CN113214382B - Acylated GLP-1 derivatives - Google Patents

Acylated GLP-1 derivatives Download PDF

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CN113214382B
CN113214382B CN202110217138.7A CN202110217138A CN113214382B CN 113214382 B CN113214382 B CN 113214382B CN 202110217138 A CN202110217138 A CN 202110217138A CN 113214382 B CN113214382 B CN 113214382B
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CN113214382A (en
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许铮
李峰
宋瑞
郭万军
潘海
冯静
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Hangzhou Xianweida Biotechnology Co ltd
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The invention provides GLP-1 (7-37) polypeptide analogues, fatty acid modified derivatives of the analogues and medicaments comprising the derivatives. In addition, the invention also provides a preparation method of the derivative and application of the derivative in preparing medicines.

Description

Acylated GLP-1 derivatives
The present application is a divisional application of the Chinese patent application with the application number 201910317953.3 and the invention name of 'acylated GLP-1 derivative'.
Technical Field
The invention belongs to the technical field of polypeptides. In particular, the invention relates to fatty acid modified derivatives of GLP-1 (7-37) polypeptide analogs. In addition, the invention also relates to a preparation method of the peptide derivative, a medicine containing the peptide derivative, application in preparing the medicine and the like.
Background
Diabetes mellitus is a sugar metabolism disorder disease caused by various factors such as heredity, environment and the like, and is now the third major disease which threatens human health and life safety after tumor and cardiovascular and cerebrovascular diseases. Diabetes itself does not necessarily cause harm, but long-term blood sugar is increased, large blood vessels and micro blood vessels are damaged and endanger heart, brain, kidney, peripheral nerves, eyes, feet and the like, and according to statistics of world health organization, diabetes complications are up to more than 100, and the diabetes is a disease with the most known complications at present. More than half of the deaths from diabetes are due to cardiovascular and cerebrovascular diseases, and 10% are due to renal changes. The amputation of diabetes is 10-20 times of that of non-diabetes. For this reason, the treatment of diabetes and thus the prevention of its complications is a critical social problem.
Diabetes mellitus can be classified into several types due to the different mechanisms of disease. Most of these are of type II diabetes (about 90%), mainly due to overweight and lack of physical activity. Type II diabetics often have abnormalities in both insulin resistance and insulin hyposecretion, and islet beta cell apoptosis often occurs in middle and late stages of the onset. At present, the action mechanism of clinically used oral hypoglycemic agents is mostly to enhance insulin sensitivity or promote insulin secretion to stabilize blood sugar, and the problem of beta cell apoptosis cannot be solved. And glucagon-like peptide-1 (GLP-1) and analogues thereof have the effects of slowing down beta cell apoptosis, promoting regeneration and promoting differentiation and proliferation of islet beta cells, so that the glucagon-like peptide-1 (GLP-1) and analogues thereof become the research focus for treating type II diabetes.
In 1983, BELL et al found GLP-1 (BELL G I, SANCHEZ-PESCADOR, LAYBOOURN P J, et al Exon duplication and divergence in the human preproglucagon gene [ J ]. Nature,1983, 304 (5924): 368-371) as a glucagon-like peptide-1 when analyzing the gene sequence of Proglucagon (PG). The PG gene sequence consists of 6 exons and 5 introns, comprising 3 major domains: glucagon (33-61), GLP-1 (72-108) and GLP-2 (126-158). mRNA of PG is expressed in pancreatic A cells, intestinal L cells and brain, and specific translational modifications are performed in these tissue cells, ultimately forming different end products.
GLP has two subtypes, GLP-1 analog and GLP-2 analog, which are nearly half identical to the amino acid sequence of glucagon and have about 35% homology therebetween. GLP-1 analogue is a polypeptide hormone secreted by Langerhans 'scell (Langerhans' scell) of jejunum, ileum and colon at the tail end, and has multiple functions of promoting insulin secretion and biosynthesis, inhibiting glucagon secretion, inhibiting gastric emptying and the like in a glucose-dependent manner. GLP-2 analogs are synthesized in both the intestinal tissue and brain stem of the central nervous system and in the hypothalamic neurogenic cells, mainly promoting normal small intestine growth and repair of intestinal mucosal lesions (Tar, gong, xu Weiren glucagon-like peptide 1 and its receptor agonist research progress [ J ]. Tianjin medicine, 2012, 40 (2): 181-184.).
GLP-1 is an endogenous insulin secretion-promoting hormone, mainly secreted by the intestinal L-cells, and plays a role in balancing insulin and glucose levels.
The primary structure of GLP-1 is: histidine (His) -alanine (Ala) -glutamic acid (Glu) -phenylalanine (Phe) -glutamic acid (Glu) -arginine (Arg) -histidine (His) -alanine (Ala) -glutamic acid (Glu) -glycine (Gly) -threonine (Thr) -phenylalanine (Phe) -threonine (Thr) -serine (Ser) -aspartic acid (Asp) -valine (Val) -serine (Ser) -tyrosine (Tyr) -leucine (Leu) -glutamic acid (Glu) -glycine (Gly) -glutamine (gin) -alanine (Ala) -lysine (Lys) -glutamic acid (Glu) -phenylalanine (Phe) -isoleucine (Ile) -alanine (Ala) -tryptophan (Trp) -leucine (Leu) -valine (Val) -lysine (Lys) -glycine (Gly) -arginine (Arg) -glycine (Gly). DDP-IV can rapidly degrade histidine (H) -alanine (A) at positions 7-8 of the N-terminus, and the DDP-IV mainly plays a role of peptide chain end hydrolase, and if the enzyme is alanine or proline at position 8, the enzyme can play a role in degradation, so that GLP-1 is rapidly deactivated (AERTGEERTS K, YE S, TENNANT M G, et al Crystal structure of human dipeptidyl peptidase IV in complex with a dipeptide peptidase reveals details on substrate specificity and tetrahedral intermediate [ J) ]Protein Sci,2004, 13 (2): 412-421). SARRAUSTE DE MENTHIERE et al model GLP-1 and observe changes in affinity and intrinsic activity of GLP-1 analogs to the receptor after amino acid substitution, histidine at position 7 being the affinity and intrinsic activityThe determinant of intrinsic activity, on which the aromatic ring is smaller than tryptophan and is free of any polar substituents; the side chain of alanine at the 8 th position has a polar group which can influence the activity of GLP-1, the volume of the side chain cannot be excessively large, and the activity can be reduced when the volume exceeds a certain limit; glutamic acid at position 9 is replaced by certain amino acids, such as acidic, polar and hydrophobic amino acids, the activity is unchanged, and the activity is reduced or even inactivated when replaced by basic amino acids; GLP-1 is in a binding state with a receptor, if an amino acid residue in the GLP-1 has an ionic bond effect, the GLP-1 is formed between 7-15 amino acids to form a ring structure, ala8-Glu9-Gly10-Thr11 can form a beta corner, so that 3 aromatic nuclei such as histidine at the 7 th position, phenylalanine at the 12 th position and tyrosine at the 19 th position interact with each other, and the interaction corresponds to an aromatic hydrophobic pocket existing on the receptor, and is presumed to play a role in activating the receptor; glycine at 22 nd position is flexible amino acid, and has flexible connection function to maintain spiral curl shape. Disruption of glycine clusters all aromatic amino acids but reduces the affinity for the receptor by 1/40 (SARAUSTE DE MENTHIEREC, CHAVANIEUA, GRASSYG, et al Structure requirements of the N-terminal region of GLP-1- [ 7-37) ]-NH 2 for receptor interaction and cAMP production[J].Eur J Med Chem，2004，39(6):473-480)。
GLP-1 includes GLP-1 (1-37), GLP-1 (1-36), GLP-1 (7-37) glycine derivatives and GLP-1 (7-36) NH 2 And an equimolecular form. The latter two are generally considered to have the same biological activity. GLP-1 (1-37) secreted by intestinal mucosa L cells is inactive, and further hydrolysis and excision of 6 amino acids at the N-terminal are required to obtain active GLP-1 (7-37). GLP-1 (7-37) is present in vivo for a relatively short period of time and is rapidly degraded. Thus, various studies have been made on GLP-1 analogs having DPP IV resistance, for example, U.S. Pat. No. 5545618 describes modification of the N-terminus with an alkyl or acyl group, and Gallwitz et al describes N-methylation or a-methylation of His at position 7, or substitution of the entire His with imidazole to increase resistance to DPP-IV and maintain physiological activity.
In addition to these modifications, exenatide-4 (exendin-4), a GLP-1 analog purified from the salivary glands of Hiragana, has resistance to DPP IV and has a higher physiological activity than GLP-1. Thus, it has an in vivo half-life of 2 to 4 hours longer than that of GLP-1. However, only the method of increasing DPP IV resistance is applicable, physiological activity cannot be sufficiently maintained, and in the case of using commercially available exenatide-4 (exenatide), it requires injection twice a day to a patient, which is still painful for the patient.
These insulinotropic peptides have a small molecular weight and are therefore rapidly excreted from the kidney. Scientists use chemical methods to add high solubility polymers such as polyethylene glycol to the surface of peptides to inhibit their loss in the kidneys. For example, U.S. patent No. 692464 describes that PEG binds to lysine residues of exenatide-4 to increase its in vivo residence time, however, this approach, while increasing the in vivo residence time of the peptide drug, at the same time the concentration of the peptide drug decreases significantly with increasing molecular weight, and the reactivity to the peptide decreases.
In addition, there are a number of other methods for modifying the structure of glucagon-like peptide-1 compounds in an attempt to extend their duration of action. For example, WO96/29342 discloses peptide hormone derivatives modified by introducing a lipophilic substituent at the C-terminal amino acid residue or at the N-terminal amino acid residue of a parent peptide hormone. WO98/08871 discloses GLP-1 derivatives (liraglutide) in which at least one amino acid residue of a parent peptide is linked to a lipophilic substituent. WO99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives having a lipophilic substituent attached to the C-terminal amino acid residue. WO00/34331 discloses bisacylated GLP-1 analogues. WO 00/69911 discloses activated insulinotropic peptides for injection which are believed to react with blood components to form conjugates in the patient's body, prolonging the duration of action in vivo.
WO2006/097537 discloses another acylated GLP-1 analogue (semaglutine) having a longer half-life than acylated GLP-1 (liragutine) of WO98/08871 by mutating amino acid 8 to an unnatural amino acid.
WO02/046227 discloses the use of genetic recombination techniques to prepare fusion proteins by binding GLP-1, exenatide-4 or an analogue thereof to human serum albumin or immunoglobulin region (Fc), which can solve problems such as low pegylation yield and non-specificity, but their effect of increasing half-life in blood is still not as pronounced as expected, and the desired effect is not achieved even unlike somalupeptide in the combined hypoglycemic effect. In order to maximize the effect of increasing half-life in blood, various types of peptide linkers have been attempted, but this approach faces the problem of possibly eliciting an immune response.
CN107033234A discloses fatty acid modified conjugates of GLP-1 analogs, the fatty acid modification site being at Lys 26 In the above, the early animal experiments show that the hypoglycemic effect is superior to that of the somalunin, and the mode can properly prolong the in vivo acting time of the GLP-1 analogue, but the prolonged time is still not ideal.
Currently commercially available GLP-1 drugs are mainly Exenatide-4 (Exenatide-4) isolated from lizard saliva, and human GLP-1 analogues modified with fatty acids, antibody Fc fragments or serum albumin. Exenatide-4 has a half-life that is too short and requires at least two injections per day for only 2-4 hours. Norand nod corporation's fatty acid modified liraglutide is the most effective in reducing the glycosylation of hemoglobin and has fewer side effects, but it has the disadvantage that it has an in vivo half-life of only 13 hours and requires daily administration. In order to further extend the in vivo half-life and reduce the frequency of administration, amino acid sequence mutants and modified long acting GLP-1 analogues such as FC, fatty acids or albumin have been developed in recent years. Such as dolalundin from gill corporation and somalundin (semaglide) from norand nod corporation. The half-life of these long-acting GLP-1 analogs in humans can be extended to varying degrees, and at best the dosing frequency of once-a-week dosing can be achieved.
The inventor of the application develops a novel GLP-1 analogue and a derivative thereof through long-term research, and compared with the prior acknowledged best drug, the cable-MALUO peptide, the in-vitro activity of the novel GLP-1 analogue and the derivative thereof is equivalent to that of the cable-MALUO peptide under the same experimental condition; in normal mice and diabetic mice models, the duration of hypoglycemic activity in the body can be increased by about 1-fold, meaning that the frequency of administration can be achieved in humans at least weekly intervals, even every two weeks or more. And when the dosage is 1/10 of the dosage of the somalundum, the hypoglycemic effect is not lower than that of the somalundum, and the somalundum has better application prospect.
Disclosure of Invention
The object of the present invention is to provide a novel GLP-1 (7-37) analogue, an acylated derivative of the analogue. In addition, the invention also provides a preparation method of the analogue or the derivative, a pharmaceutical composition containing the analogue or the derivative, a product and application of the analogue or the derivative in preventing and treating diseases.
In particular, in one aspect, the invention provides a derivative of a GLP-1 (7-37) analog or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analog comprises a polypeptide consisting of the amino acid sequence of the formula:
HX 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37
wherein X is 8 Selected from V, T, I, L, G or S, X 19 Is Y or K, X 23 Q or K, X 27 Is E or K, X 30 Is A or K, X 34 R or K, X 36 R or K, X 37 In the presence of a compound which is G or K,
provided that at X 19 、X 23 、X 27 、X 30 、X 34 、X 36 Or X 37 Only one of which is a K residue,
the derivative comprises an extension linked to the K residue, wherein the extension is
Wherein x is an integer from 4 to 38.
Wherein the extension is preferably: HOOC (CH) 2 ) 14 CO-、HOOC(CH 2 ) 15 CO-、HOOC(CH 2 ) 16 CO-、HOOC(CH 2 ) 17 CO-、HOOC(CH 2 ) 18 CO-、HOOC(CH 2 ) 19 CO-、HOOC(CH 2 ) 20 CO-、HOOC(CH 2 ) 21 CO-and HOOC (CH) 2 ) 22 CO-, more preferably HOOC (CH) 2 ) 16 CO-。
In a preferred embodiment, the extension of the derivative of the GLP-1 analog or a pharmaceutically acceptable salt thereof according to the invention is linked to the K residue of GLP-1 via a linker. The joint may be of the following structure:
Wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; p is any integer from 1 to 8.
Preferably, the joint is:
wherein m is 1 or 2; n is 1 or 2; p is any integer from 1 to 5.
More preferably: the joint is as follows:
wherein m is 1 and n is 1 or 2.
The invention also relates to GLP-1 (7-37) analogues comprising
HX 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37 A sequence comprising a mutation at one or more of the following positions:
bit 8, bit 19, bit 23, bit 27, bit 30, bit 34, bit 36 and bit 37. In a preferred embodiment, the amino acid residue at position 8 is selected from V, T, I, L, G or S, the amino acid residue at position 19 is Y or K, the amino acid residue at position 23 is Q or K, the amino acid residue at position 27 is E or K, the amino acid residue at position 30 is A or K, the amino acid residue at position 34 is R or K, the amino acid residue at position 36 is R or K, the amino acid residue at position 37 is G or K, provided that only one of positions 19, 23, 27, 30, 34, 36 or 37 is a K residue.
The in vitro binding activity of the acylated derivative of the GLP-1 analog shows that the binding affinity of the derivative with the GLP-1R receptor is greater than that of the somalunin or M0 (the 26 th position is Lys, disclosed in CN 107033234A). In vivo hypoglycemic experiments also prove that compared with the GLP-1 product, namely the somalunin, the acylated derivative of the GLP-1 analogue can obtain longer hypoglycemic activity duration in a normal mouse body; the effect of the derivatives on reducing blood glucose and improving glucose tolerance is obviously better than that of the somalundum peptide in diabetic mice, and the effect of reducing blood glucose is not lower than that of the somalundum peptide or M0 when the dosage is only 1/10 of that of the somalundum peptide or M0. Meanwhile, the research of the invention proves that the derivative of the GLP-1 (7-37) analogue has better anti-enzymatic degradation property compared with the commercial somalunin.
In particular, the invention relates to:
1. a derivative of a GLP-1 (7-37) analogue or a pharmaceutically acceptable salt thereof, wherein the GLP-1 (7-37) analogue comprises an amino acid sequence of the formula:
HX 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37 ，
wherein X is 8 Selected from V, T, I, L, G or S, X 19 Is a group of Y or K,X 23 q or K, X 27 Is E or K, X 30 Is A or K, X 34 R or K, X 36 R or K, X 37 In the presence of a compound which is G or K,
provided that at X 19 、X 23 、X 27 、X 30 、X 34 、X 36 Or X 37 Only one of which is a K residue,
the derivative comprises an extension linked to the K residue of the GLP-1 (7-37) analog, wherein the extension is
Wherein x is an integer from 4 to 38.
2. The derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the extension is selected from the group consisting of:
HOOC(CH 2 ) 14 CO-、HOOC(CH 2 ) 15 CO-、HOOC(CH 2 ) 16 CO-、HOOC(CH 2 ) 17 CO-、HOOC(CH 2 ) 18 CO-、HOOC(CH 2 ) 19 CO-、HOOC(CH 2 ) 20 CO-、HOOC(CH 2 ) 21 CO-and HOOC (CH) 2 ) 22 CO-。
3. The derivative according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the extension is linked to the K residue of a GLP-1 (7-37) analogue by a linker.
4. A derivative according to claim 3, or a pharmaceutically acceptable salt thereof, wherein the linker is:
wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; p is any integer from 1 to 8.
Preferably, the joint is:
wherein m is 1 or 2; n is 1 or 2; p is any integer from 1 to 5.
5. The derivative or pharmaceutically acceptable salt thereof according to claim 4, wherein the linker is:
wherein m is 1 and n is 1 or 2. 6. The derivative of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, which is any one of the derivatives selected from the group consisting of: n-epsilon 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M2), N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M4), N- ε 34 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Arg 26 Lys 34 -GLP-1 (7-37)) peptide (M5), N- ε 37 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Arg 26,34 Lys 37 -GLP-1 (7-37)) peptide (M7), N- ε 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino) ]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ile 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M9) \N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Thr 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M13) \N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ile 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M14).
7. A process for preparing the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, comprising:
(1) Mixing a solution in which a GLP-1 analogue of any one of the preceding claims is dissolved with a solution in which an extension of any one of the preceding claims is dissolved;
(2) Regulating pH to 4-5, stopping reaction, standing until precipitation is generated, and collecting the precipitate; and
(3) TFA was added to the precipitate and the reaction was stopped by adjusting the pH to 7.5-8.5.
8. The method of claim 7, further comprising adding triethylamine to the solution in which the GLP-1 analogue is dissolved prior to mixing with the solution in which the extension of any one of the preceding claims is dissolved.
9. The method of claim 7 or 8, wherein the solution of the extension of any of the preceding claims is acetonitrile-soluble.
10. A pharmaceutical composition comprising the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
11. Use of a derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of diabetes (including type I diabetes and type II diabetes) or diabetic complications.
12. The use of claim 11, wherein the diabetic complication is diabetic nephropathy.
13. Use of a derivative according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for reducing blood glucose, increasing glucose tolerance, reducing islet β -cell apoptosis, enhancing islet β -cell function, increasing islet β -cell number and/or restoring glucose sensitivity to islet β -cells.
14. The use of claim 13, wherein said lowering blood glucose comprises lowering fasting blood glucose and/or postprandial blood glucose.
15. A method of preventing and/or treating diabetes (including type I diabetes and type II diabetes) or diabetic complications, comprising administering to a subject a prophylactically or therapeutically effective amount of the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof.
16. The method of claim 15, wherein the diabetic complication is diabetic nephropathy.
17. A method of reducing blood glucose, increasing glucose tolerance, reducing islet β -cell apoptosis, enhancing islet β -cell function, increasing islet β -cell number, and/or restoring glucose sensitivity to islet β -cells, comprising administering to a subject a therapeutically effective amount of the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof.
18. The use of claim 17, wherein said lowering blood glucose comprises lowering fasting blood glucose and/or postprandial blood glucose.
19. A GLP-1 (7-37) analogue comprising a polypeptide consisting of the amino acid sequence:
HX 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37
wherein X is 8 Selected from V, T, I, L, G or S, X 19 Is Y or K, X 23 Q or K, X 27 Is E or K, X 30 Is A or K, X 34 R or K, X 36 R or K, X 37 Is G or K, and, in X 19 、X 23 、X 26 、X 27 、X 30 、X 34 、X 36 Or X 37 Only one of which is K.
20. A pharmaceutical composition comprising the analog of claim 19.
21. Use of the analogue of claim 19 in the manufacture of a medicament for the prevention or treatment of diabetes mellitus, complications of diabetes mellitus.
22. An article of manufacture comprising a container having the pharmaceutical composition of claim 10 or claim 20 contained therein and a package insert, wherein the package insert carries instructions for use of the pharmaceutical composition.
23. The article of manufacture of claim 22, further comprising a container containing one or more other medicaments.
24. The article of manufacture of claim 23, wherein the one or more additional drugs are additional drugs for treating diabetes or diabetic complications.
"fasting blood glucose" refers to a blood glucose level measured on a subject (e.g., a human) on a fasting basis, e.g., on a fasting basis overnight, with the exception of drinking water without any food, for at least 6 hours, e.g., 6-8 hours, after 8-10 hours.
"postprandial blood glucose" means a blood glucose level measured after a meal, for example, a blood glucose level measured 15 minutes to 2 hours, 30 minutes to 2 hours, 1 hour to 2 hours, and 2 hours after a meal.
One aspect of the invention relates to a method for the preparation of a GLP-1 (7-37) analogue, comprising expressing a DNA sequence encoding the polypeptide in a host cell under conditions allowing expression of the peptide, and recovering the resulting peptide.
The medium used to culture the cells may be any conventional medium used to culture the host cells, such as minimal medium or complex medium with suitable additives. Suitable media may be obtained commercially or may be prepared according to published procedures. The polypeptide produced by the host cell may then be recovered from the culture medium by conventional methods, for example, precipitation of the protein component in the supernatant or filtrate with a salt such as ammonium sulfate, and further purification may be performed by various chromatographic methods such as, for example, exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the kind of the target peptide.
The above-described coding DNA sequences may be inserted into any suitable vector. In general, the choice of vector will often depend on the host cell into which the vector is to be introduced, and thus the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be of a type that, when introduced into a host cell, will integrate into the host cell genome and replicate with the chromosome into which it has integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments (e.g., promoters) required for transcription of the DNA. Examples of promoters suitable for directing transcription of DNA encoding the peptides of the invention in a variety of host cells are well known in the art, see, e.g., sambrook, J, fritsch, EF and Maniatis, T, molecular cloning: instructions for experimental operation, cold Spring Harbor Laboratory Press, new york, 1989.
The vector may also contain a selectable marker, e.g., a gene whose gene product will complement a defect in the host cell or which confers resistance to a drug such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin, streptomycin, or methotrexate.
To introduce the peptides expressed in the invention into the secretory pathway of a host cell, a secretory signal sequence (also referred to as a leader sequence) may be provided in the recombinant vector. The secretion signal sequence is linked in correct reading frame to the DNA sequence encoding the peptide. The secretion signal sequence is typically located 5' to the DNA sequence encoding the peptide. The secretion signal sequence may be a secretion signal sequence normally associated with the peptide, or may be derived from a gene encoding another secreted protein.
Methods for ligating the DNA sequence encoding the peptide of the invention, the promoter and optionally the terminator and/or secretion signal peptide sequence, respectively, and inserting them into a suitable vector containing the information necessary for replication are known to the person skilled in the art.
The host cell into which the DNA sequence or recombinant vector is to be introduced may be any cell capable of producing the peptide of the present invention, including bacterial, yeast, fungal and higher eukaryotic cells. Examples of suitable host cells well known and used by those skilled in the art include, but are not limited to: coli, saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
The invention relates to a medicament or a pharmaceutical composition comprising the GLP-1 (7-37) analogue, and further relates to application of the analogue in preparing medicaments, such as application in preparing medicaments for preventing or treating diabetes (preferably type 2 diabetes), diabetic complications (such as diabetic nephropathy and diabetic heart disease), reducing blood sugar or improving sugar tolerance.
In another aspect, the invention also relates to a method of preventing or treating diabetes (e.g., type I and type II diabetes), diabetic complications (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic ocular disease, diabetic nephropathy, diabetic heart disease), lowering blood glucose (e.g., fasting blood glucose and postprandial blood glucose), or increasing glucose tolerance by administering to a subject the above GLP-1 (7-37) analog or a derivative of the above GLP-1 (7-37) analog. In another aspect, the invention also relates to the use of the above GLP-1 (7-37) analogue or a derivative of the above GLP-1 (7-37) analogue for the preparation of a medicament for preventing or treating diabetes (e.g. type I and type II diabetes), diabetic complications (e.g. diabetic vasculopathy, diabetic neuropathy, diabetic ocular disease, diabetic nephropathy, diabetic heart disease), lowering blood glucose or increasing glucose tolerance.
In another aspect, the invention relates to a pharmaceutical composition, article of manufacture or kit comprising a GLP-1 (7-37) analog as described above.
The invention also relates to a pharmaceutical composition, article of manufacture or kit comprising a derivative of the above GLP-1 (7-37) analogue.
The pharmaceutical composition of the invention comprises pharmaceutically acceptable auxiliary materials besides active ingredients GLP-1 (7-37) analogues or derivatives or salts of GLP-1 (7-37) analogues. Pharmaceutically acceptable excipients, such as non-toxic fillers, stabilizers, diluents, carriers, solvents, or other formulation excipients, are well known to those skilled in the art. For example, diluents, excipients, such as microcrystalline cellulose, mannitol, and the like; fillers such as starch, sucrose, etc.; binders such as starch, cellulose derivatives, alginates, gelatin and/or polyvinylpyrrolidone; disintegrants, such as calcium carbonate and/or sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants such as cetyl alcohol; carriers, solvents such as water, physiological saline, kaolin, bentonite, and the like; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. In addition, the pharmaceutical composition of the present invention is preferably an injection.
The invention also relates to a method of reducing islet β -cell apoptosis, enhancing islet β -cell function, increasing islet β -cell number, and/or restoring glucose sensitivity to islet β -cells, comprising administering to a subject in need thereof an effective amount of an analog, derivative or pharmaceutical, pharmaceutical composition as described above.
The invention also relates to application of the analogue, derivative or medicine composition in preparing medicines for reducing islet beta-cell apoptosis, enhancing islet beta-cell function, increasing islet beta-cell number and/or restoring glucose sensitivity of islet beta-cells.
In the present invention, GLP-1 (7-37) polypeptide analogue, GLP-1 (7-37) analogue can be used interchangeably, meaning comprising the amino acid sequence: HX (HX) 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37 Wherein X is 8 Selected from V, T, I, L, G or S, X 19 Is Y or K, X 23 Q or K, X 27 Is E or K, X 30 Is A or K, X 34 R or K, X 36 R or K, X 37 G or K. The GLP-1 (7-37) polypeptide analogue is linked to an extension to form the GLP-1 (7-37) polypeptide analogueDerivatives of the same. In particular, the invention relates to acylated derivatives of GLP-1 (7-37) analogues. The acylated derivatives not only have a remarkable therapeutic effect, but also have an in vivo activity duration which can be increased by about 1 time as compared with the currently recognized best drug, somalunin, meaning that the administration frequency of at least weekly intervals, even every two weeks or more, can be achieved in humans.
The derivatives of GLP-1 (7-37) analogues, acylated derivatives of GLP-1 (7-37) analogues, GLP-1 (7-37) derivatives, GLP-1 derivatives of the invention may be used interchangeably.
In another aspect, the invention also relates to a process for preparing the above derivatives or pharmaceutically acceptable salts thereof, comprising:
(1) Mixing a solution in which the above GLP-1 analog is dissolved with a solution in which an extension (e.g., fatty acid) is dissolved;
(2) Regulating pH to 4-5, stopping reaction, standing until precipitation is generated, and collecting the precipitate; and
(3) TFA was added to the precipitate and the reaction was stopped by adjusting the pH to 7.5-8.5.
In a preferred embodiment, the above method comprises adding triethylamine to a solution of the GLP-1 analog.
In a preferred embodiment, the extension (e.g., fatty acid) is an acetonitrile-dissolved solution.
An exemplary method of preparation of the present invention comprises (1) providing a GLP-1 (7-37) analog solution, adjusting the pH to 9-12;
(2) Then adding triethylamine into the solution obtained in the step (1);
(3) Weighing a 2-fold or more (molar ratio) of fatty acid of the structure, preferably a 3-fold or more of GLP-1 analogue, of the GLP-1 analogue, and dissolving the fatty acid in acetonitrile;
(4) Mixing the GLP-1 analogue solution obtained in the step (2) with the fatty acid solution obtained in the step (3), and standing at a low temperature, for example, for one hour;
(5) Regulating pH to 4-5, stopping reaction, standing at low temperature, precipitating with acid, and collecting precipitate;
(6) Adding TFA to the acid precipitation sample obtained in the step (5) until the final concentration of the polypeptide is 5-15mg/ml, standing for 0.5-2 hours, dripping an alkaline solution such as NaOH into the reaction solution, and regulating the pH to 7.5-8.5 to terminate the reaction;
(7) And separating and purifying the obtained product.
The present invention relates to formulations of pharmaceutical compositions comprising derivatives of GLP-1 (7-37) analogues or pharmaceutically acceptable salts thereof. In some embodiments, the derivative of the invention comprising a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, is present at a concentration of 0.1mg/ml to 25mg/ml, preferably at a concentration of 0.1mg/ml to 10.0 mg/ml. In a preferred embodiment, the pharmaceutical composition has a pH of 3.0 to 9.0. In preferred embodiments, the pharmaceutical composition may further comprise a buffer system, a preservative, a surface tension agent, a chelating agent, a stabilizer, and a surfactant. In some embodiments, the medicaments or formulations described herein are aqueous medicaments or formulations, e.g., they may generally be solutions or suspensions. In a specific embodiment of the invention, the drug or formulation is a stable aqueous solution. In other embodiments of the invention, the drug or formulation is a lyophilized formulation to which the solvent and/or diluent is added prior to use.
The invention also relates to a kit or a kit containing the pharmaceutical composition, the preparation and the medicine. In addition to the above-mentioned drugs or formulations, other drugs, drug compounds or compositions that may be used in combination with the drug compositions, formulations, drugs are included in the kit or kit, for example, the other drugs, drug compounds or compositions may be selected from antidiabetic drugs, drugs for treating and/or preventing complications arising from or associated with diabetes. Examples of such drugs include: insulin, sulfonylurea, biguanide, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in stimulating gluconeogenesis and/or glycogenolysis, glucose uptake modulators, NPY antagonists, PYY agonists, PYY2 agonists, PYY4 agonists, TNF agonists, corticotropin releasing factor agonists, 5HT, bombesin agonists, ganglion peptide antagonists, growth hormone, thyroid stimulating hormone releasing hormone agonists, trβ agonists; histamine H3 antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogues, and the like. In some embodiments, the pharmaceutical compositions, formulations, medicaments, and other medicaments, pharmaceutical compounds, or compositions of the present invention are placed in separate containers.
The present invention also relates to a method of preventing or treating diabetes (e.g., type I and type II diabetes), diabetic complications (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic ocular disease, diabetic nephropathy, diabetic heart disease), lowering blood glucose (e.g., fasting blood glucose and postprandial blood glucose), comprising administering to a subject in need thereof an analog, derivative or pharmaceutical composition as described above, wherein the analog, derivative or pharmaceutical composition is used in combination with other drugs, pharmaceutical compounds or compositions, e.g., the other drugs, pharmaceutical compounds or compositions may be selected from antidiabetic drugs, drugs for treating and/or preventing complications arising from diabetes or associated therewith. Examples of such drugs include: insulin, sulfonylurea, biguanide, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in stimulating gluconeogenesis and/or glycogenolysis, glucose uptake modulators; CART agonists, NPY antagonists, PYY agonists, PYY2 agonists, PYY4 agonists, TNF agonists, corticotropin releasing factor agonists, 5HT, bombesin agonists, ganglion peptide antagonists, growth hormone, thyroid stimulating hormone releasing hormone agonists, trβ agonists; histamine H3 antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogues, and the like. In a preferred embodiment, the diabetes is type 2 diabetes or diabetic nephropathy.
The term "diabetic complication" as used herein refers to a disease caused by damage or dysfunction of other organs or tissues of the body due to poor glycemic control during diabetes, including damage or dysfunction of liver, kidney, heart, retina, nervous system, etc. Complications of diabetes can be divided into five aspects: 1. cardiovascular lesions: including microvascular lesions, cardiomyopathy, and cardiac autonomic neuropathy on the heart and large vessels, leading causes of death in diabetics. 2. Cerebrovascular disease: is mainly characterized by cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, cerebral atrophy and the like. 3. Renal vascular lesions: the main surface is diabetic nephropathy, which is one of the most important complications of diabetics. 4. Lower limb arterial lesions: mainly represented by diabetic foot. 5. Fundus microvascular lesions: mainly manifested by diabetic retinopathy.
The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of protection of this patent, however, the features disclosed in the foregoing description and in the following examples (individually and in any combination thereof) may be materials used to carry out the invention in substantially different forms, which may be combined in any desired manner. In addition, the present invention refers to publications that are incorporated herein by reference in their entirety for the purpose of more clearly describing the invention as if repeated herein were set forth in their entirety.
Brief Description of Drawings
FIG. 1 shows the hypoglycemic effect of different acylated GLP-1 derivative molecules on type II diabetic db/db mice.
Figure 2 shows a trend graph of the effect of different doses of M0, M4 and somalunin on fasting blood glucose in diabetic mice.
Figure 3 shows the effect of different doses of M0, M4 and somalunin on random blood glucose in diabetic mice.
Figure 4 shows the effect of different doses of M0, M4 and somalunin on the area under the blood glucose curve of diabetic mice.
FIG. 5 shows a graph of the tendency of M4 and somalunin molecules to resist pepsin degradation.
Figure 6 shows a trend of M4 and somalupeptide molecules against trypsin degradation.
Examples
Hereinafter, the invention will be described by means of specific examples. Unless otherwise indicated, the method may be carried out according to the methods listed in the laboratory manuals such as "molecular cloning laboratory Manual" and "cell laboratory Manual" and the like, and the laboratory guidelines of CFDA and the like, which are familiar to those skilled in the art. Wherein, the raw materials of the used reagents are all commercial products and can be purchased through public channels.
EXAMPLE 1 construction of GLP-1 analog expression plasmids
Construction of Val 8 Glu 22 Lys 23 Arg 26,34 DNA of GLP-1 (7-37)
6-His tag, SUMO tag and Val 8 Glu 22 Lys 23 Arg 26,34 The GLP-1 (7-37) coding gene sequences (SEQ ID NO: 7) are fused in sequence in tandem and the gene fragment (SEQ ID NO: 18) is obtained using chemical synthesis. The above fragment was inserted into prokaryotic expression plasmid pET-24 (+) through BamHI and XhoI sites and verified by sequencing. The resulting expression plasmid for transformation assay, called pET-24 (+) -His-SUMO-Val 8 Glu 22 Lys 23 Arg 26,34 -GLP-1(7-37)。
According to the method, val is constructed in turn 8 Glu 22 Lys 26 Arg 34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 3), val 8 Glu 22 Lys 30 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 11), val 8 Glu 22 Lys 19 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 5), val 8 Glu 22 Lys 27 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 9), val 8 Glu 22 Lys 34 Arg 26 GLP-1 (7-37) (the coding gene is SEQ ID NO: 13), val 8 Glu 22 Arg 26,34 Lys 36 GLP-1 (7-37) (the coding gene is SEQ ID NO: 15), val 8 Glu 22 Arg 26,34 Lys 37 GLP-1 (7-37) (the coding gene is SEQ ID NO: 17), thr 8 Glu 22 Lys 23 Arg 26,34 -GLP-1(7-37 (the coding gene is SEQ ID NO: 20), ile 8 Glu 22 Lys 23 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 22), leu 8 Glu 22 Lys 23 Arg 26,34 GLP-1 (7-37) (coding gene is SEQ ID NO: 24), gly 8 Glu 22 Lys 23 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 26), ser 8 Glu 22 Lys 23 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 28), thr 8 Glu 22 Lys 30 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 30), ile 8 Glu 22 Lys 30 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 32), leu 8 Glu 22 Lys 30 Arg 26,34 GLP-1 (7-37) (SEQ ID NO: 34), gly 8 Glu 22 Lys 30 Arg 26,34 GLP-1 (7-37) (the coding gene is SEQ ID NO: 36), ser 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37) (encoding gene SEQ ID NO: 38) corresponding expression plasmid.
Example 2 fusion protein expression
Expression of the fusion protein was performed using the DNA construct described in example 1, and the protein of interest was obtained by expression cell BL21 (trabsgenbiotech., catalog #cd601). BL21 competent cells were thawed by 50. Mu.l on an ice bath, target DNA was added, gently shaken, and left in the ice bath for 30 minutes. The centrifuge tube was then rapidly transferred to an ice bath for 2 minutes without shaking the tube after a subsequent heat shock in a 42℃water bath for 30 seconds. 500. Mu.l of sterile LB medium (without antibiotics) was added to the centrifuge tube, and after mixing, the mixture was incubated at 37℃for 1 hour at 180rpm to resuscitate the bacteria. Mu.l of transformed competent cells were aspirated and plated on LB agar medium plates containing kanamycin resistance, and the cells were spread evenly. The plate was placed at 37℃until the liquid was absorbed, the plate was inverted, and incubated overnight at 37 ℃. The following day, monoclonal colonies in transformation plates were picked using an inoculating loop and inoculated in 15ml of sterile LB medium (containing antibiotics) and incubated overnight at 30 ℃.
EXAMPLE 3 fermentation of recombinant GLP-1 analog
To 50ml of LB medium, 50. Mu.l of a bacterial liquid (GLP-1 expressing bacterial liquid) was added, and simultaneously 50. Mu.l of kanamycin was added, and after mixing, the mixture was placed in a constant temperature shaker at 30℃and inoculated overnight. 10ml of the overnight inoculated broth was added to 1000ml of LB medium, together with 1000. Mu.l of kanamycin. Shaking, placing in a shaking table at 37 ℃ at 200rpm, inoculating 4 hours later, inoculating IPTG with the final concentration of 0.1mol/L into the culture medium, shaking, placing in a shaking table at 30 ℃ at 180rpm, and inducing expression overnight. The overnight expressed bacterial liquid was centrifuged at 13000g for 60min. The yield of the bacterial cells is about 4g of bacterial/L fermentation liquor, and the expression level of the target protein can reach about 40% by SDS-PAGE measurement.
EXAMPLE 4 purification of recombinant GLP-1 analog
100g of the cell slurry was weighed and resuspended in 500ml of 50mM Tris-HCl, pH8.0, 50mM NaCl and sonicated in a sonicator for 30min to disrupt the cells. The homogenate is centrifuged for 60min at 13000g at 4 ℃, and the supernatant is collected after the centrifugation is finished, namely the Ni column chromatography sample.
The resulting supernatant was concentrated by Chelating Sepharose FF equilibrated with 50mM Tris-HCl, pH8.0, 500mM NaCl,10mM imidazole (equilibration solution 1). After rinsing with the equilibration solution 1, the solution was eluted with 50mM Tris-HCl, pH8.0, 50mM NaCl,0.3M imidazole (eluent). The purity of GLP-1 intermediate product generated by the purification process is higher than 70% through SDS-PAGE analysis.
Sumo tag sequence excision using ULP enzyme: the intermediate was diluted three-fold by adding 20mM PB, pH7.4 buffer, following ULP enzyme at 4 ℃): the intermediate product was digested overnight after 1:150 ULP addition and mixing. The cleavage efficiency was approximately 100% by SDS-PAGE analysis.
Precision of GLP-1 analogues: the product obtained after the cleavage was subjected to a pretreatment with 20mM Na 2 HPO 4 The Tosoh Butyl 550C medium equilibrated with 0.7M NaCl (equilibration solution 2) was concentrated. After rinsing the equilibrium liquid 2, the solution was eluted with 20% ethanol, and the purity was about 90% by SDS-PAGE.
Adding 0.2M Na into the eluted sample 2 HPO 4 To a final concentration of 20mM Na 2 HPO 4 The pH was adjusted to 4.8-5.0,4 ℃with 1M citric acid and acid precipitated overnight. SDS-PAGE detectionThe yield is more than 90 percent. Centrifuging at 13000g at 4deg.C for 30min, collecting precipitate, and storing at-20deg.C.
EXAMPLE 5 preparation of derivatives of GLP-1 analog
Derivatives of GLP-1 analogues, N- ε, as shown below 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Lys 23 Arg 26,34 Preparation of-GLP-1 (7-37)) peptide (abbreviated as M2)
1. Fatty acid modification: val prepared and collected in the above examples 8 Glu 22 Lys 23 Arg 26，34 Adding water into GLP-1 (7-37) precipitate to prepare 4-6 mg/ml solution, adding 1M sodium hydroxide to adjust pH to 11.0-11.5, shaking to completely dissolve protein, and quantifying polypeptide concentration by HPLC. The fatty acid powder is weighed according to the mol ratio of polypeptide to fatty acid (structure is as follows) of 1:4 and dissolved in acetonitrile. Triethylamine was added to the polypeptide solution in an amount of two thousandths, and mixed with a fatty acid solution, and the mixed solution was allowed to stand at 4℃for one hour.
Diluting the sample with water 5 times, adjusting pH to 4.8 with 1M citric acid (or 10% acetic acid), stopping reaction, standing at 4deg.C, precipitating with acid for 10min, centrifuging 13000g, centrifuging at 4deg.C for 30min, and storing the precipitate at-80deg.C.
2. Deprotection and purification of fatty acids: adding TFA to the acid precipitation sample with the final concentration of the polypeptide of about 10mg/ml, oscillating to dissolve the precipitate, standing at room temperature for deprotection for 30min, and dripping 4M NaOH into the reaction solution to adjust the pH to 7.5-8.5 to terminate the reaction.
The reaction mixture after termination was concentrated by a preparative liquid phase apparatus (Shimadzu LC-8A) at a flow rate of 4ml/min by pumping UniSil 10-120C18 (available from Soy micro technologies Co., ltd.) previously equilibrated with 10mM ammonium acetate, 20% ethanol (equilibration solution 3). After rinsing the equilibrium solution 3, the solution is eluted with a gradient of 0-100% eluent (10 mM ammonium acetate, 80% ethanol), and the elution peak is collected and detected to have a purity of about 90% by RP-HPLC.
Diluting the eluting peak with water for 3 times, and acid precipitating to adjust pH to 4.80,4 deg.C for 30min. Adding PBST buffer (pH 7.0) into the precipitate after centrifugation, re-dissolving, and freezing at-80 ℃.
According to the method, N-epsilon is prepared in turn 26 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy ]Ethoxy) acetyl]Val 8 Glu 22 Lys 26 Arg 34 -GLP-1 (7-37) (M0) peptide, N-epsilon 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M4), N- ε 19 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Lys 19 Glu 22 Arg 26 34-GLP-1 (7-37)) peptide (M1), N- ε 27 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Lys 27 Arg 26,34 -GLP-1 (7-37)) peptide (M3), N- ε 34 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Arg 26 Lys 34 -GLP-1 (7-37)) peptide (M5), N- ε 36 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Val 8 Glu 22 Arg 26,34 Lys 36 -GLP-1 (7-37)) peptide (M6), N- ε 37 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl ](Val 8 Glu 22 Arg 26,34 Lys 37 -GLP-1 (7-37)) peptide (M7); n-epsilon 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Thr 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M8), N- ε 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ile 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M9), N- ε 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Leu 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M10), N- ε 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Gly 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M11), N- ε 23 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ser 8 Glu 22 Lys 23 Arg 26,34 -GLP-1 (7-37)) peptide (M12); n-epsilon 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Thr 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M13), N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ile 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M14), N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy group) Acetyl group](Leu 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M15), N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Gly 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M16), N- ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl](Ser 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7-37)) peptide (M17).
TABLE 1 GLP-1 (7-37) analog and corresponding derivative lookup table
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Example 6 determination of in vitro Activity of derivatives of GLP-1 analog in RIN-m5F cells
RIN-m5F cells with good culture state are selected. The cells were collected, counted and prepared into 1X 10 with RPMI1640 basal medium 5 Cell suspensions of individual cells/ml. Inoculating the cell suspension into 96-well cell culture plate, 100 μl/well, 37deg.C, 5% CO 2 The culture was carried out overnight under the conditions. In vitro activity of derivatives of GLP-1 analogs was detected using the cAMP detection kit (Promega): preparing diluted samples (Aib, M0, M1, M2, M3, M4, M5, M6, M7) of the assay broth to 300ng/ml, followed by 3-fold gradient dilution in 96-well plates for a total of 8 concentrations, 2 multiplex wells per dilution, wherein M0, M1, M2, M3, M4, M5, M6, M7 are prepared as described above, aib is
N-ε 26 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutyrylamino)]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl][Aib 8 ，Arg 34 ]GLP-1- (7-37) peptide (see CN101133082B example 4), commercially available as somalu peptide, was prepared according to the method disclosed in patent CN 101133082B.
The prepared cell plates were removed, the medium was discarded and blotted dry on filter paper. The sample solution was correspondingly transferred into the cell plate, 40. Mu.l/well. At 37℃with 5% CO 2 And (5) uncovering treatment for 15min under the condition. The cell culture plate was removed from the incubator, 10. Mu.l of CD solution (cAMP detection kit (Promega)) was added to each well, and the cell plate was placed at 22℃to 25℃with horizontal shaking at 500rpm for 20 minutes. 50 μl KG solution (cAMP detection kit (Promega)) was added to each well and the wells were left in the dark at 22℃to 25℃with horizontal shaking at 500rpm for 10min. The chemiluminescent value is read by a Molecular Devices SpectraMax L chemiluminescent instrument, and the detection is completed within 30 minutes. Sample EC50 was calculated using four parameter regression in softmax Pro software software.
TABLE 2 results of in vitro Activity experiments
Sample of Aib M0 M1 M2 M3 M4 M5 M6 M7
EC50 2.437 10.68 5.386 1.996 5.387 2.322 3.043 7.650 3.208
RIN-M5F cells showed comparable in vitro activity of the Somarlutide, M2, M4, M5 and M7, overall slightly higher than M0, M1, M3 and M6.
EXAMPLE 7 determination of in vitro Activity of derivatives of GLP-1 analog in HEK293/CRE-Luc/GLP1R cells
According to the GLP-1 can be combined with a receptor on a cell membrane, a HEK293/CRE-Luc/GLP1R cell line is constructed, a CAMP Response Element (CRE) is activated through a series of signal transduction, the expression of downstream luciferase is started, the expression quantity is positively correlated with the biological activity of the GLP-1, and after a luciferase substrate is added, the luminescence intensity is measured by chemiluminescent detection, so that the biological activity of the GLP-1 is measured.
Experimental materials
96-well cell culture plates (white opaque), DMEM medium (GIBCO), 0.05% trypsin-EDTA (GIBCO), fetal bovine serum (GIBCO), G418, hygromycin B, bright-GloTM Luciferase Assay System kit (Promega), HEK293/CRE-luc/GLP1R cells.
Experimental operation
(1) Cell preparation: the cells are cultured to a state of vigorous growth and in sufficient quantity. The culture medium in the flask was discarded, 3ml of Versene solution was added and washed 1 time with shaking, 2ml of TRYPSIN-EDTA digest was added, the flask was capped and left to stand for 1 minute, then 6ml of assay medium was added to terminate the digestion, centrifugation was performed for 3 minutes at 1000r/min, the supernatant was removed, and cells were resuspended in 5ml of assay medium and counted with a hemocytometer. The cell density was adjusted to a proper range by DMEM assay medium for use.
(2) Sample preparation: derivatives of the different GLP-1 analogs of Table 1 were diluted to 20ng/ml with assay medium, followed by a gradient of 8 dilutions in 96-well plates, and the assay medium was used as a cell blank instead of the sample, with 2 multiplex wells per dilution.
(3) Sample adding and culturing: transferring the prepared control and test solution into 96-well cell culture plate (white plate), adding 50 μl of the prepared cell suspension into each well, and placing at 37deg.C under 5% CO 2 Culturing under the condition for a certain time.
(4) Chemiluminescent detection: substrate was added and 96 well cell culture plates were removed, 100 μl Bright Glo reagent was added to each well and left in the dark for 3min.
(5) Reading: the measurement is carried out by using a chemiluminescent enzyme-labeled spectrometer SpectraMax L, the plate is read within 30 minutes, and the measurement result is recorded.
TABLE 3 results of in vitro Activity experiments of HEK293/CRE-Luc/GLP1R cells
HEK293/CRE-Luc/GLP1R cell pharmacodynamics shows that the in vitro activities of the somalundin, M2, M4, M9, M11, M14, M16 and M17 are equivalent and slightly higher than that of M13 as a whole.
EXAMPLE 8 blood glucose reduction studies of fatty acid modified derivatives of GLP-1 analogs in normal mice
28 healthy CD-1 female mice with the ages of 4 to 6 weeks are selected and divided into 4 groups, and M2, M4, M0 and somalunin (Aib) are respectively injected subcutaneously, wherein the dosages are respectively 0.15mg/kg body weight. The glucose inhibition rate was calculated by feeding 20% glucose to the stomach at intervals of 6 hours, 1 day, 2 days, 3 days, and 4 days, 2g/kg body weight, feeding 6 hours before feeding, taking blood from the tail tip after feeding 0, 0.5, 1, and 2 hours, and measuring the blood glucose level in real time using roche blood glucose test paper, and calculating the blood glucose AUC (area under blood glucose-time curve) within 0 to 120 minutes (table 4).
TABLE 4 comparison of in vivo hypoglycemic Effect in normal mice
P value: comparison with blood sugar before administration
As can be seen from table 4, the hypoglycemic activity of the somalupeptide in the normal mice lasted about 2 days, the hypoglycemic activity of M0 in the normal mice lasted about 3 days, while the hypoglycemic activities of M2 and M4 in the normal mice still showed significant activity on day 4, the sustained hypoglycemic activity was maintained in vivo for significantly longer than that of the somalupeptide or M0, and the hypoglycemic effects of both M2 and M4 were significantly stronger than that of the somalupeptide or M0 at each time point after the 3 rd day of administration.
28 healthy CD-1 female mice with the ages of 4 to 6 weeks are selected and divided into 4 groups, and M4, M5, M7 and M0 are injected subcutaneously, wherein the dosages are respectively 0.15mg/kg body weight. The glucose inhibition rate was calculated by feeding 20% glucose to the stomach at intervals of 6 hours, 1 day, 2 days, 3 days, and 4 days, 2g/kg body weight, feeding 20% glucose, fasted for 6 hours, taking blood from the tail tip after feeding 0, 0.5, 1, and 2 hours, and measuring the blood glucose level in real time using roche blood glucose test paper, and calculating the blood glucose AUC (area under the blood glucose-time curve) within 0 to 120 minutes (table 5).
TABLE 5 comparison of in vivo hypoglycemic Effect in normal mice
From the results shown in tables 4 and 5, the effects of M2 and M4 are superior to those of M0 and the effects of the somalunin, and the effects of M2, M4, M5 and M7 are equivalent without significant difference.
EXAMPLE 9 blood glucose reducing Effect study Using ICR mice
ICR mice OGTT test: 30 ICR mice with 4-6 weeks of age are selected and divided into 6 groups, 5 ICR mice are respectively subcutaneously injected with M0, somalunin, M2, M4, M5 and M7, and the dosages are respectively 0.15mg/kg body weight, and the ICR mice are administered in a single dose. According to the time of 4h, 1d, 2d, 3d, 4d and 5d, the glucose is infused into the stomach for 20% every day, the dosage is 2g/kg body weight, the blood is fasted for 6h before the glucose is infused, and blood is taken from the tail tip after 0, 0.5, 1 and 2 hours after the glucose is infused into the stomach, and blood glucose values are detected in real time by using a Roche blood glucose test paper. Blood was taken from the tail tip, the blood glucose level was measured in real time using a roche blood glucose test paper, the blood glucose AUC (area under the blood glucose-time curve) was calculated for 0 to 120 minutes, and the blood glucose inhibition rate was calculated (table 6).
TABLE 6 comparison of in vivo hypoglycemic effects in ICR mice
As can be seen from the results in table 6, the blood glucose lowering maintenance effect: the hypoglycemic effects of M4, M5, M2 and M7 can be maintained for at least 4 days, which is far superior to that of M0 (maintained for only 3 days) and that of the somalundin (maintained for only 2 days), and the hypoglycemic effects of the somalundin are statistically significant.
EXAMPLE 10 blood glucose reducing efficacy test on type II diabetes db/db mice
50 db/db mice, females, 8-9 weeks old, were equally divided into 10 groups according to pre-dose body weight, fasting Blood Glucose (FBG), 5 animals/group, each injected with vehicle, M2, M4, somalunin, M9, M11, M13, M14, M16 and M17, each administered at a dose of 0.05mg/kg according to 10ml/kg, and the administration time was set to 0h. Mice were fasted for 6-8 hours daily and were tested for fasting blood glucose daily after dosing until the animals of each test group recovered to the end of pre-dosing. The blood glucose level detected before administration was referred to as the basal blood glucose level and was set to 0.
Change in fasting blood glucose (delta: delta) =post-dose blood glucose value-pre-dose basal blood glucose value.
As shown in fig. 1, it can be seen from days 4 and 5 that M9, M13, M14 had a hypoglycemic effect superior to that of somalundin, nor less than M2, while M11, M16 and M17 showed a hypoglycemic effect lower than that of somalundin on day 2.
EXAMPLE 11 hypoglycemic effects of different doses of somalundin, M0 and M4 on type II diabetes db/db mice
35 db/db mice, females, 8-9 weeks old, were equally divided into 7 groups, 5 animals/group, each injected with vehicle, M4 (0.15, 0.015 mg/kg), somalunin (0.15, 0.015 mg/kg) and M0 (0.15, 0.015 mg/kg) in a single subcutaneous injection, based on pre-dose body weight, area under blood glucose curve (G-AUC), and administered at 10 ml/kg. The administration time was set to 0h, and after mice fasted for 7-8h each day, fasting blood glucose and OGTT (oral glucose tolerance measurement) were measured, and then 10% glucose was infused in an amount of 1g/kg body weight, and blood glucose values were measured in real time by taking blood from the tail tips at 0, 0.5, 1, 2h after the sugar load. Blood glucose was measured daily after dosing and prior to fasting, referred to as random blood glucose, until the animals in each test group had recovered to the end of pre-dosing levels. The basal blood glucose level, the random blood glucose level and the area under blood glucose curve (G-AUC) measured before administration were all the bases for measuring the efficacy and were set to 0.
Blood glucose change amount (delta: delta) =post-dose blood glucose value-pre-dose basal blood glucose value;
area change under blood glucose curve (delta: delta) =area under blood glucose curve after administration-area under blood glucose curve before administration.
The results are shown in tables 7, 8 and 9 and figures 2, 3 and 4.
TABLE 7 fasting blood glucose change table for mice of each test group
Note that: "-21h" represents fasting glycemic basal before administration.
TABLE 8 average random blood glucose variation for mice of each test group
Note that: "-5h" represents the random glycemic base 5h prior to dosing.
TABLE 9 area under the blood glucose curve (G-AUC) for mice of each test group
Note that: "-21h" represents the area base under the pre-dosing glycemic curve.
The results in tables 7-9 and FIGS. 2-4 show that:
fasting blood glucose: m4.15 mg/kg dose group was returned to the pre-dose basal blood glucose level at 123h post-dose, and 0.015mg/kg dose group was returned to the pre-dose basal blood glucose level at 99h post-dose; the 0.15mg/kg dose group of somalupeptide was restored to the pre-dose basal blood glucose level 51h after dosing, and the 0.015mg/kg dose group was restored to the pre-dose basal blood glucose level 27h after dosing; the M0.15 mg/kg dose group recovered to the pre-dose basal blood glucose level 75h after dosing, and the 0.015mg/kg dose group recovered to the pre-dose basal blood glucose level 51h after dosing; wherein, the fasting blood glucose reduction value of each detection time point of the 0.015mg/kg dose group of M4 is not lower than that of the 0.15kg/kg dose group of the somalundin or M0.
Random blood glucose: the M4.15 mg/kg dose group was returned to the pre-dose randomized blood glucose base 115h after dosing, and the 0.015mg/kg dose group was returned to the pre-dose randomized blood glucose base 115h after dosing; the 0.15mg/kg dose group of somalupeptide was restored to the pre-dose randomized blood glucose base 67h after dosing, and the 0.015mg/kg dose group was restored to the pre-dose randomized blood glucose base 67h after dosing; the M0.15 mg/kg dose group was returned to the pre-dose randomized blood glucose base 67h after dosing, and the 0.015mg/kg dose group was returned to the pre-dose randomized blood glucose base 67h after dosing; wherein, the inhibition effect of each detection time point of the 0.015mg/kg dose group of M4 on random blood sugar is not lower than that of the 0.15kg/kg dose group of the somalundin or M0.
Area under blood glucose curve (G-AUC): the area base number under the blood glucose curve before the administration is recovered in the M4.15 mg/kg dose group at 99h after the administration, and the area base number under the blood glucose curve before the administration is recovered in the 0.015mg/kg dose group at 99h after the administration; the area base number of the cable-marlutide 0.15mg/kg dose group under the blood glucose curve before the administration is recovered at 51h after the administration, and the area base number of the cable-marlutide 0.015mg/kg dose group under the blood glucose curve before the administration is recovered at 51h after the administration; the area base number under the blood glucose curve before administration is recovered in the M0.15 mg/kg dose group at 51h after administration, and the area base number under the blood glucose curve before administration is recovered in the 0.015mg/kg dose group at 27h after administration; wherein, the area under the blood glucose curve of each detection time point of the 0.015mg/kg dosage group of M4 is not lower than that of the 0.15kg/kg dosage group of the somalundin or M0.
The results in terms of these hypoglycemic effects demonstrate that each group shows a pronounced hypoglycemic effect after a single subcutaneous injection of M4 or somalunin or M0, but the M4 hypoglycemic effect is the best. The hypoglycemic effect of the 0.015mg/kg dose of M4 corresponds to the hypoglycemic effect of the 0.15mg/kg dose of the somalundin or the 0.15mg/kg dose of M0.
EXAMPLE 12 study of stability of M4 and somalunin to enzymatic degradation
Pepsin (3200-4500U/mg protein, from Sigma, cat# P6887), trypsin (about 10000AEE U/mg protein, from Sigma, cat# T8003).
(1) Reaction solution
A: pepsin reaction buffer: three different pH (2.6, 4.0, 7.4) of 20mM citric acid-phosphate buffer solution, and adding 0.005% Tween 20 and 0.001% BSA as pepsin buffer solution.
B: trypsin reaction buffer: three different pH (4.0, 6.8, 8.0) of 20mM citric acid-phosphate buffer solution, and adding 0.005% Tween 20 and 0.001% BSA as pepsin buffer solution.
C: pepsin-containing Simulated Gastric Fluid (SGF): taking 5ml of 0.1M hydrochloric acid, adding 0.019g pepsin to dissolve the hydrochloric acid, thus obtaining the product.
D: simulated Intestinal Fluid (SIF) containing trypsin: taking 0.0684g of monopotassium phosphate, adding 2.5ml of water to dissolve the monopotassium phosphate, adding 0.77ml of 0.2M sodium hydroxide solution and 5ml of water, adding 0.1001g of trypsin to dissolve the monopotassium phosphate, measuring the pH value to be 6.82, and adding water to dilute the solution to 10ml to obtain the finished product.
(2) Sample configuration
M4 and somalunin samples were diluted to 1.33mg/ml with PB buffer pH7.4 to obtain mother liquor as test sample.
(3) Pepsin degradation experiments
Respectively taking a proper amount of mother solution of a test sample, diluting the mother solution to 0.06mg/ml by using pepsin reaction buffer solutions with different pH values, dividing each group of reaction solutions into 1 ml/tube and 7 tubes in total, uniformly mixing, and then placing the mixed solution in a water bath at 37 ℃ for incubation for 30min. Taking out 1 tube without adding SGF as non-enzymatic reaction 0 point (marked as-5 min point), taking out 6 tubes, adding SGF respectively, mixing well, immediately adding 1M NaOH with proper volume into one tube to stop the reaction, taking out the other 5 tubes as 0 point (marked as 0min point) after enzyme addition, continuously placing the other 5 tubes at 37 ℃ for reaction, and taking out a group of 1M NaOH with proper volume respectively at 5min, 10min, 20min, 35min and 50min to stop the reaction. All experimental groups were given tubes to ensure consistent total volume after termination of the reaction.
(4) Trypsin degradation experiment
And respectively taking a proper amount of sample mother solution, diluting the sample mother solution to 0.06mg/ml by using trypsin reaction buffer solutions with different pH values, dividing each group of reaction solutions into 1 ml/tube and 7 tubes in total, uniformly mixing, and then placing the mixed solution in a water bath at 37 ℃ for incubation for 30min. Taking out 1 tube, adding no SIF as non-enzyme reaction 0 point (marked as-5 min point), taking out 6 tubes, adding SIF respectively, mixing, adding 6M HCl with proper volume immediately into one tube to stop reaction, taking out the other 5 tubes as enzyme-added 0 point (marked as 0min point), continuously placing the other 5 tubes at 37 ℃ to react, and taking out a group of 6M HCl with proper volume respectively at 5min, 10min, 20min, 35min and 50min to stop reaction. All experimental groups were given tubes to ensure consistent total volume after termination of the reaction.
The sample of the enzymatic degradation experiment is subjected to HPLC detection, the main peak area of the sample without the enzyme reaction at the 0 point (marked as the point of-5 min) is taken as the basic peak area, and the residual percentages of the main peak areas at different time points obtained after the enzyme addition are calculated.
Pepsin degradation experimental data (n=3) showed (fig. 5) that the degradation rates of M4 and somalundum molecules were comparable under acidic conditions (pH 2.6) due to the highest pepsin activity at this pH; at neutral pH7.4, both molecules are essentially undegraded, with minimal gastric protein activity; at pH4.0, the degradation rate of the somalundum is obviously higher than that of M4, the t1/2 of the former is about 10min, and the t1/2 of the latter is about 45min, which shows that the resistance of the M4 to pepsin degradation is obviously better than that of the somalundum.
The trypsin degradation experimental data (n=4) show (fig. 6) that the rates of degradation are substantially consistent at both pH6.8 and 8.0 conditions, since this pH range is the highest activity range of trypsin; m4 and somalunin also showed resistance to trypsin degradation at pH4.0, and there was no substantial difference between the two.
Sequence listing
<110> Hangzhou first reaches biotechnology Co., ltd
<120> acylated GLP-1 derivatives
<130> PC00515D4
<160> 38
<170> PatentIn version 3.5
<210> 1
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 1
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
10 15 20
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
25 30 35
<210> 2
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 2
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 3
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 3
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctaaa 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 4
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 4
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Lys Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 5
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 5
cacgttgaag gtaccttcac ctctgacgtt tcttctaaac tggaagaaca ggctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 6
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 6
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 7
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 7
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 8
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 8
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Lys Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 9
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 9
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
aaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 10
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 10
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 11
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 11
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93
<210> 12
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 12
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
25 30 35
<210> 13
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 13
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatcg cttggctggt taaaggtcgt ggt 93
<210> 14
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 14
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly
25 30 35
<210> 15
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 15
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatcg cttggctggt tcgtggtaaa ggt 93
<210> 16
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 16
His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Lys
25 30 35
<210> 17
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 17
cacgttgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt aaa 93
<210> 18
<211> 421
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 18
attttgttta actttaataa ggagatatac catgcatcac catcatcacc acgctaaacc 60
ggaagttaaa ccggaagtta aaccggaaac ccacatcaac ctgaaagttt ctgacggttc 120
ttctgaaatc ttcttcaaaa tcaaaaaaac caccccgctg cgtcgtctga tggaagcttt 180
cgctaaacgt cagggtaaag aaatggactc tctgcgtttc ctgtacgacg gtatccgtat 240
ccaggctgac cagaccccgg aagacctgga catggaagac aacgacatca tcgaagctca 300
ccgtgaacag atcggtggtc acgttgaagg taccttcacc tctgacgttt cttcttacct 360
ggaagaaaaa gctgctcgtg aattcatcgc ttggctggtt cgtggtcgtg gttaataata 420
a 421
<210> 19
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 19
His Thr Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 20
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 20
cacaccgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 21
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 21
His Ile Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 22
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 22
cacattgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 23
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 23
His Leu Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 24
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 24
cacctggaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 25
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 25
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 26
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 26
caccgcgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 27
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 27
His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Lys Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 28
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 28
cacagcgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaaa agctgctcgt 60
gaattcatcg cttggctggt tcgtggtcgt ggt 93
<210> 29
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 29
His Thr Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 30
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 30
cacaccgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93
<210> 31
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 31
His Ile Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 32
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 32
cacattgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93
<210> 33
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 33
His Leu Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 34
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 34
cacctggaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93
<210> 35
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 35
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 36
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 36
cacggcgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93
<210> 37
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 37
His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
10 15 20
Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
25 30 35
<210> 38
<211> 93
<212> DNA
<213> artificial sequence
<220>
<223> Artificial
<400> 38
cacagcgaag gtaccttcac ctctgacgtt tcttcttacc tggaagaaca ggctgctcgt 60
gaattcatca aatggctggt tcgtggtcgt ggt 93

Claims (16)

1. A derivative of a GLP-1 (7-37) analogue or a pharmaceutically acceptable salt thereof, wherein the GLP-1 (7-37) analogue has an amino acid sequence of the formula:
HX 8 EGTFTSDVSSX 19 LEEX 23 AARX 27 FIX 30 WLVX 34 GX 36 X 37，
wherein X is 8 Is V, X 19 Is Y, X 23 Q or K, X 27 Is E, X 30 Is A or K, X 34 R or K, X 36 Is R, X 37 In the presence of a compound which is G or K,
provided that at X 23 、X 30 、X 34 Or X 37 Only one of which is a K residue,
the derivative means that the K residue of the GLP-1 (7-37) analog is linked to an extension moiety, wherein the extension moiety is selected from the group consisting of:
HOOC(CH 2 ) 16 CO-、HOOC(CH 2 ) 17 CO-、HOOC(CH 2 ) 18 CO-、HOOC(CH 2 ) 19 CO-、HOOC(CH 2 ) 20 CO-、HOOC(CH 2 ) 21 CO-and HOOC (CH) 2 ) 22 CO-；
The extension is linked to the K residue of a GLP-1 (7-37) analog by a linker;
wherein the joint is:wherein m is 0, 1, 2 or 3; n is 1.
2. The derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein the extension is HOOC (CH 2 ) 16 CO-；
The joint is as follows:wherein m is 1 and n is 1.
3. The derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein X 23 K is the number.
4. The derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein X 30 K is the number.
5. The derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein X 34 K is the number.
6. The derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein X 37 K is the number.
7. A process for preparing the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, comprising:
(1) Mixing a solution in which a GLP-1 analogue of any one of the preceding claims is dissolved with a solution in which an extension of any one of the preceding claims is dissolved;
(2) Regulating pH to 4-5, stopping reaction, standing until precipitation is generated, and collecting the precipitate; and
(3) TFA was added to the precipitate and the reaction was stopped by adjusting the pH to 7.5-8.5.
8. The method of claim 7, further comprising adding triethylamine to the solution in which the GLP-1 analogue is dissolved prior to mixing with the solution in which the extension of any one of the preceding claims is dissolved.
9. The method of claim 7 or 8, wherein the solution of the extension of any of the preceding claims is acetonitrile-soluble.
10. A pharmaceutical composition comprising the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
11. Use of a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of diabetes or diabetic complications, wherein the diabetes is selected from type I diabetes and type II diabetes;
the diabetic complication is diabetic nephropathy.
12. Use of a derivative according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for lowering blood glucose, increasing glucose tolerance, decreasing islet β -cell apoptosis, increasing islet β -cell function, increasing islet β -cell number and/or restoring glucose sensitivity to islet β -cells.
13. The use of claim 12, wherein said lowering blood glucose comprises lowering fasting blood glucose and/or postprandial blood glucose.
14. An article of manufacture comprising a container having the pharmaceutical composition of claim 10 contained therein and a package insert, wherein the package insert carries instructions for use of the pharmaceutical composition.
15. The article of manufacture of claim 14, further comprising a container containing one or more other medicaments.
16. The article of manufacture of claim 15, wherein the one or more additional drugs are additional drugs for treating diabetes or diabetic complications.
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