RU2773242C2 - Acylated derivative of glp-1 - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to an acylated derivative of GLP-1(7-37), intended for preventing and/or treating diabetes. Proposed are an analogue of the polypeptide GLP-1(7-37), a fatty acid-modified derivative of said analogue, and a medicinal product containing said derivative. Also proposed is a method for producing said derivative and an application thereof for producing a medicinal product.

EFFECT: production of an acylated derivative of GLP-1 suitable for preventing and/or treating diabetes.

14 cl, 6 dwg, 9 tbl, 12 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of polypeptide technology. In particular, the present invention relates to fatty acid modified derivatives of GLP-1(7-37) polypeptide analogs. In addition, the invention also relates to a method for producing a peptide derivative, a drug containing a peptide derivative, and its use for producing a drug.

BACKGROUND OF THE INVENTION

Diabetes is a disorder of glucose metabolism caused by genetic and environmental factors. It has become the third most important disease after tumors, cardiovascular and cerebrovascular diseases, threatening the health and safety of human life. Diabetes itself is not necessarily harmful, but prolonged high blood glucose levels damage large blood vessels and microvessels and endanger the heart, brain, kidneys, peripheral nerves, eyes, legs, and more. According to the statistics of the World Health Organization, there are more than 100 complications of diabetes - diseases with the most known complications. More than half of deaths from diabetes are caused by cardiovascular and cerebrovascular diseases, and 10% by nephropathy. The number of amputations due to diabetes is 10-20 times higher than in the absence of diabetes. Therefore, the treatment of diabetes and the prevention of its complications are vital social issues.

Diabetes can be divided into several types depending on the pathogenesis. Most of them are type II diabetes (about 90%), mainly due to overweight and lack of physical activity. Patients with type II diabetes often have pathologies of insulin resistance and insufficient secretion of insulin, in addition, apoptosis of islet β-cells often occurs in the middle and late stages of this disease. At present, the mechanism of action of oral hypoglycemic drugs for clinical use is mainly to increase insulin sensitivity or stimulate insulin secretion to stabilize blood glucose levels, which cannot solve the problem of β-cell apoptosis. Drugs based on glucagon-like peptide-1 (GLP-1) and its analogue slow down β-cell apoptosis, promoting their regeneration and promoting the differentiation and proliferation of islet β-cells, which makes it the center of research regarding the treatment of type II diabetes.

In 1983 Bell et al. discovered glucagon-like peptide-1 (GLP-1) when analyzing the sequence of the proglucagon (PG) gene (Bell G. I., Sanchez-Pescador R., Laybourn P. J., et al., Exon duplication and divergence in the human preproglucagon gene [J]. Nature, 1983, 304(5924): 368-371). The PG gene sequence consists of 6 exons and 5 introns, including 3 main domains: glucagon (33-61), GLP-1 (72-108) and GLP-2 (126-158). PG mRNA is expressed in A-cells of the pancreas, L-cells of the intestine and the brain, and in the cells of these tissues a certain translational modification is carried out with the formation of different end products.

There are two subtypes of GLP, GLP-1 analog and GLP-2 analog. Their amino acid sequence is almost half identical to glucagon, and there is also about 35% homology between them. GLP-1 analog is a polypeptide hormone secreted by Langerhans cells of the terminal jejunum, ileum and colon, which has multiple functions such as glucose-dependent stimulation of insulin secretion and biosynthesis, inhibition of glucagon secretion and gastric emptying. GLP-2 analog is synthesized in intestinal tissue and neurons of the brainstem and hypothalamus of the central nervous system, and mainly contributes to the normal growth of the small intestine and the repair of damage to the intestinal mucosa (Fu Gang, Gong Min, Xu Weiren; Research progress of glucagon-like peptide 1 and its receptor agonists [J] Tianjin Medical Journal, 2012, 40(2): 181-184).

GLP-1 is an endogenous hormone that stimulates the secretion of insulin, primarily secreted by intestinal L cells, and plays a role in regulating 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 (Clu)-glycine (Gly)-threonine (Thr)-phenylalanine (Phe)-threonine (Thr)-serine (Ser)-aspartic acid (Asp)-valine (Val)-serine (Ser)-serine (Ser )-tyrosine (Tyr)-leucine (Leu)-glutamic acid (Glu)-glycine (Gly)-glutamine (Gln)-alanine (Ala)-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 cleave histidine (H)-alanine (A) at positions 7-8 at the N-terminus. DDP-IV mainly mediated the hydrolysis of the end of the peptide chain where position 8 is alanine or proline, the enzyme will cleave it and cause a rapid loss of GLP-1 activity (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. proposed models of GLP-1 to observe changes in receptor affinity and intrinsic activity of GLP-1 analogues by amino acid substitution. Histidine at position 7 is a determining factor for affinity and intrinsic activity, the aromatic ring of histidine is smaller than that of tryptophan and it has no polar substituent; the alanine side chain at position 8 has a polar group that affects GLP-1 activity; the size of the side chain should not be too large, if a certain limit is exceeded, the activity will decrease; when replacing the glutamic acid at position 9 with certain amino acids, such as acidic, polar and hydrophobic amino acids, the activity will not change, but will be reduced or even lost when it is replaced with basic amino acids. Once GLP-1 binds to its receptor, an ionic ring structure is formed between the amino acids at positions 7-15 and Ala8-Glu9-Gly10-Thr11 will form a β-turn, these conformational changes will result in the formation of three aromatic nuclei, so histidine at position 7, phenylalanine at position 12 and tyrosine at position 19 interact with each other, corresponding to hydrophobic pockets of aromatic clusters present on the receptor; they are supposed to activate the receptor; the glycine at position 22 is a flexible amino acid that acts as a flexible linker to maintain helix bending. The destruction of glycine will cause clustering of all aromatic amino acids, resulting in a decrease in affinity for the receptor by 1/40 (Sarauste De Menthierec, Chavanieua, Grassyg, et al. Structural 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 and GLP-1(7-36) NH ₂ derivatives, and other molecular forms. According to popular belief, the last two have the same biological activity. GLP-1(1-37) secreted by intestinal mucosal L cells is inactive and further hydrolysis and removal of 6 amino acids at the N-terminus is required for it to become active GLP-1(7-37). GLP-1(7-37) exists in the body for a relatively short time and is rapidly degraded. Therefore, various studies have been conducted on GLP-1 analogs with anti-DPP IV function. For example, US Pat. No. 5,545,618 describes modification of the N-terminus with an alkyl or acyl group, and Gallwitz et al. describe N-methylation or alpha-methylation of His at position 7, or replacement of all of His with imidazole to increase resistance to DPP-IV and maintain physiological activity.

In addition to these modifications, the GLP-1 analogue exendin-4 (US Patent No. 5,424,686), isolated from the salivary glands of the Arizona lizard (Heloderma suspectum), is resistant to DPP IV and has a higher physiological activity than GLP-1. Therefore, it has an in vivo half-life of 2-4 hours, which is longer than that of GLP-1. However, this is only applicable to the DPP IV resistance method, but the physiological activity cannot be sufficiently maintained, and in the case of using commercially available exendin-4 (exenatide), it must be administered to the patient twice a day, which is still very painful for the patient.

These insulinotropic peptides have a very low molecular weight and are therefore rapidly excreted by the kidneys. Some scientists use chemical methods to add highly soluble polymers (such as polyethylene glycol) to the surface of the peptide to inhibit renal clearance. For example, US Pat. No. 692,464 describes binding of PEG to a lysine residue of exendin-4, which increases retention time in the body. However, although this method increases the retention time of peptide drugs in the body, it also leads to an increase in molecular weight, the concentration of the peptide drug is significantly reduced, and the reactivity towards peptides is also reduced.

In addition, there are a number of other methods for modifying the structure of GLP-1 compounds in an attempt to increase their duration of action. For example, WO 96/29342 describes peptide hormone derivatives modified by introducing a lipophilic substituent at the C-terminal amino acid residue or N-terminal amino acid residue of the parent peptide hormone. WO98/08871 discloses a GLP-1 derivative (liraglutide) in which at least one amino acid residue of the parent peptide is linked to a lipophilic substituent. WO 99/43708 discloses derivatives of GLP-1(7-35) and GLP-1(7-36) with lipophilic substituents attached to the C-terminal amino acid residue. WO 00/34331 discloses doubly acylated GLP-1 analogs. WO 00/69911 discloses activated insulinotropic peptides for injection and is believed to react with blood components to form conjugates in patients, prolonging the duration of action in the body.

WO 2006/097537 discloses another acylated GLP-1 analog (semaglutide) obtained by mutating the amino acid at position 8 to increase the half-life compared to the acylated GLP-1 (liraglutide) in WO 98/08871.

WO 02/046227 discloses the preparation of fusion proteins by combining GLP-1, exendin-4 or its analogs with human serum albumin or an immunoglobulin (Fc) region using genetic recombination technology, this can solve problems such as low yield and non-specificity of PEGylation, but their effect of increasing the half-life from the blood, however, is not as significant as expected. With regard to the complex glucose-lowering effect, the expected effect is not achieved, and this drug does not even achieve the result of semaglutide. Various types of peptide linkers have been tried to extend the blood half-life, but the problem with this method is that it can elicit an immune response.

CN 107033234 A discloses a fatty acid modified GLP-1 analogue conjugate. The fatty acid modification site is at Lys ²⁶ . Early animal experiments showed that its glucose-lowering effect was superior to that of semaglutide. This method can adequately extend the in vivo duration of action of GLP-1 analogs, but the plasma half-life is still not ideal.

The GLP-1-based medicinal products currently available on the market mainly include exendin-4 isolated from lizard saliva and human GLP-1 analogues modified with fatty acids or fused with Fc or human serum albumin. The half-life of exendin-4 is too short (only 2-4 hours), so injections are required twice a day. Novo Nordisk's fatty acid-modified liraglutide is the most effective drug for lowering glycated hemoglobin (HbA1c) levels with fewer side effects, however, because the half-life is only 13 hours, once daily administration is required. In order to further increase the in vivo half-life and reduce the frequency of administration, amino acid sequence mutant GLP-1 analogs and long-acting GLP-1 analogs modified with Fc, fatty acids or albumin, etc. have been developed in recent years, for example, dulaglutide from Eli Lilly and Company and semaglutide from Novo Nordisk. The half-life of these long-acting GLP-1 analogs from the human body can be extended to varying degrees, and a once-weekly administration frequency can be achieved as the longest-acting maximum.

After long research, the authors of the present application have developed a new GLP-1 analog and its derivatives, under similar experimental conditions, they have equivalent in vitro activity compared to semaglutide, which is currently considered the best drug; in both normal and diabetic mouse models, the duration of the in vivo glucose-lowering effect can be extended by about 1-fold, meaning that dosing rates of at least once a week, twice a week, or even at longer intervals. Moreover, when the dosage is 1/10 of the dosage of semaglutide, its galucose-lowering effect is comparable to that of semaglutide, so it is more promising for use.

BRIEF DESCRIPTION OF THE INVENTION

The aim of the present invention is to provide a novel GLP-1(7-37) analogue and an acylated derivative of said analogue. In addition, the present invention also relates to a method for preparing said analog or derivative, a pharmaceutical composition and a product containing said analog or derivative, and their use in the prevention and treatment of diseases.

In particular, in one aspect, the present invention provides a derivative of a GLP-1(7-37) analog or a pharmaceutically acceptable salt thereof, wherein said GLP-1 analog comprises a peptide consisting of the amino acid sequence of the following formula:

where X ₈ is selected from V, T, I, L, G or S; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; and X ₃₇ is G or K;

with the proviso that only one of X ₁₉ , X ₂₃ , X ₂₇ , X ₃₀ , X ₃₄ , X ₃₆ or X ₃₇ is a K residue, and

the derivative contains an extension linked to the K moiety of the GLP-1(7-37) analog, where the extension is

where x is an integer from 4 to 38.

While the extension part 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 portion of the GLP-1 analog derivative or pharmaceutically acceptable salt thereof according to the invention is linked to the K moiety of GLP-1 via a linker. A linker contains any of the following structures:

где m равно 0, 1, 2 или 3; n равно 1, 2 или 3; s представляет собой любое целое число от 0 до 6; и р представляет собой любое целое число от 1 до 8.

where m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; and p is any integer from 1 to 8.

Preferably the linker is:

where m is 1 or 2; n is 1 or 2; and p is any integer from 1 to 5.

More preferably the linker is:

где m равно 1, и n равно 1 или 2.

where m is 1 and n is 1 or 2.

The invention also relates to a GLP-1(7-37) analogue containing the following sequence:

которая содержит мутации, выбранные из одного или более из следующих положений:

which contains mutations selected from one or more of the following positions:

positions 8, 19, 23, 27, 30, 34, 36, and 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 derivatives of the above GLP-1 analogs indicates that the binding affinity for the GLP-1R receptor is higher than that of semaglutide or MO (Lys at position 26, disclosed in CN 107033234 A). The in vivo glucose lowering experiment also proves that, compared to the acylated GLP-1 product semaglutide, the acylated derivatives of the above GLP-1 analogs can exhibit a longer-lasting halyco-lowering effect in normal mice; in diabetic mice, the above derivatives have a significantly better glucose-lowering and glucose-tolerance-increasing effect than semaglutide, and when the dose is only 1/10 of that of semaglutide or M0, its halyco-lowering effect is comparable to that of semaglutide or M0. At the same time, studies carried out by the present inventors prove that derivatives of the above GLP-1(7-37) analogs have better resistance to enzymatic degradation compared to commercially available semaglutide. In particular, the present invention relates to the following: 1. A derivative of a GLP-1(7-37) analog or a pharmaceutically acceptable salt thereof, wherein the GLP-1(7-37) analog contains the amino acid sequence of the following formula:

where X ₈ is selected from V, T, I, L, G or S; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; and X ₃₇ is G or K;

with the proviso that only one of X ₁₉ , X ₂₃ , X ₂₇ , X ₃₀ , X ₃₄ , X ₃₆ or X ₃₇ is a K residue, and

the derivative contains an extension linked to the K moiety of the GLP-1(7-37) analog, where the extension is

where x is an integer from 4 to 38.

2. A derivative or pharmaceutically acceptable salt thereof according to claim 1, where the extension part is selected from:

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-, HOOS (CH ₂ ) ₂₀ CO-, HOOS (CH ₂ ) ₂₁ CO- and HOOS (CH ₂ ) ₂₂ CO-.

3. A derivative or pharmaceutically acceptable salt thereof according to claim 1 or claim 2, wherein the extension portion is linked to the K moiety of the GLP-1(7-37) analog via a linker.

4. A derivative or pharmaceutically acceptable salt thereof according to claim 3, where the linker is:

где m равно 0, 1, 2 или 3; n равно 1, 2 или 3; s представляет собой любое целое число от 0 до б; и р представляет собой любое целое число от 1 до 8.

where m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to b; and p is any integer from 1 to 8.

Preferably the linker is:

where m is 1 or 2; n is 1 or 2; and p is any integer from 1 to 5.

и где m равно 1, и n равно 1 или 2.5. A derivative or pharmaceutically acceptable salt thereof according to claim 4, where the linker is:

and where m is 1 and n is 1 or 2.

6. Derivative or pharmaceutically acceptable salt according to any one of pi. 1-5, which is any derivative selected from the group consisting of: peptide

N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl ](Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (M2), peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2- [4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-3 7)) (M4)

peptide N-ε ³⁴ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37)) (M5),

peptide N-ε ³⁷ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl])(Val ⁸ Glu ²² Arg ^26.34 Lys ³⁷ -GLP-1(7-3 7))(M7),

peptide N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Ile ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37))(M9) \

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Thr ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37))(M13) \

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37)) (M14), or a pharmaceutically acceptable salt thereof.

7. The method of obtaining a derivative or its pharmaceutically acceptable salt according to any one of paragraphs. 1-6, including:

(1) mixing a solution in which the GLP-1 analog according to any one of paragraphs is dissolved. 1-6, with a solution in which the extension according to any one of paragraphs is dissolved. 1-6;

(2) adjusting the pH to 4-5 to stop the reaction, holding until a precipitate forms, and then collecting the precipitate; and

(3) adding TFA (trifluoroacetic acid) to the precipitate and adjusting the pH to 7.5-8.5 to stop the reaction.

8. The method according to p. 7, further comprising: adding triethylamine to a solution in which the GLP-1 analog is dissolved, followed by mixing with a solution in which the extension part according to any one of paragraphs is dissolved. 1-7.

9. The method according to p. 7 or p. 8, where the solution of the extension part according to any one of paragraphs. 1-8 was obtained in acetonitrile.

10. Pharmaceutical composition containing a derivative or a pharmaceutically acceptable salt according to any one of paragraphs. 1-6 and a pharmaceutically acceptable excipient.

11. The use of a derivative or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-6 for the manufacture of a medicament for the prevention and/or treatment of diabetes (including type I diabetes and type II diabetes) or complications of diabetes.

12. Use according to claim 11 wherein the complication of diabetes is diabetic nephropathy.

13. The use of a derivative or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-6 for the preparation of a medicament for lowering blood glucose, improving glucose tolerance, reducing islet β cell apoptosis, enhancing islet β cell function, increasing islet β cell numbers, and/or restoring glucose sensitivity of islet β cells .

14. Use according to claim 13, wherein said blood glucose lowering comprises lowering fasting blood glucose and/or postprandial blood glucose.

15. A method for preventing and/or treating diabetes (including type I diabetes and type II diabetes) or complications of diabetes, comprising administering a prophylactically or therapeutically effective amount of a derivative or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-6 subject.

16. The method of claim 15 wherein the complication of diabetes is diabetic nephropathy.

17. A method for lowering blood glucose levels, increasing glucose tolerance, reducing islet β-cell apoptosis, enhancing islet β-cell function, increasing the number of islet β-cells, and/or restoring glucose sensitivity of islet β-cells, comprising administering a therapeutically effective the amount of a derivative or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-6 subject.

18. Use according to claim 17, wherein said blood glucose lowering comprises lowering fasting blood glucose and/or postprandial blood glucose.

19. GLP-1(7-37) analogue containing a polypeptide consisting of the following amino acid sequence:

where X ₈ is selected from V, T, I, L, G or S; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; X ₃₇ is G or K; and only one of X ₁₉ , X ₂₃ , X ₂₆ , X ₂₇ , X ₃₀ , X ₃₄ , X ₃₆ or X ₃₇ is K.

20. Pharmaceutical composition containing an analogue according to claim 19.

21. The use of an analogue according to claim 19 for the preparation of a medicament for the prevention or treatment of diabetes and complications of diabetes.

22. A product containing: a container containing a pharmaceutical composition according to claim 10 or claim 20, and a package insert containing instructions for use of the pharmaceutical composition.

23. A product according to claim 22, further comprising a container containing one or more other drugs.

24. The product of claim 23 wherein the one or more other drugs are other drugs for the treatment of diabetes or complications of diabetes.

"Fasting blood glucose level" refers to a blood glucose value determined after a subject (e.g., human) fasting and, for example, a blood glucose value measured after an overnight fast, fasting (without any food other than drinking water ) for at least 6 hours, eg 6-8 hours, 8-10 hours.

"Post-meal blood glucose" refers to a blood glucose level determined after a meal, such as a blood glucose value measured after 15 minutes - 2 hours, 30 minutes - 2 hours, 1 hour - 2 hours, or after 2 hours after eating.

One aspect of the present invention relates to a method for producing a GLP-1(7-37) analogue, comprising expression of a peptide by a host cell containing a DNA sequence encoding a polypeptide under conditions allowing expression of the peptide, and then isolating the resulting peptide.

The medium used for culturing cells can be any conventional medium used for culturing host cells, such as a minimal medium or a complex medium containing suitable additives. A suitable culture medium can be purchased from the commercial market, or a suitable culture medium can be prepared according to the preparation method described. The polypeptide produced by the host cells can then be isolated from the culture medium by conventional methods, for example, the protein component in the supernatant or filtrate is precipitated with a salt such as ammonium sulfate, and further purified by various chromatographic methods such as ion exchange column chromatography, gel filtration chromatography , affinity chromatography, etc., depending on the type of peptides.

The above DNA coding sequence may be inserted into any suitable vector. In general, the choice of vector often depends on the host cell into which the vector is to be introduced. Therefore, the vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid. Alternatively, the vector may be of a type that, when introduced into a host cell, will integrate into the host cell's genome and replicate along with the chromosome into which it is inserted.

The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments required for DNA transcription (such as a promoter). Examples of promoters suitable for directing the transcription of DNA encoding the peptides of the present invention in various host cells are well known in the art, see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York, 1989.

The vector may also contain a selection marker, such as a gene whose product will repair a defect in the host cell, or may confer resistance to drugs such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin, streptomycin, or methotrexate, and the like.

In order to introduce a peptide expressed according to the present invention into the secretory pathway of a host cell, a secretion signal sequence (also referred to as a leader sequence) can be provided in the recombinant vector. The secretion signal sequence is linked to the DNA sequence encoding the peptide in the correct reading frame. The secretion signal sequence is usually located at the 5' end of the DNA sequence encoding the peptide. The secretion signal sequence may be a secretion signal sequence, typically associated with a peptide, or may be derived from a gene encoding another secreted protein.

A person skilled in the art knows a method of separating the DNA sequence encoding the peptide of the present invention, the promoter and optional terminator, and/or the secretion signal peptide sequence, and inserting it into a suitable vector containing information necessary for replication.

The host cell into which the DNA sequence or recombinant vector will be introduced can be any cell capable of producing the peptide of the present invention, including bacteria, yeast, fungi, and higher eukaryotic cells. Examples of suitable host cells that are well known and used by those skilled in the art include, but are not limited to: E. coli, S. cerevisiae, or mammalian BHK or CHO cell lines.

The present invention relates to a medicament or pharmaceutical composition containing the above analogue of GLP-1(7-37), and also relates to the use of said analogue for the preparation of a medicament, for example, the use for the preparation of a medicament for the treatment or prevention of diabetes (preferably diabetes II type), complications of diabetes (eg, diabetic nephropathy, diabetic heart disease), and lowering blood glucose levels or improving glucose tolerance.

In yet another aspect, the invention also relates to a method for preventing or treating diabetes (e.g., type I and II diabetes), complications of diabetes (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic ophthalmopathy, diabetic nephropathy, diabetic heart disease), lowering blood glucose (eg, fasting blood glucose and postprandial blood glucose) or increasing glucose tolerance by administering the aforementioned GLP-1(7-37) analog or derivative thereof to the subject. In another aspect, the present invention also relates to the use of the aforementioned GLP-1(7-37) analog or derivative thereof for the manufacture of a medicament for the prevention or treatment of diabetes (e.g., type I and II diabetes), complications of diabetes (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic ophthalmopathy, diabetic nephropathy, diabetic heart disease), decreased blood glucose levels (eg, fasting blood glucose and postprandial blood glucose), or increased glucose tolerance.

In yet another aspect, the present invention relates to a pharmaceutical composition, preparation or kit containing the above analogue of GLP-1(7-37).

The present invention also relates to a pharmaceutical composition, formulation or kit containing a derivative of the above GLP-1(7-37) analogue.

The pharmaceutical composition according to the present invention, in addition to the active ingredient, which is a GLP-1(7-37) analogue or its derivative or salt, also contains a pharmaceutically acceptable excipient. Those skilled in the art are familiar with pharmaceutically acceptable excipients such as non-toxic excipients, stabilizers, diluents, carriers, diluents, or other formulation adjuvants. For example, diluents, excipients such as microcrystalline cellulose, mannitol, etc.; fillers such as starch, sucrose, etc.; binders such as starch, cellulose derivatives, alginate, gelatin and/or polyvinylpyrrolidone; disintegrants such as calcium carbonate and/or sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; carriers, solvents such as water, saline, kaolin, bentonite, etc.; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc. In addition, the pharmaceutical composition according to the present invention is preferably an injectable dosage form.

The present invention also relates to a method for reducing islet β cell apoptosis, enhancing islet β cell function, increasing the number of islet β cells, and/or restoring glucose sensitivity of islet β cells, comprising administering an effective amount of the above analog, derivative or drug, pharmaceutical composition to a subject in need.

The present invention also relates to the use of the above analogue, derivative or drug, a pharmaceutical composition for the preparation of a medicament for reducing islet β-cell apoptosis, enhancing islet β-cell function, increasing the number of islet β-cells and/or restoring the sensitivity of islet β-cells. to glucose.

In the present invention, the terms "GLP-1(7-37) polypeptide", "GLP-1(7-37) polypeptide analogue" and "GLP-1(7-37) analogue" may be used interchangeably and refer to a polypeptide containing the following amino acid sequence:

where X ₈ is selected from V, T, I, L, G or S; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; X ₃₇ is G or K. The GLP-1(7-37) polypeptide analog is linked to the extension to form a GLP-1(7-37) polypeptide analog derivative. In particular, the invention relates to an acylated derivative of a GLP-1(7-37) analogue. Compared with semaglutide, which is currently considered the best drug, the acylated derivative not only has a significant therapeutic effect, but also shows an approximately 1-fold increase in in vivo duration of activity, which means that in humans, the dosing frequency is at least weekly. intervals, even two-week intervals or longer intervals can be achieved.

The terms "derivative of GLP-1(7-37) analog", "acylated derivative of GLP-1(7-37) analog", "derivative of GLP-1(7-37)" and "derivative of GLP-1" according to the present invention may 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 extension portion (eg, a fatty acid) is dissolved;

(2) adjusting the pH to 4-5 to stop the reaction, holding until a precipitate forms, and then collecting the precipitate; and

(3) adding TFA to the precipitate and adjusting the pH to 7.5-8.5 to stop the reaction.

In a preferred embodiment, the above method comprises adding triethylamine to the GLP-1 analogue solution.

In a preferred embodiment, the above extension (for example, a fatty acid) is dissolved in a solution of acetonitrile.

An exemplary production method according to the present invention includes:

(1) providing a solution of the GLP-1(7-37) analog and adjusting the pH to 9-12;

(2) then adding triethylamine to the solution obtained in step (1);

(3) weighing a fatty acid of the following structure, and taking an amount not less than 2 times the amount of the GLP-1 analog (molar ratio), preferably not less than 3 times the amount of the GLP-1 analog, and dissolving it in acetonitrile;

(4) mixing the GLP-1 analog solution obtained in step (2) with the fatty acid solution obtained in step (3) and keeping at a low temperature, for example, for one hour;

(5) adjusting the pH to 4-5 to stop the reaction, keeping at a low temperature for acid precipitation, and then collecting the precipitate;

(6) adding TFA to the acid-precipitated sample obtained in step (5) to a final polypeptide concentration of 5-15 mg/mL, holding for 0.5-2 hours, and dropping an alkaline solution such as NaOH into the reaction solution, adjusting the pH to 7.5-8.5 to stop the reaction;

(7) isolation and purification of the obtained product.

The present invention relates to the preparation of a pharmaceutical composition containing a GLP-1(7-37) analogue derivative or a pharmaceutically acceptable salt thereof. In some embodiments, the GLP-1(7-37) analog derivative or pharmaceutically acceptable salt thereof according to the present invention is present at a concentration of 0.1-25 mg/mL, preferably present at a concentration of 0.1-10.0 mg/mL. In a preferred embodiment, the pharmaceutical composition has a pH of 3.0-9.0. In a preferred embodiment, the pharmaceutical composition may further comprise a buffer system, a preservative, a surface tension modifying agent, a chelating agent, a stabilizer, and a surfactant. In some embodiments, the drug or formulation described herein is an aqueous drug or formulation, for example, typically a solution or suspension. In a particular embodiment of the present invention, the drug or preparation is a stable aqueous solution. In other particular embodiments of the present invention, the drug or preparation is a lyophilized preparation, and a solvent and/or diluent is added to it before use.

The present invention also relates to a medical box or kit containing the above pharmaceutical composition, preparation or drug. In addition to the above drug or preparation, the medical box or kit also contains another drug, pharmaceutical compound or composition that can be used in combination with the specified pharmaceutical composition, drug or drug, for example, another drug, pharmaceutical compound or composition may be selected from antidiabetic drugs, drugs for the treatment and/or prevention of complications caused by or associated with diabetes. Examples of these drugs include: insulin, sulfonylureas, biguanides, 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, agonists TNF, corticotropin-releasing factor agonists, 5HT, cerulein agonists, ganglion peptide antagonists, growth hormone, thyrotropin-releasing hormone agonists, TRβ agonists; histamine H3 receptor antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogs, etc. In some embodiments, the implementation of the pharmaceutical composition, drug, drug and other drugs, pharmaceutical compounds or compositions according to the present invention are placed in separate containers.

The present invention also relates to a method for preventing or treating diabetes (e.g., type I and II diabetes), complications of diabetes (e.g., diabetic vasculopathy, diabetic neuropathy, diabetic ophthalmopathy, diabetic nephropathy, diabetic heart disease), lowering blood glucose levels (e.g., fasting blood glucose and postprandial blood glucose) comprising: administering the above analog, derivative or drug, pharmaceutical composition to a subject in need, wherein the analog, derivative or drug, pharmaceutical composition and other drug, pharmaceutical compound or composition are used in combinations, for example, another drug, pharmaceutical compound or composition may be selected from antidiabetic drugs, drugs for the treatment and/or prevention of complications caused by or associated with diabetes. Examples of these drugs include: insulin, sulfonylurea, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of liver enzymes involved in stimulating gluconeogenesis and/or glycogenolysis, modulators of glucose uptake, CART agonists; NPY antagonists, PYY agonists, PYY2 agonists, PYY4 agonists, TNF agonists, corticotropin-releasing factor agonists, 5HT, cerulein agonists, ganglion peptide antagonists, growth hormone, thyrotropin-releasing hormone agonists, TRβ agonists; histamine H3 receptor antagonists, lipase/amylase inhibitors, gastric inhibitory polypeptide agonists or antagonists, gastrin and gastrin analogs, etc. In preferred embodiments, the diabetes is type II diabetes or diabetic nephropathy.

"Diabetes complication" in the present invention refers to a disease associated with damage or dysfunction of other organs or tissues of the body caused by poor control of blood glucose in diabetes, including damage or dysfunction of the liver, kidneys, heart, retina, nervous system, etc. . Complications of diabetes can be divided into five aspects: 1. cardiovascular disease: includes microvascular lesions of the heart and large vessels, cardiomyopathy, cardiac autonomic neuropathy, which is the main cause of death in patients with diabetes; 2. cerebrovascular disease: refers to intracranial macrovascular and microvascular diseases caused by diabetes, mainly manifested as cerebral atherosclerosis, ischemic cerebrovascular disease, intracerebral hemorrhage, cerebral atrophy, etc.; 3. renal vascular disease: mainly manifested as diabetic nephropathy, which is one of the most serious comorbidities in diabetic patients; 4. arterial disease of the lower extremities: mainly manifested as diabetic foot; 5. microvascular disease of the fundus: mainly manifests as diabetic retinopathy.

The present invention is further illustrated by the following examples. However, the described examples should not be construed as limiting the scope of patent protection. The features (singly and in any combination) disclosed in the foregoing description and the following examples may be materials used to implement the present invention in completely different forms, and they can be combined in an arbitrary manner. In addition, public documents are cited in the present invention, and these documents are intended to clearly describe the present invention. Their entire contents are incorporated herein by reference as if they were reproduced in their entirety in this document.

BRIEF DESCRIPTION OF GRAPHICS

1 shows the glucose-lowering effects of various GLP-1 acylated derivative molecules in type II diabetic db/db mice.

Figure 2 shows a trend plot of the effects of various doses of M0, M4 and semaglutide on fasting blood glucose levels in diabetic mice.

Figure 3 shows the effects of different doses of M0, M4 and semaglutide on random blood glucose in diabetic mice.

Figure 4 shows the effects of various doses of M0, M4 and semaglutide on the area under the blood glucose curve in diabetic mice.

Figure 5 shows a trend graph of the resistance of M4 molecules and semaglutide to degradation by pepsin.

Figure 6 shows a trend graph of the resistance of M4 molecules and semaglutide to degradation by trypsin.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

The invention will now be described with specific examples. Unless otherwise indicated, it can be implemented according to the methods listed in experimental manuals such as "Molecular Cloning: A Laboratory Manual" and "Cells: A Laboratory Manual" familiar to those skilled in the art, as well as manuals for conducting CFDA (China State Food and Drug Administration) experiments. Among them, all of the raw materials used are commercially available products that can be purchased through public channels.

Example 1 Construction of GLP-1 Analog Expression Plasmids

DNA construction Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)

The 6-His tag, the SUMO tag, and the sequence of the gene encoding Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37) (SEQ ID NO: 7) were sequentially hybridized in order, and the gene fragment (SEQ ID NO: 18) was obtained by chemical synthesis. Through the BamHI and XhoI sites, the above fragment was inserted into the prokaryotic expression plasmid pET-24(+) and confirmed by sequencing. The resulting expression plasmid for transformation analysis was named pET-24(+)-His-SUMO-Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37).

According to the method described above, the corresponding expression plasmids of the following peptides were sequentially constructed:

Val ⁸ Glu ²² Lys ²⁶ Arg ³⁴ -GLP-1(7-37) (coding gene - SEQ ID NO: 3),

Val ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37) (coding gene - SEQ ID NO: 11),

Val ⁸ Glu ²² Lys ¹⁹ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 5),

Val ⁸ Glu ²² Lys ²⁷ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 9),

Val ⁸ Glu ²² Lys ³⁴ Arg ²⁶ -GLP-1(7-37) (coding gene - SEQ ID NO: 13),

Val ⁸ Glu ²² Arg ^26.34 Lys ³⁶ -GLP-1(7-37) (coding gene - SEQ ID NO: 15),

Val ⁸ Glu ²² Arg ^26.34 Lys ³⁷ -GLP-1(7-37) (coding gene - SEQ ID NO: 17),

Thr ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 20),

Ile ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 22),

Leu ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37) (coding gene - SEQ ID NO: 24),

Gly ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 26),

Ser ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37) (coding gene - SEQ ID NO: 28),

Thr ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 30),

Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 32),

Leu ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37) (coding gene - SEQ ID NO: 34),

Gly ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 36),

Ser ⁸ Gm ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37) (coding gene - SEQ ID NO: 38).

Example 2 Expression of Fusion Proteins

The DNA construct described in Example 1 was transformed into BL21 host cells (TrabsGenBiotech., catalog number CD601) to obtain the target protein of the present invention. 50 µl of competent BL21 cells were placed in an ice bath to thaw, then DNA of interest was added and gently shaken, and placed in an ice bath for 30 min. A heat shock was then performed in a water bath at 42° C. for 30 seconds, followed by a rapid transfer of the centrifuge tube to an ice bath for 2 minutes without shaking the centrifuge tube during this process. 500 µl of sterile LB medium (without antibiotics) was added to a centrifuge tube, then mixed and cultured at 37°C, 180 rpm for 1 hour to isolate the bacteria. 200 µl of transformed competent cells were pipetted and added to a kanamycin-resistant LB agar plate, distributing the cells evenly; placed the plate at 37°C until the liquid was absorbed, then turned the plate over and incubated at 37°C overnight. The next day, monoclonal colonies in the transformation dish were collected using an inoculation ring to inoculate 15 ml of sterile LB medium (containing antibiotics), then cultured at 30° C. overnight.

Example 3 Fermentation of Recombinant GLP-1 Analogs

50 μl of bacterial suspension (bacterial suspension expressing GLP-1) was added to 50 ml of LB medium while adding 50 μl of kanamycin, mixed and placed in a shaker at a constant temperature of 30°C, then inoculated overnight. 10 ml of bacterial suspension, inoculated overnight, was added to 1000 ml of LB medium, while adding 1000 μl of kanamycin; then shaken and placed in a shaker at 37° C. at 200 rpm. After inoculation for 4 hours, IPTG (isopropyl-β-D-1-thiogalactopyranoside) was added to the medium at a final concentration of 0.1 mol/l, then shaken and placed in a shaker at 30°C and 180 rpm to induce expression during the night. The bacterial suspension, after overnight expression, was centrifuged at 13,000 g for 60 min. The yield of bacteria was about 4 g of bacteria/l of fermentation broth, and the expression of the protein of interest, determined using SDS-PAGE (protein electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate), was up to about 40%.

Example 4 Purification of Recombinant GLP-1 Analogs

100 g of the cell suspension was weighed and resuspended in 500 ml of 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, then sonicated in an ultrasonic cell disruptor for 30 minutes to disrupt the cells. The homogenate was centrifuged at 13000 g for 60 min at 4°C. After centrifugation, the supernatant was collected as a sample for Ni column chromatography.

The resulting supernatant was concentrated with Chelating Sepharose FF pre-equilibrated with 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole (equilibration fluid 1); after washing with equilibrate liquid 1, it was eluted with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl and 0.3 M imidazole (eluent). According to SDS-PAGE analysis, the purity of the GLP-1 intermediate obtained by the above purification process exceeded 70%.

Excision of the Sumo tag sequence with ULP enzyme: Add 20 mM buffer PB, pH 7.4 to intermediate to make a 3-fold dilution, add ULP enzyme to mix and digest overnight at 4°C at a ratio of ULP enzyme intermediate equal to 1 :150. The degree of cleavage according to the analysis of SDS-PAGE was almost 100%.

Purification of GLP-1 analog: product obtained after digestion was concentrated using Tosoh Butyl 550C medium pre-equilibrated with 20 mM Na ₂ HPO ₄ , 0.7 M NaCl (equilibrium liquid 2), after washing with equilibration liquid 2 it was eluted with 20% ethanol , and the purity according to the analysis of SDS-PAGE was about 90%.

0.2 M Na2HP04 was added to the eluted sample to achieve a final concentration of 20 mM Na ₂ HPO ₄ , then the pH was adjusted to 4.8-5.0 with 1 M citric acid for acid precipitation at 4° C. overnight. The yield was over 90% according to SDS-PAGE analysis. Spent centrifugation at 13000 g for 30 minutes at 4°C, then collected the precipitate and stored at -20°C.

Example 5 Derivation of GLP-1 Analogs

Preparation of GLP-1 analog derivative as shown below of N-ε peptide ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino ]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (abbreviated as M2)

1. Fatty acid modification: To the precipitate of Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37) obtained and collected in the above example, water was added to prepare a solution with a concentration of 4-6 mg/ml, then, 1M sodium hydroxide was added to adjust the pH to 11.0-11.5 while shaking to completely dissolve the protein, and the peptide concentration was quantified by HPLC (high performance liquid chromatography). The fatty acid powder was weighed and dissolved in acetonitrile at a molar ratio of peptide to fatty acid of 1:4 (the structure was as follows). Two thousandths of triethylamine was added to this polypeptide solution, mixed with the fatty acid solution, and the mixture was left at 4° C. for one hour.

The sample was diluted five times with water, then adjusted to pH 4.8 with 1 M citric acid (or 10% acetic acid) to stop the reaction, kept at 4°C for acid precipitation for 10 minutes, centrifuged at 13,000 g after acid precipitation and centrifuged at 4°C for 30 min, and then stored the precipitate at -80°C.

2. Fatty acid deprotection and purification: TFA was added to the acid-precipitated sample to a final peptide concentration of about 10 mg/mL, then shaken to dissolve the precipitate, left at room temperature to deprotect for 30 minutes, and 4 M NaOH in reaction solution to adjust the pH to 7.5-8.5 to stop the reaction.

Using preparative HPLC (Shimadzu LC-8A), the reaction liquid was pumped after stopping the reaction into UniSil 10-120 C18 (purchased from Suzhou Nanomicro Technology Co., Ltd.) pre-equilibrated with 10 mM ammonium acetate, 20% ethanol (equilibrium liquid 3) , for concentration. After washing with equilibrate liquid 3, a gradient of 0-100% eluent (10 mM ammonium acetate, 80% ethanol) was used for elution. The elution peak was collected and the purity according to RP-HPLC (reverse phase high performance liquid chromatography) analysis was about 90%.

The elution peak was diluted three times with water, then the pH of the acid precipitation was adjusted to 4.80 and the acid precipitation was carried out at 4° C. for 30 minutes. After centrifugation, PBST buffer (pH 7.0) was added to the sediment to restore it, then frozen and stored at -80°C.

In accordance with the above method, the following peptides were sequentially obtained:

peptide N-ε ²⁶ -[2-(2-[2-(2-[2-(2[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl ]Val ⁸ Glu ²² Lys ²⁶ Arg ³⁴ -GLP-1(7-37) (M0),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl])(Val ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-3 7))(M4),

peptide N-ε ¹⁹ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Lys ¹⁹ Glu ²² Arg ^26.34 LP-1(7-37))(M1),

peptide N-ε ²⁷ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Lys ²⁷ Arg ^26.34 -GLP-1(7-37))(M3),

peptide N-ε ³⁴ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37)) (M5),

peptide N-ε ^3b -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Arg ^26.34 Lys ³⁶ -GLP-1(7-37))(M6),

peptide N-ε ³⁷ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Arg ^26.34 Lys ³⁷ -GLP-1(7-37))(M7);

peptide N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Thr ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37))(M8),

peptide N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Ile ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (M9),

peptide N-ε ²³ -(2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Leu ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37))(M10),

peptide N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Gly ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (M11),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl)](Ser ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (M12);

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(8)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl])(Thr ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37))(M13),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Ile ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37) )(M14),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Leu ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37))(M15),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Gly ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37))(M16),

peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Ser ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37))(M17).

Example 6 In Vitro Determination of the Activity of GLP-1 Analog Derivatives in RIN-m5F Cells

RIN-m5F cells with good culture status were selected. Cells were then harvested, counted, and cell suspension was prepared from them at 1×10 ⁵ cells/ml in RPMI1640 minimal medium. The cell suspension was seeded into a 96-well cell culture plate at 100 μl per well, then incubated overnight at 37° C. and 5% CO ₂ . The in vitro activity of derivatives of GLP-1 analogs was measured using a cAMP (cyclic adenosine monophosphate) assay kit (Promega): samples diluted with assay medium to 300 ng/ml were prepared (Aib, M0, M1, M2, M3, M4, M5, M6 and M7), then a 3-fold serial dilution was performed in 96-well plates, giving a total of 8 concentrations, and making 2 duplicate wells for each dilution, where M0, M1, M2, M3, M4, M5, M6 and M7 were obtained, as described above and Aib is:

peptide N-ε ²⁶ -[2-(2-[2-(2-[2-(2[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl ][Aib ⁸ , Arg ³⁴ ]GLP-1-(7-37) (see CN 101133082 B, example 4), the trade name is semaglutide and was prepared according to the method disclosed in CN 101133082 B.

The resulting cell culture plate was taken out, then the medium was discarded and blotted dry on filter paper. Sample solutions were respectively transferred to a cell culture plate at 40 μl/well; treated with open lid for 15 min at 37°C and 5% CO ₂ . The cell culture plate was removed from the incubator, then 10 μl of CD solution (cAMP assay kit, Promega) was added to each well, the cell culture plate was kept at 22-25° C. and shaking horizontally at 500 rpm for 20 minutes. 50 μl of KG solution (cAMP assay kit, Promega) was added to each well, then shaken horizontally at 22-25°C, 500 rpm and hidden from light for 10 minutes. The chemiluminescence value was read using a Molecular Devices SpectraMax L chemiluminescence instrument, completing the measurement in 30 minutes. Sample EC50 was calculated using four parameter regression in softmax Pro software.

In vitro pharmacodynamics in PJN-m5F cells shows that the in vitro activities of semaglutide, M2, M4, M5 and M7 are comparable and generally slightly higher than those of M0, M1, M3 and M6.

Example 7 In Vitro Determination of the Activity of GLP-1 Analog Derivatives in HEK293/CRE-Luc/GLPlPv Cells

Based on the fact that GLP-1 can bind to a receptor on the cell membrane, the HEK293/CRE-Luc/GLP1R cell line was constructed, cAMP response elements (CRE) are activated through a series of signaling, downstream luciferase expression is initiated, and the magnitude of this expression positively correlates with the biological activity of GLP-1. After adding the luciferase substrate, a chemiluminescent assay was performed to determine the luminous intensity, thereby determining the biological activity of GLP-1.

Materials for the experiment

96-well cell culture plate (white and opaque), DMEM (Dulbecco's modified Eagle's medium) (GIBCO), 0.05% TRIPSIN-EDTA (GIBCO), fetal bovine serum (GIBCO), G418, hygromycin B, Bright-Glo™ Luciferase Assay System Kit (Promega) and HEK293/CRE-luc/GLP1R cells.

Operations during the experiment

(1) Cell preparation: Cells were cultured until they grew rapidly and reached a sufficient number, discarded the culture medium in the culture flask, added 3 ml of Versene's solution, and shook once; then 2 ml of 0.05% TRIPSIN-EDTA digestion solution was added, the flask was covered and held for 1 minute, and then 6 ml of assay medium was added to stop the digestion; after centrifugation at 1000 rpm for 3 minutes, the supernatant was discarded, the cells were resuspended in 5 ml of assay medium and counted on a hemocytometer. Cell density was adjusted to a suitable range for later use with DMEM assay medium.

(2) Sample preparation: Derivatives of various GLP-1 analogs in Table 1 were diluted to 20 ng/ml in assay medium, then serially diluted to give 8 concentrations in 96-well plates, and assay medium was used instead of sample as a blank control well. For each diluted concentration, 2 duplicate wells were made.

(3) Supplementation culture: prepared solutions of control and test samples were transferred to a 96-well cell culture plate (white) by adding 50 μl of the solution to each well; then the prepared cell suspension was added, adding 50 μl of the suspension to each well; and then incubated for a certain period of time under conditions of 37°C and 5% CO ₂ .

(4) Chemiluminescent assay: Substrate was added, then the 96-well cell culture plate was taken out, 100 μl of Bright Glo reagent was added to each well, and left in the dark for 3 minutes.

(5) Reading: The determination was carried out with a SpectraMax L microplate chemiluminescence analyzer, then the plate was read for 30 minutes and the results recorded.

Pharmacodynamics in HEK293/CRE-Luc/GLP1R cells show that the in vitro activities of semaglutide, M2, M4, M9, M11, M14, M16 and M17 are comparable and generally slightly higher than those of M13.

Example 8 Study of the Glucose-Lowering Effect of Fatty Acid Modified GLP-1 Analog Derivatives in Normal Mice

Twenty-eight healthy female CD-1 mice aged 4-6 weeks were selected and divided into 4 groups, which were respectively injected subcutaneously with M2, M4, M0 and semaglutide (Aib) at a dose of 0.15 mg/kg body weight. 20% glucose intragastrically was administered before administration and at intervals of 6 hours, 1 day, 2 days, 3 days and 4 days after administration at a dose of 2 g/kg of body weight and fasting 6 hours before administration of glucose; then blood was taken from the tip of the tail at 0 hr, 0.5 hr, 1 hr, and 2 hr, respectively, after glucose administration, and the real-time blood glucose value was measured using blood glucose test strips from Roche; and the blood glucose AUC (area under the blood glucose versus time curve) was calculated within 0-120 minutes to obtain the degree of inhibition of blood glucose (Table 4).

Degree of blood glucose inhibition = [(AUC of mice blood glucose before administration - AUC of mice blood glucose after administration) / AUC of mice blood glucose before administration] × 100%

P-value: compared with blood glucose level before administration

From Table 4, it can be seen that the glucose-lowering effect of semaglutide in normal mice lasts about 2 days, and the glucose-lowering effect of M0 in normal mice lasts about 3 days; however, the glucose-lowering effects of M2 and M4 in normal mice are still evident at day 4, and the duration of their sustained glucose-lowering action in the body is significantly longer than that of semaglutide or M0, and at various time points after day 3 administration, the glucose-lowering effects of both M2 and M4 are also significantly stronger than semaglutide or M0.

Twenty-eight healthy female CD-1 mice aged 4-6 weeks were selected and divided into 4 groups, which were injected subcutaneously with M4, M5, M7 and M0 at a dose of 0.15 mg/kg body weight. 20% glucose was administered intragastrically before administration and at intervals of 6 hours, 1 day, 2 days, 3 days and 4 days after administration at a dose of 2 g/kg of body weight and fasting 6 hours before intragastric administration of 20% glucose; and draw blood from the tip of the tail at 0h, 0.5h, 1h and 2h after glucose administration, respectively, then measure the real-time blood glucose value with Roche blood glucose test strips, and calculate AUC of blood glucose (area under the blood glucose-time curve) within 0-120 min, obtaining the degree of inhibition of blood glucose (Table 5).

Degree of blood glucose inhibition = [(AUC of mice blood glucose before administration - AUC of mice blood glucose after administration) / AUC of mice blood glucose before administration] × 100%

From the results shown in tables 4 and 5, it can be seen that the glucose-lowering effects of M2 and M4 are better than those of M0 and semaglutide; and glucose-lowering effects of M2, M4, M5 and M7 are comparable and there is no significant difference between them.

Example 9 Glucose-Lowering Study Using ICR Mice

PTTH test on ICR mice: 30 ICR mice aged 4-6 weeks were selected and divided into 6 groups, 5 mice per group, and injected subcutaneously with M0, semaglutide, M2, M4, M5 and M7, respectively, at a dose of 0 15 mg/kg of body weight by a single injection. 20% glucose intragastrically was administered every day according to the schedule of 4 hours, 1 day, 2 days, 3 days, 4 days and 5 days, at a dose of 2 g/kg of body weight and fasting 6 hours before glucose administration; and bled from the tip of the tail at 0 hr, 0.5 hr, 1 hr, and 2 hr, respectively, after administration of 20% glucose, then measured the real-time blood glucose value using Roche blood glucose test strips. Blood was taken from the tip of the tail, the blood glucose value was measured in real time using blood glucose test strips from Roche, and the blood glucose AUC (area under the blood glucose versus time curve) was calculated in the range of 0-120 min, obtaining the degree of inhibition of glucose in the blood (table 6).

Degree of blood glucose inhibition = [(AUC of mice blood glucose before administration - AUC of mice blood glucose after administration) / AUC of mice blood glucose before administration] × 100%

From the results in Table 6, it can be seen that the glucose level is maintained constant: the glucose-lowering effects of M4, M5, M2 and M7 can be maintained for at least 4 days, which is much better than that of M0 (only maintained for 3 days ) and semaglutide (only persists for 2 days), all of which are statistically significant.

Example 10 Glucose Lowering Pharmacokinetic Test in Type II Diabetic db/db Mice

Fifty 8-9 week old female db/db mice were divided into 10 equal groups based on body weight and pre-treatment fasting blood glucose (FBG) levels, 5 mice per group; and they were respectively administered by a single subcutaneous injection of vehicle, M2, M4, semaglutide, M9, M11, M13, M14, M16 and M17 at a dose of 10 ml/kg. The dosage was 0.05 mg/kg for each, and the administration time was set to 0 hours. The fasting blood glucose level was measured every day after fasting for 6-8 hours, and the fasting blood glucose level after administration was measured every day until the fasting blood glucose value of each animal of the test group returned to the value measured before introduction. The blood glucose value measured prior to administration is called the basal blood glucose value and was set to 0.

Change in fasting blood glucose (Δ: delta) = post-administration blood glucose value - pre-administration basal blood glucose value.

The results are shown in figure 1, it can be seen that from the 4th and 5th day, the glucose-lowering effects of M9, M13 and M14 are better than that of semaglutide, and also not lower than that of M2; however, the glucose-lowering effects of M11, M16 and M17 are lower than those of semaglutide on day 2.

Example 11 Glucose-lowering effect of various doses of semaglutide, M0 and M4 in type II diabetic db/db mice

Thirty-five 8-9 week old female db/db mice were divided into 7 equal groups based on body weight and area under the blood glucose curve (G-AUC) prior to administration, 5 mice per group; and they were respectively administered by a single subcutaneous injection of vehicle, M4 (0.15, 0.015 mg/kg), semaglutide (0.15, 0.015 mg/kg) and M0 (0.15, 0.015 mg/kg) at a dose of 10 ml /kg. The administration time was set to 0 hours, and every day after fasting for 7-8 hours, fasting blood glucose and OGTT (oral glucose tolerance test) were determined, then 10% glucose was administered intragastrically at a dose of 1 g / kg body weight and then bled from the tip of the tail to measure real-time blood glucose levels at 0, 0.5, 1, and 2 h after glucose loading. After administration, the blood glucose level was measured every day before fasting as a random blood glucose level until the fasting blood glucose value of each animal in the test group returned to the pre-administration value. All of the basal blood glucose value, the random blood glucose value and the blood glucose area under the curve (G-AUC) value determined prior to administration were used as baselines for evaluating drug efficacy and were set to 0 .

Change in blood glucose (Δ: delta) = post-injection blood glucose value - pre-injection basal blood glucose value.

Change in area under the blood glucose curve (Δ: delta) = area under the blood glucose curve after administration - area under the blood glucose curve before administration.

The results are presented in tables 7, 8 and 9 and also in FIG. 2, 3 and 4.

The results in Tables 7-9 and Figures 2-4 indicate the following: Fasting blood glucose: For M4, 123 hours after administration, this level in the 0.15 mg/kg dose group returns to baseline basal blood glucose before administration, and 99 hours after administration, this level in the 0.015 mg/kg dose group returns to the baseline basal blood glucose level before administration; for semaglutide, 51 hours after administration, this level in the 0.15 mg/kg dose group returns to the baseline basal blood glucose level before administration, and after 27 hours, this level in the 0.015 mg/kg group returns to the baseline basal glucose level in the blood before administration; for M0, 75 hours after administration, this level in the 0.15 mg/kg dose group returns to the baseline basal blood glucose level before administration, and 51 hours after administration, this level in the 0.015 mg/kg group returns to baseline basal the level of glucose in the blood before administration; where all fasting blood glucose reduction values in the 0.015 mg/kg M4 dose group at each measurement time point are not lower than those reported in the 0.15 mg/kg semaglutide or M0 dose group.

Random blood glucose level: for M4, 115 hours after administration, this level in the 0.15 mg/kg dose group returns to the baseline random blood glucose level before administration, and 115 hours after administration, this level in the 0.015 mg dosage group /kg returns to the baseline random blood glucose level before administration; for semaglutide, 67 hours after administration, this level in the 0.15 mg/kg dose group returns to the baseline random blood glucose level before administration, and 67 hours after administration, this level in the 0.015 mg/kg dose group returns to the baseline random the level of glucose in the blood before administration; for M0, 67 hours after administration, this level in the 0.15 mg/kg dose group returns to the base random blood glucose level before administration, and 67 hours after administration, this level in the 0.015 mg/kg dose group returns to the base random the level of glucose in the blood before administration; where all inhibitory effects on random blood glucose in the 0.015 mg/kg M4 dose group at each measurement time point are not lower than those in the 0.15 mg/kg semaglutide or M0 dose group.

Area under the blood glucose curve (G-AUC): for M4, 99 hours after administration, this parameter in the 0.15 mg/kg dose group returns to the baseline value of the area under the blood glucose curve before administration, and after 99 hours after administration, this parameter in the 0.015 mg/kg dose group returns to the base value of the area under the blood glucose level curve before administration; for semaglutide, 51 hours after administration, this parameter in the 0.15 mg/kg dose group returns to the baseline value of the area under the blood glucose level curve before administration, and 51 hours after administration, this parameter returns in the 0.015 mg/kg group to the base value of the area under the blood glucose level curve before administration; for M0, 51 hours after administration, this parameter in the 0.15 mg/kg dose group returns to the baseline value of the area under the blood glucose level curve before administration, and 27 hours after administration, this parameter returns in the 0.015 mg/kg group to the base value of the area under the blood glucose level curve before administration; where all values of the area under the blood glucose curve in the group with a dosage of 0.015 mg/kg M4 at each time point of the measurement are not lower than the indicated values in the group with a dosage of 0.15 mg/kg of semaglutide or M0.

These glucose lowering results show that after a single subcutaneous injection of M4, or semaglutide, or M0, each group shows a significant glucose-lowering effect, however, M4 has the best glucose-lowering effect. The glucose-lowering effect at a dosage of 0.015 mg/kg M4 is comparable to that for a dosage of 0.15 mg/kg semaglutide or a dosage of 0.15 mg/kg M0.

Example 12 Enzymatic Degradation Stability Study of M4 and Semaglutide

Pepsin (3200-4500 U/mg protein, from Sigma, catalog number: P6887), trypsin (approximately 10,000 AEU/mg protein, from Sigma, catalog number: T8003).

(1) Reaction solution

A: Pepsin reaction buffer: three 20 mM citrate phosphate buffers were prepared at different pH (2.6, 4.0 and 7.4), then 0.005% Tween 20 and 0.001% BSA (bovine serum albumin) were added to prepare the reaction buffer pepsin.

B: Trypsin reaction buffer: Three 20 mM citrate phosphate buffers were prepared at different pH (4.0, 6.8 and 8.0), then 0.005% Tween 20 and 0.001% BSA were added to prepare the trypsin reaction buffer.

C: Artificial gastric juice containing pepsin (SGF): was obtained by taking 5 ml of 0.1 M hydrochloric acid and adding and dissolving 0.019 g of pepsin.

D: Artificial intestinal juice containing trypsin (SIF): was obtained by taking 0.0684 g of potassium dihydrogen phosphate, adding 2.5 ml of water to dissolve it, adding 0.77 ml of 0.2 M sodium hydroxide solution and 5 ml of water, and then adding and dissolving 0.1001 g of trypsin; measured pH was 6.82, then diluted by adding water to 10 ml.

(2) Sample preparation

Samples of M4 and semaglutide were taken and respectively diluted to 1.33 mg/ml with buffer PB at pH 7.4 as test sample stock solutions.

(3) Pepsin degradation experiments

An appropriate amount of stock solution of each test sample was taken, respectively, then diluted to 0.06 mg/ml with different pH pepsin reaction buffers; the reaction solution of each group was divided into 1 ml/tube, for a total of 7 tubes, then thoroughly mixed and incubated in a water bath at 37°C for 30 minutes. 1 tube without SGF was taken out as the 0 point of the reaction without enzyme (recorded as point -5 min), and then another 6 tubes were taken out, SGF was added to them separately and mixed well, to one of them immediately added the appropriate volume of 1 M NaOH to stop the reaction as the 0 point after adding the enzyme (recorded as the 0 min point), and the remaining 5 tubes were kept at 37°C for the reaction to proceed; and one group was taken out after 5 min, 10 min, 20 min, 35 min and 50 min, respectively, and an appropriate volume of 1 M NaOH, respectively, was added to stop the reaction. All tubes in all experimental groups were checked that the total volume after the termination of the reaction was the same.

(4) Trypsin decomposition experiments

Take the appropriate amount of the test sample stock solution, respectively, then dilute it to 0.06 mg/ml with trypsin reaction buffers of different pH; the reaction solution of each group was divided into 1 ml/tube, for a total of 7 tubes, then thoroughly mixed and incubated in a water bath at 37°C for 30 minutes. 1 tube without SIF was taken out as the 0 point of the enzyme-free reaction (recorded as point -5 min), and then the other 6 tubes were taken out to separately add SIF to them, and mixed well, the appropriate volume of 6 was immediately added to one of them M HCl to stop the reaction as 0 point after the addition of the enzyme (was recorded as point 0 min), and the remaining 5 tubes were kept at 37°C for the reaction to proceed; and one group was taken out, respectively, after 5 minutes, 10 minutes, 20 minutes, 35 minutes and 50 minutes, to add the appropriate volume of 6 M HCl, respectively, to stop the reaction. All tubes in all experimental groups were checked that the total volume after the termination of the reaction was the same.

Samples from the enzymatic degradation experiment were subjected to HPLC analysis. The peak area of the main peak of the sample without enzyme reaction at point 0 (written as -5 min) was used as the base peak area, and the remaining percentage of the main peak area at various time points after the addition of the enzyme was calculated.

Experimental data on the decomposition of pepsin (n=3) indicate (figure 5) that the rate of decomposition of molecules M4 and semaglutide under acidic conditions (pH 2.6) are comparable, which is associated with the highest activity of pepsin at this pH; at neutral pH 7.4, both molecules practically do not decompose, and under these conditions, the activity of the gastric protein is the lowest; while at pH 4.0 the degradation rate of semaglutide is significantly faster than that of M4, the t1/2 of the former is about 10 minutes and the t1/2 of the latter is about 45 minutes, indicating that M4's ability to resist pepsin degradation is significantly better than semaglutide.

Experimental data on decomposition by trypsin (n=4) indicate (figure 6) that the decomposition rates of these two substances are almost the same under conditions of pH 6.8 and 8.0, since trypsin has the highest activity in this pH range; M4 and semaglutide also show the ability to resist trypsin degradation under pH 4.0 conditions, and there is practically no difference between them.

--->

Sequence listing

<110> Hangzhou Sciwind Biosciences Co., Ltd.

<120> GLP-1 acylated derivative

<130> None

<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 2

<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 1520

Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

253035

<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 4

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 5

<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 6

<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

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>7

<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 9

<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>11

<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 1520

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

<210> 39

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> formula of the amino acid sequence contained in the GLP-1 analog

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa select from Val, Thr, Ile, Leu, Gly or Ser

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> Xaa is Tyr or Lys

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> Xaa is Gln or Lys

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> Xaa is Glu or Lys

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> Xaa is Ala or Lys

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> Xaa is Arg or Lys

<220>

<221> MISC_FEATURE

<222> (30)..(30)

<223> Xaa is Arg or Lys

<220>

<221> MISC_FEATURE

<222> (31)..(31)

<223> Xaa is Gly or Lys

<400> 39

His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Glu

1 5 10 15

Xaa Ala Ala Arg Xaa Phe Ile Xaa Trp Leu Val Xaa Gly Xaa Xaa

20 25 30

<210> 40

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> semaglutide sequence

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Aib

<400> 40

His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<---

Claims (28)

1. A derivative of the GLP-1(7-37) analog or a pharmaceutically acceptable salt thereof, wherein the GLP-1(7-37) analog contains the amino acid sequence of the following formula: HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ (SEQ ID NO: 39), where X ₈ is selected from V; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; and X ₃₇ is G or K; with the proviso that only one of X ₁₉ , X ₂₃ , X ₂₇ , X ₃₀ , X ₃₄ , X ₃₆ or X ₃₇ is a K moiety, and the derivative contains an extension linked to the K residue of the GLP-1(7-37) analog via a linker, where the extension is 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-, where the linker is

, where m is equal to 1; n is 1. 2. A derivative or a pharmaceutically acceptable salt thereof according to claim 1, which is any derivative selected from the group consisting of: peptide N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Lys ²³ Arg ^26.34 -GLP-1(7-37)) (M2), peptide N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Lys ³⁰ Arg ^26.34 -GLP-1(7-37)) (M4), peptide N-ε ³⁴ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl](Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37)) (M5), peptide N-ε ³⁷ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) acetyl])(Val ⁸ Glu ²² Arg ^26.34 Lys ³⁷ -GLP-1(7-37)) (M7), or a pharmaceutically acceptable salt thereof. 3. A method for producing a derivative or a pharmaceutically acceptable salt thereof according to claim 1, including: (1) mixing the solution in which the GLP-1 analog is dissolved with the solution in which the extension is dissolved; (2) adjusting the pH to 4-5 to stop the reaction, holding until a precipitate forms, and then collecting the precipitate; and (3) adding TFA (trifluoroacetic acid) to the precipitate and adjusting the pH to 7.5-8.5 to stop the reaction. 4. The method of claim 3, further comprising: adding triethylamine to the solution in which the GLP-1 analog is dissolved, followed by mixing with the solution in which the extension is dissolved. 5. The method according to claim 3, where the solution of the extension part is obtained in acetonitrile. 6. Pharmaceutical composition for the prevention and/or treatment of diabetes or complications of diabetes, containing a therapeutically effective amount of a derivative or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable excipient. 7. A method for preventing and/or treating diabetes or complications of diabetes, comprising administering a prophylactically or therapeutically effective amount of a derivative or a pharmaceutically acceptable salt thereof according to claim 1 to a subject. 8. The method of claim 7 wherein the complication of diabetes is diabetic nephropathy. 9. A method for lowering blood glucose levels, increasing glucose tolerance, reducing islet β-cell apoptosis, enhancing islet β-cell function, increasing the number of islet β-cells, and/or restoring the sensitivity of islet β-cells to glucose, comprising administering a therapeutically effective the amount of a derivative or a pharmaceutically acceptable salt thereof according to claim 1 subject. 10. The method of claim 9 wherein said blood glucose lowering comprises lowering fasting blood glucose and/or postprandial blood glucose. 11. GLP-1(7-37) analogue containing a polypeptide consisting of the following amino acid sequence: HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ (SEQ ID NO: 39), where X ₈ is selected from V; X ₁₉ is Y or K; X ₂₃ is Q or K; X ₂₇ is E or K; X ₃₀ is A or K; X ₃₄ is R or K; X ₃₆ is R or K; X ₃₇ is G or K; and only one of X ₁₉ , X ₂₃ , X ₂₇ , X ₃₀ , X ₃₄ , X ₃₆ or X ₃₇ is K. 12. A product for the prevention and/or treatment of diabetes or complications of diabetes, containing: a container containing a pharmaceutical composition according to claim 6, and a package insert containing instructions for using the pharmaceutical composition. 13. A product according to claim 12, further comprising a container containing one or more other drugs. 14. The product of claim 13 wherein the one or more other drugs are other drugs for the treatment of diabetes or complications of diabetes.

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