CN109021093B - Polyethylene glycol modified GLP-1 derivatives and medicinal salts thereof - Google Patents
Polyethylene glycol modified GLP-1 derivatives and medicinal salts thereof Download PDFInfo
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Abstract
The invention provides polyethylene glycol modified GLP-1 derivatives and pharmaceutically acceptable salts thereof. The sequence of the GLP-1 derivative is HX1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3‑NH2Wherein X is1Selected from Aib, Ala, Ser or D-Ala, X2Is Gly or Glu, and X3 is Gly or nothing. The modified GLP-1 derivative has excellent hypoglycemic effect, long half-life period in vivo, difficult elimination by kidney or enzyme degradation, small human body heterogeneity, difficult immune response induction, small physiological toxicity, higher safety and the like.
Description
Technical Field
The invention relates to GLP-1 derivatives and medicinal salts thereof, in particular to modified GLP-1 derivatives and medicinal salts thereof and a preparation method thereof.
Background
Glucagon-like peptide-1 (GLP-1) is an intestinal peptide hormone secreted by L cells at the far end of the human small intestine and having the efficacy of reducing blood sugar, and the amino acid sequence of the intestinal peptide hormone is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. The main physiological mechanisms of action of GLP-1 include enhancement of blood glucose dependent insulin secretion, inhibition of glucagon secretion and slowing of the rate of gastric emptying. The insulinotropic action of GLP-1 is also blood glucose dependent, avoiding the risk of hypoglycemia. The rate of gastric emptying decreases, the rate of glucose absorption also slows, and the amplitude of postprandial glucose excursions decreases, so GLP-1 plays a more important role in controlling postprandial glucose than insulin. In addition, GLP-1 can also increase satiety, reduce appetite, and thereby reduce weight. Moreover, during the treatment of type II diabetes, weight gain can aggravate insulin resistance in patients, leading to various metabolic disorders, so the unique advantage of GLP-1 in reducing weight makes it very competitive in the treatment of type II diabetes. However, GLP-1 has a prominent hypoglycemic activity, but its half-life in vivo is short and it cannot be a good drug for type II diabetes. Besides regulating blood sugar, the GLP-1 receptor agonist also has the effects of stimulating beta cell proliferation, inhibiting beta cell apoptosis, relieving insulin resistance, protecting cardiovascular system and the like.
GLP-1 sequences have been modified at present, and although the GLP-1 derivatives have longer in vivo half-life compared with unmodified sequences, the GLP-1 derivatives are still easy to be cleared from the kidney and degraded by diacyl peptidase-4 and NEP-24.11 enzymes which are widely present in vivo, so the in vivo half-life is not ideal. In addition, existing methods to improve half-life in vivo generally alter the GLP-1 sequence to increase half-life in vivo. However, the greater the sequence heterogeneity with respect to the human body, the stronger the immune response of the human body, so that the existing derivatives with improved GLP-1 sequence and thus improved half-life in vivo cannot meet the therapeutic needs. Thus, the half-life of the GLP-1 derivatives currently available in vivo is still not ideal for use in the treatment of type ii diabetes. For example, the commercially available drug, leuprolide, is a chemically synthesized exenatide, which is administered twice a day.
In view of the above, there is an urgent need in the art to develop modified GLP-1 derivatives and pharmaceutically acceptable salts thereof having excellent hypoglycemic effects and having an ideal and less heterogeneous in vivo half-life.
Disclosure of Invention
The present invention aims to provide a modified GLP-1 derivative and a pharmaceutically acceptable salt thereof having excellent hypoglycemic effects and an ideal half-life in vivo and having little heterogeneity.
In a first aspect of the invention, modified GLP-1 derivatives and pharmaceutically acceptable salts thereof are provided, wherein the sequence of the modified GLP-1 derivative is:
HX1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3-NH2，
wherein, X1Selected from Aib, Ala, Ser or D-Ala, X2Is Gly or Glu, X3 is Gly or nothing;
and one or more (1, 2 or 3) amino acid residues in the sequence are modified with polyethylene glycol (PEG).
In another preferred embodiment, the polyethylene glycol is a branched polyethylene glycol.
In another preferred embodiment, the polyethylene glycol is maleimide polyethylene glycol.
In another preferred embodiment, the molecular weight of the polyethylene glycol is 10 KD-60 KD.
In another preferred embodiment, the molecular weight of the polyethylene glycol is 20 KD-40 KD.
In another preferred embodiment, the molecular weight of the polyethylene glycol is 10 KD-30 KD.
In another preferred embodiment, the molecular weight of the polyethylene glycol is 30 KD-50 KD.
In another preferred embodiment, the polyethylene glycol is a two-branched polyethylene glycol.
In another preferred embodiment, said polyethylene glycol is modified to said GLP-1 derivative by a cysteine (Cys) residue of said GLP-1 derivative.
In another preferred embodiment, the structure of the polyethylene glycol is:
in another preferred embodiment, X1Aib, Ala or Ser.
In another preferred embodiment, the modified 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
In a second aspect the present invention provides a process for the preparation of a modified GLP-1 derivative and pharmaceutically acceptable salts thereof, comprising the steps of:
GLP-1 derivatives and polyethylene glycol are used for coupling reaction to obtain the modified GLP-1 derivatives.
In another preferred example, the coupling reaction is carried out in a buffer solution with the pH value of 6-9, preferably 7-8.
In another preferred embodiment, the reaction time of the coupling reaction is 0.5-6 h; preferably, the reaction time of the coupling reaction is 3-5 h.
In another preferred example, the reaction temperature of the coupling reaction is 0-25 ℃; preferably, the temperature is 0-15 ℃; more preferably, it is 1 to 6 ℃.
In another preferred embodiment, the molar ratio of the GLP-1 derivative to the polyethylene glycol is 1: 0.5-1: 5.
In another preferred embodiment, the molar ratio of the GLP-1 derivative to the polyethylene glycol is 1: 1-1: 4, preferably 1: 2-1: 3.
In another preferred embodiment, the method comprises the following steps:
a. taking the GLP-1 derivative and polyethylene glycol for modification; wherein the molar ratio of the GLP-1 derivative to the polyethylene glycol for modification is 1: 0.5-1: 5;
b. dissolving the GLP-1 derivative and the polyethylene glycol for modification in a buffer solution with the pH of 6-9 to obtain a reaction mixture;
c, reacting the reaction mixture for 0.5-6 hours under stirring at the temperature of 0-25 ℃;
d. isolating to obtain the modified GLP-1 derivative.
In another preferred example, in the step b, the concentration of the GLP-1 derivative in the reaction mixture is 0.3-5 mg/ml, preferably 0.5-2 mg/ml.
In another preferred embodiment, in step b, the buffer solution is phosphate buffer.
In another preferred embodiment, in step c, the reaction mixture is reacted at 0-15 ℃, preferably at 1-6 ℃.
In another preferred example, the polyethylene glycol modification rate of the GLP-1 derivative is 70-99%.
A third aspect of the invention provides a pharmaceutical composition comprising a modified GLP-1 derivative as provided in the first aspect of the invention and pharmaceutically acceptable salts thereof.
In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for treating type two diabetes.
In a fourth aspect the present invention provides the use of a modified GLP-1 derivative as defined in the first aspect and pharmaceutically acceptable salts thereof and a pharmaceutical composition as defined in the third aspect for the treatment of diabetes, preferably type two diabetes.
A fifth aspect of the invention provides an in vitro non-therapeutic method of controlling and/or lowering blood glucose concentration, said method comprising: administering to a non-human target a modified GLP-1 derivative according to the first aspect and pharmaceutically acceptable salts thereof or a pharmaceutical composition according to the third aspect.
In another preferred embodiment, the method is to administer the modified GLP-1 derivative and its pharmaceutically acceptable salt or the pharmaceutical composition to a non-human target in an amount of 100-1000 nmol/kg (preferably 200-600 nmol/kg) (modified GLP-1 derivative and its pharmaceutically acceptable salt/target weight).
In another preferred example, the blood glucose concentrations include fasting blood glucose concentration and acute blood glucose concentration.
A sixth aspect of the invention provides a method of treating diabetes, preferably type ii diabetes, the method comprising: administering a modified GLP-1 derivative according to the first aspect and pharmaceutically acceptable salts thereof or a pharmaceutical composition according to the third aspect.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the results of acute blood glucose measurements in unmodified GLP-1 derivative mice.
FIG. 2 is an EC50 curve for an unmodified GLP-1 derivative.
FIG. 3 shows PEG of the present invention20KDEC50 curve for GLP-1.
FIG. 4 shows the present inventionPEG40KDEC50 curve for GLP-1.
FIG. 5 shows the results of acute blood glucose assay in mice.
FIG. 6 is a mass spectrum of modified GLP-1 derivative of the invention
FIG. 7 is an HPLC chromatogram of a modified GLP-1 derivative of the invention
Detailed Description
After extensive and intensive studies, the inventors have unexpectedly found a GLP-1 derivative which is less heterologous to the human body but has an excellent half-life and therapeutic effect. In addition, GLP-1 derivatives (with the sequence of HX) shown in the formula (I) are modified by high molecular weight branched maleimide polyethylene glycol1EGTFTSDVSSYLEX2QAAKEFIAWLVKGRGCX3-NH2(ii) a Wherein, X1Is Abi/Ala/Ser/D-Ala, X2Gly or Glu, and X3 is Gly or none), the in vivo half-life period of the GLP-1 derivative is greatly prolonged under the condition of not influencing the hypoglycemic activity, and thus the long-time hypoglycemic effect is obtained. Based on this, the present invention has been completed.
Specifically, the C-terminal of the polypeptide chain employed in the present invention has a cysteine residue with a free thiol group. And branched polyethylene glycol with maleimide activation is adopted. In a buffer solution with pH of 6.5-7.5, an active group in a maleimide molecule and a free sulfhydryl on a polypeptide chain form a stable covalent bond to obtain the PEG-GLP-1 compound. In an acidic buffer solution, the PEG-GLP-1 can be adsorbed on a cation exchange chromatography column, and finally, the linear gradient elution is carried out through a salt solution, so as to obtain the PEG-GLP-1 compound (namely the modified GLP-1 derivative of the invention).
The main advantages of the invention include:
(a) the modified GLP-1 derivative has long half-life period and low administration frequency.
(b) The sequence used by the modified GLP-1 derivative has small heterogeneity with human body, and is not easy to generate immune reaction.
(c) The modified GLP-1 derivative of the invention is not easily degraded by enzymes in the human body or excreted from the kidney.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The invention relates to a GLP-1 derivative synthesized by Hangzhou Zhongtai peptide Biochemical company Limited, wherein the branched maleimide polyethylene glycol is purchased from Sonopongg biological corporation
Example 1
Test of acute blood sugar in vivo of GLP-1 derivative monomer healthy Kunming mouse
Weighing and numbering healthy female Kunming mice (with the weight of 20-22 g). Fasting (about 12 h) was performed overnight before the experiment, and only drinking water was given. The basal blood glucose level was measured the next morning and recorded as the blood glucose level at 0 min. Selecting mice with similar blood sugar values to randomly group:
1. control 6nmol/kg glucose (n ═ 10);
2、A8GLP-1 group 6nmol/kg A8GLP-1(n＝10)；A8GLP-1 sequences such as HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2(SEQ ID NO. 1); namely X in the sequence shown in formula (I)1Is Ala, X2Is Gly and X3Is absent.
3、S8GLP-1 group 6nmol/kg S8GLP-1(n＝10)；S8GLP-1 sequences such as HSEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2(SEQ ID NO. 2); namely X in the sequence shown in formula (I)1Is Ser, X2Is Gly and X3Is absent.
4、dA8GLP-1 group 6nmol/kg dA8GLP-1(n＝10)；dA8GLP-1 sequence such as HdAEGTFTSDVSSYLEGQAAKEFAWLVKGRGC-NH2(substantially as shown in SEQ ID NO.1, wherein the second position is D-Ala); namely X in the sequence shown in formula (I)1Is D-Ala, X2Is Gly and X3Is absent.
Injecting glucose and A into abdominal cavity according to mouse weight8GLP-1、S8GLP-1 and dA8GLP-1 samples. At 30min, 100min, 165min,Blood is taken from the tail of the mouse at 250min and 350min, and the blood glucose value is measured by a blood glucose test strip. 200 μ L of 37% glucose was supplemented 30min before each blood glucose measurement. The data were subjected to t-test using GraphPad Prism5 statistical software and graphed. The statistical results are shown in table 1 and fig. 1:
TABLE 1
Mass spectrum data of the GLP-1 derivative monomer (SEQ ID NO.1) used in the examples of the present invention are shown in FIG. 6, and a high pressure liquid chromatography is shown in FIG. 7 and Table 2.
TABLE 2
The High Pressure Liquid Chromatography (HPLC) conditions were:
mobile phase A of water and 0.1% trifluoroacetic acid
B (80% acetonitrile + 20% water) + 0.09% trifluoroacetic acid
Flow rate 1.0mL/min
Detection of 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 x 150mm
Example 2
Reaction of branched 20KD PEG-maleimide with GLP-1 derivatives
20KD branched PEG activated with maleimide in GLP-1 derivatives (SEQ ID NO.1, i.e. X in the sequence shown in formula (I))1Is Ala, X2Is Gly and X3None) to form a stably associated thioether covalent bond. Chemically synthesized 10mg of GLP-1 derivative and 100mg of branched 20KD PEG-maleimide were weighed and dissolved in 5ml of 200mM phosphate buffer pH7.4, and reacted at 4 ℃ with stirring for 4hr (molar ratio of polypeptide to PEG: 1: 2).
Separating the PEGylated compound from free PEG and free polypeptide by cation exchange chromatography using an acidic pH NaCl gradient on a MacroCap SP column (GE) to obtain PEG20KD-GLP-1. The pegylated compounds were qualitatively analyzed by RP-HPLC, SEC-HPLC and tested for activity.
Example 3
Reaction of branched 40KD PEG-maleimide with GLP-1 derivatives
40KD branched PEG activated with maleimide in GLP-1 derivatives (SEQ ID NO.1, i.e. X in the sequence shown in formula (I))1Is Ala, X2Is Gly and X3None) to form a stably associated thioether covalent bond. 10mg of chemically synthesized GLP-1 derivative 200mg of branched chain 40KD PEG-maleimide is weighed and dissolved in 5ml of 200mM phosphate buffer solution with pH7.4, and stirred at 4 ℃ and then put into reactionShould be 4hr (polypeptide to PEG molar ratio of 1: 2).
Separating the PEGylated compound from free PEG and free polypeptide by cation exchange chromatography using an acidic pH NaCl gradient on a MacroCap SP column (GE) to obtain PEG40KD-GLP-1. The pegylated compounds were qualitatively analyzed by RP-HPLC, SEC-HPLC and tested for activity.
Example 4
In vitro activity assay of PEG modified products of GLP-1 derivatives
According to a GLP-1R signal transduction pathway, an HEK293 cell line transiently transfected with an alkaline phosphatase reporter gene expressing GLP-1R and driven by cAMP is established and used for screening GLP-1R agonist. When GLP-1R binds to the agonist, cellular cAMP concentration increases and the expression of the alkaline phosphatase reporter gene, driven by cAMP, is upregulated. The ability of the compound to agonize GLP-1R activity can be judged by detecting the activity of alkaline phosphatase.
Seeding HEK-293 cells into 24-well plates at a cell density of 1.0 x 105One per ml. Plasmids expressing the GLP-1R and SEAP reporter genes were transfected into cells. After 6 hours of transfection, the medium was replaced with fresh medium and samples to be tested were added with different concentration gradients. After 44 hours, cell supernatants were removed to 96-well plates and placed in a 65 ℃ oven for 30 minutes to remove endogenous alkaline phosphatase. 80ul of cell supernatant was taken to a 96-well plate and 120ul of premixed assay working solution was added to each well. And detecting the light absorption value of the sample at 405nm of the microplate reader and calculating the enzyme activity.
Data processing and statistical analysis
EC50 is the concentration value of 50% of the maximum effect, can reflect the agonist activity of the ligand to the receptor, and is an important index for researching the combination and the activation between the ligand and the receptor. GLP-1 derivatives (SEQ ID NO.1) and PEG can be drawn by Graphpadprism5.0 software according to the drug stimulation concentration corresponding to the expression amount of SEAP20KDGLP-1 (prepared in example 2) and PEG40KDEC50 curve for GLP-1 (prepared in example 3), as shown in FIG. 2, in which the EC50 curve for unmodified GLP-1 derivative is shown, whereas the EC50 curves for the products in examples 2, 3 are shown in FIG. 3 and in FIG. 3, respectivelyAs shown in fig. 4.
The calculation method comprises the following steps: EC (EC)50＝Bottom+(Top-Bottom)/(1+10^((LogEC50-X)))
The results of the in vitro activity assay are shown in table 3:
TABLE 3
| GLP-1 derivatives | PEG20KD-GLP-1 | PEG40KD-GLP-1 | |
| EC50(Log(nM)) | 0.7321 | 1.349 | 1.519 |
Example 5
PEG (polyethylene glycol) modified product of GLP-1 derivative for acute blood sugar reduction experiment in healthy Kunming mouse body
Weighing and numbering healthy female Kunming mice (with the weight of 20-22 g). Fasting (about 12 h) was performed overnight before the experiment, and only drinking water was given. The basal blood glucose level was measured the next morning and recorded as the blood glucose level at 0 min. Selecting mice with similar blood sugar values to randomly group:
1) a glucose control group;
2)10nmol/kg GLP-1 derivative group (GLP-1 derivative sequence is shown in SEQ ID NO. 1);
3)10nmol/kg PEG20KDgroup of GLP-1 (PEG)20KDGLP-1 example 2 preparation);
4)10nmol/kg PEG40KDgroup of GLP-1 (PEG)40KDGLP-1 preparation example 3).
Injecting glucose, GLP-1 derivative and PEG into abdominal cavity according to mouse weight20KD-GLP-1、PEG40KD-a GLP-1 sample. Blood was collected from the tail of the mouse at 2h, 5h, 8h, 11h, and 13h, and the blood glucose value was measured with a blood glucose test strip. 200 μ L of 37% glucose was supplemented 30min before each blood glucose measurement. The data were subjected to t-test using GraphPad Prism5 statistical software and graphed.
TABLE 4
As shown in table 4, table 5 and fig. 5, the PEG-modified product of GLP-1 derivative of the present invention has no change in hypoglycemic effect within 11 hours and is capable of maintaining blood glucose concentration substantially identical to that after 12h fasting. The hypoglycemic effect of the unmodified GLP-1 derivative is already reduced after 2 hours. The modified GLP-1 derivative of the invention has a reduced effect at hour 13 to that of the unmodified GLP-1 derivative after 2 hours, and at this time (13 hours), the unmodified GLP-1 derivative has little effect on blood glucose concentration. Specifically, the hypoglycemic effect of the modified product of the GLP-1 derivative PEG of the present invention is shown in table 5, and has a 6.5-fold improvement effect in terms of half-life as compared with that of the unmodified GLP-1.
TABLE 5
Example 6
Single administration of PEG-modified products of GLP-1 derivatives on daily fasting plasma glucose in db/db mice
25 male db/db mice at 6 weeks of age were housed individually in cages and given daily ration of normal diet, and the experiment was started by 8 weeks of age. One day before the administration, 9 am were fasted (without water deprivation), and after 6h, the fasting blood glucose of the mice was measured, which was between 15-30 mmol/L. Mice were randomly grouped according to the fasting blood glucose values:
1) a PBS control group;
2)50nmol/kg PEG40KD-group GLP-1;
3)250nmol/kg PEG40KD-group GLP-1;
4)500nmol/kg PEG40KD-group GLP-1. (PEG)40KDGLP-1 prepared as described in example 3)
Samples were injected subcutaneously according to mouse body weight (40 g. + -.3 g). Blood is taken from the tail of the mouse at 1h, 2h, 3h, 4h, 24h and 48h, and the blood glucose value is measured by a blood glucose test strip. The data were subjected to t-test using GraphPad Prism5 statistical software and graphed.
TABLE 6
As shown in Table 6, db/db mice were injected subcutaneously with three different doses of PEG at low, medium and high levels in a single injection40KD-GLP-1. The 50nmol/kg low dose group did not exhibit hypoglycemic effects, but both the medium dose group and the high dose group exhibited significant hypoglycemic effects 1 hour after administration, and 500nmol/kg PEG40KDThe GLP-1 dose group still showed significant hypoglycemic effect at 24 hours. Thus, PEG40KDGLP-1 has obvious blood sugar reducing advantage, and the blood sugar reducing effect has concentration dependence, and the higher the administration concentration is, the more obvious the blood sugar reducing effect is, and the longer the time is.
Example 7
Acute toxicity test of high dose subcutaneous administration to db/db mice
GLP-1 derivatives (SEQ ID NO.1) modified by 40KD branched chain PEG activated by maleimide according to the method of example 3 react with the Exenatide sequence of comparative example, and high-purity PEG is obtained after purification40K-GLP-1 with PEG40KExenatide, for subsequent animal experiments.
Wherein the Exenatide sequence of the experimental comparative example was synthesized by Hangzhou Zhongji peptide Biochemical Co., Ltd and had a purity of > 95%, and the Exenatide sequence was 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 weeks old db/db mice 20 were randomized into two groups:
1)1000nmol/kg PEG40KD-group GLP-1;
2)1000nmol/kg PEG40KD-Exenatide group.
A single abdominal subcutaneous dose was administered according to mouse body weight (40 g. + -.3 g). Mice were observed for mortality within 14 days of dosing. Until the end of the observation period, none of the mice in the group of the present invention died and were freely moving, while 2 mice died in the comparative group.
The results show that: the physiological toxicity of the comparative example group was greater than that of the group of the present invention.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Bioproduct research institute, LLC
<120> polyethylene glycol-modified GLP-1 derivatives and pharmaceutically acceptable salts thereof
<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 (5)
1. A modified GLP-1 derivative and a medicinal salt thereof are characterized in that the sequence of the GLP-1 derivative is as follows:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC-NH2；
and the polyethylene glycol is modified on the GLP-1 derivative through the cysteine residue of the GLP-1 derivative;
wherein, the structure of the polyethylene glycol is as follows:
and the molecular weight of the polyethylene glycol is 40 KD.
2. The modified GLP-1 derivative of claim 1, wherein said modified GLP-1 derivative is
3. A process for the preparation of a modified GLP-1 derivative according to claim 1 and pharmaceutically acceptable salts thereof, comprising the steps of:
GLP-1 derivatives and polyethylene glycol are used for coupling reaction to obtain the modified GLP-1 derivatives.
4. A pharmaceutical composition comprising a modified GLP-1 derivative according to claim 1 and pharmaceutically acceptable salts thereof.
5. Use of a modified GLP-1 derivative of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutical composition of claim 4 for the preparation of a medicament for the treatment of diabetes.
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