Long-acting GLP-1 polypeptide compound, composition and use thereof

Abstract

Provided are a long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, a composition, and a use. The amino acid sequence thereof is Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS. By means of amino acid replacement at a specific site of the main chain of the polypeptide, the long-acting GLP-1 polypeptide compound noticeably exhibits good polypeptide activity, hydrolysis resistance, and stability, has a long half-life and efficacy in treating diabetes and reducing body weight.

Description

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

[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 biochemistry 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 with significant health consequences, posing a serious threat to patients' lives. The majority of diabetic patients suffer from type 2 diabetes (T2DM), also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. It is a chronic metabolic disease that typically develops after the age of 35 to 40 and accounts for over 90% of diabetic patients. Type 2 diabetes is characterized by high blood sugar, relative insulin deficiency, and insulin resistance, and may also include increased appetite, fatigue, or aches and pains (Report of a WHO Consultation, 2011).

[0005] In addition, type II diabetes is also accompanied by a large number of serious complications and increases the incidence of other diseases. For example, patients with type II diabetes are at high risk of malignant tumors (Epidemiological analysis of patients with type II diabetes and malignant tumors. China Chronic Disease Prevention and Control. 2018(07)); type II 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 II diabetes. China Contemporary 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 and needs to be injected at least once a day, or as many as 2 to 4 times a day. It may also need to be combined with oral hypoglycemic drugs (Research and Development and Clinical Research Progress of Weekly Basal Insulin Preparations. Drug Evaluation. 2022, 19(12)). GLP-1 receptor agonists, hypoglycemic drugs, 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 blood 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 and can improve blood sugar in adult patients with type II diabetes, but the cost of medication is relatively high.

[0007] Diabetes is difficult to cure and typically requires lifelong medication to control blood sugar. However, currently available antidiabetic drugs all have limitations. Therefore, research and development of drugs with ideal blood sugar-lowering effects and minimal adverse reactions is crucial for diabetic patients, a key challenge facing those skilled in the art.

[0008] Summary of the Invention

[0009] 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.

[0010] 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:

[0011] Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS(SEQ ID NO:26),

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X29 is G.

[0016] 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.

[0017] In some optional embodiments, the amino acid sequence of the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is as follows:

[0018] Y-X2-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:27),

[0019] wherein X2 represents an amino acid selected from Aib or Iva.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In another aspect, the present disclosure provides use of the aforementioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating diabetes or obesity.

[0024] 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 chemical synthesis.

[0025] The long-acting GLP-1 polypeptide compound disclosed herein, by replacing amino acids at specific sites in the polypeptide main chain, significantly exhibits good activity, hydrolysis resistance and stability of the polypeptide. Through a large number of pharmacodynamic activity tests, the data show that: when the 2nd and / or 13th amino acid sites 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 site 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 site with E and the 29th amino acid site with G, V or A amino acids will also significantly enhance the half-life and stability of the polypeptide molecule.

[0026] Currently reported peptide drugs in this field have a half-life of less than 10 hours in rats, allowing only a weekly dosing frequency. The long-acting peptide compound disclosed herein significantly extends its half-life, reaching over 24 hours in rats. This achieves ultra-long-acting peptide drugs, enabling dosing frequencies of two weeks or more in humans. It demonstrates excellent efficacy in treating diabetes and reducing weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] FIG1 shows the effect of compound 1-25 on blood glucose in db / db mice within 0-8 hours in Example 3.

[0029] FIG2 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.

[0030] FIG3 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.

[0031] FIG4 shows the effect of continuous administration of compounds 11-13, 15-19, and 21-24 on blood glucose in ob / ob mice in Example 5. ...

[0032] FIG5 shows the effect of continuous administration of compounds 11-13, 15-19, and 21-24 on the body weight of ob / ob mice in Example 5.

[0033] FIG6 shows the chromatograms of compound 12 at 0d, 5d, and 10d in Example 7.

[0034] FIG7 shows the chromatograms of compound 21 at 0d, 5d, and 10d in Example 7.

[0035] FIG8 shows the chromatograms of the reference compound at 0d, 5d, and 10d in Example 7. DETAILED DESCRIPTION

[0036] I. Definition

[0037] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0038] The term "amino acid" refers to a molecule containing both amino and carboxyl functional groups. In α-amino acids, the amino and carboxyl groups are attached to the same carbon atom (the α carbon). The α carbon may also have one or two organic substituents. Amino acids include L and D isomers, as well as racemic mixtures. Unless otherwise specified, amino acid residues in polypeptide sequences disclosed herein are L isomers, i.e., L-amino acids. D-amino acids are indicated by the lowercase letter "d" preceding the amino acid name or abbreviation, e.g., dK.

[0039] The amino acid sequences disclosed herein contain conventional one-letter or three-letter codes for naturally occurring amino acids, as well as generally recognized three-letter codes for other amino acids, such as Iva (Isovaline); Cba (1-Aminocyclobutanecarboxylic acid); Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid); Aib (α-Aminoisobutyric acid); or GABA (γ-aminobutyric acid). Commonly used molecular structure abbreviation codes include:

[0040] hGlu is homoglutamate;

[0041] α-hGlu is the L isomer of -HNCH(CO-)CH2CH2CH2COOH;

[0042] δ-hGlu is the L isomer of -HNCH(COOH)CH2CH2CH2CO-;

[0043] α-Glu is the L isomer of -HNCH(CO-)CH2CH2COOH;

[0044] γ-Glu or gGlu is the L isomer of -HNCH(COOH)CH2CH2CO-;

[0045] α-Asp is the L isomer of -HNCH(CO-)CH2COOH;

[0046] β-Asp is the L isomer of -HNCH(COOH)CH2CO-;

[0047] β-Ala is -HN-CH2-CH2-COOH;

[0048] PEG2 is 2-(2-(2-aminoethoxy)ethoxy)acetic acid (CAS No. 134978-97-5).

[0049] The amino acid composition of the polypeptides disclosed herein can be altered without substantially affecting their biological activity. For example, a polypeptide sequence may 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 based on the properties of their 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; amino acid residues containing small aliphatic, non-polar, or weakly polar side chains include glycine, alanine, threonine, serine, and proline; amino acid residues containing large aliphatic, non-polar side chains include leucine, isoleucine, and valine; aromatic amino acid residues include phenylalanine, tryptophan, and tyrosine; and amino acid residues containing sulfur side chains include cysteine ​​and methionine.

[0050] As used herein, the term "treat" includes inhibiting, slowing, halting, or reversing the progression or severity of existing symptoms or conditions. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or the development of the disease. As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that modifies, typically improves, or ameliorates the symptoms of a disease or condition, or cures the disease or condition. As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, upon administration to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition. As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur through the administration of a single dose and may occur only after a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0051] As used herein, the term "patient" refers to a mammal, such as a human.

[0052] 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. Additionally, certain compounds of the present disclosure can be administered once a week.

[0053] It should be understood that the therapeutic agent according to the embodiment will be used together with pharmaceutically acceptable suitable carriers, excipients, and other reagents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A large number of suitable formulations are 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 (such as Lipofectin™), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semisolid gels, and semisolid 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 tolerant to the route of administration.

[0054] 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 the administration of the drug. 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.

[0055] Preparations to be used for clinical in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes.

[0056] II. Detailed description of specific implementation plan

[0057] 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:

[0058] Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS(SEQ ID NO:26),

[0059] 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.

[0060] 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.

[0061] 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.

[0062] In some optional embodiments, the above-mentioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein X29 is G.

[0063] 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.

[0064] In some optional embodiments, the amino acid sequence of the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof is as follows:

[0065] Y-X2-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:27),

[0066] wherein X2 represents an amino acid selected from Aib or Iva.

[0067] In some optional embodiments, the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof has an amino acid sequence selected from the following:

[0068] Compound 1: Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO: 1);

[0069] Compound 2: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO: 2);

[0070] Compound 3: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEVGPSSGAPPPS (SEQ ID NO: 3);

[0071] Compound 4: Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLEAGPSSGAPPPS (SEQ ID NO: 4);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <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">

[0073] <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">

[0074] <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">

[0075] <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">

[0076] <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">

[0077] <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">

[0078] <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"> 18 <h2 style=";text-align:left;direction:ltr"> CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:11);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] <h2 style=";text-align:left;direction:ltr"> Figure 12: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:12);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> Figure 13: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSG-γGlu-CO(CH2)<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:13);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] Compound 14: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:14);

[0082] Compound 15: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:15);

[0083] Compound 16: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEVGPSSGAPPPS(SEQ ID NO:16);

[0084] Compound 17: Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS(SEQ ID NO:17);

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

[0086] Compound 19: Y-Aib-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS(SEQ ID NO:19);

[0087] Compound 20: Y-Cba-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:20);

[0088] Compound 21: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:21);

[0089] Compound 22: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQ WLLEGGPSSGAPPPS(SEQ ID NO:22);

[0090] Compound 23: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2) 18 CO2H)AFVQWLLEVGPSSGAPPPS(SEQ ID NO:23);

[0091] Compound 24: Y-Iva-EGTFTSDYSL-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSGAPPPS(SEQ ID NO:24); or

[0092] Compound 25: Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEAGPSSGAPPPS (SEQ ID NO: 25).

[0093] 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.

[0094] 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.

[0095] In another aspect, the present disclosure provides use of the aforementioned long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating diabetes or obesity.

[0096] 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 chemical synthesis.

[0097] 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, and the like.

[0098] 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 paraben, 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 base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations 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 a replacement cartridge. 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, the cartridge can be replaced and the pen can be used again.

[0099] Pharmaceutical compositions suitable for injection 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 EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluidity should reach a level that allows for easy injection. 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, a polyol (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 dispersions, and utilizing a surfactant, suitable fluidity can be maintained. 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.

[0100] As needed, sterile injection solutions can be prepared by incorporating the disclosed compounds into a suitable solvent having one or a combination (as needed) of the ingredients listed above in the desired amount, 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 ingredients required as 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 a powder comprising the active ingredient and any other desired ingredients from a sterile filtered solution of the aforementioned ingredients.

[0101] 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.

[0102] Systemic administration can also be achieved through transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the permeability barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, one or more of the disclosed compounds can be formulated into ointments, unguents, gels, or creams as are generally known in the art.

[0103] 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.

[0104] 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 such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0105] 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.

[0106] In some embodiments, the disclosed compounds can be prepared into sustained-release formulations. Examples of suitable sustained-release formulations 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. Patent 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 DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin 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 of research in recent years. In addition, there are albumin microspheres, chitosan microspheres, gelatin microspheres, etc.

[0107] Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0108] 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 dosages for the 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 the required pharmaceutical carrier. The specifications of the dosage unit forms of the embodiments are dictated by and directly dependent on 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.

[0109] The pharmaceutical compositions can be placed in a container, pack, or dispenser together with instructions for administration.

[0110] The present disclosure provides a method for treating diabetes or obesity in a patient, including administering 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 administering 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 administration is subcutaneous. The present disclosure also provides a method for treating diabetes or obesity in a patient, including administering 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 administering 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 administration (determined by industry guidelines such as the American Diabetes Association).

[0111] 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 insulin-requiring diabetes; treating metabolic syndrome, 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 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.

[0112] Peptide chemical synthesis method

[0113] Solid phase chemical synthesis of peptides is a well-developed methodology, and references include, for example, RC Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989.

[0114] Linear peptides are synthesized using either the Boc solid-phase peptide synthesis method or the Fmoc solid-phase peptide synthesis method. If Fmoc chemistry is used to synthesize peptides with a carboxyl group at the C-terminus, Wang resin is usually selected; peptides with an amide group at the C-terminus are usually selected using Rink amide resin (including Rink Amide-AM resin, Rink Amide-MBHA resin, etc.). If Boc chemistry is used to synthesize peptides with a carboxyl group at the C-terminus, Pam resin is usually selected; peptides with an amide group at the C-terminus are usually selected using MBHA resin. Commonly used condensing agents and activating agents are DIC and HOBT. Other optional peptide bond condensing agents include EDC, BOP, HBTU, DEPBT, TBTU, etc. Depending on the difficulty of the reaction, the amino acid can be used in 1.1-10 equivalents, and the reaction time can be 15 minutes to 24 hours. Peptides can be synthesized manually or using a peptide solid-phase synthesizer.

[0115] 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).

[0116] For solid-phase synthesis, resins preloaded with C-terminal amino acids or resins without amino acid loading can be used.

[0117] The method for loading the first amino acid onto Rink Amide resin can be referenced by common industry practices. A common method is briefly described as follows: Weigh an appropriate amount of resin, remove the Fmoc protecting group using 20% ​​piperidine / DMF in a solid-phase synthesis tube (15 mL / g resin, 30 minutes x 2), and wash the resin with DMF. Weigh 5 equivalents of Fmoc amino acid, HATU, HOAT, and 10 equivalents of NMM to the resin's amino group, add DMF, mix well, and transfer to a 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.

[0118] When using the Fmoc solid-phase peptide synthesis method, the commonly used amino acids and protecting groups are as follows:

[0119] 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

[0120] The synthesis process utilizes appropriately protected building blocks, such as the aforementioned standard amino acids, Fmoc-8-amino-3,6-dioxaoctanoic acid (CAS No. 166108-71-0), and Fmoc-Glu-OtBu (CAS No. 84793-07-7). Fatty acid moieties can be introduced using building blocks such as, but not limited to, mono-tert-butyl octadecanoate. After each coupling step, unreacted peptide intermediates can be capped with acetic anhydride (10 equivalents) and excess collidine (20 equivalents).

[0121] After solid-phase Fmoc chemistry for peptide synthesis, TFA is a commonly used cleavage reagent. Place the dry resin in a shake flask and add an appropriate amount of a cleavage solution containing 90:4:2:2:2 (v / v) trifluoroacetic acid: triisopropylsilane: 1,2-ethanedithiol: water: thioanisole (10-25 mL / g resin). Cover the flask and shake intermittently at room temperature. After 2 hours, filter the resin and rinse it 2-3 times with fresh TFA. Combine the filtrates and add 8-10 volumes of glacial ether dropwise. Finally, collect the crude peptide precipitate by centrifugation.

[0122] 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

[0123] If the side chain amino group of lysine is used for lactam synthesis or acylation, the side chain amino group of lysine can be protected with allyloxycarbonyl (aloc) or Fmoc. If the side chain carboxyl group of aspartic acid or glutamic acid is used for lactam synthesis or acylation, the carboxyl group should be converted to an allyl ester or 9-fluorenylmethyl protection, such as Boc-Glu(OAllyl)-OH, Boc-Glu(Ofm)-OH.

[0124] After solid-phase Boc chemical peptide synthesis, PAM and MBHA resins are typically cleaved with HF. 5 ml of HF is added per 0.1 mmol of resin, along with reagents such as p-cresol, p-mercaptophenol, or anisole. The mixture is stirred in an ice bath for one hour. After vacuum removal of the HF, the peptide is precipitated with glacial ether. The precipitate is collected by centrifugation, purified by HPLC, and freeze-dried to yield the final product.

[0125] purification

[0126] The crude peptide was dissolved in an appropriate 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.). Columns of varying fillers and sizes, such as C8 or C18 semi-preparative or preparative columns, were selected based on the amount of crude peptide loaded and its polarity. Buffer A consisted of 0.1% TFA in water, and buffer B consisted of 0.1% TFA in acetonitrile. Elution was performed using a rising gradient of buffer B, and relevant fractions were analyzed by analytical HPLC. A ZORBAX 300SB-C18 (4.6 x 250 mm, 5 μM) column was used, with buffer A consisting of 0.1% TFA in water and buffer B consisting of 0.1% TFA in acetonitrile. The flow rate was 1 ml / min, and detection was performed at 210 nm. Fractions containing the pure target peptide were pooled and freeze-dried to yield the peptide trifluoroacetate salt as a white solid. The product was stored in aliquots in glass vials.

[0127] Preparation method

[0128] The compounds disclosed herein are linear peptides. Each amino acid can be coupled stepwise in the order of the C-terminus to the N-terminus of the polypeptide sequence to obtain a polypeptide backbone. The process is as follows: first, an amino acid whose amino group is protected by a blocking group is covalently linked to a solid-phase support, and the amino-protecting group of the first amino acid is removed, so that the first amino acid is attached to the solid-phase support. Then, the carboxyl group of the second amino acid whose amino group is blocked is activated and reacts with the amino group of the first amino acid attached to the solid-phase support to form a peptide bond, thus generating a dipeptide with a protecting group on the solid-phase support. The above peptide bond-forming reaction is repeated 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 support is hydrolyzed to obtain the synthesized peptide.

[0129] For the specific synthesis method, please refer to patent CN2021108155833. Compound 21 is used as an example to illustrate the synthesis route and method.

[0130] Step 1: Synthesize 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;

[0131] 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;

[0132] Step 3: adding a cleavage solution to the polypeptide resin to perform a cleavage reaction, remove the full protection of the polypeptide, and extract the crude compound;

[0133] Step 4: Purify the crude compound to obtain the long-acting GLP-1 polypeptide compound 21.

[0134] 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.

[0135] Preferably, the lysis solution in step 3 is composed of TFA, DODT, m-cresol, and H2O in a volume ratio of 92.5:2.5:2.5:2.5; and the crude compound extraction method includes filtration, precipitation and / or methyl tert-butyl ether extraction.

[0136] The present disclosure also includes novel intermediates and methods that can be used to synthesize the disclosed compounds or their pharmaceutically acceptable salts. The disclosed intermediates and compounds 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 described pathway can be combined in various ways to prepare the disclosed compounds or their salts. Reagents and starting materials are readily available to those of ordinary skill in the art.

[0137] For purposes of clarity and conciseness, 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.

[0138] Example

[0139] Example 1: Synthesis of compounds

[0140] This example uses the Fmoc solid-phase peptide synthesis method, which is synthesized from the carboxyl end to the amino end. 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 liquid chromatography-mass spectrometry, 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 0.4 mL / 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 below.

[0141] Table 1 Amino acid sequence table and LC-MS identification results of compounds 1-25

[0142] Example 2: Agonist activity of compound 1-25 and Tirzepatide on GLP-1 and GIP targets, respectively

[0143] 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 from Cisbio.

[0144] Experimental methods:

[0145] 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 into a 384-well plate (containing serum-free basal medium, GIPR-HEK293, GLP-1R-Luciferase-HEK293: 3000 cells / well, 5 μl);

[0146] 2) Simultaneously, a series of peptide drug concentrations (starting at 100 nM, with nine 5-fold dilutions) were prepared in Stimulation Buffer 1 containing 0.5 mM IBMX (a negative control was prepared by adding the same volume of Stimulation Buffer 1 containing 0.5 mM IBMX and 0.1% casein). Seal the plate and incubate at room temperature for 30 min.

[0147] 3) Prepare cAMP Eu Cryptate antibody and cAMP-d2 antibody working solutions in lysis and detection buffer. After incubation, add 5 μl to each well (for negative controls, add the same volume of lysis and detection buffer instead of cAMP-d2 antibody working solution). Seal the plate and incubate at room temperature in the dark for 1 hour.

[0148] 4) In compatibility Fluorescence emission at two wavelengths (665 nm and 620 nm) was read on a microplate reader. The ratio of the emission at 665 nm to 620 nm was calculated, and the actual cAMP level (nM) in each sample well was calculated using the cAMP standard curve. The percentage of agonism at different concentrations was then calculated using 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%. The EC50 value was calculated using the "log (agonist) vs. response - Variable slope" model in GraphPad Prism 7.0. The experimental results are shown in Table 2.

[0149] Results: The EC50 of the positive control, Tirzepatide, was consistent with historical values, indicating that the experimental system was stable and reliable. All samples tested were primarily active for GIPR activity. The order of GLP-1R activity from strongest to weakest was compound 12 > 13, 17-19, 21, 24 > 11, 15, 16, 22, 23 > 1-6, 8-10 > 7, 14, 20, 25. The order of GIPR activity from strongest to weakest 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, and 25 had the worst activity.

[0150] Table 2 Agonist activity of compounds 1-25 on GLP-1 and GIP targets

[0151] Example 3: Effects of Compound 1-25 and Tirzepatide on Blood Glucose in db / db Mice

[0152] The in vivo efficacy of compound 1-25 was investigated, and Tirzepatide was used as the positive control group and PBS was used as the blank control group. The effects of compound 1-25 and Tirzepatide at the same dose on the blood glucose of high-fat-induced spontaneously diabetic db / db mice and the duration of blood glucose maintenance were studied by measuring blood glucose after a single administration.

[0153] Experimental methods: This study used 8-week-old db / db male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 7 mice per group, and the dose of all compounds and Tirzepatide was 100 nmol / kg. Groups were grouped according to random blood glucose and body weight data to ensure that the average body weight and random blood glucose values ​​of each group were similar. The drugs were prepared on the day of administration and the corresponding drugs were injected subcutaneously at the back of the neck according to the group. Before administration (0h) and 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 data were processed using GraphPadPrism software to 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 shows the changes in blood glucose of each group of mice treated with compounds 1-25 at 8 h; Figure 2 shows the changes in blood glucose of each group of mice treated with compounds 11-13, 15-19, and 21-24 from 0 to 168 h.

[0154] Table 3 Average blood glucose (mmol / L) at different time points (h) in each group of mice treated with compounds 1-25

[0155] Results analysis: From Table 3, Figures 1 and 2, we can see that:

[0156] (1) 1h results: Compounds 1-6, 8-13, 15-19, 21-24 and the positive control Tirzepatide showed significant changes in blood glucose levels, demonstrating a blood glucose-lowering effect. However, there was no significant difference in blood glucose levels between the compound 7, 14, 20, and 25 groups and the control model group.

[0157] (2) 2h results: Compounds 2, 5, 8, 9, 10, 11-13, 15-19, 21-24 and the positive control Tirzepatide had the effect of lowering blood sugar, among which compounds 11-13, 15-19, 21-24 and the positive control Tirzepatide had the best effect; the efficacy of compounds 2, 5, 8, 9, and 10 was reduced to varying degrees compared with 1h. The effect of compounds 1, 3, 4, and 6 was not significant compared with the PBS control model group.

[0158] (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 levels close to those before administration.

[0159] (4) 24h 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 24h after administration, and compounds 12 and 21 were the lowest.

[0160] (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.

[0161] (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.

[0162] (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.

[0163] (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 level.

[0164] (9) 144h results: Compounds 12, 13, 21, and 24 still had the effect of lowering blood sugar, and compounds 15 and 18 were close to the level before administration.

[0165] (9) 168h results: Compounds 12 and 21 still had the effect of lowering blood sugar, while compounds 13 and 24 were close to the level before administration.

[0166] Conclusion: Analysis of the above results revealed that Tirzepatide and other compounds exhibited similarly favorable effects in lowering blood sugar. The duration of efficacy varied among the compounds, with compounds 12 and 21 demonstrating the greatest advantages in both efficacy and duration (long-lasting effect), with efficacy lasting for over 168 hours. Compounds 13 and 24 were next, with efficacy lasting for 144 hours. Compounds 11 and 15-18, each with efficacy lasting for over 96 hours, all outperformed Tirzepatide.

[0167] Example 4: Effects of Compounds 11-13, 15-19, 21-14 and Tirzepatide on Body Weight in db / db Mice

[0168] Based on the experimental results of Example 3, the in vivo pharmacological efficacy of compounds 11-13, 15-19, and 21-14 was investigated. Tirzepatide was used as a positive control group, and PBS was used as a blank control group. The effects of compounds 11-13, 15-19, and 21-14 and Tirzepatide at the same dose on the body weight of high-fat-induced spontaneously diabetic db / db mice and the duration of body weight maintenance were studied by measuring body weight after a single dose.

[0169] Experimental methods: This experiment used 8-week-old db / db male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 7 per group, and the dose of compounds 11-13, 15-19, 21-14 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 grouping, with a single dose. The mice were weighed every day during the experiment and the weight changes from 0 to 7 days were recorded. The data were processed using GraphPadPrism software 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.

[0170] Table 4 Average body weight (g) of mice in each group at different time (d). Note: The bold words in the table are the lowest weight of the group.

[0171] Result analysis:

[0172] As shown in Table 4 and Figure 3, during the experimental period, the weight of animals in the blank control group (PBS) maintained steady growth, with the average weight increasing from 43.26g to 53.46g. After administration of compounds 11-13, 15-19, and 21-24, the weight of the animals decreased significantly. Weight began to recover after the fourth day for compounds 17, 19, 22, and Tirzepatide, and after the fifth day for compounds 13, 18, 23, and 24. Weight began to recover after the sixth day for compounds 11, 12, 15, and 16, and after the seventh day for compound 21. The weights of mice in the groups treated with compounds 12 and 21 at the end of the experiment were 37.9g ​​and 36.7g, respectively, representing a decrease of 5.4g and 6.6g, respectively, compared to their pre-dose weights. Furthermore, the lowest weights of mice in the groups treated with compounds 13, 16-19, 23, and 24 were all lower than the lowest weight of 39.98g reached on the third day with Tirzepatide.

[0173] Conclusion: Analysis of the above results revealed that compounds 11-13, 15-19, and 21-24 all demonstrated significant weight loss efficacy. Compounds 12 and 21 were the most effective in terms of weight loss efficacy and duration of efficacy, followed by compounds 13, 16, 19, 23, and 24, all of which were superior to Tirzepatide.

[0174] Example 5: Effects of Continuous Administration of Compound 12, Compound 13, Compound 21, Compound 24, and Tirzepatide on Blood Glucose in Ob / ob Mice

[0175] Based on the experimental results of Examples 2 and 3, we continued to investigate the in vivo efficacy of Compounds 12, 13, 21, and 24. Tirzepatide was used as the positive control group and PBS was used as the blank control group. The effects of Compounds 12, 13, 21, and 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 duration were studied by measuring blood glucose and body weight after continuous administration.

[0176] Experimental Methods: Ten-week-old ob / ob male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were used in this study, with six mice per group receiving 30 nmol / kg of Compound 12, Compound 13, Compound 21, Compound 24, and Tirzepatide. After four weeks of high-fat diet induction, mice were divided into groups based on random blood glucose and body weight data, ensuring that the average body weight and random blood glucose values ​​of each group were similar. Drugs were administered every six days for four consecutive weeks, with administration times on days 0, 6, 12, 18, and 24. On the day of administration, the drugs were prepared and administered subcutaneously in the back of the neck according to the group. Random blood glucose concentrations were measured using a glucometer after puncturing the tail vein with a sterile blood collection needle before administration (day 0) and every three days during the study (Table 5). The mice were weighed every three days during the study, and weight changes were recorded (Table 6). Data were processed using GraphPad Prism software, and time-glucose and time-weight curves were plotted (Figure 4) and (Figure 5).

[0177] Table 5 Average blood glucose (mmol / L) values ​​of mice treated with continuous drug administration at different time points (d)

[0178] Table 6 Average weight (g) of mice treated with continuous drug administration at different time points (d)

[0179] Result analysis:

[0180] (1) Blood glucose results As shown in Table 5 and Figure 4: After 30 nmol / kg, once every 6 days, and continuous administration for 24 days, Compound 12, Compound 13, Compound 21, and Compound 24 had a strong blood 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.

[0181] (2) Body weight results As shown in Table 6 and Figure 5, after 24 consecutive days of administration at 30 nmol / kg, once every 6 days, the body weights of the animals in the other groups were significantly reduced compared to the PBS group. The body weight of the Tirzepatide group was reduced by 6.9 g compared to the PBS group, and by 1.6 g compared to the initial body weight. The body weights of the mice in the Compound 12, Compound 13, Compound 21, and Compound 24 groups were reduced by 11.7 g, 8.5 g, 14.7 g, and 8.8 g, respectively, compared to the initial body weight, which were significantly more effective than Tirzepatide.

[0182] Example 6: Pharmacokinetic Study of Compound 12, Compound 13, Compound 21, Compound 24 and Tirzepatide in SD Rats

[0183] Sprague-Dawley rats (SPF grade, sourced from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., Laboratory Animal Production License No. SCXK (Beijing) 2022-0063, weight: 190-210 g, age: 6-8 weeks) were housed for one week to acclimate to the environment. During this period, the general condition of the animals was examined. Unqualified animals were excluded from this experiment and fed an SPF rat maintenance diet. Twenty Sprague-Dawley rats that had been properly maintained were randomly divided into five groups of four rats each, based on body weight. (Animal grouping and statistical analysis were performed using Stata 15 software.)

[0184] In this example, the compound was administered subcutaneously to the skin on 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.

[0185] Blood samples were collected from the jugular vein of rats in the single subcutaneous administration group before dosing (0 h) and at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after dosing. Approximately 0.2 mL of whole blood was collected at each time point and placed in an EDTA-K2 anticoagulant tube. Within 1 h, the blood was centrifuged at 4°C and 1800 g for 10 min. The supernatant was collected and the separated plasma was transferred to a -80°C refrigerator for storage. UPLC-MS / MS was used to establish analytical methods for the compound concentrations in SD rat plasma and determine the drug concentrations in plasma. Pharmacokinetic parameters were calculated using WinNonlin 8.1 software. The experimental results are shown in Table 7.

[0186] The experimental results show that compared with Tirzepatide, single subcutaneous administration of compounds 12, 13, 21, and 24 is absorbed more slowly in rats, and the peak absorption time of compounds 12, 13, 21, and 24 is T max Both are 24h, significantly higher than Tirzepatide's 8h, and the half-life t1 / 2 The average half-life was 23.88 h, 19.53 h, 24.35 h, and 21.36 h, respectively, which was significantly higher than Tirzepatide's 10.53 h, and the half-life of compound 21 was more than 24 h. This confirms that the long-acting polypeptide compound disclosed herein has a longer half-life.

[0187] Table 7 Pharmacokinetic results of SD rats and corresponding dosages of the compounds

[0188] Example 7: Stability study of compound 12 and compound 21

[0189] 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).

[0190] Samples of compound 12, compound 21, and the control compound were stored in a 60°C thermostat for accelerated decomposition. 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 using the following formula:

[0191] Where: Ai is the relative peak area of ​​the test result of the sample on day i, and A0 is the relative peak area of ​​the test result on day 0.

[0192] 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) is 40% acetonitrile aqueous solution, sample concentration is 0.5mg / ml; injection volume: 10μl for compound 12, 10μl for compound 21, and 20μl for the control compound.

[0193] Test results: Figure 6 is the chromatogram of compound 12 on day 0, 5, and 10; Figure 7 is the chromatogram of compound 21 on day 0, 5, and 10; Figure 8 is the chromatogram of the control compound on day 0, 5, and 10. According to the test results, it was calculated that the degradation rates of compound 12 on day 5 and 10 compared with day 0 were 3.9% and 9.9%, respectively; the degradation rates of compound 21 on day 5 and 10 compared with day 0 were 3.5% and 8.1%, respectively; and the degradation rates of the control compound on day 5 and 10 compared with day 0 were 14.8% and 22.8%, respectively.

[0194] The only difference between the control compound and compound 12 is 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.

[0195] The above are only specific embodiments of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof, wherein the amino acid sequence is as follows: Y-X2-EGTFTSDYS-X12-X13-LDKIAQ-X20-AFVQWLLE-X29-GPSSGAPPPS (SEQ ID NO: 26), in, 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.

2. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein: When X2 is Iva, X29 is G; or, when X2 is Cba, X12 is I.

3. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein: X13 is Iva.

4. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein: X29 is G.

5. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, 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.

6. The long-acting GLP-1 polypeptide compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof, wherein the amino acid sequence is as follows: Y-X2-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2) 18 CO2H)AFVQWLLEGGPSSG APPPS(SEQ ID NO:27), in, X2 represents an amino acid selected from Aib or Iva.

7. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, comprising an amino acid sequence selected from SEQ ID NOs: 1 to 25.

8. The long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, wherein the N-terminus or C-terminus thereof is modified with an auxiliary group.

9. A pharmaceutical composition comprising the long-acting GLP-1 polypeptide compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, adjuvant or excipient.

10. Use of the long-acting GLP-1 polypeptide compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for preventing or treating diabetes or obesity.

11. A method for preparing the long-acting GLP-1 polypeptide compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 8, wherein the preparation method is by chemical synthesis.

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