A long-acting GLP-1 polypeptide compound, composition and application thereof

Abstract

The present disclosure provides a long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, a composition and use thereof, wherein the amino acid sequence is as follows: Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS; the long-acting GLP-1 polypeptide compound, by replacing amino acids at specific sites of the polypeptide main chain, significantly exhibits good polypeptide activity, hydrolysis resistance and stability, has a long half-life, and has the pharmacological effects of treating diabetes and reducing body weight.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2024101571494 filed on February 4, 2024, and this application cites the full text of the above-mentioned Chinese patent application. Technical Field

[0003] The present disclosure belongs to the field of biochemical technology. Specifically, the present disclosure relates to a novel long-acting GLP-1 polypeptide compound, composition and application thereof that can be used to treat or prevent diabetes or obesity. Background Art

[0004] Diabetes is a common non-communicable disease in clinical practice, which has a great impact on physical health and seriously threatens the life and health of patients. Among them, most diabetic patients belong to type II diabetes (diabetes mellitus type 2, T2DM), which is also called non-insulin-dependent diabetes mellitus (noninsulin-dependent diabetes mellitus, NIDDM) or adult-onset diabetes. It is a chronic metabolic disease that mostly occurs after the age of 35 to 40, accounting for more than 90% of diabetic patients. Patients with type II diabetes are characterized by high blood sugar, relative lack of insulin, insulin resistance, etc., and may also include polyphagia, fatigue or soreness (Report of a WHO Consultatio. 2011).

[0005] In addition, type 2 diabetes is also accompanied by a large number of serious complications and increases the incidence of other diseases. For example, patients with type 2 diabetes are at high risk of malignant tumors (Epidemiological analysis of patients with type 2 diabetes and malignant tumors. Chinese Journal of Chronic Disease Prevention and Control. 2018(07)); type 2 diabetes is one of the important pathogenic factors of cerebral infarction, and the prevention and treatment of diabetes is of great significance in reducing the occurrence of cerebral infarction (Clinical analysis of 85 cases of acute cerebral infarction in type 2 diabetes. Contemporary Chinese Medicine. 2009(09)).

[0006] Currently, common drugs used to treat diabetes mainly include injectable and oral types. Injectable hypoglycemic drugs include insulin and GLP-1 receptor agonists. Insulin is mostly a daily preparation that needs to be injected at least once a day, and up to 2 to 4 times a day. It may also need to be combined with oral hypoglycemic drugs (Development and Clinical Research Progress of Weekly Basal Insulin Preparations. Drug Evaluation. 2022, 19(12)). GLP-1 receptor agonists can effectively treat type II diabetes and have a good effect on controlling blood sugar indicators. Tirzepatide is a new GIP / GLP-1 receptor dual agonist with strong sugar control and weight loss effects. It was approved for marketing by the US FDA in May 2022. As an adjunct to diet and exercise, it is injected once a week to improve blood sugar in adult patients with type II diabetes, but the cost of medication is relatively high.

[0007] Diabetes is not easy to cure, and usually requires lifelong medication to control blood sugar, but the currently available hypoglycemic drugs all have certain limitations. Therefore, for diabetic patients, it is very important to study and obtain drugs with ideal hypoglycemic effects and fewer adverse reactions, which is also an important problem that technicians in this field need to solve. Summary of the invention

[0008] The present disclosure relates to a novel long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, a composition and use thereof. The polypeptide compound is more long-acting and has good activity, hydrolysis resistance and stability.

[0009] In one aspect, the present disclosure provides a long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, the amino acid sequence of which is as follows:

[0010] Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS(SEQ ID NO:26),

[0011] wherein X2 represents an amino acid selected from Aib, Iva or Cba; X12 represents an amino acid selected from I or L; X13 represents an amino acid selected from Aib, Iva or Cba; X20 is selected from K, K((AEEA) a -γGlu-CO((CH2) b CO2H)、K((PEG2) c -γGlu-CO(CH2) d CO2H) or K(G e (SG) f -γGlu-CO(CH2) gCO2H), wherein a is an integer selected from 1-6, b is an integer selected from 12-20, c is an integer selected from 1-6, d is an integer selected from 12-20, e is an integer selected from 0-5, f is an integer selected from 1-5, and g is an integer selected from 12-20; X29 represents an amino acid selected from G, A or V.

[0012] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein, when X2 is Iva, X29 is G; or, when X2 is Cba, X12 is I.

[0013] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X13 is Iva.

[0014] In some optional embodiments, in the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, X29 is G.

[0015] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X20 is K(G e (SG) f -γGlu-CO(CH2) g CO2H), wherein e is 2, f is 2 or 3, and g is 16 or 18.

[0016] In some optional embodiments, the amino acid sequence of the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is as follows:

[0017] Y-X2-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:27),

[0018] wherein X2 represents an amino acid selected from Aib or Iva.

[0019] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof comprises an amino acid sequence selected from SEQ ID NO: 1-25.

[0020] In some optional embodiments, the N-terminus or C-terminus of the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is modified with an auxiliary group.

[0021] On the other hand, the present disclosure provides a pharmaceutical composition comprising the long-acting GLP-1 polypeptide compound according to any aspect above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, adjuvant or excipient.

[0022] On the other hand, the present disclosure provides use of the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate or pharmaceutical composition thereof in the preparation of a drug for preventing or treating diabetes or obesity.

[0023] On the other hand, the present disclosure provides a method for preparing the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein the preparation method is by a chemical synthesis method.

[0024] The long-acting GLP-1 polypeptide compound disclosed herein significantly exhibits good activity, hydrolysis resistance and stability of the polypeptide by replacing amino acids at specific positions in the polypeptide main chain. Through a large number of pharmacodynamic activity tests, the data show that: after the 2nd and / or 13th amino acid positions of the main peptide chain of the polypeptide compound disclosed herein are replaced with non-natural amino acids Iva, Cba or Aib; after the 12th amino acid position is replaced with I or L, the activity, hydrolysis resistance, stability and drug half-life of the GLP-1 polypeptide compound are significantly enhanced. At the same time, the replacement of the 28th amino acid position with E and the 29th amino acid position with G, V or A amino acids will also significantly enhance the half-life and stability of the polypeptide molecule.

[0025] The half-life of the peptide drugs in rats reported so far is basically less than 10 hours, and the frequency of drug administration can only be achieved once a week. The long-acting peptide compound disclosed in the present invention greatly prolongs the half-life, and the half-life of the drug in rats can reach more than 24 hours, realizing the ultra-long-acting peptide drug, and achieving the frequency of drug administration once every 2 weeks or more for human use. It has good pharmacodynamic effects in treating diabetes and reducing body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0027] Figure 1 The graph shows the effect of compound 1-25 on blood glucose in db / db mice within 0-8 hours in Example 3.

[0028] Figure 2 The graph shows the results of the effects of compounds 11-13, 15-19, and 21-24 on blood glucose in db / db mice within 0-168 hours in Example 3.

[0029] Figure 3The graph shows the results of the effects of compounds 11-13, 15-19, and 21-24 in Example 4 on the body weight of db / db mice.

[0030] Figure 4 The graph shows the effect of continuous administration of compounds 11-13, 15-19, and 21-24 in Example 5 on the blood glucose of ob / ob mice.

[0031] Figure 5 The graph shows the effect of continuous administration of compounds 11-13, 15-19, and 21-24 in Example 5 on the body weight of ob / ob mice.

[0032] Figure 6 The chromatograms of compound 12 at 0d, 5d and 10d in Example 7 are shown.

[0033] Figure 7 The chromatograms of compound 21 at 0d, 5d and 10d in Example 7 are shown.

[0034] Figure 8 The chromatograms of the reference compound at 0d, 5d and 10d in Example 7 are shown. DETAILED DESCRIPTION

[0035] I. Definitions

[0036] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0037] The term "amino acid" refers to a molecule containing both an amino group and a carboxyl functional group. The amino group and the carboxyl group of an α-amino acid are attached to the same carbon atom (the α carbon). The α carbon may have 1-2 additional organic substituents. Amino acids include L and D isomers and racemic mixtures. Unless otherwise specified, the amino acid residues in the polypeptide sequence of the present disclosure are all L isomers, i.e., L-amino acids. D-amino acids are represented by a lowercase "d" before the amino acid name or abbreviation, such as dK.

[0038] The amino acid sequences disclosed herein contain conventional single-letter or three-letter codes for naturally occurring amino acids, as well as commonly recognized three-letter codes for other amino acids, such as Iva (Isovaline); Cba (1-Aminocyclobutanecarboxylic acid, α-aminocyclobutyric acid). Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), Aib (α-Aminoisobutyric acid, α-aminoisobutyric acid) or GABA (γ-aminobutyric acid). Abbreviated codes for commonly used molecular structures include:

[0039] hGlu is homoglutamate;

[0040] α-hGlu is the L isomer of -HNCH(CO-)CH2CH2CH2COOH;

[0041] δ-hGlu is the L isomer of -HNCH(COOH)CH2CH2CH2CO-;

[0042] α-Glu is the L isomer of -HNCH(CO-)CH2CH2COOH;

[0043] γ-Glu or gGlu is the L isomer of -HNCH(COOH)CH2CH2CO-;

[0044] α-Asp is the L isomer of -HNCH(CO-)CH2COOH;

[0045] β-Asp is the L isomer of -HNCH(COOH)CH2CO-;

[0046] β-Ala is -HN-CH2-CH2-COOH;

[0047] PEG2 is 2-(2-(2-aminoethoxy)ethoxy)acetic acid (CAS No. 134978-97-5).

[0048] The amino acid composition of the polypeptides disclosed herein can be changed without substantially affecting their biological activity. For example, a polypeptide sequence can contain one or more conservative amino acid substitutions. A conservative amino acid substitution is a substitution of one amino acid residue by another amino acid residue having a similar side chain. Amino acid residues are classified in the literature according to the properties of the amino acid residue side chains. Amino acid residues containing basic side chains include lysine, arginine, and histidine; amino acid residues containing acidic side chains and their amide side chains include aspartic acid, glutamic acid, asparagine, and glutamine; small aliphatic, non-polar or weakly polar side chain amino acid residues include glycine, alanine, threonine, serine, and proline; large aliphatic, non-polar side chain amino acid residues include leucine, isoleucine, and valine; aromatic amino acid residues include phenylalanine, tryptophan, and tyrosine; sulfur-containing side chain amino acid residues include cysteine ​​and methionine.

[0049] As used herein, the term "treatment" includes inhibiting, slowing down, stopping or reversing the progression or severity of existing symptoms or illness. Therefore, treatment includes prevention, treatment and / or cure. Prevention refers to preventing potential diseases and / or preventing symptoms from worsening or disease development. As used herein, "therapeutic effect" means the effect caused by the treatment of an individual, which changes, generally improves or improves the symptoms of a disease or disease condition, or cures a disease or disease condition. As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material or composition comprising a compound that is at least sufficient to produce a therapeutic effect after being applied to an object. Therefore, it is the amount necessary to prevent, cure, improve, block or partially block the symptoms of a disease or condition. As used herein, "preventive effective amount" or "preventive effective dose" refers to the amount of a substance, compound, material or composition comprising a compound that will have the expected preventive effect when applied to an object, for example, preventing or delaying the occurrence or recurrence of a disease or symptom, reducing the possibility of the occurrence or recurrence of a disease or symptom. A completely preventive effective dose does not have to occur by administering one dose, and can occur only after a series of doses are administered. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0050] As used herein, the term "patient" refers to a mammal, such as a human.

[0051] Certain compounds of the present disclosure are generally effective over a wide dosage range. For example, a dose administered once a week can be in the range of about 0.05 to about 30 mg per person per week. Certain compounds of the present disclosure can be administered daily. In addition, certain compounds of the present disclosure can be administered once a week.

[0052] It should be understood that the therapeutic agent according to the embodiment will be administered with pharmaceutically acceptable suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in the pharmacopoeias known to all pharmaceutical chemists. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) carriers (e.g., LipofectinTM), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the aforementioned mixtures may be applied to treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerates the route of administration.

[0053] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference work in this field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0054] Preparations to be used for clinical in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes.

[0055] II. Detailed description of specific implementation scheme

[0056] In one aspect, the present disclosure provides a long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, the amino acid sequence of which is as follows:

[0057] Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS(SEQ ID NO:26),

[0058] wherein X2 represents an amino acid selected from Aib, Iva or Cba; X12 represents an amino acid selected from I or L; X13 represents an amino acid selected from Aib, Iva or Cba; X20 is selected from K, K((AEEA) a -γGlu-CO((CH2) b CO2H)、K((PEG2) c -γGlu-CO(CH2)d CO2H) or K(G e (SG) f -γGlu-CO(CH2) g CO2H), wherein a is an integer selected from 1-6, b is an integer selected from 12-20, c is an integer selected from 1-6, d is an integer selected from 12-20, e is an integer selected from 0-5, f is an integer selected from 1-5, and g is an integer selected from 12-20; X29 represents an amino acid selected from G, A or V.

[0059] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein, when X2 is Iva, X29 is G; or, when X2 is Cba, X12 is I.

[0060] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X13 is Iva.

[0061] In some optional embodiments, in the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, X29 is G.

[0062] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X20 is K(G e (SG) f -γGlu-CO(CH2) g CO2H), wherein e is 2, f is 2 or 3, and g is 16 or 18.

[0063] In some optional embodiments, the amino acid sequence of the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is as follows:

[0064] Y-X2-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:27),

[0065] wherein X2 represents an amino acid selected from Aib or Iva.

[0066] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof has an amino acid sequence selected from the following:

[0067] <h2 style=";text-align:left;direction:ltr">Image 1:Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:1);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0068] <h2 style=";text-align:left;direction:ltr"> Page 2: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:2);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] <h2 style=";text-align:left;direction:ltr"> Page 3: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEVGPSSGAPPPS(SEQ ID NO:3);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0070] <h2 style=";text-align:left;direction:ltr"> Page 4: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEAGPSSGAPPPS(SEQ ID NO:4);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] <h2 style=";text-align:left;direction:ltr"> Image 5: Y-Aib-EGTFTSDYSL-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:5);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> Image 6:Y-Aib-EGTFTSDYSL-Iva-LDKIAQKAFVQWLLEAGPSSGAPPPS(SEQ ID NO:6);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> Image 7: Y-Cba-EGTFTSDYSL-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> Image 8:Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:8);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075] <h2 style=";text-align:left;direction:ltr"> Image 9:Y-Iva-EGTFTSDYSL-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS(SEQ ID NO:9);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0076] <h2 style=";text-align:left;direction:ltr"> Image 10:Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEAGPSSGAPPPS(SEQ ID NO:10);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> Source 11: Y-Cba-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)<h2 style=";text-align:left;direction:ltr"> 18CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:11);

[0078] Compound 12: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:12);

[0079] Compound 13: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSG-γGlu-CO(CH2) 16 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:13);

[0080] Compound 14: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:14);

[0081] Compound 15: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:15);

[0082] Compound 16: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEVGPSSGAPPPS(SEQ ID NO:16);

[0083] Compound 17: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS(SEQ ID NO:17);

[0084] Compound 18: Y-Aib-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:18);

[0085] Compound 19: Y-Aib-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS(SEQ ID NO:19);

[0086] Compound 20: Y-Cba-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:20);

[0087] Compound 21: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:21);

[0088] Compound 22: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:22);

[0089] Compound 23: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQWLLEVGPSSGAPPPS(SEQ ID NO:23);

[0090] Compound 24: Y-Iva-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:24); or

[0091] Compound 25: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS (SEQ ID NO: 25).

[0092] In some optional embodiments, the N-terminus or C-terminus of the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is modified with an auxiliary group.

[0093] On the other hand, the present disclosure provides a pharmaceutical composition comprising the long-acting GLP-1 polypeptide compound according to any aspect above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, adjuvant or excipient.

[0094] On the other hand, the present disclosure provides use of the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate or pharmaceutical composition thereof in the preparation of a drug for preventing or treating diabetes or obesity.

[0095] On the other hand, the present disclosure provides a method for preparing the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein the preparation method is by a chemical synthesis method.

[0096] The compounds of the present disclosure can react with any of a variety of inorganic or organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methods for preparing them are well known in the art. Commonly used pharmaceutically acceptable salts include trifluoroacetate, acetate, citrate, hydrochloride, etc.

[0097] The pharmaceutical composition of the embodiment is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol, methyl parabens, phenol, or metacresol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for regulating osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, vials, disposable syringes, glass or plastic multidose vials, or injection pens. There are two main types of injection pens. One is a disposable pre-filled pen that contains medication and does not require replacement of the drug cartridge, so it can be thrown away after use. The other is a more commonly used durable injection pen, which consists of an injection pen and a drug cartridge. After use, you can replace the cartridge and continue to use it.

[0098] Pharmaceutical compositions suitable for injection purposes include sterile aqueous solutions (water-soluble in this case) or dispersions and sterile powders for the immediate preparation of sterile injections or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluidity should reach a degree that is easy to inject. It must be stable under manufacturing and storage conditions and must be able to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and a suitable mixture thereof. For example, by utilizing a coating such as lecithin, maintaining the desired particle size in the case of a dispersion, and utilizing a surfactant, suitable fluidity can be maintained. The prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, metacresol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0099] As required, sterile injection solutions can be prepared by incorporating the disclosed compounds in the required amount into a suitable solvent having one or a combination of the above-listed components (as required), followed by filtration sterilization. Generally speaking, dispersions are prepared by incorporating the disclosed compounds into a sterile carrier containing a dispersion medium and those other required components listed above. With regard to sterile powders for the preparation of sterile injection solutions, the preparation method is to obtain vacuum drying and freeze drying of powders comprising active ingredients and any additional desired components from the aforementioned sterile filtered solutions of these components.

[0100] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0101] Administration can also be systemically administered via mucosal or transdermal means. For mucosal or transdermal administration, a penetrant suitable for permeation of the barrier is used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts and fusidic acid derivatives such as those used for mucosal administration. Mucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the disclosed compounds can be formulated into an ointment, an ointment, a gel, or a cream as is generally known in the art.

[0102] The compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0103] In some embodiments, the disclosed compounds can be prepared with carriers that prevent them from being rapidly eliminated by the body, such as sustained release / controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

[0104] For example, the active ingredients can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions.

[0105] In some embodiments, the disclosed compounds can be prepared into sustained-release preparations. Examples of suitable sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the disclosed compounds, which are in the form of molded articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methylpropionate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (microspheres for injection composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time. Polylactic acid (PLA) and polylactic-glycolic acid copolymers (PLGA) are hot topics in recent years. In addition, there are albumin microspheres, chitosan microspheres, gelatin microspheres, etc.

[0106] Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0107] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and consistency of dosage. As used herein, dosage unit form refers to physically separable units suitable as unit doses for a subject to be treated; each unit contains a predetermined amount of one or more of the disclosed compounds calculated to produce the desired therapeutic effect in combination with a desired pharmaceutical carrier. The specifications of the dosage unit form of the embodiments are dictated by and directly dependent upon the unique characteristics of the disclosed compounds and the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such disclosed compounds for treating individuals.

[0108] The pharmaceutical compositions can be placed in a container, pack, or dispenser together with instructions for administration.

[0109] The present disclosure provides a method for treating diabetes or obesity in a patient, including applying an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof to a patient in need of such treatment. The present disclosure also provides a method for treating diabetes or obesity in a patient, including applying an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof to a patient in need of such treatment, wherein the application is subcutaneous. The present disclosure also provides a method for treating diabetes or obesity in a patient, including applying an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof to a patient in need of such treatment, and simultaneously, separately, or successively applying an effective amount of one or more other active ingredients. In one embodiment, other one or more active ingredients are currently available oral glucose-lowering drugs, and the drug is from a class of drugs that are considered as standard of care before application (determined by industry guidelines such as the American Diabetes Association (American Diabetes Association)).

[0110] The present disclosure also provides methods for treating or preventing the following diseases or conditions: impaired glucose tolerance (IGT), hyperglycemia, type I diabetes, type II diabetes, obesity, metabolic syndrome and neurodegenerative diseases, in particular for delaying or preventing disease progression in type II diabetes, delaying the progression from impaired glucose tolerance to type II diabetes; delaying the progression from type II diabetes to diabetes requiring insulin; treating metabolic syndrome, for regulating appetite, inducing satiety, reducing food intake, increasing energy expenditure, treating obesity or preventing overweight; preventing weight rebound after successful weight loss; treating diseases or conditions associated with overweight or obesity; treating bulimia; treating binge eating; treating dyslipidemia, atherosclerosis, hypertension, coronary heart disease, beta-blocker poisoning; non-alcoholic fatty liver disease (NAFLD, non-alcoholic fatty liver disease liver disease) (which can be divided into simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH) and its related cirrhosis); for inhibiting the motility of the gastrointestinal tract, for use in conjunction with gastrointestinal investigation using techniques such as X-ray, CT and NMR scanning. The method comprises administering to a patient in need of such treatment an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, and simultaneously, separately, or sequentially administering an effective amount of one or more other active ingredients.

[0111] Peptide Chemical Synthesis Methods

[0112] The solid phase chemical synthesis of peptides is a well-developed methodology, for example, see RC Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989.

[0113] Linear peptides are synthesized using Boc solid phase peptide synthesis or Fmoc solid phase peptide synthesis. If Fmoc chemistry is used to synthesize a peptide with a carboxyl group at the C-terminus, Wang resin is usually selected; Rink amide resin (including Rink Amide-AM resin, Rink Amide-MBHA resin, etc.) is usually selected for a peptide with an amide group at the C-terminus. If Boc chemistry is used to synthesize a peptide with a carboxyl group at the C-terminus, Pam resin is usually selected; MBHA resin is usually selected for a peptide with an amide group at the C-terminus. Commonly used condensing agents and activators are DIC and HOBT, and other optional peptide bond condensing agents include EDC, BOP, HBTU, DEPBT, TBTU, etc. Depending on the difficulty of the reaction, amino acids can be used in 1.1-10 times the equivalent, and the reaction time is 15 minutes to 24 hours. Peptides can be synthesized manually or using a peptide solid phase synthesizer.

[0114] The Fmoc protecting group was removed with 20% piperidine / DMF. The Boc protecting group was removed with TFA. The peptide bond condensation reaction was monitored with ninhydrin (2,2-dihydroxyindane-1,3-dione) reagent.

[0115] For solid phase synthesis, a resin preloaded with a C-terminal amino acid or a resin without an amino acid preload can be used.

[0116] The method for loading the first amino acid on the Rink Amide resin can refer to the common practice in the industry. A common method is briefly described as follows: weigh an appropriate amount of resin, remove the Fmoc protecting group with 20% piperidine / DMF in a solid phase synthesis tube (15mL / g resin, 30 minutes X2), and wash the resin with DMF. Weigh 5 times the equivalent of Fmoc amino acid, HATU, HOAT and 10 times the equivalent of NMM to the amino group of the resin, add DMF and mix well, and then transfer to the solid phase synthesis tube. After reacting overnight, wash the resin with DMF. Add 1:1 acetic anhydride / pyridine (v / v) to the solid phase synthesis tube, empty it after 30 minutes, and wash the resin with DMF. The first amino acid is loaded.

[0117] When using the Fmoc solid phase peptide synthesis method, the commonly used amino acids and protecting groups are as follows:

[0118] Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-O H, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH

[0119] The synthesis process uses appropriately protected building blocks, such as the above-mentioned standard amino acids, Fmoc-8-amino-3,6-dioxaoctanoic acid (CAS No. 166108-71-0), Fmoc-Glu-OtBu (CAS No. 84793-07-7). The introduction of fatty acid moieties can be achieved using building blocks, such as but not limited to octadecanoic acid mono-tert-butyl ester. After each coupling step, the unreacted peptide intermediate can be capped with acetic anhydride (10 equivalents) and excess collidine (20 equivalents).

[0120] After solid-phase Fmoc chemical synthesis of peptides, the commonly used cleavage reagent is TFA. Place the dry resin in a shake bottle, add an appropriate amount of 90:4:2:2:2 (v / v) trifluoroacetic acid: triisopropylsilane: 1,2-ethanedithiol: water: anisyl thioether cleavage solution (10-25mL / g resin), cover the lid, and perform intermittent rotation and shaking at room temperature. After 2 hours, filter the resin, wash the resin with new TFA 2-3 times, combine the filtrate, and add 8-10 times the volume of ice ether. Finally, centrifuge to collect the precipitated peptide crude product.

[0121] When using the Boc solid phase peptide synthesis method, commonly used amino acids and protecting groups are as follows: Boc-Cys(4-MeBzl)-OH, Boc-Asp(OcHx)-OH, Boc-Glu(OcHx)-OH, Boc-His(Bom)-OH, Boc-Lys(2-Cl-Z)-OH, Boc-Asn(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Trp(CHO)-OH and Boc-Tyr(2-Br-Z)-OH

[0122] If the side chain amino group of lysine is used for lactam synthesis or acylation reaction, the side chain amino group of lysine can be protected by allyloxycarbonyl (aloc) or Fmoc. If the side chain carboxyl group of aspartic acid or glutamic acid is used for lactam synthesis or acylation reaction, the carboxyl group should be converted into allyl ester or 9-fluorenylmethyl protection, such as Boc-Glu (OAllyl) -OH, Boc-Glu (Ofm) -OH.

[0123] After solid-phase Boc chemical synthesis of peptides, HF cleavage is usually used for PAM and MBHA resins. 5 ml of HF is added to every 0.1 mmol of resin, and reagents such as p-cresol, p-mercaptophenol or anisole are added at the same time. The mixture is stirred for 1 hour in an ice bath. After HF is vacuum-dried, the peptide is precipitated with ice ether, the precipitate is collected by centrifugation, separated and purified by HPLC, and freeze-dried to obtain the final product.

[0124] purification

[0125] The crude peptide is dissolved in a suitable mixture of water and acetonitrile (e.g., water / acetonitrile (3:1)) and purified by reverse phase preparative HPLC (e.g., AKTA purifier, Shimadzu LC-20AR, etc.), and columns of different fillers and sizes are selected according to the amount of crude peptide loaded and the polarity, such as C8 or C18 semi-preparative columns or preparative columns. Buffer A is a 0.1% TFA aqueous solution, and buffer B is 0.1% TFA in acetonitrile. Buffer B is eluted by gradient rise, and the relevant fractions are checked by analytical HPLC. A ZORBAX 300SB-C18 (4.6X250mm, 5μM) column is used, buffer A is a 0.1% TFA aqueous solution, and buffer B is 0.1% TFA in acetonitrile. The flow rate is 1ml / min, and the detection is at a wavelength of 210nm. The fractions containing the pure target peptide are mixed and freeze-dried to obtain the peptide trifluoroacetate as a white solid. The product is stored in glass vials.

[0126] Preparation method

[0127] The compounds disclosed in the present invention are linear peptides. Each amino acid can be coupled stepwise in the order from the C-terminus to the N-terminus of the polypeptide sequence to obtain the polypeptide main chain. The process is: first, an amino acid whose amino group is protected by a blocking group is covalently connected to a solid phase carrier, and the amino protecting group of the first amino acid is removed, so that the first amino acid is connected to the solid phase carrier. Then, after the carboxyl group of the second amino acid whose amino group is blocked is activated, it reacts with the amino group of the first amino acid connected to the solid phase carrier to form a peptide bond, so that a dipeptide with a protecting group is generated on the solid phase carrier. Repeat the above peptide bond formation reaction to extend the peptide chain from the C-terminus to the N-terminus until the desired peptide chain is generated. Finally, the protecting group is removed, and the covalent bond between the peptide chain and the solid phase carrier is hydrolyzed to obtain a synthesized peptide.

[0128] For the specific synthesis method, please refer to patent CN2021108155833. Compound 21 is used as an example to illustrate the synthesis route and method.

[0129] Step 1: synthesizing the main peptide resin corresponding to the main peptide chain of the long-acting GLP-1 polypeptide compound 21 according to the Fmoc / t-Bu strategy;

[0130] Step 2: Based on the main peptide resin, the "side arm" structure corresponding to the long-acting GLP-1 polypeptide compound 1 is coupled according to the Fmoc / t-Bu strategy to obtain the polypeptide resin corresponding to the long-acting GLP-1 multi-compound 21;

[0131] Step 3: adding a cleavage solution to the polypeptide resin to perform a cleavage reaction, remove the polypeptide from full protection, and extract a crude compound;

[0132] Step 4: Purify the crude compound to obtain the long-acting GLP-1 polypeptide compound 21.

[0133] Preferably, the coupling agents used in step 2 are 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide, the solvent is N,N-dimethylformamide, and the Fmoc group is removed with a 20% piperidine / N,N-dimethylformamide solution.

[0134] Preferably, the lysis solution in step 3 consists of TFA, DODT, m-cresol and H2O in a volume ratio of 92.5:2.5:2.5:2.5; the crude compound extraction method includes filtration, precipitation and / or methyl tert-butyl ether extraction.

[0135] The present disclosure also includes novel intermediates and methods that can be used to synthesize compounds disclosed herein or pharmaceutically acceptable salts thereof. The intermediates and compounds disclosed herein can be prepared by a variety of methods known in the art. In particular, methods using chemical synthesis are illustrated in the following examples. The specific synthetic steps of each approach described can be combined in different ways to prepare compounds disclosed herein or salts thereof. Reagents and raw materials are readily available to those of ordinary skill in the art.

[0136] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0137] Example

[0138] Example 1: Synthesis of compounds

[0139] This example uses the Fmoc solid phase peptide synthesis method, which is synthesized from the carboxyl end to the amino end, and the amino acids of compounds 1-25 are connected in sequence according to the aforementioned amino acid sequence. For the specific synthesis method, refer to patent CN2021108155833. The isolated product peptide was identified by LC-MS, starting with 5% ACN / H2O (containing 0.1% formic acid), with a gradient (increasing the proportion of ACN at a rate of 6% / min), a flow rate of 0.4mL / min, and elution analysis for 15 minutes to determine the target compound, theoretical molecular weight, and measured value [M+3H] 3+ As shown in Table 1.

[0140] Table 1 Amino acid sequence table of compounds 1-25 and LC-MS identification results

[0141]

[0142]

[0143] Example 2: Agonist activity of compound 1-25 and Tirzepatide on GLP-1 and GIP targets, respectively

[0144] The in vitro activity of compounds 1-25 was investigated, and the agonist effects of compounds 1-25 and Tirzepatide (powder, purchased from Hangzhou Gutuo Biotechnology Co., Ltd.) on GLP-1 and GIP at a series of concentrations were detected using the cAMP-GS Dynamic Kit of Cisbio.

[0145] Experimental methods:

[0146] 1) Digest the cells, centrifuge (1000 rpm, 5 min), remove the supernatant, wash with serum-free medium to remove serum, centrifuge again, count and then plate 384-well plates (containing serum-free basal medium, GIPR-HEK293, GLP-1R-Luciferase-HEK293: 3000 cells / well, 5 μl);

[0147] 2) At the same time, a series of peptide drug concentrations (starting from 100 nM, 9 points of 5-fold dilution) were prepared with Stimulation buffer 1 containing 0.5 mM IBMX (the negative control was added with the same volume of Stimulation buffer 1 containing 0.5 mM IBMX and 0.1% casein), and the plate was sealed and incubated at room temperature for 30 min;

[0148] 3) Prepare cAMP Eu Cryptate antibody and cAMP-d2 antibody working solutions with lysis and detection buffer, add 5 μl to each well after incubation (add the same volume of lysis and detection buffer to replace cAMP-d2 antibody working solution for negative control), seal the plate and incubate at room temperature in the dark for 1 hour;

[0149] 4) In compatible The fluorescence emission of two wavelengths (665nm and 620nm) was read on a microplate reader. The ratio of the emission light at 665nm to that at 620nm was calculated, and the actual cAMP level (nM) of each sample well was calculated by the cAMP standard curve. The agonist percentage at different concentrations was calculated according to the formula Activity (%) = (cAMP level of testing sample-average cAMP level of low control) / (average cAMP level of high control-average cAMP level of low control)*100%, and the EC50 value was calculated by fitting with the "log (agonist) vs. response--Variable slope" model in GraphPad Prism7.0. The test results are shown in Table 2.

[0150] Results analysis: The EC50 of the positive control Tirzepatide was consistent with the historical value, indicating that the experimental system was stable and reliable; the samples tested were mainly GIPR activity, and the GLP-1R activity from strong to weak was compound 12>13, 17-19, 21, 24>11, 15, 16, 22, 23>1-6, 8-10>7, 14, 20, 25; the GIPR activity from strong to weak was compound 12, 21>13, 17-19, 24>11, 15, 16, 22, 23>1-3, 5, 8-10>4, 6>7, 14, 20, 25. Overall, compounds 12 and 21 had the best activity, followed by compounds 13, 17-19, 24, and then 11, 15, 16, 22, 23; compounds 7, 14, 20, 25 had the worst activity.

[0151] Table 2 Agonist activity of compounds 1-25 on GLP-1 and GIP targets

[0152]

[0153]

[0154] Example 3: Effects of Compound 1-25 and Tirzepatide on Blood Glucose in db / db Mice

[0155] The in vivo efficacy of compound 1-25 was investigated, and Tirzepatide was used as the positive control group and PBS as the blank control group. The blood glucose effect and maintenance time of compound 1-25 and Tirzepatide at the same dose on high-fat-induced spontaneous diabetic db / db mice were studied by measuring blood glucose after a single administration.

[0156] Experimental methods: This experiment used 8-week-old db / db male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 7 mice / group, and the dosage of all compounds and Tirzepatide was 100nmol / kg. Grouping was based on random blood glucose and body weight data to ensure that the average body weight and random blood glucose average of each group were similar. The drugs were prepared on the day of administration and the corresponding drugs were injected subcutaneously in the back of the neck according to the group. Before administration (0h), 1h, 2h, 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration, the random blood glucose concentration was measured using a blood glucose meter after puncturing the mouse tail vein with a sterile blood collection needle. The software GraphPadPrism was used to process the data and draw a time-blood glucose curve. Table 3 shows the average blood glucose (mmol / L) of each group of mice at different times (h) after a single subcutaneous injection of the corresponding compounds 1-25 and Tirzepatide in the back of the neck; Figure 1 The blood glucose changes of mice in each group treated with compound 1-25 at 8 hours; Figure 2 The blood glucose changes of mice in each group treated with compounds 11-13, 15-19, and 21-24 during 0-168 hours.

[0157] Table 3 Average blood glucose (mmol / L) of each group of mice treated with compounds 1-25 at different times (h)

[0158]

[0159]

[0160] Results analysis: From Table 3, Figure 1 and Figure 2 It can be seen that:

[0161] (1) 1h results: The blood glucose levels of compounds 1-6, 8-13, 15-19, 21-24 and the positive control Tirzepatide group changed significantly, showing the effect of lowering blood glucose. However, there was no significant difference in blood glucose between the compound 7, 14, 20, and 25 groups and the control model group.

[0162] (2) 2h results: Compounds 2, 5, 8, 9, 10, 11-13, 15-19, 21-24 and the positive control Tirzepatide have the effect of lowering blood sugar, among which compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide have the best effect; the efficacy of compounds 2, 5, 8, 9, and 10 is reduced to varying degrees compared with 1h. The effect of compounds 1, 3, 4, and 6 is not obvious compared with the PBS control model group.

[0163] (3) 4h results: Compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide had the effect of lowering blood sugar. Compounds 2, 5, 8, 9, and 10 increased blood sugar to a level close to that before administration.

[0164] (4) 24 h results: Compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide had the effect of lowering blood sugar. Among them, the blood sugar levels of compounds 12, 13, 16, 21, and 24 were lower than that of the positive control Tirzepatide 24 h after administration, and compounds 12 and 21 were the lowest.

[0165] (5) 48h results: Compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide still have the effect of lowering blood sugar. Among them, compounds 12, 13, 21, and 24 have the best efficacy, which is close to the results at 24h, followed by compounds 11, 15, 16, 17, 18, 22, Tirzepatide, and finally compounds 19 and 23.

[0166] (6) 72h results: Compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide still have the effect of lowering blood sugar, among which compounds 12, 13, 21 and 24 have the best efficacy, followed by compounds 11, 16-18, and finally compounds 15, 19, 22 and Tirzepatide; compound 23 is close to the level before administration.

[0167] (7) 96h results: Compounds 12, 13, 21, and 24 had the best efficacy, followed by compounds 11, 15-18, and compounds 19, 22, and Tirzepatide were close to the pre-dose level.

[0168] (8) 120h results: Compounds 12 and 21 had the best efficacy, followed by compounds 13, 18, and 24, and compounds 11, 15-17 were close to the pre-dose levels.

[0169] (9) 144h results: Compounds 12, 13, 21, and 24 still have the effect of lowering blood sugar, and compounds 15 and 18 are close to the level before administration.

[0170] (9) 168h results: Compounds 12 and 21 still have the effect of lowering blood sugar, while compounds 13 and 24 are close to the level before administration.

[0171] Conclusion: Analysis of the above results shows that Tirzepatide and other compounds have good effects in lowering blood sugar. In terms of duration of efficacy, the duration of efficacy of each compound is different. Among them, compounds 12 and 21 have the most obvious advantages in both efficacy and duration (long-term effect) of lowering blood sugar, with efficacy duration of more than 168 hours, followed by compounds 13 and 24 with efficacy duration of 144 hours, and compounds 11, 15-18 with efficacy duration of more than 96 hours, which are better than Tirzepatide.

[0172] Example 4: Effects of Compounds 11-13, 15-19, 21-14 and Tirzepatide on Body Weight of db / db Mice

[0173] According to the experimental results of Example 3, the in vivo efficacy of compounds 11-13, 15-19, and 21-14 was investigated, and Tirzepatide was used as the positive control group and PBS was used as the blank control group. The body weight was measured after a single dose to study the effects of compounds 11-13, 15-19, 21-14 and Tirzepatide at the same dose on the body weight of spontaneously diabetic db / db mice induced by high fat and the maintenance time.

[0174] Experimental method: This experiment used 8-week-old db / db male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 7 mice / group, and the dosage of compounds 11-13, 15-19, 21-14 and Tirzepatide was 100nmol / kg. The mice were grouped according to the random blood glucose and body weight data to ensure that the average body weight and random blood glucose average of each group were similar. The drugs were prepared on the day of administration, and the corresponding drugs were injected subcutaneously in the back of the neck according to the grouping, with a single dose. The mice were weighed every day during the experiment, and the weight changes of the mice from 0 to 7 days were recorded. The data were processed using the software GraphPadPrism to obtain the time-weight curve Figure 3 Table 4 shows the average weight (g) of mice in the compound 11-13, 15-19, 21-14, Tirzepatide and PBS groups at different times.

[0175] Table 4 Average body weight (g) of mice in each group at different time (d).

[0176]

[0177]

[0178] Note: The bold words in the table are the lowest weight of the group.

[0179] Result analysis:

[0180] From Table 4 and Figure 3 It can be seen that: during the test period, the weight of animals in the blank control group (PBS) maintained a steady increase, and the average weight increased from 43.26g to 53.46g. After the administration of compounds 11-13, 15-19, and 21-24, the weight of animals decreased significantly. The weight of compounds 17, 19, 22 and Tirzepatide began to recover after the 4th day, and the weight of compounds 13, 18, 23, and 24 began to recover after the 5th day. The weight of compounds 11, 12, 15, and 16 began to recover after the 6th day, and the weight of compound 21 began to recover after the 7th day. Among them, the weights of compounds 12 and 21 at the end of the experiment were 37.9g ​​and 36.7g, respectively, which were 5.4g and 6.6g less than the weight before administration. In addition, the lowest weight of mice in the compound 13, 16-19, 23, and 24 groups was also lower than the lowest weight of 39.98g reached by Tirzepatide on the 3rd day.

[0181] Conclusion: Analysis of the above results shows that compounds 11-13, 15-19, 21-24 all have good effects in reducing body weight. In terms of weight loss efficacy and duration of efficacy, compounds 12 and 21 are the best, followed by compounds 13, 16, 19, 23, and 24, which are all better than Tirzepatide.

[0182] Example 5: Effects of continuous administration of Compound 12, Compound 13, Compound 21, Compound 24 and Tirzepatide on blood glucose in ob / ob mice

[0183] Based on the experimental results of Examples 2 and 3, we continued to explore the in vivo efficacy of Compound 12, Compound 13, Compound 21, and Compound 24. Tirzepatide was used as the positive control group and PBS was used as the blank control group. The effects of Compound 12, Compound 13, Compound 21, Compound 24 and Tirzepatide at the same dose on blood glucose and body weight in high-fat-induced spontaneously diabetic ob / ob mice and their maintenance time were studied by measuring blood glucose and body weight after continuous administration.

[0184] Experimental methods: This experiment used 10-week-old ob / ob male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 6 mice / group, and compound 12, compound 13, compound 21, compound 24 and Tirzepatide were all 30nmol / kg. First, after 4 weeks of high-fat diet induction, the mice were grouped according to the random blood glucose and body weight data to ensure that the average body weight and random blood glucose average of each group were similar. The drug was administered once every 6 days for 4 weeks, and the administration time was 0d, 6d, 12d, 18d, and 24d respectively. The drugs were prepared on the day of administration, and the corresponding drugs were injected subcutaneously in the back of the neck according to the group. Before administration (0d) and every 3 days during the experiment, the mouse tail vein was punctured with a sterile blood collection needle and the random blood glucose concentration was measured using a blood glucose meter (Table 5). The mice were weighed every 3 days during the experiment, and the weight changes of the mice were recorded (Table 6). The data were processed using the software GraphPadPrism to draw a time-blood glucose curve ( Figure 4 ), time-weight curve ( Figure 5 ).

[0185] Table 5 Mean values ​​of blood glucose (mmol / L) at different time (d) in mice treated with continuous drug administration

[0186]

[0187] Table 6 Average weight (g) of mice treated with continuous drug administration at different time (d)

[0188]

[0189] Result analysis:

[0190] (1) Blood sugar results are shown in Table 5 and Figure 4 It can be seen that: after 30 nmol / kg, administered once every 6 days for 24 consecutive days, Compound 12, Compound 13, Compound 21, and Compound 24 had a stronger glucose-control effect in ob mice, and the difference between the groups was not significant. Compared with Tirzepatide, the final blood glucose was lower, and the blood glucose fluctuation was smaller than that of Tirzepatide.

[0191] (2) The body weight results are shown in Table 6 and Figure 5 It can be seen that: 30nmol / kg, once every 6 days, after continuous administration for 24 days, the body weight of animals in other groups was significantly reduced compared with the PBS group. The body weight of the Tirzepatide group was 6.9g lower than the PBS group, and 1.6g lower than the initial body weight; the body weight of mice in the compound 12, compound 13, compound 21, and compound 24 groups was reduced by 11.7g, 8.5g, 14.7g, and 8.8g respectively compared with the initial body weight, which was a significant weight loss effect compared with Tirzepatide.

[0192] Example 6: Pharmacokinetic study of compound 12, compound 13, compound 21, compound 24 and Tirzepatide in SD rats

[0193] SD rats (SPF grade, source: Beijing Weitonglihua Experimental Animal Technology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2022-0063, weight: 190-210g, age: 6-8 weeks) were raised for one week to adapt to the environment. The general condition of the animals was checked during the adaptation period. Unqualified animals were not included in this experiment, and the feed was maintained by SPF mice. 20 SD rats that were raised and qualified were randomly divided into 5 groups according to their weight, with 4 rats in each group (using Stata 15 software for grouping and statistical analysis of animals).

[0194] In this example, the compound was administered subcutaneously once to the skin of the back of the neck at a dose of 0.3 mg / kg, a dosing volume of 2 mL / kg, a dosing concentration of 0.15 mg / mL, and a solvent of PBS.

[0195] Blood samples were collected from rats in the single subcutaneous administration group before administration (0h) and 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 32h, 48h, 72h, 96h, 120h, 144h, and 168h after administration through the jugular vein. About 0.2mL of whole blood was collected at each time point in an EDTA-K2 anticoagulant tube. Within 1h, the supernatant was taken and the separated plasma was transferred to a -80℃ refrigerator for storage. UPLC-MS / MS was used to establish the concentration analysis method of the compounds in SD rat plasma and determine the drug concentration of the compounds in plasma. WinNonlin 8.1 software was used for data processing to calculate the pharmacokinetic parameters. The experimental results are shown in Table 7.

[0196] The experimental results show that compared with Tirzepatide, single subcutaneous administration of compounds 12, 13, 21, and 24 is absorbed more slowly in rats, among which compounds 12, 13, 21, and 24 reach peak time T max Both are 24 hours, significantly higher than Tirzepatide's 8 hours, and the half-life t 1 / 2 The averages are 23.88h, 19.53h, 24.35h, and 21.36h, respectively, which are significantly higher than 10.53h of Tirzepatide, and the half-life of compound 21 can reach more than 24h. This proves that the long-acting polypeptide compound disclosed in the present invention has a longer half-life.

[0197] Table 7 Results of pharmacokinetic experiments in SD rats and corresponding dosages of the compounds

[0198] Compound <![CDATA[T 1 / 2 (h)]]> <![CDATA[T max (h)]]> Dosage mg / kg 12 23.88 24 0.3 13 19.53 24 0.3 21 24.35 24 0.3 24 21.36 24 0.3 Tirzepatide 10.53 8 0.3

[0199] Example 7: Stability study of compound 12 and compound 21

[0200] This example studies compound 12, compound 21 and a control compound (sequence: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 Stability of CO2H)AFVQWLLDGGPSSGAPPPS-NH2).

[0201] Samples of compound 12, compound 21 and control compound were stored in a 60°C thermostat for accelerated decomposition, and the purity of the samples was tested by high performance liquid chromatography (Thermo Fisher Scientific) before storage (0 day (d)), 5 days (d) and 10 days (d) after storage, and the sample degradation rate was calculated by the following formula:

[0202]

[0203] Where: Ai is the relative peak area of ​​the sample test result on day i, A0 is the relative peak area of ​​the sample test result on day 0.

[0204] Chromatographic conditions for detection: C18 chromatographic column (4.6*250mm, 5μm), 0.1% trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, flow rate of 1.0ml / min; detection wavelength of 220nm, column temperature of 30°C; sample solvent (diluent) of 40% acetonitrile aqueous solution, sample concentration of 0.5mg / ml; injection volume: 10μl for compound 12, 10μl for compound 21, and 20μl for control compound.

[0205] Test results: Figure 6 The chromatograms of compound 12 at 0d, 5d, and 10d; Figure 7 The chromatograms of compound 21 at 0d, 5d, and 10d; Figure 8 The chromatograms of the reference compound on the 0th, 5th and 10th days were calculated based on the test results: the degradation rates of compound 12 on the 5th and 10th days compared with the 0th day were 3.9% and 9.9%, respectively; the degradation rates of compound 21 on the 5th and 10th days compared with the 0th day were 3.5% and 8.1%, respectively; and the degradation rates of the reference compound on the 5th and 10th days compared with the 0th day were 14.8% and 22.8%, respectively.

[0206] The difference between the control compound and compound 12 is only the difference in the 28th amino acid. From the experimental results, it can be seen that the degradation rates of compounds 12 and compound 21 are significantly lower than those of the control compound. It can be seen that after the 28th amino acid position in the amino acid sequence of the long-acting GLP-1 polypeptide compound of the present application is replaced by E instead of D, the stability is significantly improved.

[0207] The above are only specific embodiments of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein the polypeptide compound is selected from any one of the amino acid sequences of SEQ ID NOs: 11-13, 15-19, 21-24.

2. A pharmaceutical composition comprising the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to claim 1, and a pharmaceutically acceptable carrier, adjuvant or excipient.

3. Use of the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating diabetes or obesity.

4. The method for preparing the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the preparation method is by chemical synthesis.

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