CN113024659A - Acylated GLP-1 derivatives - Google Patents

Acylated GLP-1 derivatives Download PDF

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CN113024659A
CN113024659A CN202110218043.7A CN202110218043A CN113024659A CN 113024659 A CN113024659 A CN 113024659A CN 202110218043 A CN202110218043 A CN 202110218043A CN 113024659 A CN113024659 A CN 113024659A
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CN113024659B (en
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许铮
李峰
宋瑞
郭万军
潘海
林兆生
邓建慧
冯静
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Hangzhou Xianweida Biotechnology Co ltd
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Sciwind Biosciences Co Ltd
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Abstract

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

Description

Acylated GLP-1 derivatives
The present application is a divisional application of the chinese patent application having the invention name "acylated GLP-1 derivative" with application number 201910317953.3.
Technical Field
The invention belongs to the technical field of polypeptide. In particular, the invention relates to fatty acid modified derivatives of GLP-1(7-37) polypeptide analogs. In addition, the present invention relates to a process for producing the peptide derivative, a medicament containing the peptide derivative, and use in the production of the medicament.
Background
Diabetes is a sugar metabolism disorder disease caused by a plurality of factors such as heredity and environment, and is a third serious disease which threatens human health and life safety after tumors and cardiovascular and cerebrovascular diseases. Diabetes does not necessarily cause harm per se, but blood sugar is increased for a long time, large blood vessels and micro blood vessels are damaged and endanger the heart, the brain, the kidney, peripheral nerves, eyes, feet and the like, and according to the statistics of the world health organization, the complications of diabetes can be more than 100, and the diabetes is one of the most known complications at present. More than half of the deaths due to diabetes are caused by cardiovascular and cerebrovascular diseases, and 10% of the deaths are caused by nephropathy. Because of the diabetes, amputation is 10-20 times of that of non-diabetes. Therefore, the treatment of diabetes and the prevention of its complications are vital social problems.
Diabetes can be classified into several types due to different mechanisms. The vast majority of them belong to type II diabetes (about 90%), mainly due to excess weight and lack of physical activity. In type II diabetic patients, there are abnormalities in both insulin resistance and insulin secretion deficiency, and islet beta cell apoptosis often occurs in the middle and late stages of onset. At present, the action mechanism of oral hypoglycemic drugs used clinically is mostly to enhance insulin sensitivity or promote insulin secretion to stabilize blood sugar, and the problem of beta cell apoptosis cannot be solved. Glucagon-like peptide-1 (GLP-1) and analog drugs thereof are important in the research of treating type II diabetes because of the effects of slowing down beta cell apoptosis, promoting regeneration and promoting differentiation and proliferation of islet beta cells.
In 1983, BELL et al found glucagon-like peptide-1 (glucagon-like peptide-1), GLP-1(BELL G I, SANCHEZ-PESCADOR, LAYBOURN P J, et al, Exon duration and conversion in the human preprogugagon gene [ J ]. Nature, 1983, 304(5924): 368-) -371) when analyzing the gene sequence of pro-glucagon (PG). The PG gene sequence consists of 6 exons and 5 introns, containing 3 major domains: glucagon (33-61), GLP-1 (72-108) and GLP-2 (126-158). The mRNA of PG is expressed in pancreatic A cells, intestinal L cells and brain, and specific translation modification is carried out in these tissue cells, and different final products are finally formed.
GLP has two subtypes, GLP-1 analogs and GLP-2 analogs, which are nearly half identical in amino acid sequence to glucagon and are also about 35% homologous. GLP-1 analogues are polypeptide hormones secreted by Langerhans' scells in the distal jejunum, ileum and colon, and have glucose-dependent functions of promoting insulin secretion and biosynthesis, inhibiting glucagon secretion, and inhibiting gastric emptying. GLP-2 analogs are synthesized in the intestinal tissue and the brainstem and hypothalamic neuronal cells of the central nervous system, mainly promoting the growth of the normal small intestine and the repair of the damage of the intestinal mucosa (Dugang, Long, Xun human glucagon-like peptide 1 and its receptor agonist research progress [ J ] Tianjin medicine 2012, 40(2): 181-.
GLP-1 is an endogenous hormone that promotes insulin secretion, is primarily secreted by 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 ((s))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 (Gln) -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 N end, the DDP-IV mainly plays a role of peptide chain terminal hydrolase, and the 8 th position is alanine or proline, the enzyme can play a role of degradation, so that GLP-1 is rapidly inactivated (AERTGEERTS K, YE S, TENNANT M G, J.Crystal structure of human dipeptidyl peptidase IV in complex with a dipeptide protease recovery variants on substrate specificity and tetrahydrofolate intervention [ J.]Protein Sci, 2004, 13(2): 412-. SARRAUSTE DE MENTHIERE et al create GLP-1 model, observe the change of affinity and intrinsic activity of GLP-1 analogue to receptor after amino acid substitution, histidine at position 7 is determinant of affinity and intrinsic activity, and the upper aromatic ring is smaller than tryptophan and has no polar substituent; the alanine side chain at the 8 th position has a polar group which can influence the activity of the GLP-1, the volume of the side chain cannot be too large, and the activity can be reduced when the side chain exceeds a certain limit; when the glutamic acid at the 9 th position is replaced by certain amino acids, such as acidic, polar and hydrophobic amino acids, the activity is not changed, and when the glutamic acid is replaced by basic amino acids, the activity is reduced and even inactivated; GLP-1 and a receptor are in a binding state, if amino acid residues in the GLP-1 are in ionic bond action, the GLP-1 and the receptor take place between amino acids at positions 7-15 to form a ring structure, Ala8-Glu9-Gly10-Thr11 form beta turns, so that 3 aromatic nuclei such as histidine at position 7, phenylalanine at position 12 and tyrosine at position 19 interact to correspond to aromatic cluster hydrophobic pockets existing on the receptor, and the aromatic nuclei are supposed to play a role in activating the receptor; the glycine at position 22 is a flexible amino acid, which acts as a flexible linker and maintains a helical coil. Disruption of glycine will cluster all aromatic amino acids, butIs reduced in affinity for the receptor 1/40(SARAUSTE DE MENTHIEREC, CHAVANIEUA, GRASSYG, et al. structural requirements of the N-terminal region of GLP-1- [ 7-37)]-NH2 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) NH2And are in the form of molecules. It is generally believed that the latter two 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 from the N-terminal is required, so that active GLP-1(7-37) is obtained. GLP-1(7-37) is present in vivo for a short time and is degraded quickly. Thus, various studies on GLP-1 analogs having resistance to DPP IV have been conducted, for example, U.S. Pat. No. 5545618 describes modification of the N-terminus with an alkyl group or an acyl group, and Gallwitz et al describes N-methylation or a-methylation of His at the 7-position, or substitution of the entire His with imidazole to increase resistance to DPP-IV and maintain physiological activity.
In addition to these modifications, the GLP-1 analog exenatide-4 (exendin-4) purified from the salivary gland of Hiragana lizard (U.S. Pat. No. 5424686) has resistance to DPP IV and 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 applicable to a method of increasing DPP IV resistance, physiological activity cannot be sufficiently maintained, and in the case of using commercially available exenatide-4 (exenatide), it requires twice-a-day injection to a patient, which is still painful to the patient.
These insulinotropic peptides have a small molecular weight and are therefore rapidly excreted from the kidney. There are scientists who use chemical methods to add highly soluble polymers such as polyethylene glycol to the surface of peptides to inhibit their loss in the kidney. For example, U.S. Pat. No. 692464 describes that PEG is conjugated to lysine residue of Exenatide-4 to increase its in vivo retention time, however, this method increases the retention time of peptide drug in vivo, but at the same time, the concentration of the peptide drug is significantly reduced and the reactivity to peptide is also reduced as the molecular weight is increased.
In addition, there are a number of other methods for modifying the structure of glucagon-like peptide-1 compounds in an attempt to prolong their duration of action. For example, WO96/29342 discloses peptide hormone derivatives modified by introducing lipophilic substitutions at the C-terminal amino acid residue or at the N-terminal amino acid residue of the parent peptide hormone. WO98/08871 discloses GLP-1 derivatives (liraglutides) wherein at least one amino acid residue of the 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 double acylated GLP-1 analogues. WO 00/69911 discloses activated insulinotropic peptides for injection, which are believed to react with blood components in the patient to form conjugates, prolonging the duration of action in vivo.
WO2006/097537 discloses another acylated GLP-1 analogue (semaglutide) having a longer half-life compared to the acylated GLP-1(liraglutide) of WO98/08871, by mutating the amino acid at position 8 to an unnatural amino acid.
WO02/046227 discloses that fusion proteins are prepared by combining GLP-1, exenatide-4 or an analogue thereof with human serum albumin or an immunoglobulin region (Fc) using genetic recombination techniques, which can solve problems such as low pegylation yield and non-specificity, but their effects of increasing half-life in blood are still not as pronounced as expected, and the expected effects are not achieved even as much as the somalutide in terms of combined hypoglycemic effects. In order to maximize the effect of increasing the half-life in blood, various types of peptide connectors have been tried, but this method has a problem in that immune response may be caused.
CN107033234A discloses fatty acid modified conjugates of GLP-1 analogs with the fatty acid modification site at Lys26In the former animal experiments, the glucose-reducing effect of the GLP-1 analogue is better than that of the somaglutide, and the in-vivo acting time of the GLP-1 analogue can be prolonged properly, but the prolonged time is not ideal.
GLP-1 drugs which are currently approved in the market mainly comprise Exenatide-4 (Exenatide-4) separated from lizard saliva and human GLP-1 analogues modified by fatty acid, antibody Fc segment or serum albumin. Exenatide-4 has a too short half-life of only 2-4 hours, requiring at least two injections a day. Fatty acid modified liraglutide from noh et al is most effective in reducing glycation of hemoglobin with few side effects, but has a disadvantage in that it has an in vivo half-life of only 13 hours and requires daily administration. In order to further prolong the half-life in vivo and reduce the administration frequency, amino acid sequence mutants and long-acting GLP-1 analogues modified with FC, fatty acids, albumin, or the like have been developed in succession in recent years. Such as dulaglutide from lilac and somaglutide from norand. The half-life of these long-acting GLP-1 analogs in humans can be extended to varying degrees, up to a dosing frequency of once weekly dosing.
Through long-term research, the inventor of the application develops a novel GLP-1 analogue and a derivative thereof, and under the same experimental conditions, compared with the prior acknowledged best medicament, namely the somaglutide, the in vitro activity of the GLP-1 analogue is equivalent to that of the somaglutide; on common mouse and diabetic mouse models, the duration of the in vivo hypoglycemic activity can be improved by about 1 time, which means that the administration frequency of at least weekly interval administration, even every two weeks or longer interval administration can be realized in human body. And when the dosage is the dosage of the somaglutide 1/10, the hypoglycemic effect is not lower than that of the somaglutide, so that the application prospect is better.
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.
Specifically, 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 an amino acid sequence of the formula:
HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37
wherein X8Selected from V, T, I, L, G or S, X19Is Y or K, X23Is Q or K, X27Is E or K, X30Is A or K, X34Is R or K, X36Is R or K, X37Is a group of G or K,
provided that at X19、X23、X27、X30、X34、X36Or X37Only one of which is a K residue,
the derivative comprises an extension attached to the K residue, wherein the extension is
Figure BDA0002954690860000061
Wherein x is an integer from 4 to 38.
Among them, the extension portion is preferably: HOOC (CH)2)14CO-、HOOC(CH2)15CO-、HOOC(CH2)16CO-、HOOC(CH2)17CO-、HOOC(CH2)18CO-、HOOC(CH2)19CO-、HOOC(CH2)20CO-、HOOC(CH2)21CO-and HOOC (CH)2)22CO-, more preferably HOOC (CH)2)16CO-。
In a preferred embodiment, the extending moiety of the derivative of a GLP-1 analog according to the invention, or a pharmaceutically acceptable salt thereof, is attached to the K residue of GLP-1 via a linker. The linker may be of the structure:
Figure BDA0002954690860000062
Figure BDA0002954690860000071
Figure BDA0002954690860000072
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 linker is:
Figure BDA0002954690860000073
Figure BDA0002954690860000081
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:
Figure BDA0002954690860000082
wherein m is 1 and n is 1 or 2.
The invention also relates to GLP-1(7-37) analogs comprising
HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37A sequence comprising a mutation at one or more of the following positions:
8 th bit, 19 th bit, 23 th bit, 27 th bit, 30 th bit, 34 th bit, 36 th bit and 37 th bit. 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, and the amino acid residue at position 37 is G or K, with the proviso that only one of positions 19, 23, 27, 30, 34, 36 or 37 is a K residue.
The in vitro binding activity of the derivative of the GLP-1 analogue after acylation shows that the binding affinity with a GLP-1R receptor is larger than that of the somaglutide or M0 (the 26 position is Lys, disclosed in CN 107033234A). In-vivo hypoglycemic experiments also prove that compared with the same acylated GLP-1 product, namely the somaglutide, the derivative of the GLP-1 analogue after acylation can obtain longer hypoglycemic activity duration time in a normal mouse; in diabetic mice, the derivative has the effects of reducing blood sugar and improving sugar tolerance which are obviously better than those of the somaglutide, and the blood sugar reducing effect is not lower than that of the somaglutide or M0 when the dosage is only 1/10 dosage of the somaglutide 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 commercially available somaglutide.
Specifically, the present 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:
HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37，
wherein X8Selected from V, T, I, L, G or S, X19Is Y or K, X23Is Q or K, X27Is E or K, X30Is A or K, X34Is R or K, X36Is R or K, X37Is a group of G or K,
provided that at X19、X23、X27、X30、X34、X36Or X37Only one of which is a K residue,
said derivative comprising an extension attached to the K residue of said GLP-1(7-37) analog, wherein said extension is
Figure BDA0002954690860000091
Figure BDA0002954690860000092
Wherein x is an integer from 4 to 38.
2. The derivative of claim 1, or a pharmaceutically acceptable salt thereof, wherein the protracting moiety is selected from the group consisting of:
HOOC(CH2)14CO-、HOOC(CH2)15CO-、HOOC(CH2)16CO-、HOOC(CH2)17CO-、HOOC(CH2)18CO-、HOOC(CH2)19CO-、HOOC(CH2)20CO-、HOOC(CH2)21CO-and HOOC (CH)2)22CO-。
3. The derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the protracting moiety is attached to the K residue of the GLP-1(7-37) analog via a linker.
4. The derivative of claim 3, or a pharmaceutically acceptable salt thereof, wherein the linker is:
Figure BDA0002954690860000093
Figure BDA0002954690860000101
Figure BDA0002954690860000102
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 linker is:
Figure BDA0002954690860000103
Figure BDA0002954690860000111
wherein m is 1 or 2; n is 1 or 2; p is any integer from 1 to 5.
5. The derivative of claim 4, or a pharmaceutically acceptable salt thereof, wherein the linker is:
Figure BDA0002954690860000112
wherein m is 1 and n is 1 or 2. 6. The derivative of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which is any one of the derivatives selected from the group consisting of: n-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M2), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M4), N-epsilon34- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26Lys34GLP-1(7-37)) peptide (M5), N-epsilon37- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26,34Lys37GLP-1(7-37)) peptide (M7), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys23Arg26,34-GLP-1(7-37)) peptide (M9) \ N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Thr8Glu22Lys30Arg26,34-GLP-1(7-37)) peptide (M13) \ N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys30Arg26,34-GLP-1(7-37)) peptides(M14)。
7. A process for preparing a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, comprising:
(1) mixing a solution in which a GLP-1 analogue as defined in any of the preceding claims is dissolved with a solution in which an extension as defined in any of the preceding claims is dissolved;
(2) adjusting the pH value to 4-5 to terminate the reaction, standing until a precipitate is generated, and taking 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 protracting moiety of any of the preceding claims is dissolved.
9. The process 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 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
11. Use of the derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for the preparation 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 to 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for reducing blood glucose, increasing glucose tolerance, reducing apoptosis of islet β -cells, enhancing function of islet β -cells, increasing the number of islet β -cells, and/or restoring glucose sensitivity to islet β -cells.
14. The use of claim 13, wherein the lowering of blood glucose comprises lowering fasting blood glucose and/or postprandial blood glucose.
15. A method for the prophylaxis and/or treatment of diabetes (including type I diabetes and type II diabetes) or diabetic complications, comprising administering to a subject a prophylactically or therapeutically effective amount of a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.
16. The method of claim 15, wherein the diabetic complication is diabetic nephropathy.
17. A method of lowering 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 the lowering of blood glucose comprises lowering fasting blood glucose and/or postprandial blood glucose.
19. A GLP-1(7-37) analog comprising a polypeptide consisting of the amino acid sequence:
HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37
wherein X8Selected from V, T, I, L, G or S, X19Is Y or K, X23Is Q or K, X27Is E or K, X30Is A or K, X34Is R or K, X36Is R or K, X37Is G or K, and, at X19、X23、X26、X27、X30、X34、X36Or X37Only one of which is K.
20. A pharmaceutical composition comprising the analog of claim 19.
21. Use of an analogue according to claim 19 for the manufacture of a medicament for the prevention or treatment of diabetes mellitus, diabetic complications.
22. An article of manufacture comprising a container having the pharmaceutical composition of claim 10 or claim 20 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 holding one or more additional drugs.
24. The article of manufacture of claim 23, wherein the one or more additional agents are additional agents for treating diabetes or diabetic complications.
"fasting glucose" refers to the blood glucose value measured in a subject (e.g., a human) on fasting, e.g., fasting overnight, fasting (without any food, except drinking water) for at least 6 hours, e.g., 6-8 hours, 8-10 hours.
"postprandial blood glucose" refers to blood glucose levels measured after a meal, for example, blood glucose levels 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) analog comprising expressing a DNA sequence encoding the polypeptide in a host cell under conditions permitting expression of the peptide and recovering the peptide produced.
The medium used to culture the cells can be any conventional medium used to culture the host cells, such as minimal medium or complex medium containing suitable additives. Suitable media can be obtained commercially or prepared according to published procedures. The polypeptide produced by the host cell can then be recovered from the culture medium by conventional methods, for example, by precipitating the protein component of the supernatant or filtrate with a salt such as ammonium sulfate, and further purified by various chromatographic methods such as, for example, exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the kind of the desired peptide.
The coding DNA sequence described above 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 which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments of the DNA required for transcription, such as a promoter. Examples of promoters suitable for directing transcription of DNA encoding a peptide of the invention in a variety of host cells are well known in the art, see for example Sambrook, J, Fritsch, EF and maniotis, T, molecular cloning: a guide to the experimental work, Cold Spring Harbor Laboratory Press, New York, 1989.
The vector may also contain a selectable marker, e.g., a gene the gene product of which complements a defect in the host cell or which confers resistance to a drug, e.g., ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin, streptomycin, or methotrexate.
To introduce the expressed peptides of 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 secretory signal sequence is linked in the correct reading frame to the DNA sequence encoding the peptide. The secretion signal sequence is usually located 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be one normally linked to the peptide, or may be derived from a gene encoding another secretory protein.
Methods for ligating the DNA sequence encoding the peptide of the present invention, the promoter and optionally the terminator and/or secretion signal peptide sequence, respectively, and inserting them into a suitable vector containing information necessary for replication are known to those 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 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: e.coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
The invention relates to a medicament or a pharmaceutical composition containing the GLP-1(7-37) analogue, and also relates to application of the analogue in preparing medicaments, such as 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 present invention also relates to a method for preventing or treating diabetes (e.g., type I and type II diabetes), diabetic complications (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic eye disease, diabetic nephropathy, diabetic heart disease), lowering blood glucose (e.g., fasting blood glucose and postprandial blood glucose), or increasing glucose tolerance in a subject by administering to the subject the above GLP-1(7-37) analog or a derivative of the above GLP-1(7-37) analog. In another aspect, the present invention also relates to the use of the above GLP-1(7-37) analog or a derivative of the above GLP-1(7-37) analog for the preparation of a medicament for the prevention or treatment of diabetes (e.g., type I and type II diabetes), diabetic complications (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic eye 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 the above GLP-1(7-37) analog.
The invention also relates to a pharmaceutical composition, a preparation or a kit comprising the derivative of the GLP-1(7-37) analogue.
The pharmaceutical composition comprises an active ingredient GLP-1(7-37) analogue or a derivative of the GLP-1(7-37) analogue or a salt thereof, and also comprises pharmaceutically acceptable auxiliary materials. Pharmaceutically acceptable adjuvants, such as nontoxic fillers, stabilizers, diluents, carriers, solvents or other formulation adjuvants, 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, and the like; 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, etc.; 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 for reducing islet beta-cell apoptosis, enhancing islet beta-cell function, increasing islet beta-cell number and/or restoring islet beta-cell glucose sensitivity, comprising administering to a subject in need thereof an effective amount of the above-described analog, derivative or medicament, pharmaceutical composition.
The invention also relates to the application of the analogue, the derivative or the medicine and the pharmaceutical composition in preparing medicines for reducing the apoptosis of the islet beta-cells, enhancing the function of the islet beta-cells, increasing the number of the islet beta-cells and/or restoring the glucose sensitivity of the islet beta-cells.
In the present invention, GLP-1(7-37) polypeptides, GLP-1(7-37) polypeptide analogs, GLP-1(7-37) analogs can be used interchangeably and are meant to comprise the amino acid sequence: HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37Wherein X is8Selected from V, T, I, L, G or S, X19Is Y or K, X23Is Q or K, X27Is E or K, X30Is A or K, X34Is R or K, X36Is R or K, X37Is G or K. The GLP-1(7-37) polypeptide analog is attached to the protracting moiety to form a derivative of the GLP-1(7-37) polypeptide analog. In particular, the present invention relates to acylated derivatives of GLP-1(7-37) analogs. The acylated derivative not only has remarkable therapeutic effect, but also has the in vivo activity duration which can be improved by about 1 time compared with the prior acknowledged best medicament of the somaglutide, which means that the administration frequency of the medicament at least every week interval, even every two weeks or longer intervals can be realized in human body.
The derivative of GLP-1(7-37) analogue, the acylated derivative of GLP-1(7-37) analogue, the GLP-1(7-37) derivative and the GLP-1 derivative of the invention can be used interchangeably.
In another aspect, the present invention also relates to a process for preparing the above derivative or a pharmaceutically acceptable salt thereof, comprising:
(1) mixing a solution in which the above GLP-1 analog is dissolved with a solution in which an elongation (e.g., fatty acid) is dissolved;
(2) adjusting the pH value to 4-5 to terminate the reaction, standing until a precipitate is generated, and taking 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 a solution in which acetonitrile is dissolved.
An exemplary preparation method of the invention includes (1) providing a GLP-1(7-37) analog solution, adjusting the pH to 9-12;
(2) adding triethylamine into the solution obtained in the step (1);
(3) weighing fatty acid with the following structure which is not less than 2 times (molar ratio) of the GLP-1 analogue, preferably not less than 3 times of the GLP-1 analogue, and dissolving the fatty acid in acetonitrile;
Figure BDA0002954690860000161
(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 one hour;
(5) adjusting the pH value to 4-5 to terminate the reaction, standing at low temperature for acid precipitation, and collecting the precipitate;
(6) adding TFA into the acid precipitate sample obtained in step (5) to a final concentration of 5-15mg/ml polypeptide, standing for 0.5-2 hr, adding alkaline solution such as NaOH dropwise into the reaction solution, and adjusting pH to 7.5-8.5 to terminate the reaction;
(7) separating and purifying the obtained product.
The present invention relates to formulations of pharmaceutical compositions comprising derivatives of GLP-1(7-37) analogs or pharmaceutically acceptable salts thereof. In some embodiments, the derivative comprising a GLP-1(7-37) analog of the present invention or a pharmaceutically acceptable salt thereof is present at a concentration of 0.1mg/ml to 25mg/ml, preferably 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 a preferred embodiment, the pharmaceutical composition may further comprise a buffer system, preservatives, surface tension agents, chelating agents, stabilizers and surfactants. In some embodiments, the medicaments or formulations of the present invention are aqueous medicaments or formulations, for example, they may be in the form of solutions or suspensions. In a particular embodiment of the invention, the drug or formulation is a stable aqueous solution. In other embodiments of the invention, the medicament or formulation is a lyophilized formulation to which a solvent and/or diluent is added prior to use.
The invention also relates to a kit or kit comprising the pharmaceutical composition, preparation, medicament. In addition to the above-mentioned drugs or formulations, the kit or kit may further comprise other drugs, drug compounds or compositions that may be used in combination with the pharmaceutical composition, formulation or medicament, for example, the other drugs, drug compounds or compositions may be selected from antidiabetic drugs, drugs for treating and/or preventing complications caused by or associated with diabetes. Examples of such drugs include: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in the stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, NPY antagonists, PYY agonists, PYY2 agonists, PYY4 agonists, TNF agonists, corticotropin releasing factor agonists, 5HT, bombesin agonists, ganglioside antagonists, growth hormones, thyroid stimulating hormone releasing hormone agonists, TR β agonists; histamine H3 antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogs, and the like. In some embodiments, the pharmaceutical composition, formulation, medicament and other medicament, pharmaceutical compound or composition described herein are placed in separate containers.
The present invention also relates to a method for preventing or treating diabetes (e.g. type I and type II diabetes), diabetic complications (e.g. diabetic vasculopathy, diabetic neuropathy, diabetic eye 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 analogue, derivative or medicament, pharmaceutical composition as described above, wherein the analogue, derivative or medicament, pharmaceutical composition is used in combination with a further medicament, pharmaceutical compound or composition, e.g. the further medicament, pharmaceutical compound or composition may be selected from antidiabetic medicaments, medicaments for treating and/or preventing complications caused by or associated with diabetes. Examples of such drugs include: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in the stimulation of 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, ganglioside antagonists, growth hormone, thyroid stimulating hormone releasing hormone agonists, TR β agonists; histamine H3 antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogs, and the like. In a preferred embodiment, the diabetes is type 2 diabetes or diabetic nephropathy.
The "diabetic complications" refers to damages or dysfunctional diseases of other organs or tissues of the body caused by the poor blood sugar control in the diabetes process, wherein the damages or dysfunctional diseases comprise damages or dysfunctional diseases of the liver, the kidney, the heart, the retina and the nervous system. Complications of diabetes can be divided into five areas: 1. cardiovascular disease: including microangiopathy, cardiomyopathy and autonomic neuropathy of the heart and great vessels, leading to death of diabetic patients. 2. Cerebrovascular lesion: it refers to the disease of large blood vessels and microangiopathy in the cranium caused by diabetes, and is mainly manifested by cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, and brain atrophy. 3. Renal vascular disease: the main aspect is diabetic nephropathy, which is one of the most important complications of diabetic patients. 4. Lower limb arterial lesions: diabetic foot is mainly manifested. 5. Ocular fundus microvascular lesions: mainly manifested as diabetic retinopathy.
The invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of protection of the present patent, the features disclosed in the foregoing description and in the following examples (individually and in any combination thereof), may be material for realising the invention in substantially different forms, in any combination thereof. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced 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 somaglutide on fasting plasma glucose in diabetic mice.
Figure 3 shows the effect of different doses of M0, M4 and somaglutide on random blood glucose in diabetic mice.
Figure 4 shows the effect of different doses of M0, M4, and somaglutide on the area under the blood glucose curve of diabetic mice.
Fig. 5 shows a trend graph of M4 and the somaglutide molecule against pepsin degradation.
Figure 6 shows a trend graph of M4 and the sumatriptan molecule against trypsin degradation.
Examples
The invention will be described herein below by means of specific examples. Unless otherwise specified, the method can be performed according to the protocols listed in the protocols of molecular cloning, cell assay, and CFDA, which are well known to those skilled in the art. Wherein, the used reagent raw materials are all commercial products and can be purchased and obtained through public channels.
EXAMPLE 1 construction of GLP-1 analog expression plasmid
Construction of Val8Glu22Lys23Arg26,34DNA of GLP-1(7-37)
Coupling the 6-His tag, SUMO tag and Val8Glu22Lys23Arg26,34GLP-1(7-37) encoding gene sequence (SEQ ID NO:7) are fused in series in turn, and a gene fragment (SEQ ID NO: 18) is obtained using a chemical synthesis method. The fragment is inserted into prokaryotic expression plasmid pET-24 through BamHI and XhoI sites(+) and sequencing for verification. The resulting expression plasmid, designated pET-24(+) -His-SUMO-Val, was used for the transformation assay8Glu22Lys23Arg26,34-GLP-1(7-37)。
Constructing Val in sequence according to the above method8Glu22Lys26Arg34GLP-1(7-37) (coding gene is SEQ ID NO: 3), Val8Glu22Lys30Arg26,34GLP-1(7-37) (coding gene is SEQ ID NO:11), Val8Glu22Lys19Arg26,34GLP-1(7-37) (coding gene is SEQ ID NO:5), Val8Glu22Lys27Arg26,34GLP-1(7-37) (coding gene is SEQ ID NO:9), Val8Glu22Lys34Arg26GLP-1(7-37) (coding gene is SEQ ID NO:13), Val8Glu22Arg26,34Lys36GLP-1(7-37) (coding gene is SEQ ID NO:15), Val8Glu22Arg26,34Lys37GLP-1(7-37) (coding gene is SEQ ID NO:17), Thr8Glu22Lys23Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:20), Ile8Glu22Lys23Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:22), Leu8Glu22Lys23Arg26,34GLP-1(7-37) (coding gene is SEQ ID NO:24), Gly8Glu22Lys23Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:26), Ser8Glu22Lys23Arg26,34GLP-1(7-37) (coding gene is SEQ ID NO:28), Thr8Glu22Lys30Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:30), Ile8Glu22Lys30Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:32), Leu8Glu22Lys30Arg26,34GLP-1(7-37) (encoding gene is SEQ ID NO:34), Gly8Glu22Lys30Arg26,34GLP-1(7-37) (coding gene SEQ ID NO:36), Ser8Glu22Lys30Arg26,34-GLP-1(7-37) (coding gene is SEQ ID NO: 38).
Example 2 fusion protein expression
Expression of the fusion protein was performed using the DNA construction described in example 1, and the protein of interest was obtained by expressing cell BL21(trabsgenbiotech., catalog # CD 601). 50 μ l of BL21 competent cells were thawed on an ice bath, the DNA of interest was added, shaken gently, and placed in an ice bath for 30 minutes. Followed by heat shock in a water bath at 42 ℃ for 30 seconds, and then the centrifuge tube was quickly transferred to an ice bath for 2 minutes without shaking the centrifuge tube. The tubes were mixed with 500. mu.l of sterile LB medium (containing no antibiotics) and incubated at 37 ℃ and 180rpm for 1 hour to resuscitate the bacteria. Mu.l of the transformed competent cells were pipetted onto a plate of LB agar medium containing kanamycin resistance and the cells were spread out evenly. The plate was placed at 37 ℃ until the liquid was absorbed, inverted and incubated overnight at 37 ℃. The following day, single colonies in the transformation plate were picked using an inoculating loop and inoculated in 15ml of sterile LB medium (containing antibiotics) and cultured overnight at 30 ℃.
Example 3 fermentation of recombinant GLP-1 analogs
To 50ml of LB medium, 50. mu.l of a bacterial solution (GLP-1 expressing bacterial solution) and 50. mu.l of kanamycin were added, mixed, placed in a 30 ℃ constant temperature oscillator, and inoculated overnight. 10ml of overnight inoculated broth was added to 1000ml of LB medium, together with 1000. mu.l of kanamycin. After shaking up, the mixture is put in a shaking table at 37 ℃ and 200rpm, IPTG with the final concentration of 0.1mol/L is inoculated into the culture medium after 4h inoculation, and after shaking up, the mixture is put in a shaking table at 30 ℃ and 180rpm, and induction expression is carried out overnight. Overnight expressed broth was centrifuged at 13000g for 60 min. The yield of the bacteria is about 4g bacteria/L fermentation liquor, and the expression quantity of the target protein can reach about 40 percent by SDS-PAGE.
Example 4 purification of recombinant GLP-1 analogs
100g of the cell paste 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. And centrifuging the homogenate at 13000g for 60min at 4 ℃, and collecting supernatant after centrifugation is finished, namely the Ni column chromatography sample.
The resulting supernatant was concentrated by Chelating Sepharose FF equilibrated beforehand with 50mM Tris-HCl, pH8.0, 500mM NaCl, 10mM imidazole (equilibration solution 1). After the elution with the equilibration solution 1, the elution was carried out with 50mM Tris-HCl, pH8.0, 50mM NaCl, 0.3M imidazole (eluent). The purity of the GLP-1 intermediate product generated by the purification process is higher than 70 percent through SDS-PAGE analysis.
Excision of the Sumo tag sequence using ULP enzyme: the intermediate product was diluted three times by adding 20mM PB, pH7.4 buffer, and the enzyme was expressed as ULP at 4 ℃: adding ULP into the intermediate product 1:150, uniformly mixing, and performing enzyme digestion overnight. The enzyme cleavage rate was approximately 100% as analyzed by SDS-PAGE.
Fine purification of GLP-1 analogues: the product obtained after the digestion was treated with 20mM Na2HPO40.7M NaCl (equilibration 2) equilibrated in Tosoh Butyl 550C medium. After being washed by the equilibrium solution 2, the product is eluted by 20 percent ethanol, and the purity of the product is about 90 percent by SDS-PAGE.
0.2M Na was added to the eluted sample2HPO4To a final concentration of 20mM Na2HPO4The pH was adjusted to 4.8-5.0 with 1M citric acid and acid precipitated overnight at 4 ℃. The yield of SDS-PAGE detection is more than 90%. 13000g of the suspension is centrifuged for 30min at 4 ℃, and the precipitate is collected and stored at-20 ℃.
EXAMPLE 5 preparation of derivatives of GLP-1 analogs
A derivative of a GLP-1 analog, N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys23Arg26,34Preparation of GLP-1(7-37)) peptide (abbreviated M2)
Figure BDA0002954690860000211
1. Fatty acid modification: val prepared and collected in the above examples8Glu22Lys23Arg26，34Adding water into the GLP-1(7-37) precipitate to prepare a 4-6 mg/ml solution, adding 1M sodium hydroxide to adjust the pH to 11.0-11.5, and shakingThe protein was dissolved completely and the polypeptide concentration was quantified by HPLC. The fatty acid powder is weighed according to the molar ratio of the polypeptide to the fatty acid (the structure is shown in the specification) of 1:4 and dissolved in acetonitrile. To the polypeptide solution, two thousandths of triethylamine was added, and mixed with a fatty acid solution, and the mixture was allowed to stand at 4 ℃ for one hour.
Figure BDA0002954690860000221
Diluting the sample with water 5 times, adjusting pH to 4.8 with 1M citric acid (or 10% acetic acid) to terminate the reaction, standing at 4 deg.C for 10min, centrifuging 13000g after acid precipitation, centrifuging at 4 deg.C for 30min, and storing the precipitate at-80 deg.C.
2. Deprotection and purification of fatty acid: adding TFA to the acid precipitation sample to a final concentration of about 10mg/ml of polypeptide, shaking to dissolve the precipitate, standing at room temperature for deprotection for 30min, and dropping 4M NaOH into the reaction solution to adjust the pH value to 7.5-8.5 to terminate the reaction.
The reaction mixture after termination was concentrated by pumping into UniSil 10-120C18 (available from Suzhou Naichi, Inc.) equilibrated with 10mM ammonium acetate and 20% ethanol (equilibration 3) at a flow rate of 4ml/min using a preparative liquid chromatograph (Shimadzu LC-8A). After the equilibrium solution 3 is washed, the eluent with the concentration of 0-100% (10mM ammonium acetate, 80% ethanol) is used for gradient elution, and the purity of the elution peak is collected and is about 90% by RP-HPLC.
Diluting the elution peak with water by 3 times, adjusting pH to 4.80 by acid precipitation, and performing acid precipitation at 4 ℃ for 30 min. After centrifugation, PBST buffer (pH7.0) is added into the sediment for redissolving, and then the mixture is frozen and stored at the temperature of minus 80 ℃.
The preparation of N- ε was performed in sequence according to the above method26- [2- (2- [2- (2- [2- (2[4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group]Val8Glu22Lys26Arg34GLP-1(7-37) (M0) peptide, N- ε30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M4), N-epsilon19- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Lys19Glu22Arg2634-GLP-1(7-37)) peptide (M1), N-epsilon27- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys27Arg26,34GLP-1(7-37)) peptide (M3), N-epsilon34- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26Lys34GLP-1(7-37)) peptide (M5), N-epsilon36- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26,34Lys36GLP-1(7-37)) peptide (M6), N-epsilon37- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26,34Lys37-GLP-1(7-37)) peptide (M7); n-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Thr8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M8), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M9), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Leu8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M10), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoyl) ]Amino) -4(s) -carboxybutanoylamino]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Gly8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M11), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ser8Glu22Lys23Arg26,34-GLP-1(7-37)) peptide (M12); n-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Thr8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M13), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M14), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Leu8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M15), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Gly8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M16), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ser8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M17).
TABLE 1 GLP-1(7-37) analogs and their corresponding derivatives
Figure BDA0002954690860000241
Figure BDA0002954690860000251
Example 6 in vitro Activity assay of derivatives of GLP-1 analogs in RIN-m5F cells
RIN-m5F cells in good culture were selected. Collecting cells, counting, preparing into 1 × 10 with RPMI1640 basic culture medium5Individual cells/ml of cell suspension. The cell suspension was inoculated into 96-well cell culture plates at 100. mu.l/well, 37 ℃ and 5% CO2Incubated under conditions overnight. The in vitro activity of the derivatives of the GLP-1 analogue was detected using the cAMP detection kit (Promega): preparation of assay broth dilution samples (Aib, M0, M1, M2, M3, M4, M5, M6, M7) to 300ng/ml, followed by 3-fold gradient dilution in 96-well plates for a total of 8 concentrations, 2 duplicate wells for each dilution, where M0, M1, M2, M3, M4, M5, M6, M7 are prepared as described above, Aib is
N-ε26- [2- (2- [2- (2- [2- (2[4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Aib8，Arg34]GLP-1- (7-37) peptide (see example 4 of CN 101133082B) under the trade name of somaglutide, prepared according to the method disclosed in patent CN 101133082B.
The prepared cell plate was removed, the medium was discarded, and blotted dry on filter paper. The sample solution was transferred to the cell plate at 40. mu.l/well. At 37 ℃ with 5% CO2And (5) uncovering the cover for 15min under the condition. The cell culture plate was removed from the incubator, 10. mu.l of a CD solution (cAMP detection kit (Promega)) was added to each well, and the cell plate was left to stand at 22 ℃ to 25 ℃ for 20min with horizontal shaking at 500 rpm. Mu.l KG solution (cAMP detection kit (Promega)) was added to each well, and the mixture was left to stand at 22 ℃ to 25 ℃ for 10min with horizontal shaking at 500rpm in the dark. The chemiluminescence values were read with a Molecular Devices SpectraMax L chemiluminescence apparatus and the assay was completed in 30 min. Sample EC50 was calculated using four parameter regression in softmax Pro software.
TABLE 2 results of in vitro Activity experiments
Sample (I) 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
The in vitro pharmacodynamics of RIN-M5F cells showed that the in vitro activities of somaglutide, M2, M4, M5 and M7 were comparable, and overall slightly higher than M0, M1, M3 and M6.
Example 7 in vitro Activity assay of derivatives of GLP-1 analogs in HEK293/CRE-Luc/GLP1R cells
According to the fact that GLP-1 can be combined with a receptor on a cell membrane, an HEK293/CRE-Luc/GLP1R cell line is constructed, a CAMP Reaction Element (CRE) is activated through a series of signal transduction, expression of downstream luciferase is started, the expression quantity is in positive correlation with the biological activity of GLP-1, and after a luciferase substrate is added, chemiluminescence detection is carried out to measure the luminous intensity of the GLP-1 biological activity, so that the GLP-1 biological activity is measured.
Experimental Material
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 procedures
(1) Cell preparation: the cells are cultured to a vigorous growth state and in sufficient quantity. Discarding the culture solution in the culture flask, adding 3ml of Versene solution, shaking for 1 time, adding 2ml of 0.05 percent TRYPSIN-EDTA digestive solution, covering a bottle cap, keeping flat and standing for 1 minute, adding 6ml of determination culture solution to stop digestion, centrifuging for 3min at 1000r/min, pouring off the supernatant, resuspending the cells with 5ml of determination culture medium, and counting by using a blood counting chamber. The cell density was adjusted to the appropriate range by DMEM assay.
(2) Sample preparation: the derivatives of the different GLP-1 analogues of table 1 were diluted to 20ng/ml with assay medium, followed by 8 concentrations of gradient dilution in 96-well plates and 2 replicates per dilution concentration using assay medium instead of sample as cell blank.
(3) Sample adding and culturing: transferring the prepared reference substance and test solution into 96-well cell culture plate (white plate), adding 50 μ l per well, adding the prepared cell suspension, adding 50 μ l per well, standing at 37 deg.C and 5% CO2Culturing for a certain time under the condition.
(4) And (3) chemiluminescence detection: substrate was added, 96 well cell culture plates were removed, 100. mu.l of Bright Glo reagent was added to each well, and the plates were left for 3min in the dark.
(5) Reading: and (4) measuring by using a chemiluminescence microplate reader SpectraMax L, reading the plate within 30min, and recording the measuring result.
TABLE 3 HEK293/CRE-Luc/GLP1R cell in vitro Activity test results
Figure BDA0002954690860000271
HEK293/CRE-Luc/GLP1R cell pharmacodynamics showed that the in vitro activities of somaglutide, M2, M4, M9, M11, M14, M16 and M17 were comparable, overall slightly higher than M13.
Example 8 hypoglycemic Studies of fatty acid modified derivatives of GLP-1 analogs in Normal mice
28 healthy CD-1 female mice with the week age of 4-6 weeks are selected and divided into 4 groups, and M2, M4, M0 and somaglutide (Aib) are respectively injected subcutaneously, and the dosage is 0.15mg/kg body weight respectively. Before administration, 20% glucose was gavaged at intervals of 6 hours, 1 day, 2 days, 3 days, and 4 days after administration at a dose of 2g/kg body weight, fasting was performed for 6 hours before administration, blood was taken from the tail tip at 0, 0.5, 1, and 2 hours after administration, blood glucose value was measured in real time using Roche blood glucose test paper, and blood glucose AUC (area under blood glucose-time curve) was calculated within 0 to 120 minutes, and the blood glucose inhibition ratio was calculated (Table 4).
Figure BDA0002954690860000281
TABLE 4 comparison of the hypoglycemic Effect in Normal mice
Figure BDA0002954690860000282
P value: comparison with Pre-dose blood glucose
As can be seen from table 4, the hypoglycemic activity of somatolide in normal mice lasted for about 2 days, that of M0 in normal mice lasted for about 3 days, while the hypoglycemic activity of M2 and M4 in normal mice still showed significant activity at day 4, sustained hypoglycemic activity in vivo was maintained significantly longer than that of somatolide or M0, and the hypoglycemic effects of M2 and M4 were also significantly stronger than that of somatolide or M0 at each time point after day 3 of administration.
28 healthy CD-1 female mice with the week age of 4-6 weeks are selected and divided into 4 groups, and M4, M5, M7 and M0 are respectively injected subcutaneously, and the dosage is 0.15mg/kg of body weight. Before administration, 20% glucose was gavaged at intervals of 6 hours, 1 day, 2 days, 3 days, and 4 days after administration at a dose of 2g/kg body weight, and fasted for 6 hours before gavage of 20% glucose, and blood was taken from the tip of the tail at 0, 0.5, 1, and 2 hours after administration of glucose, and blood glucose values were measured in real time using Roche blood glucose test paper, and blood glucose AUC (area under blood glucose-time curve) was calculated within 0 to 120 minutes, and the blood glucose inhibition rate was calculated (Table 5).
Figure BDA0002954690860000291
TABLE 5 comparison of the hypoglycemic Effect in Normal mice
Figure BDA0002954690860000292
From the results in tables 4 and 5, the effects of M2 and M4 are better than those of M0 and somaglutide, and the effects of M2, M4, M5 and M7 are equivalent without significant difference.
Example 9 study of blood sugar lowering Effect Using ICR mice
ICR mouse OGTT assay: 30 ICR mice with the age of 4-6 weeks are selected and divided into 6 groups and 5 mice per group, and M0, somaglutide, M2, M4, M5 and M7 are respectively injected subcutaneously, the dosage is 0.15mg/kg body weight respectively, and the administration is carried out once. And (3) intragastrically irrigating 20% glucose every day according to the time of 4h, 1d, 2d, 3d, 4d and 5d, wherein the dosage is 2g/kg of body weight, fasting is performed for 6h before sugar administration, and tail tip blood is respectively taken at 0, 0.5, 1 and 2 hours after intragastrically irrigating 20% glucose, and the blood glucose value is detected in real time by using Roche blood glucose test paper. Blood was collected from the tail tip and the blood glucose value was measured in real time using Roche's blood glucose test paper, and the blood glucose AUC (area under the blood glucose-time curve) was calculated within 0 to 120 minutes, and the blood glucose inhibition rate was calculated (Table 6).
Figure BDA0002954690860000293
TABLE 6 comparison of the hypoglycemic Effect in ICR mice
Figure BDA0002954690860000294
Figure BDA0002954690860000301
As can be seen from the results in table 6, the glucose lowering maintenance effect: the hypoglycemic effects of M4, M5, M2 and M7 can be maintained for at least 4 days, and are far superior to those of M0 (only 3 days) and somaglutide (only 2 days), and the statistical significance is achieved.
Example 10 test of hypoglycemic Effect of db/db mice with type II diabetes
50 db/db mice, female, 8-9 weeks old, were divided into 10 groups and 5 groups on average based on the weight before administration and Fasting Blood Glucose (FBG), and were administered with a single subcutaneous injection of vehicle, M2, M4, somaglutide, M9, M11, M13, M14, M16, and M17, at a dose of 0.05mg/kg and for a period of 0h, respectively, at 10 ml/kg. After fasting for 6-8h, the mice are tested for fasting blood sugar every day, and after administration, the fasting blood sugar is tested every day until the fasting blood sugar of each animal group is recovered to the state before administration. The blood glucose level detected before administration is referred to as a basal blood glucose level and is set to 0.
Fasting blood glucose change (Δ: delta) is the post-administration blood glucose value-the pre-administration basal blood glucose value.
As shown in fig. 1, from day 4 and day 5, it can be seen that M9, M13, M14, had better hypoglycemic effects than somatolide and also did not lower than M2, while M11, M16 and M17 showed lower hypoglycemic effects than somatolide on day 2.
Example 11 hypoglycemic Effect of different doses of somaglutide, M0 and M4 on type II diabetic db/db mice
35 db/db mice, female, 8-9 weeks old, were divided on average into 7 groups and 5 groups based on pre-dose body weight, area under the blood glucose curve (G-AUC), and administered with 10ml/kg of vehicle, M4(0.15, 0.015mg/kg), somaglutide (0.15, 0.015mg/kg) and M0(0.15, 0.015mg/kg), by single subcutaneous injection, respectively. The administration time is set to 0h, after fasting for 7-8h every day, the fasting blood glucose and OGTT (oral glucose tolerance measurement) are measured, then 10% glucose is gavaged with 1g/kg body weight, and then tail tip blood is respectively taken at 0, 0.5, 1 and 2h after the glucose load to detect the blood glucose value in real time. Blood glucose was measured daily after dosing before fasting and was called random blood glucose until the blood glucose returned to the pre-dose level in each test group. The basal blood glucose level, the random blood glucose level and the area under the blood glucose curve (G-AUC) measured before administration were all bases for measuring drug effects, and were all set to 0.
Blood glucose change (Δ: delta) is the post-administration blood glucose value-the pre-administration basal blood glucose value;
the change in area under the blood glucose curve (Δ: delta) is the area under the blood glucose curve after administration-the area under the blood glucose curve before administration.
The results are shown in tables 7, 8 and 9 and FIGS. 2, 3 and 4.
TABLE 7 fasting blood sugar change table for mice of each test group
Figure BDA0002954690860000311
Note: "-21 h" represents fasting glucose base before administration.
TABLE 8 mean random blood glucose Change Table for mice of each test group
Figure BDA0002954690860000312
Note: "-5 h" represents a random glycemic basis 5h prior to dosing.
TABLE 9 Change table of area under blood glucose curve (G-AUC) of mice in each test group
Figure BDA0002954690860000313
Figure BDA0002954690860000321
Note: "-21 h" represents the base of area under the blood glucose curve before administration.
The results of tables 7-9 and FIGS. 2-4 show that:
fasting blood glucose: the M40.15mg/kg dose group returned to the pre-dose base glycemic number 123h after dosing, and the 0.015mg/kg dose group returned to the pre-dose base glycemic number 99h after dosing; the somaglutide 0.15mg/kg dose group returned to the pre-dose base glycemic number 51h after dosing, and the 0.015mg/kg dose group returned to the pre-dose base glycemic number 27h after dosing; the M00.15mg/kg dose group returned to the pre-dose base blood glucose 75h after dosing, and the 0.015mg/kg dose group returned to the pre-dose base blood glucose 51h after dosing; wherein the fasting blood glucose reduction value of the 0.015mg/kg dose group of M4 at each detection time point is not lower than that of the 0.15kg/kg dose group of the somaglutide or M0.
Random blood sugar: the M40.15mg/kg dose group returned to the pre-dose random glycemic basis at 115h post-dose, the 0.015mg/kg dose group returned to the pre-dose random glycemic basis at 115h post-dose; the somaglutide 0.15mg/kg dose group returned to the pre-dose random glycemic basis 67h after dosing, and the 0.015mg/kg dose group returned to the pre-dose random glycemic basis 67h after dosing; the M00.15mg/kg dose group returned to the pre-dose random glycemic basis 67h after dosing, and the 0.015mg/kg dose group returned to the pre-dose random glycemic basis 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 somaglutide or M0.
Area under the blood glucose curve (G-AUC): the M40.15mg/kg dose group restored the base of area under the blood glucose curve before dosing 99h after dosing, and the 0.015mg/kg dose group restored the base of area under the blood glucose curve before dosing 99h after dosing; the number of areas under the blood glucose curve before the administration is recovered 51h after the administration of the 0.15mg/kg dose group of the somaglutide, and the number of areas under the blood glucose curve before the administration is recovered 51h after the administration of the 0.015mg/kg dose group; the M00.15mg/kg dose group restored the base of area under the blood glucose curve before dosing 51h after dosing, and the 0.015mg/kg dose group restored the base of area under the blood glucose curve before dosing 27h after dosing; wherein the area under the blood glucose curve at 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 somaglutide or M0.
These results in terms of lowering blood sugar indicate that after a single subcutaneous injection of M4 or somaglutide or M0, each group showed significant lowering of blood sugar, but M4 gave the best lowering of blood sugar. The hypoglycemic effect of the M4 at the dose of 0.015mg/kg is equivalent to the hypoglycemic effect of the somaglutide at the dose of 0.15mg/kg or the somaglutide at the dose of 0.15mg/kg in M0.
Example 12 stability study of M4 and Somaltulip against enzymatic degradation
Pepsin (3200. sup. 4500U/mg protein from Sigma, cat. No. P6887), trypsin (approximately 10000AEE U/mg protein from Sigma, cat. No. T8003).
(1) Reaction solution
A: pepsin reaction buffer: three 20mM citrate-phosphate buffers at different pH's (2.6, 4.0, 7.4) were prepared and 0.005% Tween 20 and 0.001% BSA were added as pepsin reaction buffers.
B: trypsin reaction buffer: three 20mM citrate-phosphate buffers at different pH's (4.0, 6.8, 8.0) were prepared and 0.005% Tween 20 and 0.001% BSA were added as pepsin reaction buffers.
C: simulated gastric fluid with pepsin (SGF): dissolving 0.019g pepsin in 5ml of 0.1M hydrochloric acid.
D: simulated intestinal fluid with trypsin (SIF): dissolving potassium dihydrogen phosphate 0.0684g in water 2.5ml, adding 0.77ml sodium hydroxide solution 0.2M and water 5ml, adding trypsin 0.1001g to dissolve, measuring pH to 6.82, and diluting with water to 10 ml.
(2) Sample configuration
M4 and somaglutide samples were taken and diluted to 1.33mg/ml with PB buffer solution of pH7.4 to obtain a sample stock solution.
(3) Pepsin degradation experiments
Taking a proper amount of test sample mother liquor respectively, diluting the test sample mother liquor to 0.06mg/ml by pepsin reaction buffer solutions with different pH values, dividing each group of reaction solution into 1 ml/tube and 7 tubes in total, uniformly mixing, and placing in a water bath at 37 ℃ for incubation for 30 min. Taking out 1 tube without SGF as a non-enzyme reaction 0 point (marked as a point of-5 min), taking out 6 tubes, respectively adding SGF, uniformly mixing, immediately adding 1M NaOH with a proper volume into one tube to terminate the reaction, taking the obtained product as the 0 point (marked as a point of 0 min) after enzyme addition, continuously placing the other 5 tubes at 37 ℃ for reaction, and respectively taking out a group of 1M NaOH with a proper volume to terminate the reaction at 5min, 10min, 20min, 35min and 50 min. The tubes of all experimental groups were kept consistent in total volume after termination of the reaction.
(4) Trypsin degradation experiments
Taking a proper amount of test sample mother liquor respectively, diluting the test sample mother liquor to 0.06mg/ml by trypsin reaction buffer solutions with different pH values, dividing each group of reaction solution into 1 ml/tube and 7 tubes in total, uniformly mixing, and placing in a water bath at 37 ℃ for incubation for 30 min. Taking out 1 tube without SIF as a non-enzyme reaction 0 point (marked as-5 min point), taking out 6 tubes, respectively adding SIF, mixing, immediately adding 6M HCl with appropriate volume to one tube to terminate the reaction, taking as the enzyme added 0 point (marked as 0min point), continuously placing the other 5 tubes at 37 ℃ for reaction, and respectively taking out a group of 6M HCl with appropriate volume to terminate the reaction at 5min, 10min, 20min, 35min and 50 min. The tubes of all experimental groups were kept consistent in total volume after termination of the reaction.
And (3) sampling an enzyme degradation experiment, carrying out HPLC (high performance liquid chromatography) detection, taking the main peak area of the sample at the non-enzyme reaction 0 point (marked as a-5 min point) as a basic peak area, and calculating the residual percentage of the main peak area at different time points obtained after enzyme addition.
The pepsin degradation experimental data (n ═ 3) show (fig. 5) that the degradation rates of M4 and the somaglutide molecule under acidic conditions (pH2.6) are comparable, due to the highest pepsin activity at this pH; at neutral ph7.4, neither molecule is substantially degraded, at which time pepsin activity is minimal; at pH4.0, the degradation rate of the somaglutide is obviously higher than that of M4, the t1/2 of the somaglutide is about 10min, and the t1/2 of the somaglutide is about 45min, which shows that the ability of M4 to resist the degradation of pepsin is obviously better than that of the somaglutide.
The trypsin degradation experimental data (n ═ 4) show (fig. 6) that the rates of degradation were essentially identical for both pH6.8 and 8.0 conditions, since this pH range is the highest activity range for trypsin; at ph4.0, M4 and somaglutide also showed resistance to trypsin degradation, with essentially no difference between the two.
Sequence listing
<110> Hangzhou Dai Biotechnology Co Ltd
<120> acylated GLP-1 derivatives
<130> PC00515D1
<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 (10)

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:
HX8EGTFTSDVSSX19LEEX23AARX27FIX30WLVX34GX36X37，
wherein X8Selected from V, T, I, L, G or S, X19Is Y or K, X23Is Q or K, X27Is E or K, X30Is A or K, X34Is R or K, X36Is R or K, X37Is a group of G or K,
provided that at X19、X23、X27、X30、X34、X36Or X37Only one of which is a K residue,
said derivative comprising an extension attached to the K residue of said GLP-1(7-37) analog, wherein said extension is
Figure FDA0002954690850000011
Figure FDA0002954690850000012
Wherein x is an integer from 4 to 38.
2. The derivative of claim 1, or a pharmaceutically acceptable salt thereof, wherein the protracting moiety is selected from the group consisting of:
HOOC(CH2)14CO-、HOOC(CH2)15CO-、HOOC(CH2)16CO-、HOOC(CH2)17CO-、HOOC(CH2)18CO-、HOOC(CH2)19CO-、HOOC(CH2)20CO-、HOOC(CH2)21CO-and HOOC (CH)2)22CO-。
3. The derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the protracting moiety is attached to the K residue of the GLP-1(7-37) analog via a linker.
4. The derivative of claim 3, or a pharmaceutically acceptable salt thereof, wherein the linker is:
Figure FDA0002954690850000013
Figure FDA0002954690850000021
Figure FDA0002954690850000022
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 linker is:
Figure FDA0002954690850000023
Figure FDA0002954690850000031
wherein m is 1 or 2; n is 1 or 2; p is any integer from 1 to 5.
5. The derivative of claim 4, or a pharmaceutically acceptable salt thereof, wherein the linker is:
Figure FDA0002954690850000032
wherein m is 1 and n is 1 or 2.
6. The derivative of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which is any one of the derivatives selected from the group consisting of: n-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys23Arg26,34GLP-1(7-37)) peptide (M2), N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxyBase of]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M4), N-epsilon34- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26Lys34GLP-1(7-37)) peptide (M5), N-epsilon37- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Val8Glu22Arg26,34Lys37GLP-1(7-37)) peptide (M7), N-epsilon23- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys23Arg26,34-GLP-1(7-37)) peptide (M9) \ N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Thr8Glu22Lys30Arg26,34-GLP-1(7-37)) peptide (M13) \ N-epsilon30- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(s) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group](Ile8Glu22Lys30Arg26,34GLP-1(7-37)) peptide (M14).
7. A process for preparing a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, comprising:
(1) mixing a solution in which a GLP-1 analogue as defined in any of the preceding claims is dissolved with a solution in which an extension as defined in any of the preceding claims is dissolved;
(2) adjusting the pH value to 4-5 to terminate the reaction, standing until a precipitate is generated, and taking 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 protracting moiety of any of the preceding claims is dissolved.
9. The process 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 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
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