WO2025252197A1 - Pharmaceutical composition - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising a polypeptide or a pharmaceutically acceptable salt thereof. The polypeptide or the pharmaceutically acceptable salt thereof comprises the N-terminus of an amino acid sequence as shown in X1-Aib-X2, wherein X1 is a natural or unnatural amino acid residue comprising an imidazole group, and X2 is a natural or unnatural amino acid residue comprising an imidazole group. The pharmaceutical composition has good chemical stability.

Description

Pharmaceutical Composition Technical Field

This disclosure pertains to the pharmaceutical field and relates to the preparation of a pharmaceutical composition comprising a polypeptide or a pharmaceutically acceptable salt thereof.

Background Technology

GLP-1 drugs have become a research hotspot in recent years. They not only have a significant effect on lowering blood sugar, but also have the effects of weight loss, lowering blood pressure, improving blood lipid profile, and protecting the heart. They have become one of the most ideal drugs for the treatment of chronic diseases such as type 2 diabetes and obesity.

Currently, most peptide products intended for subcutaneous injection are stored at 2–8°C before use. However, during use, these products may often be processed at room temperature. Therefore, clinical use studies are an indispensable part of drug development, ensuring drug quality during use and providing a basis for the preparation, storage conditions, and shelf-life information stated in the drug's instructions/label.

This disclosure provides a pharmaceutical composition comprising a polypeptide, which has excellent chemical stability and can ensure that quality requirements are met during storage and use.

Summary of the Invention

This disclosure provides a pharmaceutical composition comprising a polypeptide or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical compositions described in this disclosure further comprise metal ions.

In some embodiments, the pharmaceutical compositions described in this disclosure further comprise divalent metal ions.

In some embodiments, the metal ions described in this disclosure may be selected from divalent zinc ions, divalent calcium ions, divalent magnesium ions, and divalent iron ions.

In some embodiments, the metal ions described herein may be selected from divalent zinc ions, divalent calcium ions, and divalent magnesium ions.

In some embodiments, the metal ions described herein may be selected from divalent zinc ions.

In some embodiments, the metal ions described herein may be selected from zinc acetate or zinc chloride.

In some embodiments, the metal ion described herein may be selected from zinc acetate.

In some embodiments, the pharmaceutical composition of this disclosure comprises a polypeptide or a pharmaceutically acceptable salt thereof and divalent zinc ions; preferably, the pharmaceutical composition comprises a polypeptide or a pharmaceutically acceptable salt thereof and zinc acetate or zinc chloride.

In some embodiments, the divalent metal ions described in this disclosure are not selected from divalent iron ions.

In some embodiments, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to the metal ion in the pharmaceutical composition of this disclosure is 1:5 to 5:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to the metal ion is 1:3 to 3:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to the metal ion is 1:2 to 2:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to the metal ion is 1:1.

In some embodiments, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to divalent zinc ions in the pharmaceutical composition of this disclosure is 1:5 to 5:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to divalent zinc ions is 1:3 to 3:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to divalent zinc ions is 1:2 to 2:1; preferably, the molar ratio of the polypeptide or its pharmaceutically acceptable salt to divalent zinc ions is 1:1.

In some embodiments, the concentration of the polypeptide or its pharmaceutically acceptable salt in the pharmaceutical composition is from 0.01 mg/mL to 1000 mg/mL, for example, 0.1 mg/mL to 500 mg/mL, 0.1 mg/mL to 400 mg/mL, 0.1 mg/mL to 300 mg/mL, 0.1 mg/mL to 200 mg/mL, 0.1 mg/mL to 100 mg/mL, 0.5 mg/mL to 200 mg/mL, 0.5 mg/mL to 150 mg/mL, 0.5 mg/mL to 100 mg/mL, 0.5 mg/mL to 50 mg/mL, 0.5 mg/mL to 25 mg/mL, 1.0 mg/mL to 100 mg/mL. mg/mL, 1.0 mg/mL to 90 mg/mL, 1.0 mg/mL to 80 mg/mL, 1.0 mg/mL to 70 mg/mL, 1.0 mg/mL to 60 mg/mL, 1.0 mg/mL to 50 mg/mL, 1.0 mg/mL to 40 mg/mL, 1.0 mg/mL to 30 mg/mL, 1.0 mg/mL to 20 mg/mL, 2.0 mg/mL to 50 mg/mL, 2.0 mg/mL to 40 mg/mL, 2.0 mg/mL to 30 mg/mL, 2.0 mg/mL to 20 mg/mL, 5.0 mg/mL to 1000 mg/mL, 5.0 mg/mL to 5 0.0 mg/mL, 5.0 mg/mL to 400 mg/mL, 5.0 mg/mL to 300 mg/mL, 5.0 mg/mL to 200 mg/mL, 5.0 mg/mL to 100 mg/mL, 5.0 mg/mL to 90 mg/mL, 5.0 mg/mL to 80 mg/mL, 5.0 mg/mL to 70 mg/mL, 5.0 mg/mL to 60 mg/mL, 5.0 mg/mL to 50 mg/mL, 5.0 mg/mL to 40 mg/mL, 5.0 mg/mL to 30 mg/mL, 5.0 mg/mL to 20 mg/mL, 5.0 mg/mL to 10 mg/mL, 6.0 mg/ 6.0 mg/mL to 1000 mg/mL, 6.0 mg/mL to 500 mg/mL, 6.0 mg/mL to 400 mg/mL, 6.0 mg/mL to 300 mg/mL, 6.0 mg/mL to 200 mg/mL, 6.0 mg/mL to 100 mg/mL, 6.0 mg/mL to 90 mg/mL, 6.0 mg/mL to 80 mg/mL, 6.0 mg/mL to 70 mg/mL, 6.0 mg/mL to 60 mg/mL, 6.0 mg/mL to 50 mg/mL, 6.0 mg/mL to 40 mg/mL, 6.0 mg/mL to 30 mg/mL, 6.0 mg/mL to 20 mg/mL.

In some embodiments, the concentration of the polypeptide or its pharmaceutically acceptable salt in the pharmaceutical composition is 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10.0 mg/mL, 11.0 mg/mL, 12.0 mg/mL, 13.0 mg/mL, 14.0 mg/mL, 1 5.0mg/mL,, 16.0mg/mL, 17.0mg/mL, 18.0mg/mL, 19.0mg/mL, 20.0mg/mL, 21.0mg/mL, 22.0mg/mL, 23.0mg/mL, 24.0mg/mL, 25.0mg/mL, 26.0mg/mL, 27.0mg/mL, 28.0mg/mL, 29.0mg/mL, 30.0mg/mL.

In some embodiments, the concentration of the polypeptide or its pharmaceutically acceptable salt in the pharmaceutical composition is 12.0 mg/mL.

In some embodiments, the concentration of divalent zinc ions in the pharmaceutical composition is 0.001 mg/mL to 100 mg/mL, 0.001 mg/mL to 90 mg/mL, 0.001 mg/mL to 80 mg/mL, 0.001 mg/mL to 70 mg/mL, 0.001 mg/mL to 60 mg/mL, 0.01 mg/mL to 50 mg/mL, 0.01 mg/mL to 40 mg/mL, 0.01 mg/mL to 30 mg/mL, 0.01 mg/mL to 20 mg/mL, 0.01 mg/mL to 10 mg/mL, 0.01 mg/mL to 5 mg/mL, 0.01 mg/mL to 3 mg/mL, 0.1 mg/mL to 5 mg/mL, 0.1 mg/mL to 3 mg/mL, or 0.1 mg/mL to 1.5 mg/mL.

In some embodiments, the concentration of zinc acetate in the pharmaceutical composition is 0.001 mg/mL to 100 mg/mL, 0.001 mg/mL to 90 mg/mL, 0.001 mg/mL to 80 mg/mL, 0.001 mg/mL to 70 mg/mL, 0.001 mg/mL to 60 mg/mL, 0.01 mg/mL to 50 mg/mL, 0.01 mg/mL to 40 mg/mL, 0.01 mg/mL to 30 mg/mL, 0.01 mg/mL to 20 mg/mL, 0.01 mg/mL to 10 mg/mL, 0.01 mg/mL to 5 mg/mL, 0.01 mg/mL to 3 mg/mL, 0.1 mg/mL to 5 mg/mL, 0.1 mg/mL to 3 mg/mL, or 0.1 mg/mL to 1.5 mg/mL.

In some embodiments, the concentration of zinc acetate in the pharmaceutical composition is selected from 0.25 mg/mL, 0.5 mg/mL, and 1.0 mg/mL.

In some embodiments, the pharmaceutical compositions described in this disclosure further comprise a buffer.

In some embodiments, the buffers described in this disclosure are selected from one or more of acetate buffers, histidine buffers, phosphate buffers, succinate buffers, tris(hydroxymethyl)aminomethane buffers (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid buffers (HEPES), 3-morpholine propanesulfonic acid buffers (MOPS), diethanolamine buffers, borate buffers, tris(hydroxymethyl)methylglycine buffers (Tricine), and citrate buffers.

In some embodiments, the buffers described in this disclosure are selected from tris(hydroxymethyl)aminomethane buffers, disodium hydrogen phosphate buffers, and 4-hydroxyethylpiperazine ethanesulfonic acid buffers.

In some embodiments, the concentration of the buffer in the pharmaceutical composition is from 0.5 mM to 50.0 mM, for example, 0.5 mM to 40.0 mM, 0.5 mM to 30.0 mM, 0.5 mM to 20.0 mM, 0.5 mM to 10.0 mM, 1.0 mM to 40.0 mM, 1.0 mM to 35.0 mM, 1.0 mM to 30.0 mM, 1.0 mM to 25.0 mM, 1.0 mM to 20.0 mM. 0.0mM, 1.0mM to 15.0mM, 1.0mM to 10.0mM, 2.0mM to 40.0mM, 2.0mM to 35.0mM, 2.0mM to 30.0mM, 2.0mM to 25.0mM, 2.0mM to 20.0mM, 2.0mM to 15.0mM, 5.0mM to 15.0mM, 2.0mM to 10.0mM, or 5.0mM to 10.0mM.

In some embodiments, the concentration of the buffer in the pharmaceutical composition is 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, or 10.0 mM.

In some embodiments, the concentration of the buffer in the pharmaceutical composition is 10.0 mM.

In some embodiments, the pharmaceutical composition further comprises an osmotic pressure regulator. The osmotic pressure regulator includes, but is not limited to: salts (e.g., sodium chloride, phosphates, sodium citrate, boric acid, and sodium tartrate), sugars or sugar alcohols (lactose, trehalose, sucrose, glucose, mannitol, sorbitol, xylitol), amino acids (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), polyhydroxy sugar alcohols [e.g., glycerol, 1,2-propanediol (also referred to as propylene glycol in this disclosure), 1,3-propanediol, 1,3-butanediol], polyethylene glycol (e.g., PEG 400), or mixtures thereof.

In some embodiments, the osmotic regulator is selected from one or more of propylene glycol, mannitol, sorbitol, xylitol, glycerol, lactose, trehalose, sucrose, glucose, sodium chloride, phosphate, sodium citrate, boric acid, and sodium tartrate.

In some embodiments, the osmotic conditioner is sodium chloride, propylene glycol, mannitol, or glycerin.

In some embodiments, the osmotic pressure regulator is propylene glycol or mannitol.

In some embodiments, the concentration of propylene glycol in the pharmaceutical composition is from 10 mg/mL to 20 mg/mL, for example, 10 mg/mL to 19 mg/mL, 10 mg/mL to 18 mg/mL, 10 mg/mL to 17 mg/mL, 10 mg/mL to 16 mg/mL, 10 mg/mL to 15 mg/mL, 11 mg/mL to 20 mg/mL, 11 mg/mL to 19 mg/mL, 11 mg/mL to 18 mg/mL, 11 mg/mL to 17 mg/mL, 11 mg/mL to 16 mg/mL, 11 mg /mL to 15mg/mL, 12mg/mL to 20mg/mL, 12mg/mL to 19mg/mL, 12mg/mL to 18mg/mL, 12mg/mL to 17mg/mL, 12mg/mL to 16mg/mL, 12mg/mL to 15mg/mL, 13mg/mL to 20mg/mL, 13mg/mL to 19mg/mL, 13mg/mL to 18mg/mL, 13mg/mL to 17mg/mL, 13mg/mL to 16mg/mL, 13mg/mL to 15mg/mL.

In some embodiments, the concentration of propylene glycol in the pharmaceutical composition is from 12 mg/mL to 16 mg/mL, for example, 14 mg/mL.

In some embodiments, the sodium chloride concentration in the pharmaceutical composition is from 1 mg/mL to 20 mg/mL, for example, 1 mg/mL to 19 mg/mL, 1 mg/mL to 18 mg/mL, 1 mg/mL to 17 mg/mL, 1 mg/mL to 16 mg/mL, 1 mg/mL to 15 mg/mL, 2 mg/mL to 18 mg/mL, 2 mg/mL to 17 mg/mL, 2 mg/mL to 16 mg/mL, 2 mg/mL to 15 mg/mL, 3 mg/mL to 18 mg/mL, 3 mg/mL to 17 mg/mL, 3 mg/mL to 16 mg/mL, 3 mg/mL to 15 mg/mL, 4 mg/mL to 14 mg/mL, 5 mg/mL to 13 mg/mL, 6 mg/mL to 12 mg/mL, 7 mg/mL Up to 11 mg/mL, 7 mg/mL to 10.5 mg/mL, 7 mg/mL to 10 mg/mL, 7 mg/mL to 9 mg/mL, 7 mg/mL to 9.5 mg/mL, 7 mg/mL to 9 mg/mL, 7 mg/mL to 8.5 mg/mL, 7.5 mg/mL to 11 mg/mL, 7.5 mg/mL to 10.5 mg/mL, 7.5 mg/mL to 10 mg/mL, 7.5 mg/mL to 9.5 mg/mL, 7.5 mg/mL to 9.0 mg/mL, 7.5 mg/mL to 8.5 mg/mL, 8 mg/mL to 11 mg/mL, 8 mg/mL to 10.5 mg/mL, 8 mg/mL to 10 mg/mL, 8 mg/mL to 9.5 mg/mL, 8 mg/mL to 9 mg/mL.

In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from 2 mg/mL to 18 mg/mL.

In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from 3 mg/mL to 15 mg/mL.

In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from 7 mg/mL to 10 mg/mL, for example 7.5 mg/mL, 7.6 mg/mL, 7.7 mg/mL, 7.8 mg/mL, 7.9 mg/mL, 8 mg/mL, 8.1 mg/mL, 8.2 mg/mL, 8.3 mg/mL, 8.4 mg/mL, 8.5 mg/mL, 8.6 mg/mL, 8.7 mg/mL, 8.8 mg/mL, 8.9 mg/mL, 9.0 mg/mL, 9.1 mg/mL, 9.28 mg/mL, 9.3 mg/mL, 9.4 mg/mL, 9.5 mg/mL, 9.6 mg/mL, 9.7 mg/mL, 9.8 mg/mL, 9.9 mg/mL, or 10 mg/mL.

In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from 7.5 mg/mL to 9.5 mg/mL.

In some embodiments, the concentration of mannitol in the pharmaceutical composition is from 10 mg/mL to 60 mg/mL, for example, from 20 mg/mL to 60 mg/mL, from 20 mg/mL to 55 mg/mL, from 30 mg/mL to 55 mg/mL, from 35 mg/mL to 55 mg/mL, from 40 mg/mL to 55 mg/mL, or from 40 mg/mL to 50 mg/mL.

In some embodiments, the concentration of mannitol in the pharmaceutical composition is 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, or 50 mg/ml.

In some embodiments, the concentration of mannitol in the pharmaceutical composition is from 35 mg/ml to 50 mg/ml, preferably 46 mg/ml.

In some embodiments, the pharmaceutical composition also includes a pH adjuster, such as sodium hydroxide and/or hydrochloric acid.

In some embodiments, the pH of the pharmaceutical composition is 6.5 to 9.0, for example 6.6 to 9.0, 6.7 to 9.0, 6.8 to 9.0, 6.9 to 9.0, 7.0 to 9.0, 7.1 to 9.0, 7.2 to 9.0, 7.3 to 9.0, 7.4 to 9.0, 7.5 to 9.0, 7.6 to 9.0, 7.7 to 9.0, 7.8 to 9.0, 7.9 to 9.0, 8.0 to 9.0, 7.0 to 8.9, 7.0 to 8.8, 7.0. 7.0 to 8.6, 7.0 to 8.5, 7.0 to 8.4, 7.0 to 8.3, 7.0 to 8.2, 7.0 to 8.1, 7.0 to 8.0, 7.1 to 8.9, 7.1 to 8.8, 7.1 to 8.7, 7.1 to 8.6, 7.1 to 8.5, 7.1 to 8.4, 7.1 to 8.3, 7.1 to 8.2, 7.1 to 8.1, 7.1 to 8.0, 7.1 to 7.9, 7.1 to 7.8, or 7.1 to 7.7.

In some embodiments, the pH of the pharmaceutical composition is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

In some embodiments, the pharmaceutical composition is 7.0 to 8.0.

In some embodiments, the pH of the pharmaceutical composition is 7.1 to 7.7, such as 7.5 or 7.4.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable antibacterial agent.

In some implementations, pharmaceutically acceptable antimicrobial agents include, but are not limited to: phenol, o-cresol, m-cresol, p-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, benzyl alcohol, ethanol, chlorobutanol, thimerosal, bromonitrile, benzoic acid, imidazoline, chlorhexidine, sodium dehydroacetate, chlorocresol, ethylparaben, benzyl chloride, or mixtures thereof.

In some implementation schemes, phenol is a pharmaceutically acceptable antibacterial agent.

In some embodiments, the concentration of the antibacterial agent in the pharmaceutical composition is 4.0 mg/mL to 7.0 mg/mL, for example, 4.2 mg/mL to 6.9 mg/mL, 4.4 mg/mL to 6.9 mg/mL, 4.6 mg/mL to 6.9 mg/mL, 4.8 mg/mL to 6.9 mg/mL, 5.0 mg/mL to 6.9 mg/mL, 5.1 mg/mL to 6.9 mg/mL, 5.2 mg/mL to 6.9 mg/mL, 5.3 mg/mL to 6.9 mg/mL. g/mL, 5.4mg/mL-6.9mg/mL, 5.5mg/mL-6.9mg/mL, 4.2mg/mL-6.8mg/mL, 4.4mg/mL-6.8mg/mL, 4.6mg/mL-6.8mg /mL, 4.8mg/mL-6.8mg/mL, 5.0mg/mL-6.8mg/mL, 5.1mg/mL-6.8mg/mL, 5.2mg/mL-6.8mg/mL, 5.3mg/mL-6.8mg/ mL, 5.4mg/mL-6.8mg/mL, 5.5mg/mL-6.8mg/mL, 4.4mg/mL-6.7mg/mL, 4.6mg/mL-6.7mg/mL, 4.8mg/mL-6.7mg/ mL, 5.0mg/mL-6.7mg/mL, 5.1mg/mL-6.7mg/mL, 5.2mg/mL-6.7mg/mL, 5.3mg/mL-6.7mg/mL, 5.4mg/mL-6.7mg/m L, 5.5mg/mL-6.7mg/mL, 4.4mg/mL-6.6mg/mL, 4.6mg/mL-6.6mg/mL, 4.8mg/mL-6.6mg/mL, 5.0mg/mL-6.6mg/mL , 5.1mg/mL-6.6mg/mL, 5.2mg/mL-6.6mg/mL, 5.3mg/mL-6.6mg/mL, 5.4mg/mL-6.6mg/mL, 5.5mg/mL-6.6mg/mL.

In some implementations, the concentration of the antibacterial agent is 4.2 mg/mL to 6.9 mg/mL.

In some implementations, the concentration of the antibacterial agent is 5.5 mg/mL to 6.6 mg/mL, for example, 5.5 mg/mL.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described herein comprises an N-terminus of an amino acid sequence as shown in _X1 -Aib- _X2 ; wherein _X1 is a natural or non-natural amino acid residue containing an imidazole group, and _X2 is a natural or non-natural amino acid residue containing an imidazole group.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described herein comprises an N-terminus of an amino acid sequence as shown in R ₁ -X ₁ -Aib-X ₂ ; wherein R ₁ is hydrogen, X ₁ is a natural or non-natural amino acid residue containing an imidazole group, and X ₂ is a natural or non-natural amino acid residue containing an imidazole group.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described in this disclosure, wherein _X1 is a His residue, and/or _X2 is a His residue.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described in this disclosure comprises the N-terminus of the amino acid sequence His-Aib-His.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described herein comprises at least one Lys, Orn, Dap, Dab or Cys residue with a side chain-linked substituent.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof described in this disclosure comprises at least one Lys residue with a side-chain-linked substituent.

In some embodiments, the substituents in the polypeptide or its pharmaceutically acceptable salt described herein comprise the following structures: _{-Z1 -Z2} ;

in,

Z ₁ contains 0-10 groups independently selected from the following: Gly residue, Glu residue, γGlu residue, -C(O)-CH _2- (O-CH ₂ -CH ₂ ) _2- NH-, -C(O)-(C ₆ H ₁₀ )-CH ₂ -NH-, -C(O)-CH(NH ₂ )-(CH ₂ ) ₄ -NH-;

_Z2 is selected from _C12-32 fatty acids or _C12-32 alkyl groups.

In some embodiments, the pharmaceutical compositions of this disclosure, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises at least one Lys residue, and the ε-amino group of the Lys residue is linked to any of the following substituents:

In some embodiments, the pharmaceutical composition described herein contains a polypeptide or a pharmaceutically acceptable salt thereof that has agonistic activity against at least one of the following receptors: GLP-1 receptor, GIP receptor, or GCG receptor.

In some embodiments, the pharmaceutical composition described in this disclosure contains a polypeptide or a pharmaceutically acceptable salt thereof as a triple agonist of the GLP-1 receptor, GIP receptor, and GCG receptor.

In some embodiments, the pharmaceutical composition described in this disclosure comprises the following sequence:

H-Aib-HGTFTSDYSILLEKKAAQEFVEWLLAGGPSSGAPPPS(SEQ ID NO:1)

H-Aib-HGTFTSDYSIYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:2)

H-Aib-HGTFTSDYSIYLEKQAAQEFVQWLLEGGPSSGAPPPS(SEQ ID NO:3)

H-Aib-HGTFTSDYSIYLEKQYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:4)

H-Aib-HGTFTSDYSYYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:5)

H-Aib-HGTFTSDYSIYLEKQAAEEFVQWLLEGGPSSGAPPPS(SEQ ID NO:6)

H-Aib-HGTFTSDYSIYLDKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:7)

H-Aib-HGTFTSDYSYYLEKQYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:8)

H-Aib-HGTFTSDYSIYLEKKYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:9)

H-Aib-HGTFTSDYSYYLEKKAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:10)

H-Aib-HGTFTSDYSIYLEKKAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:11)

H-Aib-HGTFTSDYSIYLEKQKA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:12)

H-Aib-HGTFTSDYSKYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:13)

H-Aib-HGTFTSDYSIYLEKRAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:14)

H-Aib-HGTFTSDYSILLEKKYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:15)

H-Aib-HGTFTSDYSYLLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:16)

H-Aib-HGTFTSDYSILLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:17)

H-Aib-HGTFTSDYSILLEKQAAEEFVQWLLEGGPSSGAPPPS(SEQ ID NO:18)

H-Aib-HGTFTSDYSILLEKQAAQEFVQWLLEGGPSSGAPPPS(SEQ ID NO:19)

H-Aib-HGTFTSDYSILLEKQAAREFVQWLLEGGPSSGAPPPS(SEQ ID NO:20)

H-Aib-HGTFTSDYSILLEKQAAHEFVQWLLEGGPSSGAPPPS(SEQ ID NO:21)

H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKAGGHPS-Aib-KPPPK(SEQ ID NO:22)

H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKA-dAla-GHPS-Aib-KPPPK(SEQ ID NO:23)

H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKAGGPPS-Aib-KPPPK(SEQ ID NO:24)

H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:25)

H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKAGGPPS-Aib-KPPPK(SEQ ID NO:26)

H-Aib-HGTFTSDLSKLKEEQRQ-dAla-EFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:27)

H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKAGGHPS-Aib-KPPPK(SEQ ID NO:28)

H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKA-dAla-GHPS-Aib-KPPPK(SEQ ID NO:29)

H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:30)

Preferably, the polypeptide or its pharmaceutically acceptable salt has a C-terminal carboxylic acid group or an amide group, more preferably a C-terminal amide group.

In some embodiments, the pharmaceutical composition described in this disclosure comprises the polypeptide or a pharmaceutically acceptable salt thereof, containing any of the following structures:

Compound 1,

Compound 2,

Compound 3,

Compound 4,

Compound 5,

Compound 6,

Compound 7,

Compound 8,

Compound 9,

Compound 10,

Compound 11,

Compound 12,

Compound 13,

Compound 14,

Compound 15,

Compound 16,

Compound 17,

Compound 18,

Compound 19,

Compound 20,

Compound 21,

Compound 22,

Compound 23,

Compound 24,

Compound 25,

Compound 26,

Compound 27,

Compound 28,

Compound 29,

Compound 30,

Compound 31,

Compound 32,

Compound 33,

Compound 34,

Compound 35,

Compound 36,

Compound 37,

Compound 38,

Compound 39,

Compound 40,

Compound 41,

Compound 42,

Compound 43,

Compound 44,

Compound 45,

Compound 46,

Compound 47,

Compound 48,

Compound 49,

Compound 50,

Compound 51,

Compound 52,

Compound 53,

In some embodiments, the compound comprises (or is) any of the following structures:

In some embodiments, the pharmaceutical compositions described in this disclosure are selected from:

1) The aforementioned polypeptide or its pharmaceutically acceptable salt, for example comprising the structure shown in any of compounds 1-53;

2) Divalent zinc ions, such as zinc acetate or zinc chloride;

3) Buffers, such as phosphate buffers, such as sodium dihydrogen phosphate;

4) Osmotic pressure regulators, such as sodium chloride, propylene glycol, mannitol, and glycerin;

5) Optional antibacterial agents, such as phenol.

In some embodiments, the pharmaceutical compositions described in this disclosure are selected from:

1) The aforementioned polypeptide or its pharmaceutically acceptable salt, for example comprising the structure shown in any of compounds 1-53;

2) Divalent zinc ions, such as zinc acetate;

3) Buffers, such as sodium dihydrogen phosphate;

4) Osmotic pressure regulators, such as mannitol;

5) Optional antibacterial agents, such as phenol.

In some embodiments, the pharmaceutical compositions described in this disclosure are selected from:

1) The aforementioned polypeptide or its pharmaceutically acceptable salt, for example comprising the structure shown in any of compounds 1-53;

2) Divalent zinc ions, such as zinc acetate or zinc chloride;

3) Buffers, such as phosphate buffers, such as sodium dihydrogen phosphate;

4) Osmotic pressure regulators, such as sodium chloride, propylene glycol, mannitol, and glycerin;

5) Optional antibacterial agents, such as phenol;

6) pH adjusters, such as sodium hydroxide and/or hydrochloric acid;

7) Water for injection.

In some embodiments, the pharmaceutical compositions described in this disclosure are selected from:

1) The aforementioned polypeptide or its pharmaceutically acceptable salt, for example comprising the structure shown in any of compounds 1-53;

2) Divalent zinc ions, such as zinc acetate;

3) Buffers, such as sodium dihydrogen phosphate;

4) Osmotic pressure regulators, such as mannitol;

5) Optional antibacterial agents, such as phenol;

6) pH adjusters, such as sodium hydroxide and/or hydrochloric acid;

7) Water for injection.

In some embodiments, the pharmaceutical compositions described in this disclosure are selected from:

1) The aforementioned polypeptide or its pharmaceutically acceptable salt, for example comprising the structure shown in any of compounds 1-53;

2) Divalent zinc ions, such as zinc chloride;

3) Buffers, such as sodium dihydrogen phosphate;

4) Osmotic pressure regulators, such as mannitol;

5) Optional antibacterial agents, such as phenol;

6) pH adjusters, such as sodium hydroxide and/or hydrochloric acid;

7) Water for injection.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof in the pharmaceutical composition described herein comprises the structure shown in compound 1.

In some embodiments, the polypeptide or pharmaceutically acceptable salt thereof in the pharmaceutical composition described herein is selected from the structure shown in Compound 1.

In some embodiments, the pharmaceutical compositions described herein can be used to treat patients requiring such treatment via parenteral administration. Parenteral administration routes may include subcutaneous injection, intramuscular injection, or intravenous injection.

In some embodiments, the pharmaceutical composition described herein, when placed under high temperature conditions for 10 days, has a hydrolysis impurity content of no more than 5%, preferably no more than 3%, more preferably no more than 2%, and more preferably no more than 1%.

In some embodiments, the pharmaceutical composition of this disclosure, when placed under high temperature conditions for 1 month, has a hydrolytic impurity content of no more than 10%, preferably no more than 6%, preferably no more than 5%, preferably no more than 4%, preferably no more than 3%, preferably no more than 2%, and preferably no more than 1.5%.

In some embodiments, the pharmaceutical composition of this disclosure, when placed under high temperature conditions for 10 days, has a content of hydrolytic impurities generated by the cleavage of the N-terminal His-Aib not exceeding 5%, preferably not exceeding 3%, preferably not exceeding 2%, and preferably not exceeding 1%.

In some embodiments, the pharmaceutical composition of this disclosure, after being placed under high temperature conditions for 1 month, exhibits a content of hydrolytic impurities resulting from the cleavage of the N-terminal His-Aib not exceeding 10%, preferably not exceeding 6%, preferably not exceeding 5%, preferably not exceeding 4%, preferably not exceeding 3%, preferably not exceeding 2%, and preferably not exceeding 1.5%.

In some embodiments, the pharmaceutical composition of this disclosure, after being placed under high temperature conditions for 10 days, has a total impurity content of not more than 15%, preferably not more than 12%, preferably not more than 10%, preferably not more than 9%, preferably not more than 8%, preferably not more than 7%, preferably not more than 6%, preferably not more than 5%, preferably not more than 4%, and preferably not more than 3%.

In some embodiments, the pharmaceutical composition of this disclosure, when placed under high temperature conditions for one month, has a total impurity content of not more than 15%, preferably not more than 12%, preferably not more than 10%, preferably not more than 9%, preferably not more than 8%, preferably not more than 7%, preferably not more than 6%, preferably not more than 5%, preferably not more than 4%, and preferably not more than 3%.

In some embodiments, the high temperature conditions described in this disclosure are 25°C, 30°C, or 40°C; preferably, the high temperature conditions are 30°C or 40°C.

This disclosure also provides a method for preparing a lyophilized formulation containing a polypeptide or a pharmaceutically acceptable salt thereof, comprising the step of lyophilizing the aforementioned pharmaceutical composition. In an optional embodiment, the lyophilization sequentially comprises the steps of pre-freezing, primary drying, and secondary drying.

This disclosure also provides a lyophilized formulation containing a polypeptide or a pharmaceutically acceptable salt thereof, prepared by the aforementioned method for preparing a lyophilized formulation containing a polypeptide or a pharmaceutically acceptable salt thereof.

This disclosure also provides a method for preparing a reconstitution solution of a lyophilized preparation containing a polypeptide or a pharmaceutically acceptable salt thereof, including a step of reconstitution of the aforementioned lyophilized preparation, wherein the solution used for reconstitution is selected from, but is not limited to, water for injection, physiological saline or glucose solution.

This disclosure also provides a reconstitution solution of a lyophilized preparation containing a polypeptide or a pharmaceutically acceptable salt thereof, prepared by the aforementioned method of reconstitution solution of a lyophilized preparation containing a polypeptide or a pharmaceutically acceptable salt thereof.

This disclosure further provides an article or kit comprising a container holding any of the stable pharmaceutical compositions described herein. In some embodiments, the glass vial is a neutral borosilicate glass controlled injection vial.

This disclosure also provides an article comprising a container containing the aforementioned pharmaceutical composition or lyophilized formulation or a reconstituted solution of the lyophilized formulation.

This disclosure also provides pharmaceutical compositions or lyophilized formulations or reconstituted solutions of lyophilized formulations for use in methods of treating and preventing diseases or conditions.

This disclosure also provides the use of the aforementioned pharmaceutical compositions or lyophilized formulations or reconstituted solutions of lyophilized formulations in the preparation of medicaments for the treatment and/or prevention of diseases or conditions.

This disclosure also provides a method for treating and preventing diseases or conditions, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of a lyophilized preparation to a patient in need.

This disclosure provides the use of pharmaceutical compositions in the preparation of medicaments for treating non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, obesity, non-alcoholic fatty liver disease, hepatic steatosis, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes.

This disclosure provides a pharmaceutical composition for treating non-insulin-dependent diabetes mellitus/type II diabetes mellitus, insulin-dependent diabetes mellitus, obesity, non-alcoholic fatty liver disease, hepatic steatosis, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes.

This disclosure provides a method for treating non-insulin-dependent diabetes mellitus/type II diabetes mellitus, insulin-dependent diabetes mellitus, obesity, non-alcoholic fatty liver disease, hepatic steatosis, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes mellitus, comprising administering the pharmaceutical composition of this disclosure to a subject in need.

the term

To facilitate understanding of this disclosure, some technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

The amino acid sequences disclosed herein contain standard single-letter or three-letter codes for twenty amino acids. Unless otherwise specified, all amino acid residues in this disclosure are preferably configured in the L-type. Additionally, Aib is α-aminoisobutyric acid, D-Ala is D-alanine, Orn is ornithine, Dap is 2,3-diaminopropionic acid, and Dab is 2,4-diaminobutyric acid.

The term "agonist" is defined as a substance that activates the GLP-1 receptor or the GIP receptor.

As used in the context of this disclosure, the term "triple agonist of GLP-1 receptor, GIP receptor and GCG receptor" refers to a substance or ligand that can activate GLP-1 receptor, GIP receptor and GCG receptor.

In this disclosure, the term "treatment" includes suppressing, slowing down, stopping, or reversing existing symptoms or the progression or severity of a patient's condition.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 8 carbon atoms, an alkyl group containing 1 to 6 carbon atoms, or an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. Alkyl groups can be, for example, lower alkyl groups containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any accessible connection point. The substituent can be one or more groups independently selected from: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester. The substituted alkyl group disclosed herein can be methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, or hydroxy-substituted alkyl.

The term "natural GLP-1" refers to a naturally occurring molecule of the glucagon family of peptides or the venom exopeptide family, wherein: the glucagon family of peptides is encoded by the proglucagonogenamic gene and includes three highly homologous small peptides, namely glucagon (1-29), GLP-1 (1-37), and GLP-2 (1-33); and venom exopeptides are peptides expressed in lizards and, similar to GLP-1, are insulin-stimulating. In some embodiments, the term "natural GLP-1" also refers to human GLP-1 (7-37) and human GLP-1 (7-36).

The term "substitution" of an amino acid residue as used in this disclosure refers to the substitution of an amino acid residue by a different substance.

The term "fatty acid" refers to a carboxylic acid having a long fatty acid tail (chain), which may be saturated or unsaturated; in this disclosure, fatty acids are carboxylic acids having a straight-chain or branched aliphatic group with C4-C30.

As used in this disclosure, the term "peptide" refers to a sequence of two or more amino acids. "Peptide" encompasses the category of peptides having modified amino and carboxyl terms. A "peptide" may comprise a native amino acid sequence or a modified one. For example, one or more amino acid residues may be substituted by other amino acid residues, and/or one or more amino acid residues may be deleted from the peptide, and/or one or more amino acid residues may be added to the peptide, and/or one or more amino acid residues may be linked with substituents. Such modifications to amino acid residues may occur at any position at the N-terminus and/or C-terminus and/or in the middle of the peptide. For example, an amino acid chain containing a terminal carboxylic acid with an amide group substituted is also included within the amino acid sequence named as a native amino acid.

The term "amino acid" is a molecule containing an amino group and a carboxylic acid group, and optionally one or more additional groups often referred to as side chains. Amino acids include proteinogenic (or naturally occurring) amino acids (of which 20 are standard amino acids) and non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids are either not present in proteins or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modifications). Non-limiting examples of non-proteinogenic amino acids are Aib (α-aminoisobutyric acid or 2-aminoisobutyric acid), leucine (Nle), valine, and the D-isomers of proteinogenic amino acids. D-Ala is D-alanine, -αL- is alpha-methylated leucine, αS is alpha-methylated serine, αK is alpha-methylated lysine, etc.

"Natural amino acids" refers to 20 common amino acids (i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

"Non-natural amino acids" refer to amino acids that are not naturally encoded or found in the genetic code of any organism. They can be, for example, purely synthetic compounds. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azacyclobutanecarboxylic acid, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, and N-ethylasparagine. The ingredients include: high-proline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, valine, leucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline. Additionally, the C-terminal carboxyl group, N-terminal amino group, and/or their side chain functional groups of natural or non-natural amino acids are chemically modified.

All hydrogen atoms described in this disclosure can be replaced by their isotopes (protium, deuterium, tritium), and any hydrogen atom in the compounds disclosed herein can also be replaced by isotopic atoms.

"Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, that are independently substituted by a substituent. Substituents are only considered in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutical salts or prodrugs, along with other chemical components, such as physiologically/pharmaceutical carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

"Buffer" refers to a buffer that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetates, succinates, citrates, phosphates, gluconates, histidines, oxalates, lactates, phosphates, citrates, tartrates, fumarates, glycylglycine, and other organic acid buffers.

"Histidine buffers" are buffers containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate buffers, such as histidine-acetate buffers or histidine-hydrochloride buffers. Histidine-acetate buffers are prepared by reacting histidine with acetic acid, while histidine buffers are prepared by reacting histidine with hydrochloric acid.

"Citrate buffers" are buffers that contain citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A citrate buffer can be sodium citrate.

"Succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, and calcium succinate. Succinate buffers can be sodium succinate.

A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid. A phosphate buffer can be disodium hydrogen phosphate-sodium dihydrogen phosphate.

"Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. Acetate buffers can be sodium acetate.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically/pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically/pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity. In this document, "pharmaceutical composition" and "formulation" are used interchangeably.

Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

"Lyophilized formulation" refers to a formulation or pharmaceutical composition obtained by a vacuum freeze-drying step after a liquid or solution form of a pharmaceutical composition or solution preparation.

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The values in this disclosure are instrument measurements or calculated values after instrument measurement, and are subject to a certain degree of error. Generally speaking, ±10% is within the reasonable error range. Of course, the context in which the value is used needs to be considered. For example, for the content of total impurities, the value is defined as having an error variation of no more than ±10% after measurement, and can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

Attached Figure Description

Figure 1 shows the plasma concentration-time curves of pharmacokinetic data for prescriptions 26 and 27 in rats.

Detailed Implementation

The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

Example 1: Preparation of compound 0120

Compound 0120 was chemically synthesized using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t-Bu) synthesis method on a Prelude-X fully automated peptide synthesizer. The resin used was Rink-amide MBHA resin with a substitution degree of 0.54 mmol/g. The amino acid residues used for fatty acid modification were Fmoc-L-Lys(Mtt)-OH, and the N-terminal amino acid was Boc-L-His(Trt)-OH. Before the amino acid condensation step, the Fmoc group was removed using a DMF solution containing 20% 4-methylpiperidine (reaction twice, 8 minutes each). All standard amino acid condensations used equimolar ratios of Fmoc amino acids, HATU, and 2 equivalents of 4-methylmorpholine, condensed at room temperature for 25 minutes at a dose exceeding the theoretical peptide loading by 10 equivalents. Exceptions were made for coupling to Cα-methylated amino acids, which required two or three condensations, each lasting 60 minutes, to ensure complete condensation. After completing the above peptide-resin extension, the resin peptide was washed with dichloromethane and then a hexafluoroisopropanol/dichloromethane mixture (30% hexafluoroisopropanol, 10 mL) was added. The mixture was shaken at room temperature for 45 minutes, then removed. Another hexafluoroisopropanol/dichloromethane mixture (30%, 10 mL) was added, and the mixture was shaken at room temperature for 45 minutes, then removed. After the reaction, the resin was washed three times with DMF. Additional coupling/deprotection cycles to extend the lysine side chain using the Fmoc/tBu solid-phase synthesis strategy on the same peptide synthesizer involved Fmoc-AEEA-OH (twice), Fmoc-L-Glu-OtBu (once), and eicosanoic acid monotert-butyl ester (once). Condensation was performed using equimolar ratios of Fmoc amino acids, HATU, and 2 equivalents of 4-methylmorpholine at room temperature for 25 minutes at 10 equivalents above the theoretical peptide loading. The exception is the coupling of eicosanoic acid monotert-butyl ester, which requires condensation at room temperature for at least 3 hours to ensure complete condensation.

After synthesis, the obtained resin peptide was washed three times with DMF and DCM, then vacuum dried. 10 mL of freshly prepared lysis buffer (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was added, and the mixture was shaken at room temperature for 3-4 hours. After the reaction, the mixture was filtered, and the resin was washed twice with trifluoroacetic acid. The filtrates were combined, and a large amount of methyl tert-butyl ether was added to precipitate the crude peptide solid. After centrifugation to remove the supernatant, the crude peptide with compound number 0120 was obtained. The crude peptide was dissolved in a mixed solvent containing 20% acetic acid/water, filtered through a 0.22 μm membrane, and separated using a WATERS Prep150 LC reversed-phase high-performance liquid chromatography system. The mobile phases were A (0.1% trifluoroacetic acid, 10% acetonitrile, aqueous solution) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution). The chromatographic column used was an X-SELECT OBD C-18 (WATERS) reversed-phase column. During purification, the detection wavelength was set to 220 nm, and the flow rate was 15-20 mL/min. The relevant fractions of the product were collected and lyophilized to obtain the pure peptide with compound number 0120. The purity and compound identity of the pure peptide were determined by analytical ultra-high performance liquid chromatography (UHPLC) and liquid chromatography/mass spectrometry (LC/MS), with a purity of 98.03% and a determined molecular weight of 4854.2.

Example 2: Bioactivity Test of Compound 0120

The purpose of this embodiment is to detect the agonist activity of the test compound 0120 in human glucagon-like peptide-1 receptor, human glucose-dependent insulinotropic peptide receptor, and human glucagon receptor.

2.1 Experimental Methods:

Functional activity was assessed in CHO-K1 clonal cell lines expressing GIPR, GLP-1R, and GCGR. The peptides were diluted 10-fold (8–10 spots) in DMEM/F12 1:1 (HyClone, SH30023.01) supplemented with 5% FBS (fetal bovine serum, Gibco, 10099141), 0.1% Casein (Sigma, C4765), and 0.25 mM IBMX (Selleck, S5836) in 10 μL assay volume. Recipient cells (5000 cells/well) were added to SV 96-well plates (Cisbio, 66PL96025) and incubated with the prepared peptides at 37°C for 30 minutes. The increase in intracellular cAMP was quantified using the cAMP-GS dynamic HTRF assay kit (Cisbio, 62AM4PEJ). In short, intracellular cAMP levels were detected by adding a cAMP-d2 conjugate to cell lysis buffer, followed by the addition of an antibody-anti-cAMP-Eu cavitation compound (also in cell lysis buffer). The resulting competitive assay was incubated at room temperature for at least 60 minutes and then detected using a Tecan Infinite F Plex instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The measured data (emission at 665 nm/620 nm * 10000) were inversely proportional to the amount of cAMP present and converted to per-well cAMP concentration using a cAMP standard curve.

The amount of cAMP generated in each well was converted into the percentage of the maximum response observed with human GLP-1 (7-36) _-NH2 , human GCG (1-29), or human GIP (1-42) _-NH2 (purchased from Sigma). Using the percentage of maximum response versus the added peptide concentration, relative EC50 values were derived via nonlinear regression analysis and fitted to a four-parameter logarithmic equation. Specific data are shown in Table 1 below. The LY3437943 molecule is described in Example 12 of WO2019/125938, the foregoing of which is incorporated herein by reference in its entirety.

Table 1

Example 3. Detection of the pharmacokinetic properties of the compound in mice

Male C57 mice were used as test animals. The target compound was injected intravenously once, and the pharmacokinetic characteristics of compound 0120 in C57 mice (plasma) were detected.

3.1 Test Methods:

Male C57 mice, weighing approximately 25g and aged 4-6 weeks, were used. After preparing peptide compound solutions using 1×PBS, the compounds were administered intravenously at a dose of 200 nmol/kg, with three animals receiving each compound. Blood samples (60 μL) were collected at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h, and 72 h after infusion and transferred to centrifuge tubes containing EDTA-K2 anticoagulant. Whole blood samples were centrifuged at 4000g for 5 min at 4°C to obtain plasma, which was then stored at -80°C.

Sample pretreatment method: Take 30 μL of plasma sample, add 120 μL of 0.1% formic acid methanol solution containing 1 ng/mL internal standard verapamil, vortex for 5 min, centrifuge at 10000 rpm for 10 min at low temperature, take 70 μL of supernatant, add 70 μL of water, mix well, and analyze by LC-MS instrument.

LC-MS analysis method: (1) Chromatographic conditions: Mobile phases A and B were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution, respectively; gradient elution mode was used; flow rate was 0.5 mL/min; injection volume was 10 μL; chromatographic column was Nanomicro Unisil C18aq (4.6 mm × 150 mm, 5 μm); column temperature: 40 ℃. (2) Mass spectrometry conditions: Mass spectrometry was performed using electrospray ionization (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning mode.

Data processing used SCIEX OS (Version 2.1.6) software to quantify blood drug concentration data and PKSolver (Version 2.0) software to calculate pharmacokinetic parameters.

3.2 Test Results

Table 2. Comparison of pharmacokinetic parameters of compounds (200 nmol/kg) administered to mice

Example 4: Preparation process of the pharmaceutical composition

The pharmaceutical composition disclosed herein can be prepared according to the following process:

Preparation of the stock solution: Take an appropriate amount of water for injection, add an appropriate amount of buffer salt solution, weigh out the prescribed amount of antibacterial agent and osmotic pressure regulator, add them to the above solution, and stir until dissolved. Weigh out the prescribed amount of API-0120 molecules, add them to the above solution, stir until dissolved, and add an appropriate amount of water for injection.

Preparation of the formulation: Take an appropriate amount of mother liquor, add the prescribed amount of metal ion solution as needed, adjust the pH with 1M NaOH solution or 0.5M HCl solution, and add water for injection to make up the volume.

Filtration and filling: Filter the prescription through a 0.22μm PVDF filter membrane into 2mL vials, 1mL/vial.

Table 3. Formulation Information (Unit Dosage)

Example 5: Stability study of the pharmaceutical composition

The formulations 1-5 prepared in Example 4 were placed under high temperature for 1 month to examine their chemical stability. The formulation samples were analyzed for related substances by HPLC and HMPW by SEC. The results are shown in Table 4.

The HPLC column used was an Agilent Advance Bio Peptide Map, 4.6 × 150 mm, 2.7 μm; the detection wavelength was 220 nm; the mobile phases were: mobile phase A: 50 mM sodium sulfate (0.1% perchloric acid), mobile phase B: 80% acetonitrile (0.2% perchloric acid).

The chromatographic column used for SEC detection was Sepax Zenix SEC-80 (7.8*300mm, 3μm); the detection wavelength was 280nm; and the mobile phase was 50% ACN (0.1% TFA).

Table 4 Chemical stability results of formulations 3-5

ASAP stability prediction results show that the hydrolytic impurities generated by the N-terminal His-Aib breakage are low activation energy sensitive, and the increase in temperature will significantly increase the chemical instability of the impurities.

The results of the high-temperature accelerated test in Table 4 show that by adding zinc ions to the formulation to slow down the growth of hydrolytic impurities (RRT1.08) and aggregates (HMPW) in the compound, the overall degradation of impurities decreased, which meets the impurity requirements of the product at the end of the clinical use and efficacy period.

Example 6: Stability Study of Drug Compositions with Different Metal Ions

The effects of different metal ions on the stability of the pharmaceutical composition were investigated, referring to the preparation method and dosage shown in Formulation 4 of Example 4 (API-0120: 12 mg/mL, disodium hydrogen phosphate buffer: 10 mM, phenol: 5.5 mg/mL, mannitol: 46 mg/mL, pH 7.5).

Table 5. Formulation Information (Zinc Ions)

Table 6. Formulation Information (Magnesium Ions)

Table 7. Formulation Information (Calcium Ions, Iron Ions)

Formulation 6-23, prepared using the same batch of API, had the same initial impurity levels: total impurities of 3.15%, hydrolytic impurities of 0.62%, and HMPW of 0.11%. As shown in Table 8, compared to other divalent metal ions, the chemical stability of the formulation is superior when using divalent zinc ions.

Table 8 Chemical stability results

Example 7: In vitro activity evaluation of zinc-containing preparations:

The effect of zinc ion addition on API activity was investigated using the preparation method shown in Formulation 4 of Example 4.

Table 9. Formulation Information

In vitro activity tests were conducted on formulations 24 and 25. The results showed that the in vitro activity of the three targets of formulation 25 was not significantly affected compared to that of formulation 24, indicating that the addition of zinc ions did not affect the activity of the terminal amino acids.

Example 8: Investigation of PK behavior of zinc-containing preparations in rats

Following the preparation method shown in Formulation 4 of Example 4, the differences in pharmacokinetic behavior of zinc ions in rats were compared and investigated. Six- to seven-week-old male SD rats were used as test animals, and the target formulation was administered via single subcutaneous injection. The pharmacokinetic characteristics of compound 0120 in SD rat plasma were then examined.

Table 10

8.1 Test Methods:

Male SD rats, weighing approximately 200-250g and 6-7 weeks old, were used. The drug was administered subcutaneously at a dose of 1060 nmol/kg, with three animals receiving each compound. Blood samples of 500 μL were collected at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 168 h post-administration and transferred to centrifuge tubes containing EDTA-K2 anticoagulant. Whole blood samples were centrifuged at 4000g for 5 min at 4°C to obtain plasma, which was then stored at -80°C.

Sample pretreatment method: Take 30 μL of plasma sample, add 120 μL of 0.1% formic acid methanol solution containing 1 ng/mL internal standard verapamil, vortex for 5 min, centrifuge at 10000 rpm for 10 min at low temperature, take 70 μL of supernatant, add 70 μL of water, mix well, and analyze by LC-MS instrument.

LC-MS analysis method: (1) Chromatographic conditions: Mobile phases A and B were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution, respectively; gradient elution mode was used; flow rate was 0.5 mL/min; injection volume was 10 μL; chromatographic column was Nanomicro Unisil C18aq (4.6 mm × 150 mm, 5 μm); column temperature: 40 ℃. (2) Mass spectrometry conditions: Mass spectrometry was performed using electrospray ionization (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning mode.

Data processing used SCIEX OS (Version 2.1.6) software to quantify blood drug concentration data.

The results showed no significant difference in the overall PK behavior of zinc-containing preparations.

Example 9: Characterization of the 0120 high-order structure

Referring to the preparation method shown in Formulation 4 of Example 4, the effect of formulations containing different proportions of zinc ions on the higher-order structure of API was investigated.

Table 11 Formulation Information

Because mannitol and phenol cause significant interference in the far-ultraviolet region during circular dichroism spectroscopy, excipients such as mannitol and phenol were not added to the formulation for advanced structure characterization. Circular dichroism spectroscopy characterization was performed on formulations containing zinc (10 mM disodium hydrogen phosphate, pH 7.5) and those without zinc (10 mM disodium hydrogen phosphate, pH 7.5). The results are shown in Table 12. Adding zinc ions at pH 7.5 did not significantly alter the secondary structure.

Table 12 Aggregate Detection in Formulations with Different Zinc Ratios

Example 10: Stability Study of SAR441255 Molecular Formulation

Following the preparation method shown in Formulation 4 of Example 4, the effect of formulations containing different proportions of zinc ions on the chemical stability of API-SAR441255 was investigated. Four groups with different proportions of zinc ions were set up for investigation (Group 1: no zinc ions added; Group 2: API:zinc ion molar ratio 2:1; Group 3: API:zinc ion molar ratio 1:1; Group 4: API:zinc ion molar ratio 1:2). Related substances in the formulation samples were detected by HPLC, and HMPW was detected by SEC. The structure of SAR441255 is as follows:

Table 13 Chemical stability results

The formulation without zinc showed a significant increase in total impurities and hydrolytic impurities over 3 months (at 25 degrees Celsius). Adding zinc ions to the formulation can significantly reduce the generation of hydrolytic impurities and polymers.

Claims (26)

A pharmaceutical composition comprising a polypeptide or a pharmaceutically acceptable salt thereof and a divalent metal ion. The polypeptide comprises an N-terminus of an amino acid sequence as shown in _X1 -Aib- _X2 ; wherein _X1 is a natural or non-natural amino acid residue containing an imidazole group, and _X2 is a natural or non-natural amino acid residue containing an imidazole group. The pharmaceutical composition according to claim 1, wherein the polypeptide is N-terminus of an amino acid sequence as shown in _R1 - _X1 -Aib- _X2 ; wherein _R1 is hydrogen, _X1 is a natural or non-natural amino acid residue containing an imidazole group, and _X2 is a natural or non-natural amino acid residue containing an imidazole group. The pharmaceutical composition according to claim 1 or 2, wherein _X1 is a His residue, and/or _X2 is a His residue; preferably, the polypeptide comprises the N-terminus of the amino acid sequence His-Aib-His. The pharmaceutical composition according to any one of claims 1-3, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises at least one Lys, Orn, Dap, Dab or Cys residue with a side chain-linked substituent; Preferably, the polypeptide or its pharmaceutically acceptable salt comprises at least one Lys residue with a side-chain-linked substituent; Preferably, the substituent comprises the structure shown below: _{-Z1 -Z2} ; in, Z ₁ contains 0-10 groups independently selected from the following: Gly residue, Glu residue, γGlu residue, -C(O)-CH _2- (O-CH ₂ -CH ₂ ) _2- NH-, -C(O)-(C ₆ H ₁₀ )-CH ₂ -NH-, -C(O)-CH(NH ₂ )-(CH ₂ ) ₄ -NH-; _Z2 is selected from _C12-32 fatty acids or _C12-32 alkyl groups. The pharmaceutical composition according to any one of claims 1-4, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises at least one Lys residue, and the ε-amino group of the Lys residue is linked to any of the following substituents:


The pharmaceutical composition according to any one of claims 1-5, wherein the polypeptide or a pharmaceutically acceptable salt thereof has agonistic activity against at least one of the following receptors: GLP-1 receptor, GIP receptor, or GCG receptor; preferably, the polypeptide or a pharmaceutically acceptable salt thereof is a triple agonist of GLP-1 receptor, GIP receptor, and GCG receptor. The pharmaceutical composition according to any one of claims 1-6, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises the following sequence: H-Aib-HGTFTSDYSILLEKKAAQEFVEWLLAGGPSSGAPPPS(SEQ ID NO:1) H-Aib-HGTFTSDYSIYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:2) H-Aib-HGTFTSDYSIYLEKQAAQEFVQWLLEGGPSSGAPPPS(SEQ ID NO:3) H-Aib-HGTFTSDYSIYLEKQYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:4) H-Aib-HGTFTSDYSYYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:5) H-Aib-HGTFTSDYSIYLEKQAAEEFVQWLLEGGPSSGAPPPS(SEQ ID NO:6) H-Aib-HGTFTSDYSIYLDKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:7) H-Aib-HGTFTSDYSYYLEKQYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:8) H-Aib-HGTFTSDYSIYLEKKYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:9) H-Aib-HGTFTSDYSYYLEKKAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:10) H-Aib-HGTFTSDYSIYLEKKAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:11) H-Aib-HGTFTSDYSIYLEKQKA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:12) H-Aib-HGTFTSDYSKYLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:13) H-Aib-HGTFTSDYSIYLEKRAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:14) H-Aib-HGTFTSDYSILLEKKYA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:15) H-Aib-HGTFTSDYSYLLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:16) H-Aib-HGTFTSDYSILLEKQAA-Aib-EFVQWLLEGGPSSGAPPPS(SEQ ID NO:17) H-Aib-HGTFTSDYSILLEKQAAEEFVQWLLEGGPSSGAPPPS(SEQ ID NO:18) H-Aib-HGTFTSDYSILLEKQAAQEFVQWLLEGGPSSGAPPPS(SEQ ID NO:19) H-Aib-HGTFTSDYSILLEKQAAREFVQWLLEGGPSSGAPPPS(SEQ ID NO:20) H-Aib-HGTFTSDYSILLEKQAAHEFVQWLLEGGPSSGAPPPS(SEQ ID NO:21) H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKAGGHPS-Aib-KPPPK(SEQ ID NO:22) H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKA-dAla-GHPS-Aib-KPPPK(SEQ ID NO:23) H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKAGGPPS-Aib-KPPPK(SEQ ID NO:24) H-Aib-HGTFTSDLSKLKEEQRQKEFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:25) H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKAGGPPS-Aib-KPPPK(SEQ ID NO:26) H-Aib-HGTFTSDLSKLKEEQRQ-dAla-EFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:27) H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKAGGHPS-Aib-KPPPK(SEQ ID NO:28) H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKA-dAla-GHPS-Aib-KPPPK(SEQ ID NO:29) H-Aib-HGTFTSDLSKLKEEQRQ-Aib-EFIEWLKA-dAla-GPPS-Aib-KPPPK(SEQ ID NO:30) Preferably, the polypeptide or its pharmaceutically acceptable salt has a C-terminal carboxylic acid group or an amide group, more preferably a C-terminal amide group. The pharmaceutical composition according to any one of claims 1-7, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises any of the following structures: Compound 1,
Compound 2,
Compound 3,
Compound 4,
Compound 5,
Compound 6,
Compound 7,
Compound 8,
Compound 9,
Compound 10,
Compound 11,
Compound 12,
Compound 13,
Compound 14,
Compound 15,
Compound 16,
Compound 17,
Compound 18,
Compound 19,
Compound 20,
Compound 21,
Compound 22,
Compound 23,
Compound 24,
Compound 25,
Compound 26,
Compound 27,
Compound 28,
Compound 29,
Compound 30,
Compound 31,
Compound 32,
Compound 33,
Compound 34,
Compound 35,
Compound 36,
Compound 37,
Compound 38,
Compound 39,
Compound 40,

Compound 41,
Compound 42,
Compound 43,
Compound 44,
Compound 45,
Compound 46,
Compound 47,
Compound 48,
Compound 49,
Compound 50,
Compound 51,
Compound 52,
Compound 53,
The pharmaceutical composition according to any one of claims 1-8, wherein the divalent metal ion is selected from divalent zinc ions, divalent calcium ions, divalent magnesium ions, and divalent iron ions; Preferably, the metal ion is selected from divalent zinc ions; More preferably, the metal ion is selected from zinc acetate or zinc chloride; More preferably, the metal ion is zinc acetate. The pharmaceutical composition according to any one of claims 1-9, wherein the molar ratio of the polypeptide or its pharmaceutically acceptable salt to the divalent metal ion is 1:5 to 5:1; The preferred molar ratio of polypeptide or its pharmaceutically acceptable salt to divalent metal ions is 1:3 to 3:1; The preferred molar ratio of polypeptide or its pharmaceutically acceptable salt to divalent metal ions is 1:2 to 2:1; The preferred molar ratio of the polypeptide or its pharmaceutically acceptable salt to the divalent metal ion is 1:1. The pharmaceutical composition according to any one of claims 1-11, wherein the concentration of the polypeptide or its pharmaceutically acceptable salt is from 0.1 mg/mL to 500 mg/mL; Preferably, the concentration of the polypeptide or its pharmaceutically acceptable salt is from 0.1 mg/mL to 200 mg/mL; More preferably, 1.0 mg/mL to 100 mg/mL. The pharmaceutical composition according to any one of claims 1-12, wherein the concentration of the divalent zinc ions is from 0.01 mg/mL to 50 mg/mL. Preferably, the concentration of the divalent zinc ions is from 0.01 mg/mL to 30 mg/mL. More preferably, the concentration of the divalent zinc ions is from 0.01 mg/mL to 10 mg/mL. The pharmaceutical composition according to any one of claims 1-12, wherein the pharmaceutical composition further comprises a buffer; Preferably, the buffer is selected from one or more of the following: acetate buffer, histidine buffer, phosphate buffer, succinate buffer, tris(hydroxymethyl)aminomethane buffer (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), 3-morpholine propanesulfonic acid buffer (MOPS), diethanolamine buffer, borate buffer, tris(hydroxymethyl)methylglycine buffer (Tricine), and citrate buffer. More preferably, the buffer is selected from tris(hydroxymethyl)aminomethane buffer, disodium hydrogen phosphate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer. The pharmaceutical composition according to claim 13, wherein the concentration of the buffer in the pharmaceutical composition is from 0.5 mM to 50.0 mM; The preferred concentration of the buffer is from 0.5 mM to 35.0 mM; The preferred concentration of the buffer is from 0.5 mM to 25.0 mM; The preferred concentration of the buffer is 2 mM to 10.0 mM. The pharmaceutical composition according to any one of claims 1-14, wherein the pharmaceutical composition further comprises an osmotic pressure regulator; Preferably, the osmotic pressure regulator is selected from one or more of propylene glycol, mannitol, sorbitol, xylitol, glycerol, lactose, trehalose, sucrose, glucose, sodium chloride, phosphate, sodium citrate, boric acid, and sodium tartrate; More preferably, the osmosis conditioner is sodium chloride, propylene glycol, mannitol, or glycerin; More preferably, the osmotic pressure regulator is propylene glycol or mannitol. The pharmaceutical composition according to claim 15, wherein the concentration of propylene glycol is from 10 mg/mL to 20 mg/mL, preferably from 12 mg/mL to 16 mg/mL, more preferably 14 mg/mL; Alternatively, the concentration of mannitol in the pharmaceutical composition may be from 30 mg/mL to 70 mg/mL, preferably from 30 mg/mL to 50 mg/mL, and more preferably 46 mg/mL; Alternatively, the concentration of sodium chloride in the pharmaceutical composition may be from 2 mg/mL to 18 mg/mL, preferably from 7 mg/mL to 10 mg/mL, and more preferably 9 mg/mL. The pharmaceutical composition according to any one of claims 1-16, wherein the pharmaceutical composition further comprises a pH adjuster; preferably the pH adjuster is selected from sodium hydroxide and/or hydrochloric acid. The pharmaceutical composition according to any one of claims 1-17, wherein the pH of the pharmaceutical composition is 6.5 to 9.0, preferably 7.0 to 8.0; more preferably 7.1 to 7.7; and most preferably 7.5 or 7.4. The pharmaceutical composition according to any one of claims 1-18, wherein the pharmaceutical composition is selected from: 1) The polypeptide or its pharmaceutically acceptable salt as described in any one of claims 1-9; 2) Divalent zinc ions, such as zinc acetate or zinc chloride; 3) Buffers, such as phosphate buffers, such as sodium dihydrogen phosphate; 4) Osmotic pressure regulators, such as sodium chloride, propylene glycol, mannitol, and glycerin. The pharmaceutical composition according to any one of claims 1-19, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable antibacterial agent; Preferred antibacterial agents are selected from phenol, o-cresol, m-cresol, p-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, benzyl alcohol, ethanol, chlorobutanol, as well as thimerosal, bromonitrile glycol, benzoic acid, imidazoline, chlorhexidine, sodium dehydroacetate, chlorocresol, ethylparaben, benzyl chloride, or mixtures thereof; More preferably, the antibacterial agent is selected from phenol. The pharmaceutical composition according to claim 22, wherein the concentration of the antibacterial agent in the pharmaceutical composition is 4.0 mg/mL to 7.0 mg/mL; The preferred concentration is 4.4 mg/mL to 6.8 mg/mL; More preferably, the concentration is 5.5 mg/mL to 6.6 mg/mL; The optimal concentration is 5.5 mg/mL. A method for preparing a pharmaceutical composition according to any one of claims 1-21, comprising the step of dissolving the polypeptide or a pharmaceutically acceptable salt thereof. A lyophilized formulation, wherein the lyophilized formulation, after reconstitution, can form the pharmaceutical composition according to any one of claims 1-21, or the lyophilized formulation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1-21. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized formulation of claim 23. An article comprising a container containing a pharmaceutical composition as described in any one of claims 1-21, a lyophilized formulation as described in claim 23, or a reconstituted solution as described in claim 24. Use of the pharmaceutical composition according to any one of claims 1-21, the lyophilized formulation according to claim 23, the reconstituted solution according to claim 24, or the article according to claim 25 in the preparation of a medicament for treating non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, obesity, non-alcoholic fatty liver disease, hepatic steatosis, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes mellitus.