WO2019201333A1 - Glp-1 derivative and therapeutic use thereof - Google Patents

Abstract

Provided are a GLP-1(7-37) polypeptide analogue, a fatty-acid-modified derivative thereof and a drug containing the derivative. In addition, also provided are a method for preparing the derivative and the use of same in the preparation of drugs.

Description

GLP-1 derivatives and their therapeutic uses Technical field

The invention belongs to the field of polypeptide technology. In particular, the invention relates to fatty acid modified derivatives of GLP-1 (7-37) polypeptide analogs. Further, the present invention relates to a method for producing the peptide derivative, a medicament containing the peptide derivative, a use in the preparation of a medicament, and the like.

Background technique

GLP-1 is an endogenous hormone that promotes insulin secretion, mainly secreted by intestinal L-cells, and plays a role in balancing insulin and glucose levels. GLP-1 includes molecular forms such as GLP-1 (1-37), GLP-1 (1-36), GLP-1 (7-37) glycine derivatives, and GLP-1 (7-36) NH ₂ . It is generally believed that the latter two have the same biological activity. GLP-1 (1-37) secreted by intestinal mucosal L cells is inactive and requires further hydrolysis to excise the N-terminal 6 amino acids to become active GLP-1 (7-37). GLP-1 (7-37) is present in the body for a short period of time and is rapidly degraded. In order to maximize its half-life in the blood, many studies and attempts have been made. Currently approved GLP-1 drugs on the market mainly include Exenatide-4 isolated from lizard saliva, and human GLP-1 similar to fatty acid, antibody Fc fragment or serum albumin modification. Things. The half-life of Exenatide-4 is too short, only 2-4 hours, requiring at least two injections a day. Novo Nordisk's fatty acid-modified liraglutide is most effective in reducing hemoglobin glycosylation with fewer side effects, but the disadvantage is that the in vivo half-life is only 13 hours and requires daily dosing. In order to further extend the half-life in vivo and reduce the frequency of administration, in recent years, amino acid sequence mutants and modified long-acting GLP-1 analogs such as FC, fatty acid or albumin have been developed. Such as Lula Rubide from Lilly and the Semaglutide from Novo Nordisk. The half-life of these long-acting GLP-1 analogs in humans can be extended to varying degrees, up to the frequency of dosing once a week. Since GLP-1 analogs require long-term injection administration, attempts have been made to find longer-acting drugs to further improve patient compliance.

The inventors of the present application have developed a new GLP-1 analogue and its derivative under long-term research, and under the same experimental conditions, compared with the currently recognized best drug somaglutide, in vitro viability and Somaglutide is equivalent; its duration of activity in vivo can be increased by about 1 fold, meaning that the frequency of administration of at least weekly interval administration, even every two weeks interval or longer interval can be achieved in the human body, and When the dosage is reduced to 1/10 of the amount of somaglutide, the hypoglycemic and weight-reducing effects are not lower than that of somatoglutide, which has a better application prospect.

Summary of the invention

It is an object of the present invention to provide a novel GLP-1 (7-37) analog, an acylated derivative of the analog. Further, the present invention provides a method for producing the analog or derivative, a pharmaceutical composition, an article comprising the same, or a derivative thereof, and diseases thereof for preventing and treating disorders of glucose metabolism and/or disorders of fat metabolism, such as diabetes Uses in diabetic complications, fatty liver, cirrhosis, and obesity.

Specifically, in one aspect, the present invention provides a derivative of a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analogue comprises an amino acid sequence of the following formula Peptide:

H X ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇

Wherein 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, and 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,

The derivative comprises an extension joined to the K residue, wherein the extension is

Wherein x is an integer from 4 to 38.

Wherein, the extension is preferably: HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-, HOOC(CH ₂ ) ₁₈ CO -, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO- and HOOC(CH ₂ ) ₂₂ CO-, more preferably HOOC(CH ₂ ) ₁₆ CO-.

In a preferred embodiment, the extension of a derivative of a GLP-1 analogue of the invention, or a pharmaceutically acceptable salt thereof, is linked to the K residue of GLP-1 via a linker. The joint may be of the following structure:

Wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; p is an arbitrary integer from 1 to 8.

Preferably, the joint is:

Wherein m is 1 or 2; n is 1 or 2; and p is an arbitrary integer from 1 to 5.

More preferably: the joint is:

Where m is 1, and n is 1 or 2.

The invention also relates to GLP-1 (7-37) analogs, the analogs comprising

HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ sequence, which contains a mutation selected from one or more of the following positions:

8th, 19th, 23rd, 27th, 30th, 34th, 36th and 37th. In a preferred embodiment, the amino acid at position 8 is selected from V, T, I, L, G or S, the amino acid residue at position 19 is Y or K, and the amino acid residue at position 23 is Q or K, position 27 The amino acid residue 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, provided that only one of the 19th, 23rd, 27th, 30th, 34th, 36th or 37th positions is a K residue.

The acylated derivative of the above GLP-1 analogue has an in vitro binding activity indicating that the binding affinity to the GLP-1R receptor is greater than that of the somaglutide or M0 (26 is Lys, CN107033234A is disclosed), and the in vivo hypoglycemic assay is also It was demonstrated that a longer duration of activity was obtained in mice compared to the same acylated GLP-1 product somaglutide, and at a dose of only 1/10 dose of somaglutide or M0, Its hypoglycemic effect is also not lower than somaglutide or M0. At the same time, the acylated derivatives of the above GLP-1 analogues can reduce body weight, reduce food intake, treat obesity, protect the liver, prevent and treat liver cell damage, and prevent and treat fatty liver and cirrhosis. The above-mentioned GLP-1 (7-37) analog of the present invention has a longer activity duration than the commercially available somatoglutide, and has better anti-enzymatic degradation characteristics. In particular, the invention relates to:

A derivative of a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, wherein the GLP-1 (7-37) analog comprises an amino acid sequence of the formula:

HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ ,

Wherein X _{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, and 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,

The derivative comprises an extension linked to the K residue of the GLP-1 (7-37) analog, wherein the extension is

Wherein x is an integer from 4 to 38.

2. A derivative according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the extension is selected from the group consisting of:

HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-, HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO- and HOOC(CH ₂ ) ₂₂ CO-.

The derivative according to claim 1 or 2, wherein the extension moiety is linked to the K residue of the GLP-1 (7-37) analog by a linker.

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

Wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; p is an arbitrary integer from 1 to 8.

Preferably, the joint is:

Wherein m is 1 or 2; n is 1 or 2; and p is an arbitrary integer from 1 to 5.

The derivative according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the linker is:

Where m is 1, and n is 1 or 2.

The derivative according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which is any derivative selected from the group consisting of N-ε ²³ -[ 2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutanoylamino]ethoxy)ethoxy]acetyl Amino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37))peptide (M2), N-ε ³⁰ -[2-(2- [2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy ]ethoxy)acetyl](Val ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37))peptide (M4), N-ε ³⁴ -[2-(2-[2-(2) -[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) Acetyl] (Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1 (7-37)) peptide (M5), N-ε ³⁷ -[2-(2-[2-(2-[2-(2-) [4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Arg ^26,34 Lys ³⁷ -GLP-1(7-37))peptide (M7), N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17) - Heptadecane-yl amido) -4 (s) - carboxy-butyrylamino] ethoxy) ethoxy] acetylamino) ethoxy] ethoxy) ^{acetyl] (Ile 8 Glu 22 Lys 23} Arg 26, ³⁴ -GLP-1(7-37))peptide (M9)\N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoyl) Amino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Thr ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 (7-37)) Peptide (M13)\N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4) s) ^{-Carboxybutyrylamino} ]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 (7-37) ) peptide (M14).

The use of the derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for the treatment of a disorder associated with a glucose metabolism disorder, a disorder associated with a fat metabolism disorder, or a neurodegenerative disease.

8. The use of claim 7, wherein the disease is selected from one or more of the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, Cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, inflammatory bowel disease, dyspepsia and gastrointestinal ulcers.

9. The use of claim 8, wherein the diabetic complications include diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy, and distal limb necrosis of the lower extremities.

10. The use of claim 8, wherein the obesity is congenital obesity or obesity secondary to the disease.

11. The use of claim 8, wherein the fatty liver is alcoholic fatty liver or nonalcoholic fatty liver.

The use according to claim 11, wherein the derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, lowers one or more blood biochemical indicators of a fatty liver subject: blood TC, TG, ALT, AST, HDL-C and LDL-C levels.

13. The use of claim 12, wherein the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof, further improves the NAS score of a fatty liver subject.

14. The use of claim 7, wherein the neurodegenerative disease comprises Parkinson's syndrome and Alzheimer's disease.

15. Use of a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for protecting a liver in a subject with liver damage.

16. The use of claim 15, wherein the liver damage is a liver damage caused by a chemical.

17. The use of claim 16 wherein the chemical is a poison gas, a drug, a toxin or an alcohol.

18. The use of any of claims 15-17, wherein the derivative or a pharmaceutically acceptable salt thereof reduces blood ALT, AST and/or TBIL levels in a subject.

19. Use of a derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, selected from one or more of the following: lowering blood glucose, reducing body weight and protecting the liver.

20. A method of preparing the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof, comprising:

(1) mixing a solution in which the GLP-1 analogue described in any one of the preceding claims is dissolved with a solution in which the extension according to any of the preceding claims is dissolved;

(2) adjusting the pH to 4-5 to terminate the reaction, allowing to stand until the precipitation is produced, and taking a precipitate;

(3) TFA was added to the precipitate, and the pH was adjusted to 7.5-8.5 to terminate the reaction.

21. The method of claim 20, further comprising adding triethylamine to the solution in which the GLP-1 analog is dissolved prior to mixing with the solution in which the extension of any of the preceding claims is dissolved.

22. The method of claim 20 or 21, wherein the extended portion of the solution of any of the preceding claims is acetonitrile dissolved.

23. A pharmaceutical composition comprising the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

A method for treating a disorder associated with a glucose metabolism disorder, a disorder associated with a fat metabolism disorder, or a neurodegenerative disease, comprising administering to a subject an effective amount of the derivative according to any one of claims 1 to 6, or a pharmaceutical thereof Acceptable salt.

25. The method of claim 24, wherein the disease is selected from one or more of the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, Cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, inflammatory bowel disease, dyspepsia and gastrointestinal ulcers.

26. The method of claim 25, wherein the diabetic complications comprise diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy, and distal limb necrosis of the lower extremities.

27. The use of claim 25, wherein the obesity is congenital obesity or obesity secondary to the disease.

28. The method of claim 25, wherein the fatty liver is alcoholic fatty liver or nonalcoholic fatty liver.

The method of claim 28, wherein the derivative of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, lowers one or more blood biochemical indicators of a fatty liver subject: blood TC, TG, ALT, AST, HDL-C and LDL-C levels.

30. The method of claim 29, wherein the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof, further improves the NAS score of a fatty liver subject.

31. The method of claim 24, wherein the neurodegenerative disease comprises Parkinson's syndrome and Alzheimer's disease.

32. A method of protecting a liver of a subject with liver damage comprising administering to a subject an effective amount of the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof.

33. The method of claim 32, wherein the liver damage is a liver damage caused by a chemical.

34. The method of claim 33, wherein the chemical is a poison gas, a drug, a toxin or an alcohol.

35. The method of any of claims 32-34, wherein the derivative or a pharmaceutically acceptable salt thereof reduces blood ALT, AST, and/or TBIL levels in a subject.

36. A method of lowering blood glucose, reducing body weight, or protecting a liver, comprising administering to a subject an effective amount of the derivative of any one of claims 1-6, or a pharmaceutically acceptable salt thereof.

37. A GLP-1 (7-37) analog comprising a polypeptide consisting of the following amino acid sequences:

H X ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇

Wherein 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, and 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.

38. A pharmaceutical composition comprising the analog of claim 37.

39. The analog of claim 37 for preventing or treating diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fat Uses of liver, cirrhosis, osteoporosis, cognitive disorders, neurodegenerative diseases (including Parkinson's syndrome and Alzheimer's disease), inflammatory bowel disease, dyspepsia, and gastrointestinal ulcers.

40. An article comprising a container and package insert having the derivative of any of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein the package insert carries the derivative or Instructions for the use of salt.

41. The article of claim 40 further comprising a container containing one or more other drugs.

42. The article of claim 41 wherein said other agent is for the treatment of diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, Fatty liver, cirrhosis, osteoporosis, cognitive disorders, neurodegenerative diseases (including Parkinson's syndrome and Alzheimer's disease), inflammatory bowel disease, dyspepsia, other drugs for gastrointestinal ulcers.

In the present application, a "sugar metabolism disorder"-related disease is a general term for a related disease caused by a disorder of glucose metabolism, and includes, for example, 1) diabetes and a diabetic complication such as diabetic vascular disease such as a heart caused by damage of a large blood vessel and a microvascular, Brain, kidney, peripheral nerves, eyes, feet and other tissues and organs, including diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy and lower extremity limb necrosis; 2) high incidence in diabetes Diseases that occur with diabetes or are aggravated by diabetes, such as atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, osteoporosis, etc.; 3) high incidence of diabetes, accompanied by diabetes Fat metabolism disorders and related diseases that occur or are exacerbated by diabetes, including hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, cirrhosis.

"Fat metabolism disorder" is a general term for related diseases caused by disorders of fat metabolism, including, for example, hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, cirrhosis, coronary heart disease, angina pectoris, myocardial infarction Inflammatory bowel disease, indigestion and gastrointestinal ulcers.

"Obesity" or "obesity" refers to a condition or condition in which the body weight exceeds normal standards. The normal standard of body weight varies from country to country and from gender to gender. Those skilled in the art can refer to relevant diagnostic criteria for judgment. In the present application, "obesity" or "obesity" are used interchangeably, that is, include congenital obesity and secondary obesity, such as obesity secondary to disease (disease).

The present invention relates to a method of producing a GLP-1 (7-37) analog which comprises expressing a DNA sequence encoding the polypeptide in a host cell under conditions which permit expression of the peptide, and then recovering the produced peptide.

The medium used to culture the cells may be any conventional medium for culturing the host cells, such as a minimal medium or a complex medium containing suitable additives. A suitable medium can be obtained by commercially available or a suitable medium can be prepared according to the published method. The polypeptide produced by the host cell can then be recovered from the culture medium by a conventional method, for example, the supernatant of the supernatant or the protein component in the filtrate is precipitated with a salt such as ammonium sulfate, and various chromatographic methods are selected depending on the kind of the peptide of interest. Further purification is carried out by exchange chromatography, gel filtration chromatography, affinity chromatography or the like.

The above-described coding DNA sequence can be inserted into any suitable vector. Generally, the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be of a type which, when introduced into a host cell, will integrate into the host cell genome and replicate along with the chromosome into which it is integrated.

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

The vector may also contain a selection marker, such as a gene whose gene product will compensate for a defect in the host cell or confer a drug such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin Resistance to streptomycin or methotrexate.

To introduce a peptide expressed by 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 ligated in the correct reading frame to the DNA sequence encoding the peptide. The secretion signal sequence is usually located on the 5' side of the DNA sequence encoding the peptide. The secretion signal sequence may be a secretion signal sequence that is normally linked to the peptide, or may be derived from a gene encoding another secreted protein.

Method for respectively linking a DNA sequence encoding a peptide of the present invention, a promoter and a selectable terminator and/or a secretion signal peptide sequence, and inserting it into a suitable vector containing information necessary for replication, The skilled person is known.

The host cell into which the DNA sequence or recombinant vector is introduced may 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, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

The present invention relates to a pharmaceutical or pharmaceutical composition comprising the above-mentioned GLP-1 (7-37) analog, and to the use of the analog in the preparation of a medicament, for example, in the preparation of a prophylactic or therapeutic diabetes and a diabetic complication, hyperlipidemia , atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, cognitive impairment, neurodegenerative diseases, inflammatory bowel disease And use in drugs such as other gastrointestinal diseases.

In another aspect, the invention also relates to the prevention or treatment of diabetes and diabetic complications, by administering a GLP-1 (7-37) analog or a derivative of the above GLP-1 (7-37) analog as described above to a subject. Lipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, cognitive impairment, neurodegenerative diseases (eg In the case of inflammatory bowel disease and other gastrointestinal diseases and the like.

In another aspect, the invention features a pharmaceutical composition, article or kit comprising the above-described GLP-1 (7-37) analog.

The invention further relates to a pharmaceutical composition, article or kit comprising a derivative of the above GLP-1 (7-37) analog.

The pharmaceutical composition of the present invention comprises, in addition to the active ingredient GLP-1 (7-37) analog or a derivative of the GLP-1 (7-37) analog or a salt thereof, a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable excipients such as non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients are well known to those skilled in the art. For example, diluents, excipients such as microcrystalline cellulose, mannitol, etc.; fillers such as starch, sucrose, etc.; binders such as starch, cellulose derivatives, alginates, gelatin and/or polyethylene Pyrrolidone; a disintegrating agent such as calcium carbonate and/or sodium hydrogencarbonate; an absorption enhancer such as a quaternary ammonium compound; a surfactant such as cetyl alcohol; a carrier, a solvent such as water, physiological saline, kaolin, soap clay, etc.; Lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. Further, the pharmaceutical composition of the present invention is preferably an injection.

The invention also relates to disorders of fat metabolism and disorders associated with lipodystrophy, including hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver A method of cirrhosis comprising administering to a subject in need thereof an effective amount of the above analog, derivative or drug, pharmaceutical composition. Further, the present invention relates to the use of the above-mentioned analogs, derivatives or drugs, pharmaceutical compositions of the present invention for the treatment of diseases frequently associated with diabetes and lipodystrophy, such as osteoporosis, treatment of cognitive disorders, and neurodegenerative diseases ( For example, methods of Parkinson's syndrome, Alzheimer's disease, and methods of treating gastrointestinal diseases such as inflammatory bowel disease, malnutrition, and peptic ulcer.

In the present invention, a GLP-1 (7-37) polypeptide, a GLP-1 (7-37) polypeptide analog, and a GLP-1 (7-37) analog are used interchangeably to indicate an amino acid sequence: H X ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ polypeptide, wherein X _{8 is} selected from V, T, I, L, G or S, X ₁₉ is Y or K, X ₂₃ is Q or K, and 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. The GLP-1 (7-37) polypeptide analog forms a derivative of the GLP-1 (7-37) polypeptide analog by attachment to an extension. In particular, the invention relates to acylated derivatives of GLP-1 (7-37) analogs. The acylated derivative not only has a remarkable therapeutic effect, but also has an in vivo activity duration of about 1 time higher than that of the currently recognized best drug somaglutide, meaning that at least weekly can be achieved in the human body. The frequency of dosing administered at intervals, even at intervals of two or more weeks.

A derivative of the GLP-1 (7-37) analog of the present invention, an acylated derivative of the GLP-1 (7-37) analog, a GLP-1 (7-37) derivative, and a GLP-1 derivative may be mutually Change to use.

In another aspect, the present invention is also a process for the preparation of 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 extended portion (for example, a fatty acid) is dissolved;

(2) adjusting the pH to 4-5 to terminate the reaction, allowing to stand until the precipitation is produced, and taking a precipitate;

(3) TFA was added to the precipitate, and the pH was adjusted to 7.5-8.5 to terminate the reaction.

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 present invention comprises (1) providing a GLP-1 (7-37) analog solution, adjusting the pH to 9-12;

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

(3) weighing a fatty acid having a structure of not less than 2 times the molar ratio of the GLP-1 analogue, preferably not less than 3 times the amount of the GLP-1 analogue, dissolved in acetonitrile;

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

(5) adjusting the pH to 4-5 to terminate the reaction, standing at a low temperature, and collecting the precipitate;

(6) adding TFA to the acid precipitation sample obtained in the step (5) to a final concentration of the polypeptide of 5-15 mg/ml, standing for 0.5-2 hours, and dropping an alkaline solution such as NaOH into the reaction solution to adjust the pH to 7.5. -8.5 terminate the reaction;

(7) The obtained product was isolated and purified.

The present invention relates to a preparation of a pharmaceutical composition comprising a derivative of GLP-1 (7-37) analog or a pharmaceutically acceptable salt thereof. In some embodiments, a derivative of the invention comprising a GLP-1 (7-37) analog, or a pharmaceutically acceptable salt thereof, is present at a concentration of from 0.1 mg/ml to 25 mg/ml, preferably at 0.1 mg/ml It is present at a concentration of 10.0 mg/ml. In a preferred embodiment, the pharmaceutical composition has a pH of from 3.0 to 9.0. In a preferred embodiment, the pharmaceutical composition may further comprise a buffer system, a preservative, a surface tensioning agent, a chelating agent, a stabilizer, and a surfactant. In some embodiments, the medicaments or formulations of the invention are aqueous medicaments or formulations, for example, they may generally be solutions or suspensions. In a particular embodiment of the invention, the medicament or formulation is a stable aqueous solution. In other specific 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 further relates to a kit or kit comprising the above pharmaceutical composition, formulation, medicament. In the kit or kit, in addition to the above-mentioned drugs or preparations, other drugs, pharmaceutical compounds or compositions which can be used in combination with the pharmaceutical composition, preparation, or drug, for example, the other drugs and drug compounds are included. Or the composition may be selected from the group consisting of anti-diabetic 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 stimulating gluconeogenesis and/or glycogenolysis , glucose uptake regulator, NPY antagonist, PYY agonist, PYY2 agonist, PYY4 agonist, TNF agonist, cortisol releasing factor agonist, 5HT, bombesin agonist, gangliopeptide antagonist, growth hormone , thyroid stimulating hormone releasing hormone agonist, TRβ agonist; histamine H3 antagonist, lipase/amylase inhibitor, gastric inhibitory polypeptide agonist or antagonist, gastrin and gastrin analog, and the like. In some embodiments, the pharmaceutical compositions, formulations, medicaments, and other drugs, pharmaceutical compounds, or compositions of the invention are each placed in separate containers.

The present invention also relates to a method for preventing or treating diabetes (preferably type 2 diabetes), diabetic complications (such as diabetic nephropathy, diabetic heart disease), comprising administering to a subject in need thereof the above-mentioned analog, derivative or drug, a pharmaceutical composition, wherein the analog, derivative or drug, pharmaceutical composition is used in combination with other drugs, pharmaceutical compounds or compositions, for example, the other drug, pharmaceutical compound or composition may be selected from an anti-diabetic drug, A medicament 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 stimulating gluconeogenesis and/or glycogenolysis , glucose uptake regulator; CART agonist, NPY antagonist, PYY agonist, PYY2 agonist, PYY4 agonist, TNF agonist, cortisol releasing factor agonist, 5HT, bombesin agonist, ganglion peptide antagonism Agent, growth hormone, thyroid stimulating hormone releasing hormone agonist, TRβ agonist; histamine H3 antagonist, lipase/amylase inhibitor, gastric inhibitory polypeptide agonist or antagonist, gastrin and gastrin analog Wait. In a preferred embodiment, the diabetes is type 2 diabetes or diabetic nephropathy.

The "diabetic complication" as used in the present invention refers to damage or dysfunction of other organs or tissues of the body caused by poor glycemic control during diabetes, including damage or function of the liver, kidney, heart, retina, nervous system. Obstacles and so on. Complications of diabetes can be divided into five aspects: 1. Cardiovascular disease: including microvascular lesions on the heart and large blood vessels, myocardial lesions, cardiac autonomic neuropathy, the leading cause of death in diabetic patients. 2. Cerebrovascular disease: refers to intracranial large blood vessels and microvascular lesions caused by diabetes, mainly manifested as cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy. 3. Renal vascular disease: The main form is diabetic nephropathy, which is one of the most important complications of diabetic patients. 4. Lower extremity arterial disease: mainly manifested as diabetic foot. 5. Fundus microvascular disease: mainly manifested as diabetic retinopathy.

The invention is further illustrated by the following examples, however, the examples are not to be construed as limiting the scope of the invention, the features disclosed in the foregoing description and the following examples (individually and any combination thereof) may be It is a material for carrying out the invention in substantially different forms, and they can be combined arbitrarily. In addition, the present invention is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in its entirety in its entirety in the the the the the the the

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of different GLP-1 derivatives on body weight of DIO rats.

Figure 2 is a graph showing the effect of different GLP-1 derivatives on the relative body weight of DIO rats.

Figure 3 is a graph showing the effects of different administration modes of different GLP-1 derivatives on the body weight of DIO rats.

Figure 4 is a graph showing the effect of different administration modes of different GLP-1 derivatives on the relative body weight of DIO rats.

Figure 5 shows the effect of different administration modes of different GLP-1 derivatives on the food intake of DIO rats.

Figure 6. Effect of different doses of GLP-1 derivatives on body weight.

Figure 7. Effect of different doses of GLP-1 derivatives on relative body weight.

Figure 8. Effect of different doses of GLP-1 derivatives on animal food intake.

Figure 9 shows the weight loss effects of different doses of M0, M4 and somaglutide on type 2 diabetic db/db mice.

Figure 10 shows the effect of different doses of M0, M4 and somaglutide on changes in food intake in type 2 diabetic db/db mice.

Figure 11 shows the protective effect of M2 on liver injury induced by CCl4.

Figure 12 shows the trend of blood biochemical changes of the GLP-1 derivative of the present invention against NASH model mice.

Figure 13 shows the HE staining results of liver in NASH model mice (x10 times), where A is the normal control group, B is the vehicle control group, C is the M2 (0.05 mg/kg) group, and D is the M4 (0.05 mg/kg) group. E is the group of somaglutide (0.05 mg/kg).

Figure 14 shows the effect of the GLP-1 derivative of the present invention on the liver pathological NAS score of NASH model mice.

Figure 15 shows the effect of the GLP-1 derivative of the present invention on liver weight and liver weight/body weight ratio of NASH model mice.

Figure 16 shows the hypoglycemic effect of different acylated GLP molecules on type 2 diabetic db/db mice.

Figure 17 shows the effect of different doses of M0, M4 and somaglutide on fasting blood glucose in diabetic mice.

Figure 18 shows the effect of different doses of M0, M4 and somaglutide on random blood glucose in diabetic mice.

Figure 19 shows the effect of different doses of M0, M4 and somaglutide on the area under the blood glucose curve in diabetic mice.

Figure 20 shows the results of anti-pepsin degradation of M4 and somaglutide molecules.

Figure 21 shows the results of anti-trypsin degradation of M4 and somaglutide molecules.

Example

The invention will be described below by way of specific examples. Unless otherwise specified, it can be carried out according to the methods listed in the "Molecular Cloning Experimental Guide", the "Cellular Experiment Guide" and the like, and the CFDA test guides, which are familiar to those skilled in the art. Among them, the reagent raw materials used are all commercially available and can be purchased through public channels.

Example 1 Construction of GLP-1 Analog Expression Plasmid

Construction of DNA of Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37)

The 6-His tag, the SUMO tag and the Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37) coding gene sequence (SEQ ID NO: 7) were sequentially fused in tandem, and the gene was obtained by chemical synthesis. Fragment (SEQ ID NO: 18). The above fragment was inserted into the prokaryotic expression plasmid pET-24(+) by BamHI and XhoI sites and verified by sequencing. The resulting expression plasmid for transformation assay was ^designated pET-24(+)-His-SUMO-Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37).

According to the above method, sequentially constructed ^{Val 8 Glu 22 Lys 26 Arg 34} -GLP-1 (7-37) ( gene encoding SEQ ID NO: 3), Val 8 Glu 22 Lys 30 Arg 26,34 -GLP-1 (7 -37) (coding gene is SEQ ID NO: 11), Val ⁸ Glu ²² Lys ¹⁹ Arg ^{26, 34 -} GLP-1 (7-37) (coding gene is SEQ ID NO: 5), Val ⁸ Glu ²² Lys ²⁷ Arg ^26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 9), Val ⁸ Glu ²² Lys ³⁴ Arg ²⁶ -GLP-1 (7-37) (coding gene is SEQ ID NO: 13 ), Val ⁸ Glu ²² Arg ^26,34 Lys ³⁶ -GLP-1 (7-37) (coding gene is SEQ ID NO: 15), Val ⁸ Glu ²² Arg ^{26, 34} Lys ³⁷ -GLP-1 (7-37) (coding gene is SEQ ID NO: 17), Thr ⁸ Glu ²² Lys ²³ Arg ^{26, 34 -} GLP-1 (7-37) (coding gene is SEQ ID NO: 20), Ile ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 22), Leu ⁸ Glu ²² Lys ²³ Arg ^{26, 34} -GLP-1 (7-37) (coding gene is SEQ ID NO: 24), Gly ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 26), Ser ⁸ Glu ²² Lys ²³ Arg 26, ³⁴ -GLP-1 (7- 37) (coding gene is SEQ ID NO: 28), Thr ⁸ Glu ²² Lys ³⁰ Arg ^{26, 34 -} GLP-1 (7-37) (coding gene is SEQ ID NO: 30), Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 32), Leu ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 (7-37) (coding gene is SEQ ID NO: 34), Gly ⁸ Glu ²² Lys ³⁰ Arg ^{26, 34 -} GLP-1 (7-37) (coding gene is SEQ ID NO: 36), Ser ⁸ Glu ²² Lys ³⁰ Arg ^{26, 34 - GLP-} 1 (7-37) (coding gene is SEQ ID NO: 38) corresponding expression plasmid.

Example 2 fusion protein expression

Expression of the fusion protein was carried out using the DNA construction described in Example 1, and the target protein was obtained by expressing the cell BL21 (Trabs Gen Biotech., catalog #CD601). 50 μl of BL21 competent cells were thawed on an ice bath, DNA of interest was added, gently shaken, and left in an ice bath for 30 minutes. Then heat in a 42 ° C water bath for 30 seconds, then quickly transfer the tube to the ice bath for 2 minutes, do not shake the tube. 500 μl of sterile LB medium (without antibiotics) was added to the centrifuge tube, mixed and placed at 37 ° C, and cultured at 180 rpm for 1 hour to resuscitate the bacteria. 200 μl of transformed competent cells were pipetted onto LB agar medium plates containing kanamycin resistance, and the cells were evenly spread. The plate was placed at 37 ° C until the liquid was absorbed, the plate was inverted, and cultured overnight at 37 °C. The next day, monoclonal colonies in the transformation plates were picked using an inoculating loop and inoculated in 15 ml of sterile LB medium (containing antibiotics) and cultured overnight at 30 °C.

Example 3 Fermentation of Recombinant GLP-1 Analogs

50 μl of the bacterial solution (expressing GLP-1 bacterial solution) was added to 50 ml of LB medium, and 50 μl of kanamycin was added thereto, mixed, and placed in a 30 ° C constant temperature shaker, and inoculated overnight. 10 ml of the overnight inoculum was added to 1000 ml of LB medium while 1000 μl of kanamycin was added. After shaking, the cells were placed in a 37 ° C shaker at 200 rpm. After inoculation for 4 hours, IPTG was added to the medium at a final concentration of 0.1 mol/L, shaken, placed in a shaker at 30 ° C, and induced to express overnight at 180 rpm. The overnight expressed bacterial solution was centrifuged at 13,000 g for 60 min. The bacterial cell yield is about 4g bacteria/L fermentation broth, and the expression of the target protein by SDS-PAGE is about 40%.

Example 4 Purification of Recombinant GLP-1 Analogs

100 g of the cell slurry was weighed and resuspended in 500 ml of 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and sonicated for 30 min in an ultrasonic cell pulverizer to disrupt the cells. The homogenate was centrifuged at 13,000 g for 60 min at 4 ° C. After centrifugation, the supernatant was collected and the sample was a Ni column chromatography.

The resulting supernatant was concentrated by Chelating Sepharose FF previously equilibrated with 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole (Equilibration 1). After the equilibration solution 1 was rinsed, it was further eluted with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.3 M imidazole (eluent). The purity of the GLP-1 intermediate produced by the above purification process was higher than 70% by SDS-PAGE analysis.

The Sumo tag sequence was excised using the ULP enzyme: the intermediate product was diluted three times by adding 20 mM PB, pH 7.4 buffer, and mixed with ULP according to the ULP enzyme: intermediate product of 1:150 at 4 ° C, and then cleaved overnight. The enzyme digestion rate was nearly 100% by SDS-PAGE.

Purification of GLP-1 analogue: The product obtained after digestion was concentrated by Tosoh Butyl 550C medium previously equilibrated with 20 mM Na ₂ HPO ₄ , 0.7 M NaCl (equilibrium solution 2). After the equilibration solution 2 was rinsed, it was eluted with 20% ethanol, and the purity by SDS-PAGE was about 90%.

To the eluted sample, 0.2 M Na ₂ HPO ₄ was added to give a final concentration of 20 mM Na ₂ HPO ₄ , the pH was adjusted to 4.8-5.0 with 1 M citric acid, and acid precipitation was carried out overnight at 4 °C. The yield was more than 90% by SDS-PAGE. Centrifuge at 13,000 g for 30 min at 4 ° C, collect the pellet and store at -20 ° C.

Example 5 Preparation of Derivatives of GLP-1 Analogs

A derivative of the GLP-1 analog shown below, N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino))- 4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7- 37) Preparation of peptide (abbreviated as M2)

1. Fatty acid modification: Water was added to the precipitate of Val ⁸ Glu ²² Lys ²³ Arg ^{26, 34} -GLP-1 (7-37) prepared and collected in the above examples to prepare a 4-6 mg/ml solution, and 1 M hydroxide was added. The sodium was adjusted to pH 11.0-11.5, shaken to completely dissolve the protein, and the polypeptide concentration was quantified by HPLC. The fatty acid powder was dissolved in acetonitrile according to a molar ratio of the polypeptide to the fatty acid (structure as follows) 1:4. To the polypeptide solution, triethylamine in a volume of two thousandths was added and mixed with the fatty acid solution, and the mixture was allowed to stand at 4 ° C for one hour.

The sample was diluted with water 5 times, the pH was adjusted to 4.8 with 1 M citric acid (or 10% acetic acid) to terminate the reaction, and the acid solution was allowed to stand at 4 ° for 10 min, after centrifugation, centrifuged 13000 g, centrifuged at 4 ° C for 30 min, and the precipitate was placed at -80. °C save.

2. Deprotection and purification of fatty acids: TFA was added to the acid precipitation sample to a final concentration of about 10 mg/ml of the polypeptide. The precipitate was dissolved by shaking, left to stand at room temperature for 30 min, and 4 M NaOH was added dropwise to the reaction solution to adjust the pH to 7.5. -8.5 Terminate the reaction.

Using a preparative liquid phase meter (Shimadzu LC-8A), the reaction solution after termination was pumped at a flow rate of 4 ml/min into UniSil 10-120 C18 previously equilibrated with 10 mM ammonium acetate, 20% ethanol (equilibrium solution 3) (purchased from Suzhou Nawei Technology Co., Ltd.) is concentrated. After the equilibration solution 3 was rinsed, it was further eluted with a gradient of 0-100% eluent (10 mM ammonium acetate, 80% ethanol), and the eluted peak was collected by RP-HPLC to a purity of about 90%.

The elution peak was diluted 3 times with water, the acid was adjusted to pH 4.80, and the acid was precipitated at 4 ° C for 30 min. After centrifugation, the precipitate was reconstituted by adding PBST buffer (pH 7.0), and then frozen at -80 °C.

Preparation of N-ε ²⁶ -[2-(2-[2-(17-carboxyheptadecanoylamino)-4(s)-carboxybutanoyl) in order according to the above method Amino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl]Val ⁸ Glu ²² Lys ²⁶ Arg ³⁴ -GLP-1(7-37)peptide (M0), N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutanoylamino]ethoxy)ethoxy) Acetylamino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37))peptide (M4), N-ε ¹⁹ -[2-( 2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino) Oxy]ethoxy)acetyl](Val ⁸ Lys ¹⁹ Glu ²² Arg ²⁶ ,34-GLP-1(7-37)) peptide (M1), N-ε ²⁷ -[2-(2-[2- (2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy Acetyl] (Val ⁸ Glu ²² Lys ²⁷ Arg ^26,34 -GLP-1 (7-37)) peptide (M3), N-ε ³⁴ -[2-(2-[2-(2-[2 -(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl] (Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37))peptide (M5), N-ε ³⁶ -[2-(2-[2-(2-[2-(2-[4-(17) -carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl] (Val ⁸ Glu ²² Arg ^26,34 Lys ³⁶ -GLP-1(7-37)) peptide (M6), N-ε ³⁷ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecane) Amido)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl] (Val ⁸ Glu ²² Arg ^26,34 Lys ³⁷ -GLP- 1(7-37)) peptide (M7); N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4) (s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl] (Thr ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1 (7-37 )) peptide (M8), N-ε ²³ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxyl) ^Butyrylamino ]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37)) peptide (M9 , N-ε ²³ -[2-(2-[2-(2-[2-(2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]) Oxy)ethoxy]acetylamino)ethoxy]ethoxy ) ^{Acetyl] (Leu 8 Glu 22 Lys 23} Arg 26,34 -GLP-1 (7-37)) peptide ^{(M10), N-ε 23} - [2- (2- [2- (2- [2- (2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]( Gly ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37))peptide (M11), N-ε ²³ -[2-(2-[2-(2-[2-(2-[4 -(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ser ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37))peptide (M12); N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyl) Heptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Thr ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37)) peptide (M13), N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino) -4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 ( 7-37)) Peptide (M14), N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4) )-carboxybutyrylamino]ethoxy)ethoxy]acetyl Amino)ethoxy]ethoxy)acetyl](Leu ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37)) peptide (M15), N-ε ³⁰ -[2-(2- [2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy ]ethoxy)acetyl](Gly ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37)) peptide (M16), N-ε ³⁰ -[2-(2-[2-(2) -[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) Acetyl] (Ser ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1 (7-37)) peptide (M17).

Table 1 GLP-1 (7-37) analogues and their corresponding derivatives comparison table

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

RIN-m5F cells in good culture were selected. The cells were collected, counted, and RPMI1640 base medium was used to prepare a cell suspension of 1 × 10 ⁵ cells/ml. The cell suspension was seeded in a 96-well cell culture plate at 100 μl per well, and cultured overnight at 37 ° C under 5% CO ₂ . Detection of in vitro activity of derivatives of GLP-1 analogues using the cAMP assay kit (Promega): preparation of assay medium dilution samples (Aib, M0, M1, M2, M3, M4, M5, M6, M7) to 300 ng/ml Then, a 3-fold gradient dilution was performed in a 96-well plate for a total of 8 concentrations, and 2 replicate wells were made for each dilution, wherein M0, M1, M2, M3, M4, M5, M6, M7 were prepared as described above, Aib for

N-ε ²⁶ -[2-(2-[2-(2-[7-carboxyheptanoylamino)-4(s)-carboxybutyrylamino]ethoxy) Ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib ⁸ ,Arg ³⁴ ]GLP-1-(7-37) peptide (see CN101133082B Example 4) under the trade name Somadupide Prepared according to the method disclosed in the patent CN101133082B.

The prepared cell plate was taken out, the medium was discarded, and the filter paper was blotted dry. The sample solution was correspondingly transferred into the cell plate at 40 μl/well. The lid was opened for 15 min at 37 ° C under 5% CO ₂ . The cell culture plate was taken out from the incubator, 10 μl of CD solution (cAMP detection kit (Promega)) was added to each well, and the cell plate was placed at 22 ° C - 25 ° C and shaken at 500 rpm for 20 min. 50 μl of KG solution (cAMP detection kit (Promega)) was added to each well, and the cells were shaken at a temperature of 22 ° C to 25 ° C and 500 rpm for 10 min. The chemiluminescence values were read using a Molecular Devices SpectraMax L chemiluminometer and the assay was completed in 30 min. Sample EC50 was calculated using a four parameter regression in softmax Pro software software.

Table 2 Results of in vitro activity of RIN-m5F cells

sample	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 somaglutide, M2, M4, M5 and M7 were comparable in vitro and were generally slightly higher than M0, M1, M3 and M6.

Example 7 In vitro Activity Determination of Derivatives of GLP-1 Analogs in HEK293/CRE-Luc/GLP1R Cells

The HEK293/CRE-Luc/GLP1R cell line was constructed based on the binding of GLP-1 to receptors on the cell membrane. The cAMP response element (CRE) was activated by a series of signal transductions to initiate the expression of downstream luciferase. It is positively correlated with the biological activity of GLP-1, and after adding a luciferase substrate, the luminescence intensity is measured by chemiluminescence detection to determine the biological activity of GLP-1.

Experimental Materials

96-well cell culture plate (white opaque), DMEM medium (GIBCO), 0.05% TRYPSIN-EDTA (GIBCO), fetal bovine serum (GIBCO), G418, hygromycin B, Bright-GloTM Luciferase Assay System kit (Promega) ), HEK293/CRE-luc/GLP1R cells.

Experimental operation

(1) Cell preparation: The cells were cultured until the growth state was vigorous and sufficient. Discard the culture solution in the culture flask, add 3 ml of Versene solution and shake once, add 2 ml of 0.05% TRYPSIN-EDTA digestive solution, cover with a cap and let stand for 1 minute, then add 6 ml of the assay medium to terminate digestion, 1000r/ After centrifugation for 3 min at min, the supernatant was discarded, and the cells were resuspended in 5 ml assay medium and counted using a hemocytometer. The culture medium was adjusted by DMEM to adjust the cell density to a suitable range, and was used.

(2) Sample preparation: Dilute the derivatives of different GLP-1 analogues in Table 1 to 20 ng/ml with the assay medium, then serially dilute 8 concentrations in 96-well plates, and replace the sample with the assay medium as a cell blank. , make 2 duplicate wells for each dilution.

(3) Adding culture: Transfer the prepared control and test solution into a 96-well cell culture plate (white plate), add 50 μl per well, add the prepared cell suspension, add 50 μl per well, and place Incubate for a certain period of time at 37 ° C, 5% CO ₂ .

(4) Chemiluminescence detection: The substrate was added, and a 96-well cell culture plate was taken out, and 100 μl of Bright Glo reagent was added to each well, and left to stand for 3 minutes in the dark.

(5) Reading: The measurement was performed with a chemiluminescent microplate reader SpectraMax L, and the plate was read within 30 minutes, and the measurement results were recorded.

Table 3 Results of in vitro activity of HEK293/CRE-Luc/GLP1R cells

The pharmacodynamics of HEK293/CRE-Luc/GLP1R cells showed that the in vitro activities of somaglutide, M2, M4, M9, M11, M14, M16 and M17 were comparable and generally slightly higher than M13.

Example 8 Derivatives of GLP-1 Analogs

In this experiment, male rats were used to establish the experimental animal DIO model (high fat diet induced obese rat model) (Misawa E, Tanaka M, Nabeshima K, et al. Administration of dried Aloe vera gel powder reduced body fat mass in diet-induced obesity (DIO) rats [J]. J Nutr Sci Vitaminol (Tokyo), 2012; 58(3): 195-201). Forty male rats of 6 weeks old were acclimated for 1 week in SPF breeding environment. 36 rats were randomly selected to feed high fat diet (Rodent diets with 60 kcal% Fat), and 10 were fed with common vocabulary. All animals were free to drink water and ingest food. After 10 weeks of feeding, the body weight of the normal diet was increased by 20% as the standard of obese rats. The model rats were randomly divided into 7 groups, normal control group, high fat diet control group, Somaru. Peptide group (0.025 mg/kg), M0 group (0.025 mg/kg), M2 group (0.025 mg/kg), M4 group (0.025 mg/kg), and M7 group (0.025 mg/kg). The above experimental group was injected subcutaneously once a day, weighing 3 times a week, and the inhibition rate of body weight growth and the inhibition rate of relative body weight growth were calculated according to the following formula (Table 4-5, Figure 1-2):

Table 4 compares the effects of body weight on DIO rats

Note: D0 is the weight of each group before each group.

Table 5 compares the effects of relative weight on DIO rats

Note: D0 is 1 for each component group.

To sum up: M2, M4 and somaglutide all showed significant effects on reducing DIO body weight (P<0.05). M0 had no inhibitory effect on DIO rat weight gain, and M7 had weaker inhibition on DIO rat weight gain. M2 and M4 reduced the body weight of DIO rats, which was stronger than that of somatoglutide, which was statistically significant.

Example 9 Inhibition of body weight and feeding by derivatives of GLP-1 analogues

In this experiment, male rats were used to establish the DIO model of experimental animals (Misawa E, Tanaka M, Nabeshima K, et al. Administration of dried Aloe vera gel powder reduced body fat mass in diet-induced obesity (DIO) rats [J]. J Nutr Sci Vitaminol (Tokyo), 2012; 58(3): 195-201). Forty male rats of 6 weeks old were acclimated for 1 week in SPF breeding environment. 36 rats were randomly selected to feed high fat diet (Rodent diets with 60 kcal% Fat), and 10 were fed with common vocabulary. All animals were free to drink water and ingest food. After 10 weeks of feeding, the body weight was increased by 20% compared with the normal diet-fed group as the standard of obese rats, and the model rats were selected. Randomly divided into 4 groups, high fat diet control group, somaglutide + M4 group (0.025 + 0.025mg / kg), the dosing regimen was cross-administration, that is, the first 21 days of injection of somaglutide, from the 22nd day Start to stop the injection of somaglutide, start M4 injection until the end of the 28th day; test group M2 (0.025mg / kg), M2 injection from beginning to end; M4 + somalupin group (0.025 + 0.025mg / kg), dosing The protocol was cross-administration, ie M4 was injected for the first 21 days, M4 was stopped from day 22, and somatoglutide was started until the end of day 28.

The body weight of the animal was weighed daily, and the inhibition rate of weight growth and the inhibition rate of relative body weight growth were calculated according to the following formula:

At the same time, the food intake of the rats was measured to observe the feeding inhibition effect.

Table 6 DIO rat body weight inhibition rate

Table 7 Relative weight inhibition rate of DIO rats

From the results in Tables 6-7 and 3-4, it can be seen that the first 21 days of weight loss, M2 and M4 are stronger than somatoglutide. In the somaglutide + M4 group, after stopping the administration of somaglutide on the 22nd day to M4, the body weight can maintain a relatively stable level, that is, M4, you can continue to maintain the weight loss level of the previous somatoglutide In the M4+somaglutide group, after stopping the administration of M4 to domaline on day 22, the body weight of the rats increased, and somaglutide could not continue to maintain the weight loss level of M4. Rats in the M2 group have been given the weight loss effect at the level of the weight platform after reaching a platform.

Table 8 Effect of DIO rats on food intake

As can be seen from Table 8 and Figure 5, the food intake of the three drug-administered groups was significantly less than that of the control group, and the mice administered to the M2 and M4 groups had less food intake than the Aib group (the first 21 days), and the M4 was given in the previous phase. The food intake of the rats given the cross-administration of the semaglutide group rebounded, which was higher than that of the M2 group, and also higher than the food intake of the M4 group after the prior administration of somatoglutide.

Example 10 Effect of Derivatives of GLP-1 Analogs on Weight Growth Inhibition Rate

In this experiment, male SD rats were used to establish the experimental animal DIO model (Misawa E, Tanaka M, Nabeshima K, et al. Administration of dried Aloe vera gel powder reduced body fat mass in diet-induced obesity (DIO) rats [J]. Nutr Sci Vitaminol (Tokyo), 2012; 58(3): 195-201). Thirty male rats of 6-8 weeks old were acclimated for 1 week in SPF breeding environment. 30 rats were fed with high fat diet (Rodent diets with 60 kcal% Fat). All animals were given free access to water and food. After 10 weeks of feeding, they were randomly divided into 5 groups: the somaglutide group (0.025 mg/kg), the M2 high dose group (0.025 mg/kg), the M2 low dose group (0.0125 mg/kg), and the M4 high dose group ( 0.025 mg/kg), M4 low dose group (0.0125 mg/kg). The subcutaneous injection was performed once a day for 28 days, and the animal body weight was weighed 2-3 times a week.

And calculate the weight growth inhibition rate and the relative weight growth inhibition rate according to the following formula:

At the same time, the food intake of the rats was measured to observe the effect on the food intake inhibition.

Table 9: Comparison of body weight inhibition rates

Note: D0 is the weight before administration, and D0 is the first day of administration, and so on.

Table 10 Comparison of relative body weight inhibition rates

Note: D0 is the relative body weight before administration, defined as 1.000, relative body weight = day weight / D0 body weight.

Through the above body weight and relative body weight data (Fig. 6 and Fig. 7 and Table 9-10), it can be concluded that M2 and M4 have the same body weight inhibition effect on DIO rats under the same dosage conditions, and the dose-effect relationship between high and low doses is obvious, and Both showed significant inhibition of weight gain.

Compared with the same doses of M2 and M4, somaglutide showed a certain weight loss effect, but was significantly inferior to the same doses of M2 and M4. At 1 week before dosing, the body weight inhibition rate of the somaglutide group was close to that of the M2 and M4 low dose groups; in the later stage of the experiment (week 4), the weight of the somaglutide group was significantly higher than that of the M2 and M4 groups, while M2 and The inhibitory effect of weight gain on animals in the M4 high dose group remained basically unchanged.

Table 11: Comparison of rat food intake

The above data (Fig. 8 and Table 11) showed that the M2 and M4 animals in the high dose group had the lowest food intake; the M2 and M4 animals in the same dose group had similar food intake; the same dose (0.025 mg/kg), the M2 and M4 animals consumed slightly. Less than the sumaglutide group.

Example 11 Effect of different doses of semaglutide, M0 and M4 on weight loss efficacy of type 2 diabetes db/db mice

50 db/db mice, female, 8-9 weeks old, divided into 10 groups according to body weight, 5/group, single subcutaneous injection of vehicle, M4 (0.15, 0.03, 0.015 mg/kg), Soma Rupeptide (0.15, 0.03, 0.015 mg/kg) and M0 (0.15, 0.03, 0.015 mg/kg) were administered at 10 ml/kg body weight. The administration time was set to 0h, and the body weight of the animals was weighed every day. The body weight of the animals before the administration was set to 0. In the evening, the feeds of each group were quantified to 50 g, and the food intake and body weight of the mice were weighed the next morning.

Weight change (Δ: delta) = body weight after administration - base weight before administration;

Food intake / only = (quantitative 50g - remaining food) / 5;

Cumulative food intake / only: Accumulate the daily food intake.

The results are shown in Table 12, Table 13, and Figures 9, and 10.

Table 12 Changes in body weight of mice in each test group (mean ± standard deviation)

Note: Compared with the control group, "*" P < 0.05, "***" P < 0.001.

Table 13 Table of changes in average cumulative food intake of mice in each test group

In summary, from Table 12, Table 13, and Figure 9, Figure 10, after a single subcutaneous injection of each test substance (M4, somaglutide or M0) for 60h, the body weight showed significant weight loss, each The dose-effect relationship of the test group was obvious, and the M4 weight loss effect was the best, and the 0.015 mg/kg dose group of M4 was equivalent to the 0.15 mg/kg dose group of the sumaglutide or M0 group, and the mice of each group were fed. The amount was also positively correlated with body weight, and the cumulative food intake of the three groups was basically the same.

Example 12 Protection of Liver by Derivatives of GLP-1 Analogs

Thirty C57BL/6 mice were randomly divided into three groups according to body weight: normal group, vehicle control group and M2 group (0.05 mg/kg). Except the normal group, the other two groups were induced by carbon tetrachloride (CCl4), and 10% CCl4 was injected intraperitoneally with a 1 ml syringe at a dose of 2 μl/g body weight (Monday, Wednesday, and Friday). ), the normal group is injected with the same amount of olive oil. The treatment group was given 0.05 mg/kg of M2 once a day, and the vehicle control group was injected with an equal volume of vehicle (PBS solution) for 28 days. The normal group received no treatment for routine feeding. After the last injection, the mice were sacrificed after fasting for 24 hours, and serum samples were taken. According to the alanine aminotransferase (ALT) test kit, aspartate aminotransferase (AST) test kit, and total bilirubin (TBIL) detection reagent. The box instructions detect serum ALT, AST, and TBIL levels. The above kits were purchased from Beijing Lederman Biochemical Co., Ltd.

The experimental results (see Figure 11) showed that serum levels of ALT, AST, and TBIL were significantly higher in the CCl4 model group than in the normal group (P < 0.01), and ALT, AST, and TBIL levels were significantly lower in the M2 treatment group. Significance (P < 0.05), indicating that M2 has a significant protective effect on liver function.

Example 13 Efficacy of M4 on high-fat, high-sugar and high-cholesterol feed for induction of C57BL/6 mouse NASH

Fifty male C57BL/6 mice were randomly divided into two groups for modeling: normal group and model group. The normal group was fed with normal maintenance feed as a normal control. The model group used high fat, high glucose and high cholesterol diet (FFC) to induce nonalcoholic fatty hepatitis (NASH) in mice (Clapper JR1, Hendricks MD, Gu G, et al. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis Amjin Physiol progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol. 2013 Oct 1; 305(7): G483-95), modeled for 25 weeks, weighing once a week. After modeling, the model group was divided into 4 groups according to body weight: vehicle control group, M2 group (0.05 mg/kg), M4 group (0.05 mg/kg), and somaglutide group (0.05 mg/kg). The body weight was weighed 2-3 times a week, and the experimental data was recorded in time, and continuous subcutaneous injection was administered for 8 weeks.

After the last dose, fasting overnight (free drinking water), taking the eyeball to take blood, the blood sample is not anticoagulated, and the blood sample is stored on ice. After taking the blood, the serum is separated by centrifugation at 4000 rpm/min for 20 min, and the serum is separated according to total cholesterol (TC). Test kit, triglyceride (TG) detection kit, alanine aminotransferase (ALT) detection kit, aspartate aminotransferase (AST) detection kit, high density lipoprotein cholesterol (HDL-C) detection kit and low density The serum lipoprotein cholesterol (LDL-C) test kit is used to detect serum TC, TG, ALT, AST, HDL-C, and LDL-C levels. The above kits were purchased from Beijing Lederman Biochemical Co., Ltd.

Liver tissue was stained with HE and Sirius red. Grading of the degree of liver fibrosis was taken after Sirius staining. After taking pictures of liver slices of each mouse, NAS scores (NAS score = steatosis score + inflammation score) and fibrosis score were performed. (The results are shown in Figure 12-15)

The results of Fig. 12 to Fig. 15 show that: (1) Compared with the vehicle control group, M2, M4 and somaglutide have significant improvement on blood biochemistry; at the same time, M4 has better effect on AST and ALT. Marupeptide (P<0.05); (2) The NAS score of the model group was significantly higher than that of the healthy control group. The NAS score and hepatic steatosis in the M4 group were significantly improved (P<0.05), but the inflammatory cell infiltration was improved. However, the difference was not significant; (3) Compared with the vehicle control group, the liver weight of each test group showed a significant decrease, which was statistically significant (P<0.05).

Example 14 Hypoglycemic Study of Fatty Acid Modified Derivatives of GLP-1 Analogs in Normal Mice

Twenty-eight healthy CD-1 female mice aged 4-6 weeks were divided into 4 groups, subcutaneously injected with M2, M4, M0 and somaglutide (Aib) at a dose of 0.15 mg/kg body weight. According to the pre-dose, 20% glucose was administered at intervals of 6h, 1 day, 2 days, 3 days and 4 days after the administration, the dose was 2g/kg body weight, fasted for 6h before sugar feeding, and 0 after sugar feeding. At 0.5, 1, and 2 h, blood was taken from the tip of the tail, and the blood glucose level was measured in real time using Roche blood glucose test paper, and the blood glucose AUC (area under the blood glucose to time curve) was calculated within 0 to 120 minutes, and the blood glucose suppression rate was calculated (Table 11).

Table 14 Comparison of hypoglycemic effects in normal mice

P value: compared with pre-dose blood glucose

As can be seen from Table 14, the hypoglycemic activity of somaglutide in normal mice lasted about 2 days, and the hypoglycemic activity of M0 in normal mice lasted about 3 days, while M2 and M4 were in normal mice. On day 4, it still showed significant hypoglycemic activity, and the time to maintain sustained hypoglycemic activity in the body was significantly longer than that of somaglutide or M0, and the hypoglycemic effect of M2 and M4 was also observed at various time points after the third day of administration. Both are significantly stronger than somatoglutide or M0.

Twenty-eight healthy CD-1 female mice, aged 4-6 weeks, were divided into 4 groups, subcutaneously injected with M4, M5, M7 and M0, respectively at a dose of 0.15 mg/kg body weight. According to the pre-dose, 20% glucose was administered at intervals of 6h, 1 day, 2 days, 3 days and 4 days after the administration, the dose was 2g/kg body weight, fasted for 6h before sugar feeding, and 0 after sugar feeding. At 0.5, 1, and 2 h, blood was taken from the tip of the tail, and the blood glucose level was measured in real time using Roche blood glucose test paper, and the blood glucose AUC (area under the blood glucose to time curve) was calculated within 0 to 120 minutes, and the blood glucose suppression rate was calculated (Table 15).

Table 15 Comparison of hypoglycemic effects in normal mice

From the results of Table 14 and Table 15, M2 and M4 were better than M0 and Aib, and the effects between M2, M4, M5 and M7 were comparable, and there was no significant difference.

Example 15 Study on Hypoglycemic Effect of ICR Mice

ICR mouse OGTT test: 30 ICR mice aged 4-6 weeks were divided into 6 groups, 5 rats/group, subcutaneously injected with M0, somaglutide, M2, M4, M5 and M7, respectively. A single dose of 0.15 mg/kg body weight. According to the time of 4h, 1d, 2d, 3d, 4d, 5d, 20% glucose was administered daily, the dose was 2g/kg body weight, fasted for 6h before sugar, and 0, 0.5, 1, 2 hours after sugar supply. Blood was taken from the tip of the tail and blood glucose was measured in real time using Roche blood glucose test strips. The tail tip was taken and the blood glucose level was measured in real time using a Roche blood glucose test strip, and the blood glucose AUC (area under the blood glucose to time curve) in 0 to 120 minutes was calculated, and the blood glucose suppression rate was calculated (Table 16).

Table 16 Comparison of hypoglycemic effects in ICR mice

From the results in Table 16, it can be seen that the hypoglycemic maintenance effect: M4, M5, M2, M7 can maintain hypoglycemic effect for at least 4 days, far better than M0 (only maintained for 3 days) and somaglutide (only maintain 2 Day), both have statistical significance.

Example 16 Pharmacodynamic test of db/db fractional hypoglycemic effect on type II diabetes

50 db/db mice, female, 8-9 weeks old, were divided into 10 groups according to the pre-dose weight and fasting blood glucose level (FBG), 5/group, respectively, a single subcutaneous injection of vehicle, M2, M4, Somatoglutide, M9, M11, M13, M14, M16 and M17 were administered at 10 ml/kg at a dose of 0.05 mg/kg. The administration time was set to 0h, and the fasting blood glucose was measured after 6-8 hours of fasting in the mice every day. Fasting blood glucose was measured every day after administration until the fasting blood glucose of each test group returned to the end before administration. The blood glucose level detected before administration is called the basic blood glucose level and is set to zero.

Fasting blood glucose change value (Δ: delta) = blood glucose level after administration - basic blood glucose level before administration.

The results are shown in Fig. 16. From the 4th and 5th days, it can be seen that M9, M13, M14 have lower hypoglycemic effect than somadutide and not lower than M2, while M11, M16 and M17 are in the first It showed lower hypoglycemic effect than somatoglutide in 2 days.

Example 17 Hypoglycemic effect test of different doses of somaglutide, M0 and M4 on type 2 diabetes db/db mice

35 db/db mice, female, 8-9 weeks old, were divided into 7 groups according to the weight of the pre-dose weight and blood glucose curve (G-AUC), 5/group, respectively, a single subcutaneous injection of vehicle, M4 (0.15, 0.015 mg/kg), somaglutide (0.15, 0.015 mg/kg) and M0 (0.15, 0.015 mg/kg) were administered at 10 ml/kg. The administration time was set to 0h, and after fasting for 7-8h every day, fasting blood glucose and OGTT (oral glucose tolerance test) were measured, 1g/kg body weight was administered with 10% glucose, and then 0, 0.5, 1, after sugar load. 2h, respectively, blood was taken from the tip of the tail to detect blood glucose in real time. Blood glucose was measured as random blood glucose before fasting every day after administration until the blood glucose of each test group returned to the end of the pre-dose level. 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 the bases for measuring the efficacy, and were all set to zero.

Blood glucose change (Δ: delta) = blood glucose level after administration - basic blood glucose level before administration;

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

The results are shown in Tables 17, 18 and 19 and Figures 17, 18 and 19.

Table 17 Table of fasting blood glucose changes in each test group

Note: "-21h" represents the fasting blood glucose base before administration.

Table 18 Mean randomized blood glucose changes in mice of each test group

Note: "-5h" represents the random blood glucose base 5 hours before administration.

Table 19: Changes in area under the blood glucose curve (G-AUC) of each test group

Note: “-21h” represents the area under the blood glucose curve before administration.

The results in Tables 17-19 and 17-19 show that:

Fasting blood glucose: M4 0.15mg/kg dose group recovered to the basal blood glucose base before administration at 123h after administration, and the 0.015mg/kg dose group recovered to the basal blood glucose base before administration at 99h after administration; somaglutide 0.15mg The /kg dose group returned to the basal blood glucose base before administration at 51 h after administration, and the 0.015 mg/kg dose group returned to the pre-dose basal blood glucose base at 27 h after administration; the M0 0.15 mg/kg dose group was 75 h after administration. After returning to the basal blood glucose base before administration, the 0.015 mg/kg dose group returned to the basal blood glucose base before administration at 51 h after administration; among them, the 0.015 mg/kg dose group of M4 was not low in the fasting blood glucose at each test time. In the 0.15 kg/kg dose group of somaglutide or M0.

Randomized blood glucose: M4 0.15mg/kg dose group returned to the pre-dose random blood glucose level at 115h after administration, and the 0.015mg/kg dose group returned to the pre-dose random blood glucose base at 115h after administration; somaglutide 0.15mg The /kg dose group returned to the pre-dose random blood glucose level at 67h after administration, and the 0.015mg/kg dose group returned to the pre-dose random blood glucose level at 67h after administration; M0 0.15mg/kg dose group was 67h after administration The random blood glucose level was restored to the pre-dose group. The 0.015 mg/kg dose group returned to the pre-dose random blood glucose level at 67 h after administration. Among them, the M15 0.015 mg/kg dose group inhibited the random blood glucose at each test time. Not less than the 0.15 kg/kg dose group of somaglutide or M0.

Area under the blood glucose curve (G-AUC): The M4 0.15 mg/kg dose group resumed the area under the blood glucose curve at 99 h after administration, and the 0.015 mg/kg dose group resumed the pre-dose blood glucose curve at 99 h after administration. The area base; the dose group of the 0.15 mg/kg dose of somaglutide was restored at 51 h after administration, and the area base of the blood glucose curve before the administration was resumed at 51 h after the administration of the 0.015 mg/kg dose group; M0 In the 0.15mg/kg dose group, the area under the blood glucose curve was restored at 51h after administration, and the 0.0115mg/kg dose group was restored to the area under the blood glucose curve before administration at 27h after administration; among them, M15 was 0.015mg/kg. The area under the blood glucose curve at each test time point of the dose group was not lower than the 0.15 kg/kg dose group of somatoglutide or M0.

These hypoglycemic results showed that after a single subcutaneous injection of M4 or somaglutide or M0, each group showed significant hypoglycemic effect, but M4 had the best hypoglycemic effect. The 0.015 mg/kg dose hypoglycemic effect of M4 corresponds to a hypoglycemic effect of a 0.15 mg/kg dose of somaglutide or a 0.15 mg/kg dose of M0.

Example 18 Study on Anti-enzymatic Degradation Stability of M4 and Somatoglutide

Pepsin (3200-4500 U/mg protein from sigma, Cat. No. P6887), trypsin (about 10000 AEE U/mg protein, source Sigma, Cat. No. T8003).

(1) Reaction solution

A: Pepsin reaction buffer: Three different pH (2.6, 4.0, 7.4) 20 mM citrate-phosphate buffers were prepared, and 0.005% Tween 20 and 0.001% BSA were added as pepsin reaction buffer.

B: Trypsin reaction buffer: Two 20 mM citrate-phosphate buffers of different pH (4.0, 6.8, 8.0) were placed, and 0.005% Tween 20 and 0.001% BSA were added as a pepsin reaction buffer.

C: Pepsin-containing simulated gastric juice (SGF): 5 ml of 0.1 M hydrochloric acid was taken, and 0.019 g of pepsin was added to dissolve it.

D: Simulated intestinal fluid (SIF) containing trypsin: Take 0.0684 g of potassium dihydrogen phosphate, dissolve it with 2.5 ml of water, add 0.77 ml of 0.2 M sodium hydroxide solution and 5 ml of water, and add 0.1001 g of trypsin to dissolve it. The pH was measured to be 6.82, and then diluted with water to 10 ml.

(2) Sample configuration

A sample of M4 and somaglutide was taken and diluted to 1.33 mg/ml with PB buffer of pH 7.4 as a mother liquor for the test sample.

(3) Pepsin degradation experiment

Take appropriate amount of the test solution mother liquor, dilute to 0.06mg/ml with different pH pepsin reaction buffer solution, divide each reaction solution into 1ml/tube, a total of 7 tubes, mix and incubate in 37°C water bath for 30min. Take 1 tube without SGF as the no-enzyme reaction 0 point (denoted as -5min point), then take out 6 tubes and add SGF to mix separately. One tube immediately added the appropriate amount of 1M NaOH to stop the reaction, as the 0 point after enzyme addition. (Remarked as 0min point), the remaining 5 tubes were further placed at 37 ° C, and a group of 5M, 1 min, 5 min, 35 min, 50 min, respectively, was taken to stop the reaction. All tubes in all experimental groups ensured that the total volume after termination of the reaction was consistent.

(4) Trypsin degradation experiment

Take appropriate amount of test solution mother liquor, dilute to 0.06mg/ml with different pH trypsin buffer, divide each reaction solution into 1ml/tube, a total of 7 tubes, mix and incubate in 37°C water bath for 30min. Take 1 tube without SIF as the no-enzyme reaction 0 point (denoted as -5min point), then take out 6 tubes and add SIF to mix separately. One tube immediately added the appropriate amount of 6M HCl to stop the reaction, as the 0 point after enzyme addition. (Remarked as 0min point), the remaining 5 tubes were continuously placed at 37 ° C, and a group of 5M HCl was added to remove the reaction at 5 min, 10 min, 20 min, 35 min, and 50 min, respectively. All tubes in all experimental groups ensured that the total volume after termination of the reaction was consistent.

The enzymatic degradation experiment was sampled and subjected to HPLC detection. The peak area of the main peak of the sample without the enzyme reaction 0 point (denoted as -5 min point) was used as the base peak area, and the remaining percentage of the main peak area at different time points obtained after the addition of the enzyme was calculated.

The pepsin degradation experimental data (n=3) showed (Fig. 20) that the degradation rate of M4 and somaglutide molecules under acidic conditions (pH 2.6) was comparable because the pepsin activity was highest at this pH; At neutral pH 7.4, both molecules were not degraded, and the activity of gastric protein was the lowest. At pH 4.0, the rate of degradation of somatoglutide was significantly higher than that of M4. The former was about t1/2. At 10 min, the latter t1/2 was about 45 min, indicating that the ability of M4 to resist pepsin degradation was significantly better than that of somatoglutide.

The experimental data of trypsin degradation (n=4) showed (Fig. 21). The rates of degradation were basically the same under pH 6.8 and 8.0, because the pH range was the highest activity range of trypsin; at pH 4.0. M4 and somaglutide also showed resistance to trypsin degradation, and there was essentially no difference between the two.

Claims (21)

1. A method for treating a disorder associated with a glucose metabolism disorder, a disorder associated with a fat metabolism disorder, or a neurodegenerative disease, comprising administering to a subject an effective amount of a derivative of a GLP-1 (7-37) analog or a pharmaceutically acceptable thereof a salt wherein the GLP-1 (7-37) analog comprises an amino acid sequence of the formula:

   HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇ ,

   Wherein X _{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, and 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,

   The derivative comprises an extension linked to the K residue of the GLP-1 (7-37) analog, wherein the extension is

   

   

   Wherein x is an integer from 4 to 38.
2. The method of claim 1 wherein said extension of said derivative or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

   HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-, HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO- and HOOC(CH ₂ ) ₂₂ CO-.
3. The method according to claim 1 or 2, wherein the extension in the derivative or a pharmaceutically acceptable salt thereof is linked to the K residue of the GLP-1 (7-37) analog through a linker.
4. The method of claim 3 wherein the linker in the derivative or a pharmaceutically acceptable salt thereof is:

   

   

   

   Wherein m is 0, 1, 2 or 3; n is 1, 2 or 3; s is any integer from 0 to 6; p is an arbitrary integer from 1 to 8,

   Preferably, the joint is:

   

   Wherein m is 1 or 2; n is 1 or 2; and p is an arbitrary integer from 1 to 5.
5. The method of claim 4 wherein the linker in the derivative or a pharmaceutically acceptable salt thereof is:

   

   Where m is 1, and n is 1 or 2.
6. The method according to any one of claims 1 to 5, wherein the derivative or a pharmaceutically acceptable salt thereof is selected from any one of the following derivatives or a pharmaceutically acceptable salt thereof: N-ε ²³ -[ 2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutanoylamino]ethoxy)ethoxy]acetyl Amino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37))peptide (M2), N-ε ³⁰ -[2-(2- [2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy ]ethoxy)acetyl](Val ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37))peptide (M4), N-ε ³⁴ -[2-(2-[2-(2) -[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy) Acetyl] (Val ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1 (7-37)) peptide (M5), N-ε ³⁷ -[2-(2-[2-(2-[2-(2-) [4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Val ⁸ Glu ²² Arg ^26,34 Lys ³⁷ -GLP-1(7-37))peptide (M7), N-ε ²³ -[2-(2-[2-(2-[2-( 2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Ile ⁸ Glu ²² Lys ²³ Arg ^26,34 -GLP-1(7-37))peptide (M9)\N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4- (17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl](Thr ⁸ Glu ²² Lys ³⁰ Arg ^26,34 -GLP-1(7-37))peptide (M13)\N-ε ³⁰ -[2-(2-[2-(2-[2-(2-[4-(17-carboxy- ten) Heptaylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl](Ile ⁸ Glu ²² Lys ³⁰ Arg ^26,34 - GLP-1 (7-37)) peptide (M14).
7. The method of any one of claims 1 to 6, wherein the disease is selected from one or more of the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, Cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, inflammatory bowel disease, dyspepsia and gastrointestinal ulcers.
8. The method of claim 7, wherein said diabetic complications include diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy, and distal limb necrosis of the lower extremities.
9. The method of claim 7 wherein said obesity is congenital obesity or secondary obesity.
10. The method of claim 7 wherein said fatty liver is alcoholic fatty liver or nonalcoholic fatty liver.
11. The method of claim 10, wherein the derivative or a pharmaceutically acceptable salt thereof reduces one or more blood biochemical indicators of the fatty liver subject as follows: blood TC, TG, ALT, AST, HDL-C, and LDL- C level.
12. The method of claim 11 wherein said derivative or a pharmaceutically acceptable salt thereof also improves NAS scores in fatty liver subjects.
13. The method of claim 1 wherein the neurodegenerative disease comprises Parkinson's syndrome and Alzheimer's disease.
14. A method of protecting a liver of a subject with liver damage comprising administering to a subject an effective amount of the derivative of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.
15. The method of claim 14 wherein said liver damage is a liver damage caused by a chemical.
16. The method of claim 15 wherein said chemical is a poison gas, a drug, a toxin or an alcohol.
17. The method of any one of claims 14-16, wherein the derivative or a pharmaceutically acceptable salt thereof lowers blood ALT, AST and/or TBIL levels in the subject.
18. A method of lowering blood glucose and/or reducing body weight, comprising administering to a subject an effective amount of the derivative of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.
19. A GLP-1 (7-37) analog comprising a polypeptide consisting of the following amino acid sequences:

    HX ₈ EGTFTSDVSSX ₁₉ LEEX ₂₃ AARX ₂₇ FIX ₃₀ WLVX ₃₄ GX ₃₆ X ₃₇

    Wherein 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, and 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.
20. A derivative comprising the analog of claim 19.
21. A pharmaceutical composition comprising the analog of claim 19.

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