: 5.
In another preferred example, the molar ratio of the GLP-1 derivative and the polyethylene glycol is 1: 1~1: 4, preferably
It is 1: 2~1: 3.
In another preferred example, the method includes the following steps:
A. the GLP-1 derivative and modification polyethylene glycol are taken；Wherein, the GLP-1 derivative and described
The molar ratio of modification polyethylene glycol is 1: 0.5~1: 5；
B. the GLP-1 derivative and the modification are dissolved in the buffer solution of pH=6~9 with polyethylene glycol,
Obtain reaction mixture；
~25 DEG C c.0 at, the reaction mixture reacts 0.5~6h under stiring；
D. separation obtains modified GLP-1 derivative.
In another preferred example, in step b, in the reaction mixture, the concentration of the GLP-1 derivative is
0.3~5mg/ml, preferably 0.5~2mg/ml.
In another preferred example, in step b, the buffer solution is phosphate buffer.
In another preferred example, in step c, the reaction mixture reacts at 0~15 DEG C, it is preferable that 1~6
It is reacted at DEG C.
In another preferred example, the polyethyleneglycol modified rate of the GLP-1 derivative is 70~99%.
The third aspect of the present invention provides a kind of pharmaceutical composition, and it includes repair as provided by first aspect present invention
Decorations type GLP-1 derivative and its pharmaceutical salts.
In another preferred example, the pharmaceutical composition is the pharmaceutical composition for treating type-II diabetes.
The fourth aspect of the present invention provides a kind of modification type GLP-1 derivative and its pharmaceutical salts as described in relation to the first aspect
With the purposes of the pharmaceutical composition as described in the third aspect, for treating diabetes (preferably type-II diabetes).
The fifth aspect of the present invention provides a kind of external method non-therapeutic control and/or reduce blood sugar concentration, institute
The method stated includes: to inhuman target application modification type GLP-1 derivative as described in relation to the first aspect and its pharmaceutical salts or such as the
Pharmaceutical composition described in three aspects.
In another preferred example, the method is with 100~1000nmol/kg (preferably, with 200~600nmol/
Kg) amount of (modification type GLP-1 derivative and its pharmaceutical salts/target weight) applies the modification type GLP-1 to inhuman target and spreads out
Biology and its pharmaceutical salts or the pharmaceutical composition.
In another preferred example, the blood sugar concentration includes fasting plasma glucose concentration and acute glycemic concentration.
The sixth aspect of the present invention provides a kind of method for treating diabetes (preferably, type-II diabetes), the side
Method includes: application modification type GLP-1 derivative as described in relation to the first aspect and its pharmaceutical salts or the drug as described in the third aspect
Composition.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is acute blood glucose test result in unmodified GLP-1 derivative Mice Body.
Fig. 2 is the EC50 curve of unmodified GLP-1 derivative.
Fig. 3 is PEG of the invention20KDThe EC50 curve of-GLP-1.
Fig. 4 is PEG of the invention40KDThe EC50 curve of-GLP-1.
Fig. 5 is acute glycemic experimental result in Mice Body.
Fig. 6 is modification type GLP-1 derived material spectrogram of the present invention
Fig. 7 is modification type GLP-1 derivative HPLC map of the present invention
Specific embodiment
Inventor is after after extensive and in-depth study, it has unexpectedly been found that smaller with human body heterologous but have excellent
The GLP-1 derivative of half-life period and therapeutic effect.It is additionally polyethyleneglycol modified by high molecular weight ramiform maleimide
GLP-1 derivative (the sequence HX as shown in formula (I)1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3-NH2；Wherein, X1
For Abi/Ala/Ser/D-Ala, X2It is Gly or nothing for Gly or Glu, X3), prolong significantly in the case where not influencing hypoglycemic activity
The Half-life in vivo of GLP-1 derivative is grown, to obtain prolonged blood sugar decreasing effect.Based on this, the present invention is completed.
Specifically, the C-terminal for the polypeptide chain that the present invention uses all deposits a cysteine residues, has free sulfhydryl groups.Simultaneously
Using the branched polyethylene glycol with maleimide activation.In pH 6.5-7.5 buffer solution, maleimide amine molecule
In active group and polypeptide chain on free sulfhydryl groups form stable covalent bond, obtain PEG-GLP-1 compound.It is slow in acidity
In fliud flushing, which can be adsorbed on cation-exchange chromatography post, is finally eluted, is obtained by salting liquid linear gradient again
PEG-GLP-1 compound (modification type GLP-1 derivative i.e. of the invention).
Main advantages of the present invention include:
(a) modification type GLP-1 derivative long half time of the invention, dosage rate are small.
(b) sequence and human body heterologous used in modification type GLP-1 derivative of the invention are small, are not likely to produce immune anti-
It answers.
(c) modification type GLP-1 derivative of the invention is not easy to be discharged by the enzyme degradation in human body or from kidney.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight percent and weight
Number.
The present invention relates to GLP-1 derivatives to be synthesized by Zhongtai Bio-Chem. Co., Ltd., Hangzhou, branched maleimide used
Polyethylene glycol is purchased from Sino nation lattice biology Co., Ltd
Embodiment 1
Acute glycemic is tested in GLP-1 derivatives monomer health Kunming mouse body
By Healthy female kunming mice (20~22g of weight) weighing, number.In experiment evening before that day fasting, (12h is left
It is right), it is given only drinking-water.Morning next day Fundamentals of Measurement blood glucose value, and blood glucose value when being denoted as 0min.Select mouse similar in blood glucose value
Random grouping:
1, control group 6nmol/kg glucose (n=10)；
2、A8GLP-1 group 6nmol/kg A8GLP-1 (n=10)；A8GLP-1 sequence is such as
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2Shown in (SEQ ID NO.1)；That is X in sequence shown in formula (I)1For
Ala、X2For Gly and X3For nothing.
3、S8GLP-1 group 6nmol/kg S8GLP-1 (n=10)；S8GLP-1 sequence is such as
HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2Shown in (SEQ ID NO.2)；That is X in sequence shown in formula (I)1For
Ser、X2For Gly and X3For nothing.
4、dA8GLP-1 group 6nmol/kg dA8GLP-1 (n=10)；dA8GLP-1 sequence is such as
HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2(substantially wherein with SEQ ID NO.1, second D-Ala) institute
Show；That is X in sequence shown in formula (I)1For D-Ala, X2For Gly and X3For nothing.
According to mouse weight, through glucose, A is injected intraperitoneally8GLP-1、S8GLP-1 and dA8GLP-1 sample.30min,
Mouse tail takes blood when 100min, 165min, 250min, 350min, measures blood glucose value with blood sugar test paper item.Before surveying blood glucose every time
30min supplements 200 μ L, 37% glucose.The data obtained carries out t inspection with 5 statistical software of GraphPad Prism and draws figure
Table.Statistical result is as shown in table 1 and Fig. 1:
Table 1
The mass spectrometric data of GLP-1 derivatives monomer used in the embodiment of the present invention (SEQ ID NO.1) as shown in fig. 6,
High pressure liquid chromatography map is as shown in Fig. 7 and table 2.
Table 2
High pressure liquid chromatography (HPLC) condition are as follows:
Mobile phase: A :+0.1% trifluoroacetic acid of water
+ 20% water of B:(80% acetonitrile)+0.09% trifluoroacetic acid
Flow velocity: 1.0mL/min
Detect UV 220nm
Linear gradient:
| Mobile phase A | Mobile phase B | |
| 0min | 54% | 44% |
| 20min | 44% | 54% |
| 21min | 5% | 95% |
Chromatographic column: SepaxGP-5u 120A 4.6*150mm
Embodiment 2
Ramiform 20KD PEG- maleimide is reacted with GLP-1 derivative
With the 20KD branch PEG of maleimide activation in GLP-1 derivative (SEQ ID NO.1, i.e. sequence shown in formula (I)
X in column1For Ala, X2For Gly and X3For nothing) Pegylation selective on introduced cysteine residues, it is formed and is stablized
In conjunction with thioether covalent bond.Weigh chemically synthesized 10mg GLP-1 derivative and 100mg branch 20KD PEG- maleimide
It is dissolved in 5ml 200mM pH7.4 phosphate buffer, 4hr is stirred to react under the conditions of 4 DEG C (molar ratio of polypeptide and PEG are 1:2).
Use the Nacl gradient of acid pH on MacroCap SP column (GE) by poly- second two by cation-exchange chromatography post
The compound and Free PEG and free peptide separation of alcoholization, obtain PEG20KD-GLP-1.PEGylated compounds RP-
HPLC, SEC-HPLC qualitative analysis simultaneously detect its activity.
Embodiment 3
Ramiform 40KD PEG- maleimide is reacted with GLP-1 derivative
With the 40KD branch PEG of maleimide activation in GLP-1 derivative (SEQ ID NO.1, i.e. sequence shown in formula (I)
X in column1For Ala, X2For Gly and X3For nothing) Pegylation selective on introduced cysteine residues, it is formed and is stablized
In conjunction with thioether covalent bond.It is molten to weigh chemically synthesized 10mg GLP-1 derivative 200mg branch 40KD PEG- maleimide
4hr is stirred to react under the conditions of 5ml 200mM pH7.4 phosphate buffer, 4 DEG C (molar ratio of polypeptide and PEG are 1:2).
Use the Nacl gradient of acid pH on MacroCap SP column (GE) by poly- second two by cation-exchange chromatography post
The compound and Free PEG and free peptide separation of alcoholization, obtain PEG40KD-GLP-1.PEGylated compounds RP-
HPLC, SEC-HPLC qualitative analysis simultaneously detect its activity.
Embodiment 4
The PEG modified outcome active determination in vitro of GLP-1 derivative
According to GLP-1R signal transduction pathway, the alkaline phosphatase for transiently transfecting expression GLP-1R and being driven by cAMP is established
The HEK293 cell line of enzyme reporter gene, the screening for GLP-1R agonist.When GLP-1R is in conjunction with agonist, cell cAMP
Concentration increases, and will be raised by the expression of the alkaline phosphatase reporter gene of cAMP driving.By to alkaline phosphatase activities
Detection can determine whether the active ability of compound excitement GLP-1R.
HEK-293 cell is inoculated with to 24 orifice plates, cell density 1.0*105A/ml.It will expression GLP-1R and SEAP report
The plasmid transfection of gene is into cell.Transfection 6 hours after, more renew culture medium, at the same be added various concentration gradient to test sample
Product.It takes cell conditioned medium into 96 orifice plates after 44 hours, heating 30 minutes in 65 DEG C of baking ovens is placed in, to remove endogenous alkaline phosphorus
Sour enzyme.Take 80ul cell supernatant into 96 orifice plates, 120ul premix detection working solution is added in every hole.It is detected at microplate reader 405nm
Sample light absorption value simultaneously calculates enzyme activity.
Data processing and statistical analysis
EC50 is concentration value when causing 50% ceiling effect, can reflect out ligand to the agonist activity of receptor, is to grind
Study carefully combined between ligand and receptor, excitement an important indicator.It can using the concentration that the expression quantity of SEAP corresponds to medicine irritation
To go out GLP-1 derivative (SEQ ID NO.1), PEG with GraphpadPrism5.0 Software on Drawing20KD- GLP-1 (make by embodiment 2
It is standby) and PEG40KDThe EC50 curve of-GLP-1 (preparation of embodiment 3), as shown in Fig. 2, which show unmodified GLP-1 to spread out
The EC50 curve of biology, and the EC50 curve of the product in embodiment 2,3 difference is as shown in Figures 3 and 4.
Calculation method are as follows: EC50=Bottom+ (Top-Bottom)/(1+10^ ((LogEC50-X)))
The experimental result of active determination in vitro, as shown in table 3:
Table 3
| GLP-1 derivative | PEG20KD-GLP-1 | PEG40KD-GLP-1 | |
| EC50(Log(nM)) | 0.7321 | 1.349 | 1.519 |
Embodiment 5
Acute hypoglycemic experiment in the PEG modified outcome health Kunming mouse body of GLP-1 derivative
By Healthy female kunming mice (20~22g of weight) weighing, number.In experiment evening before that day fasting, (12h is left
It is right), it is given only drinking-water.Morning next day Fundamentals of Measurement blood glucose value, and blood glucose value when being denoted as 0min.Select mouse similar in blood glucose value
Random grouping:
1) glucose control group；
2) 10nmol/kg GLP-1 derivative group (GLP-1 derivative sequence is as shown in SEQ ID NO.1)；
3)10nmol/kg PEG20KD- GLP-1 group (PEG20KDPrepared by-GLP-1 embodiment 2)；
4)10nmol/kg PEG40KD- GLP-1 group (PEG40KDPrepared by-GLP-1 embodiment 3).
According to mouse weight, through glucose, GLP-1 derivative, PEG is injected intraperitoneally20KD-GLP-1、PEG40KD- GLP-1 sample
Product.In 2h, 5h, 8h, 11h, 13h, mouse tail takes blood, measures blood glucose value with blood sugar test paper item.30min before survey blood glucose every time
Supplement 200 μ L, 37% glucose.The data obtained carries out t inspection with 5 statistical software of GraphPad Prism and draws a diagram.
Table 4
As shown in table 4, table 5 and Fig. 5, the PEG modified outcome of GLP-1 derivative of the invention hypoglycemic effect in 11 hours
It is constant and can make blood sugar concentration keep with after fasting 12h blood sugar concentration it is almost the same.And unmodified GLP-1 derivative exists
Hypoglycemic effect has just weakened after 2 hours.Modification type GLP-1 derivative of the invention just weakens in the 13rd hour effect to 2h
The effect of unmodified GLP-1 derivative afterwards, and GLP-1 derivative (13h) unmodified at this time does not almost have blood sugar concentration
Have an impact.Specifically, the modified outcome blood sugar decreasing effect of GLP-1 derivative PEG of the invention is as shown in table 5, relatively unmodified
GLP-1 in terms of half-life period with 6.5 times of promotion effect.
Table 5
Embodiment 6
The PEG modified outcome single-dose of GLP-1 derivative influences the daily fasting blood-glucose of db/db mouse
The raising of the 6 male db/db mouse single cages of week old 25, every ration are fed with chow diet, until 8 week old start to test.
One day before administration, morning, 9 fasting (can't help water), mouse fasting blood-glucose were measured after 6h, fasting blood-glucose is between 15-
Between 30mmol/L.Mouse is grouped at random according to above-mentioned fasting blood sugar:
1) PBS control group；
2)50nmol/kg PEG40KD- GLP-1 group；
3)250nmol/kg PEG40KD- GLP-1 group；
4)500nmol/kg PEG40KD- GLP-1 group.(PEG40KDIt is prepared in-GLP-1 embodiment 3)
According to mouse weight (40g ± 3g), subcutaneous injection sample.1h, 2h, 3h, 4h, for 24 hours, 48h when mouse tail
Blood is taken, measures blood glucose value with blood sugar test paper item.The data obtained carries out t inspection with 5 statistical software of GraphPad Prism and draws
Chart.
Table 6
As shown in table 6, the PEG of basic, normal, high three various doses of db/db mouse single subcutaneous injection40KD-GLP-1。
50nmol/kg low dose group does not show blood sugar decreasing effect, but middle dose group and high dose group all shown after 1h is administered it is bright
Aobvious blood sugar decreasing effect, and 500nmol/kg PEG40KD- GLP-1 dosage group still showed significant blood sugar decreasing effect at 24 hours.
It can be seen that PEG40KD- GLP-1 has apparent hypoglycemic advantage, and its hypoglycemic effect has concentration dependent, is administered dense
Degree is higher, and blood sugar decreasing effect is more obvious, and the time is more lasting.
Embodiment 7
Acute toxicity test of the high dose subcutaneous administration to db/db mouse
GLP-1 derivative (SEQ is modified respectively according to the 40KD branch PEG of method maleimide activation in embodiment 3
ID NO.1) and comparative example Exenatide serial response, the PEG of high-purity is obtained after purification40K- GLP-1 and PEG40K-
Exenatide is used for subsequent zoopery.
Wherein, the Exenatide sequence of experiment comparative example is synthesized by Zhongtai Bio-Chem. Co., Ltd., Hangzhou, and purity is >
The sequence of 95%, Exenatide are as follows:
His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Nle-Glu-Glu-
Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Gln-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
Pro-Pro-Pro-Cys-NH2。
About 12 week old db/db mouse 20 are only randomly divided into two groups:
1)1000nmol/kg PEG40KD- GLP-1 group；
2)1000nmol/kg PEG40KD- Exenatide group.
According to mouse weight (40g ± 3g), the administration of single subcutaneous abdomen.Dead mouse situation in observation administration 14 days.Directly
Terminate to the observation period, of the present invention group freely, and 2 dead mouses then occurs without an example dead mouse, equal activity in comparative example group.
The result shows that: the physiological-toxicity of comparative example group is greater than of the present invention group.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Shanghai Institute of Biological Products Co., Ltd.
<120>polyethyleneglycol modified GLP-1 derivative and its pharmaceutical salts
<130> P2017-2344
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 32
<212> PRT
<213>artificial sequence (Artificial Sequence)
<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 Ala Trp Leu Val Lys Gly Arg Gly Cys
20 25 30
<210> 2
<211> 32
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
His Ser 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 Gly Cys
20 25 30
Claims (10)
1. a kind of modification type GLP-1 derivative and its pharmaceutical salts, which is characterized in that the sequence of the GLP-1 derivative are as follows:
HX1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3-NH2 (I)
Wherein, X1Selected from Aib, Ala, Ser or D-Ala, X2For Gly or Glu, X3For Gly or nothing；
And one or more amino acid residues in sequence are through polyethyleneglycol modified.
2. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that the polyethylene glycol
For branched polyethylene glycol.
3. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that the polyethylene glycol
For maleimide polyethylene glycol.
4. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that the polyethylene glycol
Molecular weight be 10KD~60KD.
5. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that the polyethylene glycol
It is modified by the cysteine residues of GLP-1 derivative on the GLP-1 derivative.
6. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that the polyethylene glycol
Structure are as follows:
7. modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that X1For Aib, Ala or
Ser。
8. a kind of method for preparing modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts, which is characterized in that
Comprising steps of
Coupling reaction is carried out with GLP-1 derivative and polyethylene glycol, obtains modified GLP-1 derivative.
9. a kind of pharmaceutical composition, which is characterized in that include modification type GLP-1 derivative as described in claim 1 and its medicine
Use salt.
10. a kind of modification type GLP-1 derivative as described in claim 1 and its pharmaceutical salts and medicine as claimed in claim 9
The purposes of compositions, which is characterized in that for treating diabetes.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006121860A2 (en) * | 2005-05-06 | 2006-11-16 | Bayer Pharmaceuticals Corporation | Glucagon-like peptide 1 (glp-1) receptor agonists and their pharmacological methods of use |
| CN1904150A (en) * | 2006-08-01 | 2007-01-31 | 华东师范大学 | Human glucagon peptide/derivative and its solid phase chemical synthesis |
| CN102083854A (en) * | 2008-06-17 | 2011-06-01 | 大塚化学株式会社 | Glycosylated GLP-1 peptide |
| WO2011140638A1 (en) * | 2010-05-10 | 2011-11-17 | Corporation De L'ecole Polytechnique De Montreal | Gene therapy for diabetes with chitosan-delivered plasmid encoding glucagon-like peptide 1 |
| CN102920658A (en) * | 2012-11-02 | 2013-02-13 | 艾韦特(溧阳)医药科技有限公司 | Liposome combined with GLP-1 (Glucagon-Like Peptide-1) analogue and polyethylene glycol and preparation method of liposome |
| CN104402990A (en) * | 2014-11-22 | 2015-03-11 | 马海龙 | Polypeptide for treating diabetes |
| CN105801705A (en) * | 2014-12-31 | 2016-07-27 | 天视珍生物技术（天津）有限公司 | Glucagon-like peptide-1 and immunoglobulin hybrid Fc fusion polypeptide and use thereof |
| CN107266557A (en) * | 2016-04-06 | 2017-10-20 | 天津药物研究院有限公司 | A kind of polyethyleneglycol modified glucagon-like peptide 1 analog |
-
2018
- 2018-08-29 CN CN201810995139.2A patent/CN109021093B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006121860A2 (en) * | 2005-05-06 | 2006-11-16 | Bayer Pharmaceuticals Corporation | Glucagon-like peptide 1 (glp-1) receptor agonists and their pharmacological methods of use |
| CN1904150A (en) * | 2006-08-01 | 2007-01-31 | 华东师范大学 | Human glucagon peptide/derivative and its solid phase chemical synthesis |
| CN102083854A (en) * | 2008-06-17 | 2011-06-01 | 大塚化学株式会社 | Glycosylated GLP-1 peptide |
| WO2011140638A1 (en) * | 2010-05-10 | 2011-11-17 | Corporation De L'ecole Polytechnique De Montreal | Gene therapy for diabetes with chitosan-delivered plasmid encoding glucagon-like peptide 1 |
| CN102920658A (en) * | 2012-11-02 | 2013-02-13 | 艾韦特(溧阳)医药科技有限公司 | Liposome combined with GLP-1 (Glucagon-Like Peptide-1) analogue and polyethylene glycol and preparation method of liposome |
| CN104402990A (en) * | 2014-11-22 | 2015-03-11 | 马海龙 | Polypeptide for treating diabetes |
| CN105801705A (en) * | 2014-12-31 | 2016-07-27 | 天视珍生物技术（天津）有限公司 | Glucagon-like peptide-1 and immunoglobulin hybrid Fc fusion polypeptide and use thereof |
| CN107266557A (en) * | 2016-04-06 | 2017-10-20 | 天津药物研究院有限公司 | A kind of polyethyleneglycol modified glucagon-like peptide 1 analog |
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|---|---|
| CN109021093B (en) | 2021-09-07 |
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