Pharmaceutical composition of long-acting GLP-1 / GIP dual agonist

Abstract

A pharmaceutical composition comprising a long-acting GLP-1 / GIP dual agonist, a kit comprising the pharmaceutical composition, and use of the pharmaceutical composition in the preparation of a drug for treating diabetes or obesity.

Description

Pharmaceutical composition of long-acting GLP-1 / GIP dual agonist Technical Field

[0001] The present invention belongs to the field of polypeptide drug preparations. Specifically, the present invention provides a pharmaceutical composition comprising a long-acting GLP-1 / GIP dual agonist, a kit comprising the pharmaceutical composition, and the use of the pharmaceutical composition for preparing a drug for treating diabetes or obesity. Background Art

[0002] Glucagon-like peptide (GLP-1) is a polypeptide hormone secreted by the intestine in response to food. GLP-1 stimulates insulin secretion and reduces glucagon secretion in a glucose-dependent manner. The glucose-dependent insulinotropic hormone (GIP) is currently believed to be secreted primarily by enteroendocrine K cells in the duodenum and upper jejunum. Similar to GLP-1, GIP can stimulate insulin secretion. However, native GLP-1 and GIP have very short half-lives in humans and animals, only a few minutes.

[0003] Drug developers have developed a series of GLP-1R / GIPR dual agonists designed to exert the same biological effects as native GLP-1 and GIP, while also prolonging their duration of action. Peptide GLP-1 and GIP receptor agonists are commonly administered via injection or parenteral routes. However, peptides are susceptible to undesirable product properties such as chemical degradation, aggregation, and physical aggregation, depending on their amino acid sequence, net charge, secondary structure, and chemical modifications, as well as the media environment in which they are placed, such as excipients, pH, and temperature. Adverse media conditions such as buffers, osmotic pressure regulators, and pH, as well as environmental factors such as temperature and light, can affect or even destroy the stability of peptide compounds. This can lead to adverse chemical reactions such as peptide isomerization, deamidation, amide bond cleavage, and aggregation, as well as physical aggregation between peptide molecules. Furthermore, increasing peptide concentration exacerbates the tendency for peptides to physically aggregate.

[0004] Therefore, there is still a need to develop alternative pharmaceutical compositions containing GLP-1 receptor agonists or GIP receptor agonists, in the hope that the pharmaceutical compositions can exert good and lasting therapeutic effects in lowering blood sugar or reducing weight, and maintain stability during production, storage, and use, and can withstand certain abnormal temperatures without deterioration, thereby increasing the safe and effective use of users in different scenarios. Summary of the Invention

[0005] In one aspect of the present invention, there is provided a compound of the following formula I, or a pharmaceutically acceptable salt thereof, or a physiologically acceptable salt thereof, or a precursor compound thereof:

[0006] Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK Formula I (SEQ ID NO: 12);

[0007] in,

[0008] One of the K positions at positions 16, 24, 28, and 40 is selected by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by conjugation to the ε-amino group of the K side chain, wherein each a is independently selected from an integer of 1, 2, or 3, each b is independently selected from an integer of 1, 2, or 3, and each c is independently selected from an integer of 16, 18, or 20, wherein Z is independently selected from -CH3, a carboxylic acid or a carboxylic acid bioisostere, a phosphonate or a sulfonate, preferably -COOH; and the C-terminal amino acid is amidated to a C-terminal primary amide.

[0009] In one aspect of the present invention, a pharmaceutical composition is provided, comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a physiologically acceptable salt thereof, or a precursor compound thereof.

[0010] In one aspect of the present invention, there is provided a use of the pharmaceutical composition according to the present invention for preparing a medicament for treating a disease, such as diabetes or obesity and other related diseases.

[0011] In one aspect of the present invention, there is provided a pharmaceutical composition according to the present invention for use in treating a disease, such as diabetes or obesity.

[0012] In one aspect of the present invention, a method for preventing or treating a disease is provided, comprising administering an effective amount of the pharmaceutical composition according to the present invention to a human or animal in need thereof, wherein the disease is, for example, diabetes or obesity.

[0013] The compound of Formula I of the present invention has a dual agonist effect on both the GLP-1 receptor and the GIP receptor, and can be used as a GLP-1 / GIP dual agonist. It exhibits excellent efficacy in lowering blood sugar and / or reducing weight, and has a long half-life. Pharmaceutical compositions containing the compound of the present invention maintain excellent stability during storage at elevated temperatures, room temperature, and under refrigeration, as well as during use, particularly with respect to clarity, color, purity, polymer content, and pH, and are able to withstand certain abnormal temperatures without deterioration. DETAILED DESCRIPTION

[0014] Unless otherwise specified, in the context of the present invention, all scientific and technical terms should have the same meaning as known to those skilled in the art. In the event of any inconsistency, the definitions provided in the present invention shall prevail.

[0015] It should be understood that all detailed descriptions of materials, methods, and examples are for illustrative purposes only and are not to be construed as limiting the present invention unless otherwise expressly stated.

[0016] As used herein, the terms "comprising," "including," and "including" are synonymous and indicate that other ingredients or steps that do not affect the end result may be included. This term encompasses the terms "consisting of" and "consisting essentially of." Products and methods according to the present invention may include, consist of, or consist essentially of the basic technical features and / or limitations of the present invention described herein, as well as any additional and / or optional ingredients, components, steps, or limitations described herein.

[0017] Unless otherwise stated, in the context of the present invention each numerical range is meant to include both endpoints and any number and sub-range within the numerical range.

[0018] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for a particular value determined by one of ordinary skill in the art, which value depends, to some extent, on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" or "substantially comprising" can mean a floating range of up to ±20%. For example, a pH value of about 5.5 means a pH value of 5.5 ± 1.1. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0019] Unless otherwise specified, all materials and reagents used in the present invention are commercially available.

[0020] [Pharmaceutical composition and preparation method thereof]

[0021] The present invention discloses a pharmaceutical composition comprising a compound of the following formula I, or a pharmaceutically acceptable salt thereof, or a physiologically acceptable salt thereof, or a precursor compound thereof:

[0022] Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK Formula I (SEQ ID NO: 12);

[0023] in,

[0024] One of the K positions at positions 16, 24, 28, and 40 is optionally replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by conjugation to the ε-amino group of the K side chain, wherein each a is independently selected from an integer of 1, 2, or 3, each b is independently selected from an integer of 1, 2, or 3, and each c is independently selected from an integer of 16, 18, or 20, wherein Z is independently selected from -CH3, a carboxylic acid or a carboxylic acid bioisostere, a phosphonate or a sulfonate, preferably -COOH; and the C-terminal amino acid is amidated to a C-terminal primary amide.

[0025] The compounds of formula I of the present invention have the same polypeptide backbone: Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK, thus having the physicochemical stability characteristics associated with a common polypeptide backbone, including the isoelectric point and solubility characteristics of the polypeptide backbone, the chemical reactivity of specific amino acid residue functional groups and their amide bonds in a medium environment. In the compound of formula I of the present invention, the polypeptide backbone is substituted by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is used to chemically modify the side chain, giving the polypeptide main chain charge, hydrophilicity and hydrophobicity, but retaining the common characteristics of the polypeptide main chain itself.

[0026] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more polypeptide compounds of Formula I of the present invention, or pharmaceutically acceptable salts thereof, or physiologically acceptable salts thereof, or precursor compounds thereof, and optionally other components. The other components include physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating the absorption of the polypeptide compound of Formula I of the present invention as a pharmaceutically active ingredient, thereby exerting its biological activity.

[0027] In the context of the present invention, unless otherwise specified or clearly contradictory, the terms "pharmaceutical composition", "composition", and "formulation" are used interchangeably.

[0028] The compound of Formula I of the present invention has a dual agonist effect on both the GLP-1 receptor and the GIP receptor, and can be used as a GLP-1 / GIP dual agonist. It exhibits excellent efficacy in lowering blood sugar and / or reducing weight, and has a long half-life. Pharmaceutical compositions containing the compound of Formula I maintain excellent stability during storage at elevated temperatures, room temperature, and under refrigeration, as well as during use, particularly with respect to clarity, color, purity, polymer content, and pH, and are able to withstand certain abnormal temperatures without deterioration.

[0029] In some embodiments, the pharmaceutical composition is in the form of an aqueous solution.

[0030] In some embodiments, the pharmaceutical composition is injectable.

[0031] In some embodiments, the pharmaceutical composition is a purified liquid formulation.

[0032] In some embodiments, the purified liquid formulations include subcutaneous injection formulations, intravenous injection formulations, intraperitoneal administration formulations, intramuscular injection formulations, intravenous / subcutaneous injection formulations, and intravitreal injection formulations.

[0033] In some embodiments, the purified liquid preparation includes an aqueous injection preparation and a prefilled injection preparation, preferably an aqueous injection preparation.

[0034] In some embodiments, the purified liquid preparation includes an aqueous injection preparation and a prefilled injection preparation. The container of the prefilled injection preparation can be a vial, a cartridge, or a prefilled syringe.

[0035] In some embodiments, the pharmaceutical composition is a subcutaneous injection preparation.

[0036] In some embodiments, the pharmaceutical composition comprises a polypeptide compound of Formula I, a buffer, an osmotic pressure regulator, water, and optionally a pH regulator and other excipients and / or carriers. The other excipients and / or carriers include one or more of the following: preservatives, stabilizers, and antioxidants.

[0037] In some embodiments, the water is preferably purified water, deionized water, or water for injection.

[0038] In some embodiments, the preservative includes phenol, m-cresol, cresol, phenoxyethanol, chlorobutanol, benzyl alcohol, benzoate derivatives, and any combination thereof; preferably, the preservative is phenol, m-cresol, benzyl alcohol, and any combination thereof.

[0039] In the context of the present invention, cresol includes m-cresol, o-cresol, p-cresol, or any combination thereof.

[0040] In the context of the present invention, there is no particular limitation on benzoate derivatives, and any benzoate derivative commonly used in the art can be used in the present invention. In some embodiments, the benzoate derivative can be a paraben, such as a C1-C10 ester of p-hydroxybenzoic acid, preferably a C1-C6 ester of p-hydroxybenzoic acid, and more preferably a C1-C4 ester of p-hydroxybenzoic acid, such as methyl paraben, ethyl paraben, propyl paraben, t-butyl paraben, and the like.

[0041] In some embodiments, the method for preparing the pharmaceutical composition comprises:

[0042] providing a buffer, an osmotic pressure regulator, and optionally other excipients and / or carriers to obtain a mixture,

[0043] mixing the mixture with water to obtain an aqueous mixture,

[0044] Adding the polypeptide compound of formula I or an aqueous solution of the polypeptide compound of formula I to the aqueous mixture to obtain a mixture containing the polypeptide compound of formula I,

[0045] According to the difference between the pH value of the mixture and the target pH value, a pH regulator is optionally added to adjust the pH value of the mixture to the target pH value, and

[0046] Add additional water to bring to volume.

[0047] Optional addition of a pH adjuster refers to testing the pH of the mixture containing the polypeptide compound of Formula I. If the pH test value deviates from the target pH value, a pH adjuster is added to adjust the pH of the polypeptide-containing aqueous mixture to the target pH value. If the pH test value is the target pH value, no pH adjuster is added.

[0048] Those skilled in the art will appreciate that the water added last is used to adjust the volume, so there is no need to limit the amount of water used.

[0049] In some embodiments, the pharmaceutical composition comprises a polypeptide compound of Formula I, a buffer, an osmotic pressure regulator, water, and optionally a pH regulator.

[0050] In some embodiments, the pharmaceutical composition contains:

[0051] A polypeptide compound of formula I,

[0052] pH regulator, wherein the pH regulator is selected from aqueous sodium hydroxide solution or hydrochloric acid,

[0053] a buffer selected from disodium hydrogen phosphate, tromethamine, trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof;

[0054] an osmotic pressure regulator selected from mannitol, sodium chloride, propylene glycol, glycerol, and combinations thereof,

[0055] a preservative selected from phenol, m-cresol, cresol, phenoxyethanol, chlorobutanol, benzyl alcohol, benzoate derivatives, and any combination thereof, and

[0056] water.

[0057] In some embodiments, the pharmaceutical composition according to the present invention is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition comprises:

[0058] about 0.1 to about 50 mg / mL of a compound of formula I, for example, about 1 to about 40 mg / mL of compound P016 or P014 or P020,

[0059] The total molar concentration is about 0.5 to about 50 mM citric acid and trisodium citrate, preferably about 1.0 to about 10 mM citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4-50, preferably about 1:4-20.

[0060] about 0 to about 50 mg / mL, preferably about 0.1 to about 50 mg / mL, more preferably about 1 to about 10 mg / mL, and even more preferably about 2 to about 6 mg / mL of a preservative,

[0061] about 1 to about 100 mg / mL of an osmotic pressure regulator, preferably about 1 to about 100 mg / mL of mannitol, 1,2-propylene glycol, 1,3-propylene glycol, glycerol, sodium chloride, or any combination thereof,

[0062] optionally aqueous sodium hydroxide or hydrochloric acid,

[0063] water,

[0064] The pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to about 8.8, and more preferably about 6.5 to about 7.4.

[0065] In some embodiments, the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition contains:

[0066] about 0.1 to about 50 mg / mL, e.g., about 0.1 to about 40 mg / mL of a polypeptide compound of Formula I,

[0067] The total molar concentration of citric acid and trisodium citrate is about 0.5 to about 50 mM, preferably about 1.0 to about 10 mM citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4-50, preferably about 1:4-20,

[0068] about 0.1 to about 50 mg / mL, preferably about 1 to about 10 mg / mL, more preferably about 2 to about 6 mg / mL of a preservative,

[0069] about 1 to about 100 mg / mL of mannitol,

[0070] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0071] water,

[0072] The pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to 8.8, and more preferably about 6.5 to about 7.4.

[0073] In some embodiments, the pharmaceutical composition contains:

[0074] A polypeptide compound of formula I,

[0075] pH regulator, wherein the pH regulator is selected from aqueous sodium hydroxide solution or hydrochloric acid,

[0076] a buffer selected from disodium hydrogen phosphate, tromethamine, trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof;

[0077] an osmotic pressure regulator selected from the group consisting of mannitol, sodium chloride, propylene glycol, glycerol, and combinations thereof, and

[0078] water.

[0079] In some embodiments, the osmotic pressure regulating agent is mannitol and the pharmaceutical composition is free of preservatives.

[0080] In some embodiments, the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition contains:

[0081] about 0.1 to about 50 mg / mL, e.g., about 0.1 to about 40 mg / mL of a polypeptide compound of Formula I,

[0082] The total molar concentration of citric acid and trisodium citrate is about 0.5-about 50 mM, preferably about 1.0-10 mM citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4-50, preferably about 1:4-20,

[0083] about 1 to about 100 mg / mL of mannitol,

[0084] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0085] water,

[0086] The pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to 8.8, and more preferably about 6.5 to about 7.4.

[0087] In some embodiments, the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition comprises:

[0088] about 0.1 to about 50 mg / mL of a compound of formula I, for example, about 1 to about 40 mg / mL of compound P016 or P014 or P020,

[0089] The total molar concentration of citric acid and trisodium citrate is about 0.5-about 50 mM, preferably about 1.0-10 mM citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4-50, preferably about 1:4-20,

[0090] about 1 to about 100 mg / mL of mannitol,

[0091] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0092] water,

[0093] The pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to about 8.8, and more preferably about 6.5 to about 7.4.

[0094] In some embodiments, the content or concentration of the polypeptide compound of Formula I is about 0.1-about 50 mg / mL, for example, about 0.1-about 40 mg / mL, about 0.2 mg / mL-about 40 mg / mL, about 0.3 mg / mL-about 40 mg / mL, about 0.4 mg / mL-about 40 mg / mL, about 0.5 mg / mL-about 40 mg / mL, about 0.6 mg / mL-about 40 mg / mL, about 0.7 mg / mL-about 40 mg / mL, about 0.8 mg / mL-about 40 mg / mL, about 0.9 mg / mL-about 40 mg / mL, about 1-about 40 mg / mL, about 1.2-about 40 mg / mL, about 1.5-about 40 mg / mL, about 2.0-about 40 mg / mL, about 2.4-about 40 mg / mL, about 3.0-about 40 mg / mL, about 4.0-about 40 mg / mL, about 4.8 to about 40 mg / mL, about 5.0 to about 40 mg / mL, about 1 to about 30 mg / mL, about 1.2 to about 30 mg / mL, about 1.5 to about 30 mg / mL, about 2.0 to about 30 mg / mL, about 2.4 to about 30 mg / mL, about 3.0 to about 30 mg / mL, about 4.0 to about 30 mg / mL, about 4.8 to about 30 mg / mL, about 5.0 to about 30 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 24 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.1 to about 10 mg / mL, about 1 to about 30 mg / mL, about 1 to about 24 mg / mL, about 1 to about 20 mg / mL, about 1 to about 10 mg / mL, or about 20 to about 30 mg / mL.

[0095] In some embodiments, the pH value of the aqueous solution of the pharmaceutical composition is about 5.0 to about 9.0, for example, about 5.8 to about 9.0, preferably about 6.0 to about 9.0, for example, about 6.0 to about 8.8, about 6.1 to about 9.0, about 6.1 to about 8.8, about 6.2 to about 9.0, about 6.2 to about 8.8, about 6.3 to about 9.0, about 6.4 to about 9.0, about 6.5 to about 9.0, about 6.5 to about 8.9, about 6.5 to about 8.8, about 6.5 to about 8.7, about 6.5 to about 8.6, about 6.5 to about 8.5, about 6.5 to about 8.4, about 6.5 to about 8.3, about 6.6 to about 8.7. from about 6.5 to about 8.2, from about 6.5 to about 8.1, from about 6.5 to about 8.0, from about 6.5 to about 7.9, from about 6.5 to about 7.8, from about 6.5 to about 7.7, from about 6.5 to about 7.6, from about 6.5 to about 7.5, from about 6.5 to about 7.4, from about 6.6 to about 7.4, from about 6.7 to about 7.4, from about 6.8 to about 7.4, more preferably from about 6.5 to about 8.5, from about 6.5 to about 8.0, from about 6.3 to about 7.5, from about 6.5 to about 7.5, from about 6.8 to about 7.5, from about 6.8 to about 7.4, from about 7.1 to about 7.5, from about 7.1 to about 7.7, and from about 7.2 to about 7.5. In some embodiments, the pH of the aqueous solution of the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5, e.g., about 7.5 or about 7.4.

[0096] The pharmaceutical composition of the present invention exhibits stability in the following parameters after storage for a period of time at elevated temperature, room temperature, and / or low temperature: clarity, color, pH, API purity, polymer content, and biological activity. After storage for a period of time at elevated temperature, room temperature, and / or low temperature, no significant changes in properties, pH, osmolarity, insoluble particulates, active pharmaceutical ingredient, polymer impurities, physical aggregates, precipitation, and / or denaturation of the pharmaceutical composition are observed when tested for color and / or clarity, or when tested using a pH meter, osmotic pressure meter, particle counter, HPLC-UV, or size exclusion chromatography (SEC).

[0097] Furthermore, the polypeptide pharmaceutical composition of the present invention exhibits acceptable stability for various indicators, including appearance, concentration, clarity, pH, osmotic pressure, purity of the active pharmaceutical ingredient (API), polymer impurity content, and insoluble particulate matter, for up to one month at 40°C or three to six months at 25°C. These indicators meet the current quality standards for GLP-1 / GIP dual agonist drugs and exhibit good stability even when stored at high temperatures.

[0098] A stable pharmaceutical formulation is one in which no significant change is observed when stored at refrigerated temperature (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, and up to 3 years. Stable liquid formulations include liquid formulations that exhibit the desired quality profile after being stored at 25°C for a period of 1 month, 3 months, 6 months, or 1 month at 40°C. Typical acceptance criteria for stability are as follows: the polymer content does not exceed about 3% as measured by SEC-HPLC, and the content of the formulation or the total amount of related substances (total impurities) does not exceed about 10% change as measured by HPLC-UV. Visual inspection shows that the pharmaceutical formulation is a colorless to light yellow clear or almost clear liquid, and visible foreign matter and insoluble particles meet the pharmacopoeial requirements for safety of parenteral administration, and the biological activity is within the acceptable range of 60-140%.

[0099] In some embodiments, the pharmaceutical composition is stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months when stored at about 2 to about 8° C. In some embodiments, the pharmaceutical composition is stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months when stored at about 25° C. In some embodiments, the pharmaceutical composition is stable for at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months when stored at about 40° C.

[0100] [Peptide compounds]

[0101] According to the present invention, the active pharmaceutical ingredient in the pharmaceutical composition is a compound of the following formula I, or a pharmaceutically acceptable salt thereof, or a physiologically acceptable salt thereof, or a precursor compound thereof:

[0102] Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK formula I (SEQ ID NO: 12);

[0103] in,

[0104] One of the K positions at positions 16, 24, 28, and 40 is optionally replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c-Z is chemically modified by conjugation to the ε-amino group of the K side chain, wherein each a is independently selected from an integer of 1, 2, or 3, each b is independently selected from an integer of 1, 2, or 3, and each c is independently selected from an integer of 16, 18, or 20, wherein Z is independently selected from -CH3, a carboxylic acid or a carboxylic acid bioisostere, a phosphonate or a sulfonate, preferably -COOH; and the C-terminal amino acid is amidated to a C-terminal primary amide.

[0105] In the context of the present invention, the term "active pharmaceutical ingredient" (API) is a polypeptide compound of formula I, which is a GLP-1 / GIP dual agonist.

[0106] In some embodiments, the API refers to the polypeptide compounds P007, P008, P014, P019, P013, P015, P016, P017, P018, P020, and combinations thereof of the present invention.

[0107] In some embodiments, the API refers to the polypeptide compound P016 of the present invention.

[0108] In the context of the present invention, unless otherwise specified or clearly contradictory, the terms "polypeptide compound" and "polypeptide", "pharmaceutical active ingredient" and "GLP-1 / GIP dual agonist" are used interchangeably.

[0109] In some embodiments, in the compound of Formula I,

[0110] At position 24, K is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is conjugated to the ε-amino group of the K side chain for chemical modification; or

[0111] At position 28, K is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification.

[0112] In some embodiments, in the compound of formula I, at position 24, K is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c-CO2H is chemically modified by conjugating to the ε-amino group of the K side chain, wherein each a is independently selected from an integer of 2 or 3, each b is independently 1, and each c is independently selected from an integer of 16, 18, or 20, wherein Z is independently -COOH.

[0113] In some embodiments, in the compound of formula I, at position 28, K is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by conjugating to the ε-amino group of the K side chain, wherein each a is independently selected from an integer of 2 or 3, each b is independently selected from an integer of 1 or 3, each c is independently selected from an integer of 16 or 20, and Z is independently -COOH.

[0114] In some embodiments, the compound of Formula I is one or more compounds selected from:

[0115] Compound P007 (SEQ ID NO: 2): K at position 24 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0116] Compound P013 (SEQ ID NO: 4): K at position 24 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 18 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0117] Compound P014 (SEQ ID NO: 5): K at position 24 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0118] Compound P015 (SEQ ID NO: 6): K at position 24 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16-CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0119] Compound P016 (SEQ ID NO: 7): K at position 24 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0120] Compound P008 (SEQ ID NO: 3): K at position 28 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0121] Compound P017 (SEQ ID NO: 8): K at position 28 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0122] Compound P018 (SEQ ID NO: 9): K at position 28 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0123] Compound P019 (SEQ ID NO: 10): K at position 28 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0124] and

[0125] Compound P020 (SEQ ID NO: 11): K at position 28 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H is conjugated to the ε-amino group of the K side chain and chemically modified; and the C-terminal amino acid is amidated to form a C-terminal primary amide, with the following structure:

[0126] In some embodiments, the compound of Formula I is compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 12), molecular formula C 243 H 382 N 52 O 73 , the structure is as follows:

[0127] In some embodiments, the concentration of the polypeptide compound of Formula I in the pharmaceutical composition is about 0.1 to about 50 mg / mL, for example, about 0.1 to about 40 mg / mL, about 0.2 mg / mL to about 40 mg / mL, about 0.3 mg / mL to about 40 mg / mL, about 0.4 mg / mL to about 40 mg / mL, about 0.5 mg / mL to about 40 mg / mL, about 0.6 mg / mL to about 40 mg / mL, about 0.7 mg / mL to about 40 mg / mL, about 0.8 mg / mL to about 40 mg / mL, about 0.9 mg / mL to about 40 mg / mL, about 1 to about 40 mg / mL, about 1.2 to about 40 mg / mL, about 1.5 to about 40 mg / mL, about 2.0 to about 40 mg / mL, about 2.4 to about 40 mg / mL, about 3.0 to about 40 mg / mL, about 4. 0-about 40 mg / mL, about 4.8-about 40 mg / mL, about 5.0-about 40 mg / mL, about 1-about 30 mg / mL, about 1.2-about 30 mg / mL, about 1.5-about 30 mg / mL, about 2.0-about 30 mg / mL, about 2.4-about 30 mg / mL, about 3.0-about 30 mg / mL, about 4.0-about 30 mg / mL, about 4.8-about 30 mg / mL , about 5.0 to about 30 mg / mL, about 0.1 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 24 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.1 to about 10 mg / mL, about 1 to about 30 mg / mL, about 1 to about 24 mg / mL, about 1 to about 20 mg / mL, about 1 to about 10 mg / mL, or about 20 to about 30 mg / mL.

[0128] In some embodiments, the concentration of the polypeptide compound of Formula I in the pharmaceutical composition is at least about 0.1 mg / mL, at least about 1 mg / mL, at least about 10 mg / mL, more preferably at least about 20 mg / mL, even more preferably at least about 30 mg / mL, and even more preferably at least about 50 mg / mL.

[0129] In some embodiments, the concentration of the polypeptide compound of Formula I is about 0.1 to about 50 mg / mL, for example, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 1.2 mg / mL, about 2 mg / mL, about 2.4 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / About 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, or about 30 mg / mL.

[0130] [pH adjusters and buffers]

[0131] In some embodiments, the pharmaceutical composition comprises a pH adjusting agent.

[0132] In some embodiments, the pharmaceutical composition does not contain a pH adjusting agent.

[0133] In some embodiments, the pharmaceutical composition comprises a buffer.

[0134] In some embodiments, during the preparation of the pharmaceutical composition, after the active ingredient and the excipients other than the pH adjuster are mixed, the pH value of the mixture is detected. If the pH value detected is the target pH value or close to the target pH value, no additional pH adjuster is needed. If the pH value detected deviates from the target pH value, a small amount of pH adjuster is slowly added until the pH value reaches the target value. Even in the case of using a pH adjuster, those skilled in the art understand that there is no need to specifically limit the amount of the pH adjuster, because the amount of pH adjuster added is very small and can be adjusted according to the target pH value.

[0135] In the context of the present invention, both pH adjusters and buffers are used to adjust the pH value of a pharmaceutical composition. For the sake of distinction, in the context of the present invention, "buffer" specifically refers to a substance that has the ability to maintain a relatively stable pH value through acid-base conjugation, and "pH adjuster" specifically refers to a substance other than "buffer" that has a pH-adjusting effect.

[0136] Buffers and pH regulators commonly used in medicines can be used in the present invention as long as they do not affect the therapeutic effect and stability of the pharmaceutical composition of the present invention.

[0137] In some embodiments, the buffering agent comprises an inorganic acid, an organic acid, an inorganic base, an organic base, a combination of an inorganic acid and a salt thereof, a combination of an organic acid and a salt thereof, a combination of an inorganic base and a salt thereof, and a combination of an organic base and a salt thereof.

[0138] In the context of the present invention, acids, bases and salts may be in the form of themselves or of their hydrates.

[0139] In some embodiments, the salt of an inorganic acid is composed of an inorganic acid radical ion and an alkali metal ion or an ammonium ion, preferably sodium or potassium.

[0140] In some embodiments, the salt of the organic acid is composed of an organic acid radical ion and an alkali metal ion or an ammonium ion, preferably sodium or potassium.

[0141] In some embodiments, the buffer comprises acetic acid, succinic acid, citric acid, phosphoric acid, gluconic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), tromethamine (Tris), glycine, histidine, glycylglycine, glutamic acid, acetate, succinate, citrate, gluconate, histidine, oxalate, lactate, tartrate, fumarate, phosphate, citric acid-citrate, hydrates thereof, and any combination thereof.

[0142] In the context of the present invention, the same substance can be classified as different components because it has different functions at the same time. For example, glycine can act as a buffer, an osmotic pressure regulator, and a stabilizer.

[0143] In some embodiments, the buffer is selected from one or more of glycylglycine, histidine, glutamic acid, tromethamine (Tris), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), citric acid, citrate, phosphate, hydrates thereof, and any combination thereof.

[0144] In some embodiments, the buffer is selected from one or more of: disodium hydrogen phosphate, tromethamine, trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof.

[0145] In some embodiments, the molar concentration of the buffer in the pharmaceutical composition is about 0.5 to about 50 mM, for example, about 0.5 mM to about 40.0 mM, about 0.5 mM to about 30.0 mM, about 0.5 mM to about 20.0 mM, about 0.5 mM to about 10.0 mM, about 1.0 mM to about 40.0 mM, about 1.0 mM to about 35.0 mM, about 1.0 mM to about 30.0 mM, about 1.0 mM to about 25.0 mM, about 1.0 mM to about 20.0 mM, about 1.0 mM to about 15.0 mM, about 1.0 mM to about 10.0 mM, about 1.0 mM to about 40.0 mM, about 1.0 mM to about 35.0 mM, about 1.0 mM to about 30.0 mM, about 1.0 mM to about 25.0 mM, about 1.0 mM to about 20.0 mM, about 1.0 mM to about 15.0 mM, about 1.0 mM to about 10.0 In some embodiments, the present invention provides a method for providing a soluble fiber containing at least one molecule of the present invention to be used in combination with other molecule(s) of the present invention to provide ...

[0146] In some embodiments, the molar concentration of the buffer in the pharmaceutical composition is about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11 mM, about 12.0 mM, about 15.0 mM, about 18.0 mM, or about 20.0 mM.

[0147] In some embodiments, the buffer is a phosphate, including one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid.

[0148] In some embodiments, the phosphate is selected from disodium hydrogen phosphate or its hydrate, sodium dihydrogen phosphate or its hydrate, dipotassium hydrogen phosphate or its hydrate, potassium dihydrogen phosphate or its hydrate.

[0149] In some embodiments, the phosphate is a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, or a phosphate buffer formed by a combination of other phosphoric acids or pharmaceutically acceptable salts or hydrates thereof.

[0150] In some embodiments, the phosphate is sodium hydrogen phosphate and / or sodium hydrogen phosphate hydrate. Its molar concentration in the pharmaceutical composition is about 0.5 to about 50 mM, for example, about 0.5 mM to about 40.0 mM, about 0.5 mM to about 30.0 mM, about 0.5 mM to about 20.0 mM, about 0.5 mM to about 10.0 mM, about 1.0 mM to about 40.0 mM, about 1.0 mM to about 35.0 mM, about 1.0 mM to about 30.0 mM, about 1.0 mM to about 25.0 mM, about 1.0 mM to about 20.0 mM, about 1.0 mM to about 15.0 mM, about 1.0 mM to about 10.0 mM, about 2. In some embodiments, the present invention provides a method for producing an aqueous solution of at least one amino acid sequence of the present invention. The aqueous solution of the present invention can be substituted with an amino acid sequence of at least one amino acid sequence of the present invention. In some embodiments, the aqueous solution of the present invention can be substituted with an amino acid sequence of at least one amino acid sequence of the present invention.

[0151] In some embodiments, the molar concentration of sodium phosphate dibasic and / or sodium phosphate dibasic hydrate in the pharmaceutical composition is about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11 mM, about 12.0 mM, about 15.0 mM, about 18.0 mM, or about 20.0 mM.

[0152] In some embodiments, the molar concentration of tromethamine in the pharmaceutical composition is about 0.5 to about 50 mM, for example, about 0.5 mM to about 40.0 mM, about 0.5 mM to about 30.0 mM, about 0.5 mM to about 20.0 mM, about 0.5 mM to about 10.0 mM, about 1.0 mM to about 40.0 mM, about 1.0 mM to about 35.0 mM, about 1.0 mM to about 30.0 mM, about 1.0 mM to about 25.0 mM, about 1.0 mM to about 20.0 mM, about 1.0 mM to about 15.0 mM, about 1.0 mM to about 10.0 mM. In some embodiments, the present invention provides a method for providing a soluble fiber containing at least one molecule of the present invention to be used in combination with other molecule(s) of the present invention to provide ...

[0153] In some embodiments, the molar concentration of tromethamine in the pharmaceutical composition is about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11 mM, about 12.0 mM, about 15.0 mM, about 18.0 mM, or about 20.0 mM.

[0154] In some embodiments, the buffer is a citrate, including trisodium citrate, tripotassium citrate, disodium citrate, calcium citrate, magnesium citrate, hydrates thereof, or any combination thereof.

[0155] In some embodiments, the buffer is citric acid-citrate salt, including citric acid-trisodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.

[0156] In the context of the present invention, "citric acid" and "citric acid" have the same meaning and can be used interchangeably. "Sodium citrate", "sodium citrate" and "trisodium citrate" have the same meaning and can be used interchangeably.

[0157] In some embodiments, the citric acid-citrate buffer is citric acid-trisodium citrate or a hydrate thereof, and its molar concentration in the pharmaceutical composition is about 0.5 to about 50 mM, based on the total molar amount of citric acid and trisodium citrate, for example, about 0.5 mM to about 40.0 mM, about 0.5 mM to about 30.0 mM, about 0.5 mM to about 20.0 mM, about 0.5 mM to about 10.0 mM, about 1.0 mM to about 40.0 mM, about 1.0 mM to about 35.0 mM, about 1.0 mM to about 30.0 mM, about 1.0 mM to about 25.0 mM, about 1.0 mM to about 20.0 mM, about 1.0 mM to about 30.0 mM. - about 15.0 mM, about 1.0 mM to about 10.0 mM, about 2.0 mM to 40.0 mM, about 2.0 mM to about 35.0 mM, about 2.0 mM to about 30.0 mM, about 2.0 mM to about 25.0 mM, about 2.0 mM to about 20.0 mM, about 2.0 mM to about 15.0 mM, about 2.0 mM to about 10.0 mM, about 5.0 mM to about 20.0 mM, about 5.0 mM to about 15.0 mM, about 5.0 mM to about 10.0 mM, about 6.0 to about 9.0 mM, about 6.0 to about 8.5 mM, about 6.0 to about 8.0 mM, or about 6.0 to about 7.5 mM.

[0158] In some embodiments, the molar concentration of trisodium citrate in the pharmaceutical composition is about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11 mM, about 12.0 mM, about 15.0 mM, about 18.0 mM, or about 20.0 mM.

[0159] In some embodiments, the total molar concentration of citric acid-sodium citrate or its hydrate in the pharmaceutical composition, based on the total molar amount of citric acid and trisodium citrate, is about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11 mM, about 12.0 mM, about 15.0 mM, about 18.0 mM, or about 20.0 mM.

[0160] In some embodiments, in the citric acid-trisodium citrate buffer, the molar ratio of citric acid to trisodium citrate is about 1:4-50, such as about 1:5-50, preferably about 1:6-50, such as about 1:1-20. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:18. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:16. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:14. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:12. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:10. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:9. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:8. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:7. In some embodiments, the molar ratio of citric acid to trisodium citrate is about 1:6.15.

[0161] In the same solution system, the molar ratio of citric acid to trisodium citrate is equivalent to the molar concentration ratio of citric acid to trisodium citrate.

[0162] In some of the embodiments, the total molar concentration of citric acid and trisodium citrate is about 7.5 mM, and the molar ratio of citric acid to trisodium citrate is about 1:6.15.

[0163] In some of the embodiments, the total molar concentration of citric acid and trisodium citrate is about 7.5 mM, and the molar ratio of citric acid to trisodium citrate is about 1:9.

[0164] In some of the embodiments, the total molar concentration of citric acid and trisodium citrate is about 5 mM, and the molar ratio of citric acid to trisodium citrate is about 1:9.

[0165] The pH adjuster includes one or more of citric acid, sodium hydroxide, and hydrochloric acid (ie, aqueous HCl solution); preferably, sodium hydroxide and / or hydrochloric acid.

[0166] In the context of the present invention, "aqueous HCl solution" and "hydrochloric acid" have the same meaning and can be used interchangeably.

[0167] In some embodiments, the pH adjuster is about 0.1 to about 0.5 M aqueous sodium hydroxide solution or aqueous HCl solution.

[0168] In some embodiments, the pH adjuster is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, or about 0.5 M sodium hydroxide in water.

[0169] In some embodiments, the pH adjuster is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, or about 0.5 M aqueous HCl solution.

[0170] In some embodiments, during the preparation of the pharmaceutical composition, it may be necessary to alternate between aqueous HCl and aqueous sodium hydroxide solutions to adjust the pH. In other words, the pH adjuster may be a combination of aqueous HCl and aqueous NaOH. In some embodiments, the pH adjuster may be a combination of an approximately 0.1- to approximately 0.5 M aqueous HCl solution and an approximately 0.1- to approximately 0.5 M aqueous NaOH solution.

[0171] [Osmotic pressure regulator]

[0172] In some embodiments, the pharmaceutical composition, preferably an aqueous solution of the pharmaceutical composition, contains an osmotic pressure regulating agent.

[0173] In the context of the present invention, osmotic pressure regulators and isotonic pressure regulators can be used interchangeably. Any osmotic pressure regulator commonly used in medicine can be used in the present invention, as long as it does not affect the therapeutic effect and stability of the pharmaceutical composition of the present invention.

[0174] In some embodiments, osmotic pressure regulators include, but are not limited to, salts (e.g., sodium chloride, phosphate, citrate, borate, and 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), polyols (e.g., glycerol, 1,2-propylene glycol (also known as propylene glycol), 1,3-propylene glycol, 1,3-butylene glycol), polyethylene glycol (e.g., PEG 400), or any combination thereof. Phosphates include one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and disodium hydrogen phosphate-potassium dihydrogen phosphate.

[0175] In some embodiments, the osmotic pressure regulator is selected from one or more of propylene glycol, mannitol, sorbitol, xylitol, glycerol, lactose, trehalose, sucrose, glucose, sodium chloride, phosphate, trisodium citrate, boric acid, and sodium tartrate.

[0176] In some embodiments, the osmotic pressure adjusting agent includes one or more of mannitol, sodium chloride, propylene glycol, and glycerol.

[0177] In some embodiments, the osmotic pressure of the pharmaceutical composition is about 200 to about 500 mOsm / kg, preferably about 250 to about 350 mOsm / kg, and more preferably about 260 to about 320 mOsm / kg. The osmotic pressure can be measured using an osmotic pressure meter.

[0178] In some embodiments, the osmotic pressure regulating agent is sodium chloride.

[0179] In some embodiments, the sodium chloride concentration is about 1-20 mg / mL, preferably about 5-20 mg / mL, more preferably about 6-15 mg / mL, and even more preferably about 6-10 mg / mL.

[0180] In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from about 1 mg / mL to about 20 mg / mL, for example, from about 1 mg / mL to about 19 mg / mL, from about 1 mg / mL to about 18 mg / mL, from about 1 mg / mL to about 17 mg / mL, from about 1 mg / mL to about 16 mg / mL, from about 1 mg / mL to about 15 mg / mL, from about 2 mg / mL to about 18 mg / mL, from about 2 mg / mL to about 17 mg / mL, from about 2 mg / mL to about 16 mg / mL, from about 2 mg / mL to about 15 mg / mL, from about 3 mg / mL to about 18 mg / mL, from about 3 mg / mL to about 17 mg / mL, from about 3 mg / mL to about 16 mg / mL, from about 3 mg / mL to about 15 mg / mL, from about 4 mg / mL to about 14 mg / mL, from about 5 mg / mL to about 13 mg / mL, from about 6 mg / mL to about 12 mg / mL, from about 7 mg / mL From about 11 mg / mL to about 10.5 mg / mL, from about 7 mg / mL to about 10 mg / mL, from about 7 mg / mL to about 9 mg / mL, from about 7 mg / mL to about 9.5 mg / mL, from about 7 mg / mL to about 9 mg / mL, from about 7 mg / mL to about 8.5 mg / mL, from about 7.5 mg / mL to about 11 mg / mL, from about 7.5 mg / mL to about 10.5 mg / mL, from about 7.5 mg / mL to about 10 mg / mL, from about 7.5 mg / mL to about 9.5 mg / mL, from about 7.5 mg / mL to about 9.0 mg / mL, from about 7.5 mg / mL to about 8.5 mg / mL, from about 8 mg / mL to about 11 mg / mL, from about 8 mg / mL to about 10.5 mg / mL, from about 8 mg / mL to about 10 mg / mL, from about 8 mg / mL to about 9 mg / mL. In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is about 2 mg / mL to about 18 mg / mL. In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is about 3 mg / mL to about 15 mg / mL.In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from about 7 mg / mL to about 10 mg / mL, such as about 7.5 mg / mL, about 7.6 mg / mL, about 7.7 mg / mL, about 7.8 mg / mL, about 7.9 mg / mL, about 8 mg / mL, about 8.1 mg / mL, about 8.2 mg / mL, about 8.3 mg / mL, about 8.4 mg / mL, about 8.5 mg / mL, about 8.6 mg / mL, about 8.7 mg / mL, about 8.8 mg / mL, about 8.9 mg / mL, about 9.0 mg / mL, about 9.1 mg / mL, about 9.28 mg / mL, about 9.3 mg / mL, about 9.4 mg / mL, about 9.5 mg / mL, about 9.6 mg / mL, about 9.7 mg / mL, about 9.8 mg / mL, about 9.9 mg / mL, or about 10 mg / mL. In some embodiments, the concentration of sodium chloride in the pharmaceutical composition is from about 7.5 mg / mL to about 9.5 mg / mL.

[0181] In some embodiments, the osmotic pressure regulating agent is propylene glycol.

[0182] In some embodiments, the propylene glycol concentration is about 5-50 mg / mL, preferably about 10-50 mg / mL, more preferably about 15-40 mg / mL, more preferably about 20-40 mg / mL, and more preferably about 20-30 mg / mL.

[0183] In some embodiments, the concentration of propylene glycol in the pharmaceutical composition is from about 10 mg / mL to about 20 mg / mL, for example, from about 10 mg / mL to about 19 mg / mL, from about 10 mg / mL to about 18 mg / mL, from about 10 mg / mL to about 17 mg / mL, from about 10 mg / mL to about 16 mg / mL, from about 10 mg / mL to about 15 mg / mL, from about 11 mg / mL to about 20 mg / mL, from about 11 mg / mL to about 19 mg / mL, from about 11 mg / mL to about 18 mg / mL, from about 11 mg / mL to about 17 mg / mL, from about 11 mg / mL to about 16 ... In some embodiments, the concentration of propylene glycol in the pharmaceutical composition is from about 12 mg / mL to about 16 mg / mL, for example, about 14 mg / mL.

[0184] In some embodiments, the osmotic pressure regulating agent is mannitol.

[0185] In some embodiments, the concentration of mannitol in the pharmaceutical composition is from about 10 mg / mL to about 60 mg / mL, e.g., from about 15 mg / mL to about 48 mg / mL, about 20 mg / mL to about 48 mg / mL, about 20 mg / mL to about 48 mg / mL, about 25 mg / mL to about 48 mg / mL, about 30 mg / mL to about 48 mg / mL, about 15 mg / mL to about 40 mg / mL, about 15 mg / mL to about 35 mg / mL, about 20 mg / mL to about 40 mg / mL, about 20 mg / mL to about 35 mg / mL, about 25 mg / mL to about 40 mg / mL, about 25 mg / mL to about 35 mg / mL, about 30 mg / mL to about 40 mg / mL, about 35 mg / mL to about 40 mg / mL. In some embodiments, the concentration of mannitol in the pharmaceutical composition is about 25 mg / mL, about 25.5 mg / mL, about 26 mg / mL, 26.5 mg / mL, about 27 mg / mL, about 27.5 mg / mL, about 28 mg / mL, about 28.5 mg / mL, about 29 mg / mL, about 29.5 mg / mL, about 30 mg / mL, 30.5 mg / mL, about 31 mg / mL, about 31.5 mg / mL, about 32 mg / mL, about 32.5 mg / mL, about 33 mg / mL, 33.5 mg / mL, about 34 mg / mL, 34.5 mg / mL, about 35 mg / mL, 35.5 mg / mL, about 36 mg / mL, 36.5 mg / mL, About 37 mg / mL, 37.5 mg / mL, about 38 mg / mL, 38.5 mg / mL, about 39 mg / mL, 39.5 mg / mL, about 40 mg / mL, about 41 mg / mL, about 41.5 mg / mL, about 42 mg / mL, about 42.5 mg / mL, about 43 mg / mL, about 43.5 mg / mL, about 44 mg / mL, about 44.5 mg / mL, about 45 mg / mL, about 45.5 mg / mL, about 46 mg / mL, about 46.5 mg / mL, about 47 mg / mL, about 47.5 mg / mL, about 48 mg / mL, about 48.5 mg / mL, about 49 mg / mL, about 49.5 mg / mL, or about 50 mg / mL.

[0186] [preservative]

[0187] The pharmaceutical compositions of the present invention may optionally contain a preservative.

[0188] In some embodiments, the pharmaceutical composition contains a preservative.

[0189] In some embodiments, the pharmaceutical composition is preservative-free.

[0190] In the context of the present invention, preservatives and antibacterial agents can be used interchangeably. Preservatives commonly used in pharmaceuticals can be used in the present invention as long as they do not affect the therapeutic effect and stability of the pharmaceutical composition of the present invention.

[0191] In some embodiments, preservatives include phenol, m-cresol, cresol, phenoxyethanol, chlorobutanol, benzyl alcohol, benzoate derivatives, and any combination thereof.

[0192] In some embodiments, the preservative comprises phenol, m-cresol, cresol, chlorobutanol, benzyl alcohol, and any combination thereof. In some embodiments, the concentration of m-cresol in the pharmaceutical composition is from about 0.10 mg / mL to about 10.00 mg / mL, for example, from about 0.5 mg / mL to about 9.00 mg / mL, from about 1.0 mg / mL to about 8.00 mg / mL, from about 1.5 mg / mL to about 7.00 mg / mL, from about 2.0 mg / mL to about 6.00 mg / mL. In some embodiments, the concentration of phenol in the pharmaceutical composition is about 0.50 mg / mL, about 1.00 mg / mL, about 2.00 mg / mL, about 2.10 mg / mL, about 2.20 mg / mL, about 2.30 mg / mL, about 2.40 mg / mL, about 2.50 mg / mL, about 2.60 mg / mL, about 2.70 mg / mL, about 2.80 mg / mL, about 2.90 mg / mL, about 3 .00 mg / mL, about 3.50 mg / mL, about 4.00 mg / mL, about 4.50 mg / mL, about 5.00 mg / mL, about 5.50 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, or about 10.0 mg / mL.

[0193] In some embodiments, the concentration of phenol in the pharmaceutical composition is about 0.10 mg / mL to about 10.00 mg / mL, for example, about 1.0 mg / mL to about 9.00 mg / mL, about 2.0 mg / mL to about 8.00 mg / mL, about 3.0 mg / mL to about 7.00 mg / mL, about 4.0 mg / mL to about 6.00 mg / mL. In some embodiments, the concentration of phenol in the pharmaceutical composition is about 0.50 mg / mL, about 0.60 mg / mL, about 1.00 mg / mL, about 2.00 mg / mL, about 3.00 mg / mL, about 4.00 mg / mL, about 4.10 mg / mL, about 4.20 mg / mL, about 4.30 mg / mL, about 4.40 mg / mL, about 4.50 mg / mL, about 4.60 mg / mL, about 5.0 mg / mL, about 5.5 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, or about 10.0 mg / mL.

[0194] In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is about 0.10 mg / mL to about 10.00 mg / mL, for example, about 0.5 mg / mL to about 9.00 mg / mL, about 1.0 mg / mL to about 8.00 mg / mL, about 1.5 mg / mL to about 7.00 mg / mL, about 2.0 mg / mL to about 6.00 mg / mL. In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is about 0.50 mg / mL, about 1.00 mg / mL, about 2.00 mg / mL, about 2.10 mg / mL, about 2.20 mg / mL, about 2.30 mg / mL, about 2.40 mg / mL, about 2.50 mg / mL, about 2.60 mg / mL, about 2.70 mg / mL, about 2.80 mg / mL, about 2.90 mg / mL, about 3 .00 mg / mL, about 3.50 mg / mL, about 4.00 mg / mL, about 4.50 mg / mL, about 5.00 mg / mL, about 5.50 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, or about 10.0 mg / mL.

[0195] [Antioxidants]

[0196] The pharmaceutical composition of the present invention may optionally contain an antioxidant,

[0197] In some embodiments, the pharmaceutical composition contains an antioxidant.

[0198] In some embodiments, the pharmaceutical composition is free of antioxidants.

[0199] Antioxidants commonly used in medicines can be used in the present invention, as long as they do not affect the therapeutic effect and stability of the pharmaceutical composition of the present invention.

[0200] In some embodiments, the antioxidant is selected from vitamin E, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, sodium bisulfite, sodium sulfite, sodium thiosulfate, or a combination thereof.

[0201] In some embodiments, the pharmaceutical composition does not contain vitamin E, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, sodium bisulfite, sodium sulfite, or sodium thiosulfate.

[0202] [Stabilizer]

[0203] The pharmaceutical composition of the present invention may optionally contain a stabilizer,

[0204] In some embodiments, the pharmaceutical composition contains a stabilizer to inhibit chemical degradation or polymerization, physical aggregation or precipitation of the polypeptide.

[0205] In some embodiments, the pharmaceutical composition does not contain a stabilizer.

[0206] Stabilizers or surfactants commonly used in medicines can be used in the present invention, as long as they do not affect the therapeutic effect and stability of the pharmaceutical composition of the present invention.

[0207] The preparation of the present invention may include a stabilizer, which can be selected from conventional surfactants, amino acids, polysaccharides, metal chelators in the art, such as non-ionic surfactants and / or ionic surfactants, and specific examples can be lipid excipients (such as oleoyl polyoxyethylene glyceride Labrafil, caprylic capric macrogol glyceride Labrasol, propylene glycol dicaprylic capric ester Labrafac, Gelucire), polysorbates (such as polysorbate 20 and polysorbate 80), poloxamers (such as poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside, lauryl-, myristyl bean Coryl-, linoleyl-, or stearoyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearoyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroylamidopropyl-, cocoamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoylamidopropyl-, or isostearamidopropyl-betaine (e.g., lauroylamidopropyl), myristamidopropyl-, palmitoylamidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl taurate or disodium methyl oleyl taurate, and polyethylene glycol, polypropylene glycol, and ethylene glycol and propylene glycol copolymers (e.g., Pluronics PF68, etc.). Among these, polysorbate is preferred, and polysorbate 80 is more preferred. The surfactant may be included in an amount of about 0.01 mg / mL to about 50 mg / mL, preferably about 0.5 mg / mL to 50 mg / mL, based on the total volume of the pharmaceutical composition.

[0208] In some embodiments, the stabilizer is selected from Tween (eg, Tween-40), poloxamer, amino acids (eg, glycine, histidine), or any combination thereof.

[0209] In some embodiments, the pharmaceutical composition does not comprise glycine and Tween.

[0210] In some embodiments, the pharmaceutical composition does not comprise poloxamer and histidine.

[0211] [Use of the pharmaceutical composition for treating diseases, pharmaceutical use, and method for treating diseases]

[0212] In some embodiments, the present invention provides use of a pharmaceutical composition in the preparation of a medicament for preventing or treating a disease.

[0213] In some embodiments, the pharmaceutical compositions of the present invention are used to prevent or treat a disease.

[0214] In some embodiments, the present invention provides a method for preventing or treating a disease, comprising administering an effective amount of a pharmaceutical composition according to the present invention to a human or animal in need thereof.

[0215] In some embodiments, the disease comprises diabetes, obesity, a disease associated with diabetes, and / or a disease associated with obesity.

[0216] In some embodiments, the unit dose of the pharmaceutical composition is from about 0.1 mg to about 0.5 mg or from about 0.5 mg to about 50 mg, for example, from about 1 to about 40 mg, about 10-35 mg, about 20-30 mg, about 0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, or about 45 mg. In the context of the present invention, "unit dose" and "unit administration dose" are synonymous and used interchangeably to refer to the amount of the pharmaceutical composition in a single-dose administration package, or the amount of the pharmaceutical composition in an independently administrable dosage unit in a multi-dose administration package.

[0217] In some embodiments, the disease comprises one or more of: non-insulin dependent diabetes mellitus / type II 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.

[0218] In some embodiments, the disease comprises one or more of symptomatic obesity, obesity based on simple obesity, disease states or diseases associated with obesity, eating disorders, diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes, obese diabetes), hyperlipidemia (e.g., hypertriglyceridemia, hypercholesterolemia, high LDL-cholesterolemia, low HDL-cholesterolemia, postprandial hyperlipidemia), hypertension, heart failure, complications of diabetes, such as neuropathy, nephropathy, retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, infectious diseases (e.g., respiratory tract infection, urinary tract infection, gastrointestinal infection, superficial soft tissue infection, lower limb infection), diabetic gangrene, dry mouth, hearing loss, cerebrovascular disorders, peripheral blood circulation disorders, metabolic syndrome (a disease state having three or more selected from hypertriglyceridemia (TG), low HDL cholesterolemia (HDL-C), hypertension, abdominal obesity and impaired glucose tolerance), sarcopenia, and the like.

[0219] In some embodiments, the disease comprises one or more of the following: endocrine obesity (e.g., Cushing's syndrome, hypothyroidism, insulinoma, obese type II diabetes, pseudohypoparathyroidism, hypogonadism), central obesity (e.g., hypothalamic obesity, frontal lobe syndrome, Klebsiella pneumoniae syndrome), genetic obesity (e.g., Prader-Willi syndrome, Lawrence-Moore syndrome), drug-induced obesity (e.g., steroid, phenothiazine, insulin, sulfonylurea (SU) drugs, beta-blocker induced obesity), and the like.

[0220] In some embodiments, disease states or diseases associated with obesity include: glucose tolerance disorders, diabetes (especially type II diabetes, obese diabetes), lipid metabolism disorders (synonymous with hyperlipidemia above), hypertension, heart failure, hyperuricemia, fatty liver (including non-alcoholic hepatitis), coronary heart disease (myocardial infarction, angina pectoris), cerebral infarction (cerebral thrombosis, transient ischemic attack), bone / joint diseases (knee osteoarthritis, hip osteoarthritis, ankylosing spondylitis, low back pain), sleep apnea syndrome / Pickwick syndrome, menstrual disorders (abnormal menstrual cycle, amenorrhea, abnormal menstrual symptoms), metabolic syndrome, etc.

[0221] The "effective amount" or "therapeutically effective amount" of the present invention is the amount or dose of the pharmaceutical composition of the present invention, which, when administered in single or multiple doses to an animal or human, provides the desired effect in the animal being diagnosed or treated.

[0222] As used herein, "treating" includes attenuating, inhibiting, reversing, slowing, delaying or stopping the progression or severity of an existing condition, disease, disorder or symptom. As used herein, "preventing" includes reducing the risk of acquiring a particular disease, condition or disorder.

[0223] Unless otherwise specified or clearly contradictory, the pharmaceutical composition of the present invention can be administered to humans and animals (including pets), including monkeys, mice, dogs, cats, rabbits, pigs, alpacas, horses, sheep, and cattle.

[0224] [Container and kit]

[0225] In some embodiments, the present invention provides an article of manufacture or kit comprising a container, and a container filled with a pharmaceutical composition of the present invention.

[0226] In some embodiments, the container is a molded bottle or a tube bottle made of borosilicate glass sealed by an elastic sealing component, such as a vial, a cartridge, a pre-filled glass needle, or a pre-filled needle. A "pre-filled syringe" refers to a syringe that is filled with a pharmaceutical composition (i.e., a formulation containing the GLP-1 / GIP dual agonist of the present invention) before being distributed to the end user who will administer the drug to the patient. A pre-filled syringe or pre-filled needle generally includes: a drug-holding container that forms a portion of the syringe body (i.e., a syringe barrel). As used herein, the term "pre-filled glass syringe" refers to a pre-filled syringe in which at least the syringe barrel is made of glass.

[0227] In the context of the present invention, "prefilled", "prefilled" and "prefilled" have the same meaning and can be used interchangeably.

[0228] In some embodiments, the container is an injection pen or device that facilitates accurate, safe, and convenient drug administration.

[0229] [Specific embodiment of the pharmaceutical composition containing polypeptide compound P016]

[0230] Embodiment 1. A pharmaceutical composition having a pH of about 5.8 to about 9.0, and comprising about 0.1 to about 50 mg / mL of the following polypeptide compound:

[0231] Compound P016 (SEQ ID NO: 7): H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2

[0232] Embodiment 2. The pharmaceutical composition according to embodiment 1, wherein the pharmaceutical composition is an aqueous solution, and the concentration of the polypeptide compound in the aqueous solution is at least 10 mg / mL based on the total volume of the aqueous solution.

[0233] Embodiment 3. The pharmaceutical composition according to embodiment 1 or 2, wherein the pharmaceutical composition further comprises an osmotic pressure regulator, a buffer and water, and optionally a pH regulator.

[0234] Embodiment 4. The pharmaceutical composition of claim 3, wherein the buffer is selected from the group consisting of disodium hydrogen phosphate, tromethamine, trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof.

[0235] Embodiment 5. The pharmaceutical composition of embodiment 3 or 4, wherein the molar concentration of the buffer is about 0.5 to about 50 mM based on the total volume of the aqueous solution.

[0236] Embodiment 6. The pharmaceutical composition of any one of Embodiments 3-5, wherein the osmotic pressure regulator is selected from mannitol, sodium chloride, propylene glycol, and combinations thereof.

[0237] Embodiment 7. The pharmaceutical composition of any one of embodiments 3-6, wherein the content of the osmotic pressure regulator is about 1 to about 100 mg / mL based on the total volume of the aqueous solution.

[0238] Embodiment 8. The pharmaceutical composition of any one of Embodiments 3-7, wherein the pH adjuster is aqueous sodium hydroxide solution or hydrochloric acid.

[0239] Embodiment 9. The pharmaceutical composition according to any one of embodiments 1-8, wherein the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition comprises:

[0240] about 0.1 to about 50 mg / mL, for example about 1 to about 40 mg / mL of compound P016,

[0241] The total molar concentration of citric acid and trisodium citrate is 0.5 to about 50 mM, preferably about 1.0 to about 10 mM citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4-50, preferably about 1:4-20,

[0242] about 1 to about 100 mg / mL of mannitol,

[0243] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0244] water,

[0245] The pH value of the aqueous solution is about 5.8 to about 9.0, preferably about 6.0 to about 8.8, and more preferably about 6.5 to about 7.4.

[0246] Embodiment 10. A pharmaceutical kit comprising a container, and the pharmaceutical composition according to any one of embodiments 1-9 contained in the container.

[0247] Embodiment 11. Use of the pharmaceutical composition according to any one of embodiments 1-10 for preparing a medicament for treating a disease, such as diabetes or obesity.

[0248] Embodiment 12. The pharmaceutical composition according to any one of embodiments 1-10, for use in treating a disease, such as diabetes or obesity.

[0249] Embodiment 13 provides a method for preventing or treating a disease, comprising administering an effective amount of the pharmaceutical composition according to any one of embodiments 1-10 to a human or animal in need thereof, wherein the disease is, for example, diabetes or obesity.

[0250] The following examples are intended to illustrate the present invention in detail but are not intended to limit the scope of the present invention.

[0251] Example

[0252] [Preparation of polypeptide compounds]

[0253] [Compound Preparation Example 1—Preparation of Polypeptide Compound P015]

[0254] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0255] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P015, SEQ ID NO: 6).

[0256] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K24, for which the structure has been unfolded.

[0257] The preparation method adopts the Fmoc solid phase peptide synthesis strategy, and the method includes:

[0258] (1) Synthesis of peptide resin intermediate 1

[0259] Take Fmoc-Rink Linker-Nle-MBHA resin (S = 0.49 mmol / g), swell it with an appropriate amount of DCM, and wash it with DCM 2-3 times. Deprotect it with 20% PIP / DMF solution for 30 minutes, filter and wash it to obtain the Fmoc-free NH2-Rink linker-Nle-MBHA resin, and drain the solvent for use.

[0260] Take 4 equivalents of Fmoc-Lys(Boc)-OH and HOBt respectively, dissolve them in appropriate amount of DMF / DCM; take another 4 equivalents of DIC, dilute it by half with DCM, slowly add it to the DMF / DMC solution with stirring, and stir the reaction at -5~0℃ for not less than 60 minutes. After activation, set aside.

[0261] Add the activated Fmoc-Lys(Boc)-OH solution to the NH2-Rink linker–Nle-MBHA resin, control the reaction temperature at 10-30°C, and conduct the coupling reaction for 240–480 min. Filter and wash to obtain the Fmoc-Lys(Boc)-Rink linker–Nle-MBHA resin. Deprotect the resin with a 20% PIP / DMF solution for 30 min, filter and wash to obtain the de-Fmoc Lys(Boc)-Rink linker–Nle-MBHA resin.

[0262] Under the same reaction conditions, start from the second amino acid at the C-terminus and couple to the N-terminus one by one. If the coupling is incomplete (color reaction), use HBTU / DIEA for a second condensation to ensure that each amino acid is completely condensed. The coupling sequence is Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly- Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Ala-OH.H2O , Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Aib-OH, Fmoc-Tyr( tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-α-Me-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH.

[0263] get:

[0264] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln (Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Mtt)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA Resin

[0265] After the above condensation is complete, the Mtt protection is removed with 50% HFIP / DCM solution for 30 minutes, followed by washing and filtration to obtain the peptide resin intermediate 1:

[0266] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala- Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA Resin.

[0267] (2) Modification chain modification step

[0268] Dissolve 4 equivalents of Fmoc-AEEA-OH and HOBt in an appropriate amount of DMF / DCM. Dissolve another 4 equivalents of DIC in DCM by half, then slowly add it to the DMF / DCM solution with stirring. Stir and react at -5 to 0°C for at least 60 minutes. Activate and set aside.

[0269] The activated Fmoc-AEEA-OH solution was added to the previously swollen and washed peptide resin intermediate 1. The reaction temperature was controlled at 10-30°C. The coupling reaction was carried out for 240-480 minutes. The mixture was filtered and washed. The Fmoc protection was removed with 20% PIP / DMF solution for 30 minutes. The mixture was filtered and washed. According to the above reaction conditions, the activated Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH and octadecane dioic acid mono-tert-butyl ester were coupled on the resin, Fmoc was removed, and the mixture was washed with DCM and dried to obtain P015 peptide resin:

[0270] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-A ib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(tBuO -Ste-γ-Glu(α-OtBu)-γ-Glu(α-OtBu)-γ-Glu(α-OtBu)-AEEA-AEEA)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gl y-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink-Linker-Nle-MBHA Resin.

[0271] Take P015 peptide resin, add 12-15mL / g peptide resin cleavage agent (TFA:EDT:TIS:H2O, volume ratio 94:2:2:2), stir and react at 25±5℃ for 4 hours, filter the reaction mixture using a sand core funnel, collect the filtrate, wash the resin with a small amount of TFA three times, combine the filtrates and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) to precipitate, and then wash with MBTE 3-4 times. After evaporating MTBE, the crude product is dried under reduced pressure at room temperature to constant weight to obtain crude P015. After subsequent purification and drying, a P015 sample (mass spectrum MS: 5255.2) is obtained.

[0272] [Compound Preparation Example 2—Preparation of Polypeptide Compound P019]

[0273] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0274] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 16 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P019, SEQ ID NO: 10).

[0275] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K28, for which the structure has been unfolded.

[0276] The preparation method adopts the Fmoc solid phase peptide synthesis strategy, and the method includes:

[0277] (1) Synthesis of peptide resin intermediate 2

[0278] Take Fmoc-Rink Linker-Nle-MBHA resin (S = 0.49 mmol / g), swell it with an appropriate amount of DCM, and wash it with DCM 2-3 times. Deprotect it with 20% PIP / DMF solution for 30 minutes, wash and filter to obtain the Fmoc-free NH2-Rink linker-Nle-MBHA resin, and drain the solvent for use.

[0279] Take 4 equivalents of Fmoc-Lys(Boc)-OH and HOBt respectively, dissolve them in appropriate amount of DMF / DCM; take another 4 equivalents of DIC, dilute it half with DCM, slowly add it to the DMF / DMC solution with stirring, and react with stirring at -5~0℃ for not less than 60 minutes. After activation, set aside.

[0280] Add the activated Fmoc-Lys(Boc)-OH solution to the NH2-Rink linker–Nle-MBHA resin, control the reaction temperature at 10-30°C, and conduct the coupling reaction for 240–480 min. Filter and wash to obtain the Fmoc-Lys(Boc)-Rink linker–Nle-MBHA resin. Deprotect the resin with a 20% PIP / DMF solution for 30 min, filter and wash to obtain the de-Fmoc Lys(Boc)-Rink linker–Nle-MBHA resin.

[0281] Under the same reaction conditions, start from the second amino acid at the C-terminus and couple to the N-terminus one by one. If the coupling is incomplete (color reaction), use HBTU / DIEA for a second condensation to ensure that each amino acid is completely condensed. The coupling sequence is Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly- Gly-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Ala-OH.H2O , Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Aib-OH, Fmoc-Tyr( tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-α-Me-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH.

[0282] get:

[0283] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln (Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Mtt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA Resin

[0284] After the above condensation is complete, the Mtt protection is removed with 50% HFIP / DCM solution for 30 minutes, followed by washing and filtration to obtain the peptide resin intermediate 2:

[0285] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala- Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA Resin.

[0286] (2) Modification chain modification step

[0287] Dissolve 4 equivalents of Fmoc-AEEA-OH and HOBt in an appropriate amount of DMF / DCM. Dissolve another 4 equivalents of DIC in DCM by half, then slowly add it to the DMF / DCM solution with stirring. Stir and react at -5 to 0°C for at least 60 minutes. Activate and set aside.

[0288] The activated Fmoc-AEEA-OH solution was added to the previously swollen and washed peptide resin intermediate 2. The reaction temperature was controlled at 10-30°C. The coupling reaction was carried out for 240-480 minutes. The mixture was filtered and washed. The Fmoc protection was removed with a 20% PIP / DMF solution for 30 minutes. The mixture was filtered and washed. According to the above reaction conditions, the activated Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and octadecane dioic acid mono-tert-butyl ester were coupled on the resin in sequence. The Fmoc protection was removed and the mixture was washed with DCM and dried to obtain the P019 peptide resin:

[0289] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(t Bu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-V al-Lys(tBuO-Ste-γ-Glu(α-OtBu)-AEEA-AEEA-AEEA)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink-Linker-Nle-MBHA Resin.

[0290] Take the P019 peptide resin, add 12-15 mL / g of peptide resin cleavage agent (TFA:EDT:TIS:H2O, volume ratio 94:2:2:2), stir and react at 25±5°C for 4 hours, filter the reaction mixture using a sand core funnel, collect the filtrate, wash the resin with a small amount of TFA three times, combine the filtrates and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) to precipitate, and then wash with MBTE 3-4 times. After evaporating MTBE, the crude product is dried under reduced pressure at room temperature to constant weight to obtain a crude compound P019. After subsequent purification and drying, a polypeptide compound P019 sample (mass spectrum MS: 5142.0) is obtained.

[0291] [Compound Preparation Example 3—Preparation of Polypeptide Compound P007]

[0292] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0293] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P007, SEQ ID NO: 2).

[0294] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K24, for which the structure has been unfolded.

[0295] The peptide compound P007 of the present invention (mass spectrum MS: 4997.2) was synthesized using peptide resin intermediate 1 in a similar manner as described in Compound Preparation Example 1 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and octadecandioic acid mono-tert-butyl ester on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P007 peptide resin. The other steps were similar.

[0296] [Compound Preparation Example 4—Preparation of Polypeptide Compound P013]

[0297] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0298] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 18 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P013, SEQ ID NO: 4).

[0299] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K24, for which the structure has been unfolded.

[0300] The peptide compound P013 of the present invention (mass spectrum MS: 5025.2) was synthesized using peptide resin intermediate 1 in a similar manner as described in Compound Preparation Example 1 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and mono-tert-butyl eicosadioate on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P013 peptide resin. The other steps were similar.

[0301] [Compound Preparation Example 5—Preparation of Polypeptide Compound P014]

[0302] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0303] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P014, SEQ ID NO: 5).

[0304] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K24, for which the structure has been unfolded.

[0305] The peptide compound P014 of the present invention (mass spectrum MS: 5052.4) was synthesized using peptide resin intermediate 1 in a similar manner as described in Compound Preparation Example 1 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and mono-tert-butyl docosanediolate on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P014 peptide resin. The other steps were similar.

[0306] [Compound Preparation Example 6—Preparation of Polypeptide Compound P016]

[0307] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0308] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P016, SEQ ID NO: 7).

[0309] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K24, for which the structure has been unfolded.

[0310] The peptide compound P016 of the present invention (mass spectrum MS: 5198.0) was synthesized using peptide resin intermediate 1 in a similar manner to that described in Compound Preparation Example 1. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and mono-tert-butyl docosanediolate on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P016 peptide resin. The other steps were similar.

[0311] [Compound Preparation Example 7—Preparation of Polypeptide Compound P008]

[0312] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0313] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P008, SEQ ID NO: 2).

[0314] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K28, for which the structure has been unfolded.

[0315] The peptide compound P008 of the present invention (mass spectrum MS: 4996.8) was synthesized using peptide resin intermediate 2 in a similar manner as described in Example 2 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and octadecandioic acid mono-tert-butyl ester on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P008 peptide resin. The other steps were similar.

[0316] [Compound Preparation Example 8—Preparation of Polypeptide Compound P017]

[0317] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0318] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P017, SEQ ID NO: 8).

[0319] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K28, for which the structure has been unfolded.

[0320] The peptide compound P017 of the present invention (mass spectrum MS: 5052.8) was synthesized using peptide resin intermediate 2, similar to the preparation steps described in Example 2 above. The difference was that the modification process of the modified chain was to sequentially couple Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and mono-tert-butyl docosanediolate on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P017 peptide resin. The other steps were similar.

[0321] [Compound Preparation Example 9—Preparation of Polypeptide Compound P018]

[0322] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0323] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P018, SEQ ID NO: 9).

[0324] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K28, for which the structure has been unfolded.

[0325] The peptide compound P018 of the present invention (mass spectrum MS: 5255.2) was synthesized using peptide resin intermediate 2 in a similar manner as described in Example 2 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH, and octadecandioic acid mono-tert-butyl ester on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P018 peptide resin. The other steps were similar.

[0326] [Compound Preparation Example 10—Preparation of Polypeptide Compound P020]

[0327] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0328] wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is replaced by ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H was conjugated to the ε-amino group of the K side chain for chemical modification; and the C-terminal amino acid was amidated to a C-terminal primary amide (polypeptide compound P020, SEQ ID NO: 11).

[0329] The above structure contains the standard single-letter amino acid code except for residues Aib2, αMePhe6, Aib11, Aib13 and K28, for which the structure has been unfolded.

[0330] The peptide compound P020 of the present invention (mass spectrum MS: 5198.4) was synthesized using peptide resin intermediate 2 in a similar manner as described in Example 2 above. The difference was that the modification process for the modified chain involved sequentially coupling Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, and mono-tert-butyl docosanediolate on the resin, followed by Fmoc deprotection, washing with DCM, and drying to obtain the P020 peptide resin. The other steps were similar.

[0331] In some embodiments of the present invention, peptide resin intermediate 3 (a peptide resin intermediate with a modified chain at position 16 Lys) is obtained by following the preparation method of peptide resin intermediate 1 in Reference Example 1:

[0332] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys-Gln(Trt)-Ala-Gln(T rt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA Resin

[0333] Peptide resin intermediate 4 (peptide resin intermediate with a modified chain at position 40 Lys) was obtained by following the preparation method of peptide resin intermediate 1 in reference compound preparation example 1:

[0334] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala- Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys-Rink Linker-Nle-MBHA Resin

[0335] [Pharmacodynamics of polypeptide compounds]

[0336] [Pharmacodynamics Example 1] Pharmacodynamics of polypeptide compounds in db / db mice

[0337] This study used a single subcutaneous administration of the compound to db / db mice. Changes in blood glucose, food intake, and body weight were measured to elucidate the hypoglycemic effects and duration of efficacy of the compound described herein, and compared with positive controls Tirzepatide and P001. Male db / db mice, 8-9 weeks of age, were used in this study. The db / db mice were housed in individually ventilated cages in an IVC facility with controlled temperature (20-26°C) and humidity (40-70%), a 12h:12h light / dark cycle, and free access to food and water. Blood was collected from the tail tip of the mouse using a Roche blood glucose meter to measure basal blood glucose. The mice were randomly divided into groups (n=6 / group) based on their initial blood glucose and initial body weight. All groups had similar body weights and blood glucose levels.

[0338] The compound of the present invention (10 nmol / kg) or the positive control Tirzepatide (10 nmol / kg) or P001 (10 nmol / kg) were dissolved in a solvent (PBS containing 0.1% Tween 20, pH 7.2-7.4). After a single subcutaneous administration, random blood glucose levels were recorded at set time points (0-120 hours, recording of random blood glucose for the compound was stopped when the random blood glucose level was no different from that of the vehicle group), as well as daily body weight and food intake. Data were statistically analyzed using GraphPad Prism 8. Statistical differences between groups were analyzed using T-TEST, and differences were considered significant when P < 0.05.

[0339] Duration of hypoglycemic effect: the longest time during which the random blood glucose level of the compound is significantly different from that of the Vehicle group (P<0.05).

[0340] Table 1-1 Duration of hypoglycemic effect of compounds

[0341] *Compound P001 (SEQ ID NO: 1) has the same peptide chain as Formula I, but does not have chemical modifications of the side chain ε-amino group at 24K and 28K.

[0342] Compared with P001, the duration of the blood sugar lowering effect of the compounds of the present invention is 24 hours or longer (P007, P008, P014, P019), even more than 40 hours (P008, P014), and more than 72 hours (P014).

[0343] Compared with Tirzepatide, the duration of blood sugar lowering effect of the compounds of the present invention is comparable to or longer (P007, P008, P013, P014, P016, P017, P018, P019, P020), even 16 hours or more (P008, P013, P014, P016, P017, P020), 24 hours or more (P013, P014, P016, P017, P020), 48 hours or more (P014, P016, P020), and 56 hours or more (P016, P020).

[0344] Compared with P001: Based on the test described above, during the experimental period, P008 (p < 0.01), P014 (p < 0.001), and P019 (p < 0.05) showed significantly superior glucose-lowering efficacy compared to P001 (blood glucose AUC, P < 0.05). The glucose AUC inhibition rates were 2.3, 2.6, and 2.2 times that of P001, respectively. The duration of glucose-lowering effects was 40, 72, and 24 hours longer than that of P001, respectively. P007 showed comparable glucose-lowering efficacy to P001, but the duration of glucose-lowering effects was 24 hours longer than that of P001.

[0345] Compared with Tirzepatide: P014 (p < 0.05), P016 (p < 0.05), and P020 (p < 0.05) showed stronger glucose-lowering effects (blood glucose AUC, p < 0.05), with glucose AUC inhibition rates 1.7, 2.2, and 2.0 times that of Tirzepatide, respectively. Furthermore, they exhibited significantly longer durations of action (48, 56, and 56 hours longer than Tirzepatide, respectively; Table 9). P013 and P017 had comparable glucose-lowering effects to Tirzepatide, but the duration of action was 24 hours longer than Tirzepatide.

[0346] The acylated GLP-1 / GIP agonists of this invention significantly enhance the glucose-lowering effect. Compounds of this invention with different chain modifications can achieve stronger and longer-lasting glucose-lowering effects than P001, and some compounds of this invention exhibit stronger and longer-lasting glucose-lowering effects than Tirzepatide.

[0347] [Pharmacodynamic Example 2] Weight loss effect of polypeptide compound P016 in the DIO mouse obesity model

[0348] In this study, the polypeptide compound P016 of the present invention was administered subcutaneously multiple times to DIO mice. Changes in mouse body weight and blood glucose were measured to demonstrate the weight-reducing effects of the compound and compared with the positive control, Tirzepatide. 26-week-old male DIO mice were used in this study. DIO mice were housed in appropriately sized cages in an SPF-grade animal room with a controlled environment of 20-26°C, 40%-70% relative humidity, and a 12-hour light-dark cycle. Food and water were available ad libitum. DIO mice with similar randomized blood glucose (RBG) (D1) and body weight (D1) were randomly divided into groups (n=10 / group). Compound P016 of the present invention (0.3, 1, 3, or 30 nmol / kg) or the positive control, Tirzepatide (1 or 30 nmol / kg), were dissolved in a vehicle (PBS containing 0.1% Tween 20, pH 7.2-7.4). Subcutaneous injections were administered every three days for four consecutive weeks. The drug was administered by subcutaneous injection on days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28.

[0349] The animals were weighed before administration on the first day of administration (D1) and at fixed times every 3 days thereafter. Food intake was measured once a day after administration. Random blood glucose (RBG) was measured before grouping and 48 hours after each administration. On D30, after a 5-hour fast (water was not allowed), FBG and glycosylated hemoglobin (HbA1c) were measured. Blood was collected venously to separate serum. Serum insulin content was measured using a mouse insulin ELISA kit, and serum total cholesterol (TC), TG, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), free fatty acids (FFA), ALT, and aspartate aminotransferase (AST) levels were measured using a biochemical analyzer. The liver was removed, and the perirenal and peritesticular adipose tissues were separated and weighed to calculate the organ-to-body ratio. The liver was stained with HE and Oil Red O for pathological scoring. Data were analyzed using one-way analysis of variance (ANOVA). If the ANOVA was statistically significant (P ≤ 0.05) and the variances were homogeneous, intergroup comparisons were performed using the Tukey test. If the variances were unequal, intergroup comparisons were performed using the Dunnet's T3 test. Results are expressed as mean ± SEM. The p-value was 0.05, and both statistical and biological significance were considered in the analysis.

[0350] In experiments conducted as described above, the polypeptide compound P016 of the present invention significantly reduced the cumulative food intake and body weight of obese mice in a dose-dependent manner. The onset of action was at a dose of 1 nmol / kg, which was significantly more effective than the positive control, Tirzepatide, at the same dose. At a dose of 30 nmol / kg, the compound of the present invention exhibited a greater weight reduction than Tirzepatide (percentage weight reduction, 39.97% vs. 34.47%). The compound of the present invention also significantly reduced total abdominal fat (the sum of perirenal and peritesticular fat) and serum TC in DIO mice, demonstrating a significant lipid-lowering effect. At doses of 1, 3, and 30 nmol / kg, the polypeptide compound P016 of the present invention significantly reduced fasting blood glucose and insulin levels on D30, ameliorating insulin resistance. The compound of the present invention also significantly reduced liver weight and serum ALT levels, improving liver function.

[0351] Table 1-2 Cumulative food intake, body weight, fat and liver weight of DIO mice

[0352] Note: **P≤0.01, ***P≤0.001, ****P≤0.0001 compared with the Vehicle group; P≤0.01, P≤0.0001 compared with the positive control Tirzepatide group at the same dose. Results are expressed as Mean ± SEM of 10 mice.

[0353] Table 1-3 Fasting blood glucose, insulin and insulin resistance index of DIO mice

[0354] Note: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 compared with the Vehicle group; P ≤ 0.05 compared with the positive control Tirzepatide group at the same dose. Results are expressed as Mean ± SEM of 10 mice.

[0355] Table 1-4 Serum TC and ALT in DIO mice

[0356] Note: *P≤0.05, ***P≤0.001, ****P≤0.0001 compared with the Vehicle group. Results are expressed as Mean ± SEM of 10 mice.

[0357] In summary, in this experimental system, the polypeptide compound P016 of the present invention effectively reduced the body weight of DIO mice after subcutaneous injection for four weeks, with an effective dose of 1 nmol / kg. This dose significantly outperformed the positive control, Tirzepatide, at the same dose. Furthermore, the compound of the present invention achieved a greater maximum weight loss (30 nmol / kg) than Tirzepatide at the same dose (percentage weight loss, 39.97% vs. 34.47%). Furthermore, the compound of the present invention exhibited effects on lowering blood sugar, improving insulin resistance, and improving liver function.

[0358] [Method for measuring composition index]

[0359] The properties, clarity, color, osmotic pressure, pH value, purity and polymer content of the solution were used as indicators of the composition, and the temporal stability of the indicators of each composition sample under different storage conditions was measured.

[0360] [Properties]

[0361] Take an appropriate amount of the composition sample to be tested in a bright place and observe directly.

[0362] [Visible foreign matter]

[0363] Take an appropriate amount of sample of the composition to be tested and inspect it according to the visible foreign matter inspection method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0904).

[0364] [pH value]

[0365] The pH value of each composition sample was measured using a pH meter or a micro pH meter.

[0366] [Clarity and color] Visual inspection

[0367] Take an appropriate amount of the composition sample to be tested and an equal volume of turbidity standard solution, place them separately in a colorless, transparent test tube or vial, and place them vertically in a dark room under a canopy light from a clarity tester at an illumination of 1000 lx. Observe and compare them horizontally. Sample aqueous solutions whose clarity does not exceed that of turbidity standard solution No. 0.5 are defined as "clear." Turbidities between 0.5 and 1 are defined as "almost clear." Other turbidities correspond to the turbidity values ​​of the corresponding turbidity standard solutions.

[0368] Take an appropriate amount of the test composition sample and an equal volume of a standard colorimetric solution, place them separately in a Nessler colorimetric tube or a colorless, transparent vial. Place the sample and the reference standard colorimetric solution on a white background and observe the sample color by comparing them. The sample solution is defined as "colorless" if its color is the same as water or the solvent used. The sample solution is defined as "almost colorless" if its color is no darker than the corresponding hue 0.5 standard colorimetric solution. The remaining color numbers correspond to the hue values ​​of the corresponding standard colorimetric solutions.

[0369] [Insoluble particles]

[0370] Take an appropriate amount of sample of the composition to be tested and inspect it according to the insoluble particulate matter test method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0903).

[0371] [Purity or related substances] High performance liquid chromatography (HPLC)

[0372] Method 1

[0373] Dissolve an appropriate amount of the test composition sample in water and dilute to approximately 1.0 mg / mL of sample solution. Chromatographic conditions: YMC-Pack ODS-AQ 150×4.6 mm, 3.0 μm column; mobile phase A: 25 mM potassium dihydrogen phosphate aqueous solution, pH adjusted to 2.1-2.3 with sulfuric acid or phosphoric acid; mobile phase B: acetonitrile; gradient elution; detection wavelength: 220 nm; column temperature: 40-45°C; flow rate: 1.0 mL / min; injection volume: 20 μL.

[0374] Purity refers to the main peak chromatographic purity (%) of the active pharmaceutical ingredient (API) in the composition.

[0375] Method 2

[0376] Chromatographic conditions

[0377] Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-AQ 150×4.6mm 3μm chromatographic column);

[0378] Mobile phase A: 100 mM potassium dihydrogen phosphate solution-acetonitrile (90:10);

[0379] Mobile phase B: 100 mM potassium dihydrogen phosphate solution-acetonitrile (40:60);

[0380] Flow rate: 1.0 mL / min. Detection wavelength: 220 nm; column temperature: 45°C; injection volume: 10 μL.

[0381] The content of related substances (total impurities) refers to the chromatographic peak area percentage (%) of all impurities contained in the composition sample.

[0382] [Polymer] Size Exclusion Chromatography (SEC)

[0383] Take an appropriate amount of the composition sample to be tested, dissolve it in water and dilute it to prepare an aqueous solution of about 0.25 to about 0.3 mg / mL P016 sample.

[0384] System suitability solution: Take an appropriate amount of the polypeptide compound P016 reference substance of the present invention, place it at 100°C for 1 hour, then take it out, cool it naturally to room temperature, dissolve it in water and dilute it to make a system suitability solution of about 0.25 mg / mL.

[0385] Chromatographic conditions: hydrophilic modified silica as filler, TSKgel G2000SWxl, 300 mm × 7.8 mm, 5 μm chromatographic column; trifluoroacetic acid-acetonitrile-water (0.1:30:70) as mobile phase; isocratic elution for 35 min; column temperature of 35°C; detection wavelength of 210 nm; flow rate of 0.5 mL / min; injection volume of 10 or 20 μL.

[0386] System suitability requirements: In the system suitability solution chromatogram, the separation between the polymer impurity peak and the main peak of the polypeptide compound P016 (API) of the present invention should be no less than 1.5.

[0387] The polymer content refers to the percentage (%) of the chromatographic peak area of ​​polymer impurities contained in the active pharmaceutical ingredient (API) in the composition.

[0388] [Molarity Osmolarity]

[0389] Method 1

[0390] Instruments and reagents: osmotic pressure molar concentration meter, pipette, 200mOsmol / kg standard sodium chloride solution, 300mOsmol / kg standard sodium chloride solution, 400mOsmol / kg standard sodium chloride solution.

[0391] How to do it:

[0392] Zero point calibration: Use a pipette to inject 60μL of purified water into a clean, dry test tube, ensuring that there are no visible bubbles. For calibration, push the test tube into the support seat until it stops, allowing the temperature probe to completely enter the purified water in the test tube. The test result should be consistent with 0±2mOsmol / kg H2O.

[0393] Calibration using standard solutions: Take 60uL of 200mOsmol / kg, 300mOsmol / kg, and 400mOsmol / kg standard sodium chloride solutions, respectively, and place them in a clean, dry test tube (make sure there are no visible bubbles). Push the test tube into the support to the stop position, allowing the temperature probe to completely enter the standard in the test tube. Calibrate the instrument. The test results should all be within ±2mOsmol / kg H2O.

[0394] Sample measurement: Take 60 μL of the composition sample to be tested and inject it into the test tube (make sure there are no visible bubbles). Push the test tube into the support to the stop position so that the temperature probe is completely inserted into the test sample in the test tube. Measure the molar concentration and osmotic pressure of the sample solution.

[0395] Method 2

[0396] Instruments and reagents: osmotic pressure molar concentration meter, pipette, 200mOsmol / kg standard sodium chloride solution, 400mOsmol / kg standard sodium chloride solution.

[0397] How to do it:

[0398] Zero point calibration: Use a pipette to inject 60μL of purified water into a clean, dry test tube, ensuring that there are no visible bubbles. For calibration, push the test tube into the support until it stops, allowing the temperature probe to completely enter the purified water in the test tube. The test result should be consistent with 0±3mOsmol / kg H2O.

[0399] Calibration using standard solutions: Take 60μL of 200mOsmol / kg and 400mOsmol / kg standard sodium chloride solutions respectively and place them in a clean, dry test tube (make sure there are no visible bubbles). Push the test tube into the support seat to the stop position so that the temperature probe is completely immersed in the standard in the test tube. Calibrate the instrument. The test results should be consistent with ±3mOsmol / kg H2O.

[0400] Sample measurement: Take 60 μL of the composition sample to be tested and inject it into the test tube (make sure there are no visible bubbles). Push the test tube into the support to the stop position so that the temperature probe is completely inserted into the test sample in the test tube. Measure the molar concentration and osmotic pressure of the sample solution.

[0401] [Composition Example 1] pH Range Screening—Phosphate and Sodium Chloride System

[0402] According to Table 2-1 below, 80 mg of NaCl and 14.2 mg of Na₂HPO₄ were weighed and placed in a beaker. 8 mL of purified water was added and stirred to dissolve. 300 mg of polypeptide compound P016 (API) was slowly added with stirring until dissolved. The pH of the resulting mixture was tested, and then 0.5 M NaOH or 0.1 M HCl aqueous solution was added to adjust the pH to the target pH shown in Table 2-1 below. The mixture was then brought to 10 mL with purified water, mixed, and filtered through a 0.22 μm PES membrane to obtain Compositions A1, A2, and A3 having the following compositions: 30 mg / mL API, 8 mg / mL NaCl, and 10 mM Na₂HPO₄.

[0403] The solution was sterilized and filtered using a 0.22 μm PES membrane, and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotic was added and capped with an aluminum-plastic composite cap to obtain a sample of the composition to be tested.

[0404] After standing, the clarity, color, and purity of the obtained composition samples were tested at different temperatures and for different time periods according to the aforementioned test method, and the results are recorded in Tables 2-2 and 2-3 below, respectively.

[0405] Table 2-1 Combinations of phosphate and sodium chloride at different pH

[0406] Table 2-2 Clarity and color over time at different pH values

[0407] In Table 2-2 above and the tables that follow this document, the number following the color indicates the hue value of the standard colorimetric solution corresponding to the composition's color; higher numbers indicate darker colors; the number following the turbidity indicates the turbidity value of the turbidity standard solution corresponding to the composition's turbidity; higher numbers indicate more turbid solutions. The results in Table 2-2 above show that composition A1 at pH 5.8 was a suspension, with P016 precipitated, indicating that the compounds of the present invention became turbid at pH values ​​as low as 5.8, and a clear solution could not be obtained. Compositions A2 at pH 6.5 and A3 at pH 8.0 were clear solutions. Compositions A2 and A3 remained clear after 10 days at low temperatures (2-8°C) and high temperatures (40°C and 60°C). After 20 days at 40°C, compositions A2 and A3 became nearly clear. After 20 days at 60°C, the turbidity of composition A2 increased to turbidity 2, while composition A3 remained clear after 20 days at 60°C. This indicates that the clarity of the polypeptide compound solution of the present invention is affected by high temperature and has a certain pH correlation, and the turbidity is more likely to increase at pH 6.5 than at pH 8.0.

[0408] After composition A2 and composition A3 were stored at 2-8°C, 40°C and 60°C for 10 days or 20 days respectively, the color depth intensified with increasing temperature, and the solution color of the polypeptide compound of the present invention showed temperature dependence.

[0409] Table 2-3 Purity at different pH values

[0410] The results in Tables 2-3 above indicate that the purity decreases for Compositions A2 and A3 at 60°C were significantly greater than those observed for samples stored at 40°C, indicating that the purity stability of the polypeptide compound solutions of the present invention exhibits a temperature-dependent trend. After 10 and 20 days at 60°C, the purity decrease for Composition A2 at pH 8.0 was more significant than that for Composition A2 at pH 6.5, indicating that the purity stability of the polypeptide compound solutions of the present invention exhibits a pH-dependent trend at high temperatures. In summary, the purity stability of the polypeptide compound solutions of the present invention exhibits a significant pH-dependent and temperature-dependent trend.

[0411] [Composition Example 2] Buffer Type Screening - Citrate and Phosphate

[0412] According to Tables 2-4 below, 470 mg of mannitol, 29.4 mg of trisodium citrate dihydrate, or 14.2 mg of Na2HPO4 were weighed and placed in a beaker. 8 mL of purified water was added and stirred to dissolve. 300 mg of polypeptide compound P016 was slowly added to the beaker with stirring until dissolved. 0.1-0.5 M aqueous HCl was added to adjust the pH to the target pH value shown in Tables 2-4 below. Purified water was added to 10 mL, mixed, and filtered through a 0.22 μm PES membrane to obtain Compositions B1 to B3, and Compositions B4 to B6, having the following compositions: 30 mg / mL API, 47 mg / mL mannitol, and 10 mM Na2HPO4 or 10 mM trisodium citrate.

[0413] The solution was sterilized and filtered using a 0.22 μm PES membrane, and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotic was added and capped with an aluminum-plastic composite cap to obtain a sample of the composition to be tested.

[0414] After standing, the clarity, color, and purity of the obtained composition samples were tested at different temperatures and for different time periods according to the aforementioned test method, and the results were recorded in Tables 2-5 and 2-6 below, respectively.

[0415] Table 2-4 Combinations of mannitol and different buffers at different pH values

[0416] Table 2-5 Effects of different buffers on clarity and color

[0417] The results in Tables 2-5 above demonstrate that compositions B1, B2, and B3 containing disodium hydrogen phosphate + mannitol, and compositions B4, B5, and B6 containing trisodium citrate + mannitol, all remained clear after exposure to high temperatures of 40°C and 60°C for 5 and 20 days, respectively. Observation of sample color at high temperatures indicates that citrate buffers, represented by trisodium citrate, are more effective in suppressing the formation of colored degradation impurities in solutions of the compounds of the present invention than phosphate buffers, represented by Na2HPO4.

[0418] In addition, the results of composition A2 in Table 2-2 and composition B1 in Table 2-5 show that at pH 6.5, mannitol as an osmotic pressure regulator has the effect of improving the clarity stability of the composition compared to sodium chloride as an osmotic pressure regulator.

[0419] Table 2-6 Effect of different buffers on purity (%)

[0420] The results in Tables 2-6 above show that after 5 and 20 days at 60°C, the purity of compositions B1 to B6 decreased significantly. After 5 and 20 days at 40°C, compositions B1 to B6 exhibited relatively excellent purity at pH 6.5, pH 7.0, and pH 7.5. Within the pH range of 6.5-7.5, the dependence of purity stability on pH was relatively unnoticeable. This indicates that the purity stability of the polypeptide compound P016 solution of the present invention is highly dependent on temperature.

[0421] The results in Tables 2-6 above show that, compared with compositions B1 to B3 containing phosphate buffer, compositions B4 to B6 containing citrate buffer have smaller purity variation trends at high temperatures and better stability.

[0422] The results of Tables 2-5 and 2-6 show that among the compositions containing the polypeptide compound P016 of the present invention and mannitol, the compositions containing citrate buffer have a smaller decrease in purity and a smaller color deepening when stored at high temperatures than the compositions containing phosphate buffer; compositions B1 to B6 with a pH of 6.5 to pH 7.5 still have good purity and color after being placed at 40°C for 20 days, and the purity stability and color stability of compositions B4 to B6 containing citrate buffer are better than those of compositions B1 to B3 containing phosphate buffer at the same pH value.

[0423] [Composition Example 3] Screening of osmotic pressure regulators - propylene glycol, mannitol, sodium chloride

[0424] According to Tables 2-7 below, mannitol, NaCl, or propylene glycol, as well as trisodium citrate dihydrate and Na2HPO4 were weighed and placed in a beaker. 28 mL of purified water was added and stirred to dissolve. 1050 mg of polypeptide compound P016 was slowly added to the beaker with stirring until dissolved. The pH of the resulting mixture was tested, and then 0.5 M aqueous HCl or 0.5 M aqueous NaOH was added to adjust the pH to 6.5. The mixture was then brought to 35 mL with purified water, mixed, and sterile-filtered using a 0.22 μm PES membrane to obtain Compositions C1 to C7 containing 30 mg / mL of the polypeptide compound P016 (API) of the present invention.

[0425] The compositions C1-C7 were divided into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle, and the injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene film (after sterilization). The antibiotics were added and the aluminum-plastic composite cap was rolled to obtain the test composition sample.

[0426] After standing, the clarity, color, and purity of the obtained composition samples were tested at different temperatures and for different time periods according to the aforementioned test method, and the results are recorded in Tables 2-8 and 2-9 below, respectively.

[0427] Table 2-7 Combinations of different osmotic pressure regulators and different buffers

[0428] Table 2-8 Effects of different osmotic pressure regulators and buffers on clarity and color

[0429] The results in Tables 2-8 above show that, using solution clarity and color as indicators, for compositions containing 30 mg / mL of the polypeptide compound of the present invention and pH 6.5, in compositions C2 to C7 using trisodium citrate or Na2HPO4 buffer alone, the turbidity of compositions C2 and C3 using mannitol as an osmotic pressure regulator increased more slowly at room temperature (25°C) and high temperature (40°C, 60°C) compared to using sodium chloride or propylene glycol, indicating that mannitol is more suitable than sodium chloride or propylene glycol as an osmotic pressure regulator for the polypeptide compound solution of the present invention.

[0430] In summary, when trisodium citrate or disodium hydrogen phosphate is used as a buffer, mannitol as an osmotic pressure regulator is more suitable for maintaining the clarity of the solution of the pharmaceutical composition of the present invention.

[0431] Table 2-9 Effects of different osmotic pressure regulators and buffers on purity

[0432] Note: The purity of the API P016 in the C1-C5 compositions is 99.48%, and the purity of the API P016 in the C6-C7 compositions is 98.46%. The purity of P016 in the C1-C7 compositions after being stored at 2-8°C for 5 days is comparable to that of the API used in the compositions.

[0433] The results in Tables 2-9 above show that when purity is used as a stability indicator, there is little difference in the purity change trends between compositions C1 to C7 when stored at 25°C for 30 days. Among compositions C3, C5, and C7 containing trisodium citrate buffer, composition C3, which uses mannitol as an osmotic pressure regulator, exhibits a comparable or smaller decrease in purity at high temperatures (40°C and 60°C) compared to compositions C5 and C7 using sodium chloride or propylene glycol as osmotic pressure regulators, demonstrating better overall stability. This indicates that mannitol is more suitable as an osmotic pressure regulator for the solutions of the present invention than sodium chloride or propylene glycol.

[0434] It can be seen that the combination of a citrate buffer represented by trisodium citrate and mannitol is more suitable as the medium environment of the solution of the present invention than the combination of a Na2HPO4 buffer and sodium chloride or propylene glycol, and maintains the stability of the purity index at different temperatures.

[0435] Combining the results in Tables 2-8 and 2-9 above, it can be seen that among the compositions containing 30 mg / mL API and pH 6.5, the compositions using mannitol as an osmotic pressure regulator and trisodium citrate as a buffer have the best stability of solution clarity, color, and purity when stored at room temperature (25°C) and high temperature (40°C, 60°C).

[0436] [Composition Example 4] Screening of Stabilizers—Tween 20

[0437] According to Tables 2-10 below, 1645 mg of mannitol, 102.9 mg of trisodium citrate dihydrate, and 14 mg of Tween 20 were weighed and placed in a beaker. 28 mL of purified water was added and stirred thoroughly. 1050 mg of polypeptide compound P016 was slowly added to the beaker with stirring until dissolved. The pH was then adjusted to the target pH by adding 0.1 M aqueous HCl or 0.5 M aqueous NaOH. The volume was then made up to 35 mL with purified water, mixed thoroughly, and sterile-filtered through a 0.22 μm PES membrane to obtain compositions D1 to D5 containing 30 mg / mL of API.

[0438] The compositions D1 to D5 were dispensed into 2 mL neutral borosilicate glass injection vials (after sterilization), 1.0 mL / vial, and fully plugged with butyl rubber stoppers partially covered with polytetrafluoroethylene membrane (after sterilization) for injection. The antibiotics were added and capped with aluminum-plastic composite caps to obtain the test composition samples.

[0439] After standing, the clarity, color, and purity of the obtained composition samples after different temperatures and time periods were tested according to the aforementioned test method, and the results were recorded in the following Tables 2-10 and 2-11, respectively.

[0440] Table 2-10 Compositions containing the stabilizer Tween 20

[0441] Table 2-11 Clarity and color of compositions containing stabilizer Tween 20 at different pH values

[0442] The results in Table 2-11 above show that for the composition containing the polypeptide compound of the present invention, mannitol and trisodium citrate, although a clear and colorless solution containing 30 mg / mL API can be prepared within the pH range of 6.1-6.9, the addition of Tween 20 to composition D3 in Table 2-11, compared with composition C3 in Table 2-8 above, actually exacerbated the increase in turbidity, indicating that Tween 20 exacerbated the aggregation of the polypeptide compound of the present invention in the composition.

[0443] Tween 20 is not suitable as a stabilizer for the polypeptide compound of the present invention.

[0444] [Composition Example 5] Screening of Citric Acid-Trisodium Citrate Buffer Concentration, PO16 (API) Concentration, and pH Value—Citric Acid:Trisodium Citrate = 1:9 (Molar Ratio)

[0445] Prepare citric acid-trisodium citrate buffer as follows:

[0446] 40 mM citric acid aqueous solution: Weigh 8.4056 g of citric acid monohydrate (C6H8O7·H2O) and dilute with water to 1000 mL to obtain a 40 mM citric acid aqueous solution.

[0447] 40 mM trisodium citrate aqueous solution: Weigh 11.764 g of trisodium citrate dihydrate (C6H5Na3O7·2H2O) and dilute with water to 1000 mL to obtain 40 mM trisodium citrate aqueous solution.

[0448] 40mM citric acid-trisodium citrate buffer: Mix 10 mL of 40mM citric acid aqueous solution with 90 mL of 40mM trisodium citrate aqueous solution to obtain a 40mM citric acid-trisodium citrate buffer, wherein the total molar concentration of citric acid and trisodium citrate is 40mM, the molar ratio of citric acid:trisodium citrate is 1:9, the molar concentration of citric acid is 4mM, and the molar concentration of trisodium citrate is 36mM.

[0449] 20mM citric acid-trisodium citrate buffer: Mix 50mL of 40mM citric acid-trisodium citrate buffer with 50mL of purified water to obtain 20mM citric acid-trisodium citrate buffer, where the total molar concentration of citric acid and trisodium citrate is 20mM, the molar ratio of citric acid:trisodium citrate is 1:9, the molar concentration of citric acid is 2mM, and the molar concentration of trisodium citrate is 18mM.

[0450] 10mM citric acid-trisodium citrate buffer: Mix 50mL of 20mM citric acid-trisodium citrate buffer with 50mL of purified water to obtain a 10mM citric acid-trisodium citrate buffer with a total molar concentration of 10mM citric acid and trisodium citrate. The molar ratio of citric acid to trisodium citrate is 1:9, resulting in a citric acid molar concentration of 1mM and a trisodium citrate molar concentration of 9mM.

[0451] Unless otherwise specified, in Composition Example 5, the final concentration of citric acid-trisodium citrate in Compositions E1 to E14 refers to the total molar concentration of citric acid and trisodium citrate, wherein the molar concentration of citric acid is 1 / 10 of the total molar concentration, the molar concentration of trisodium citrate is 9 / 10 of the total concentration, and the molar ratio of citric acid:trisodium citrate = 1:9.

[0452] According to Tables 2-12 below, weigh the excipient solids into a container. Add an appropriate amount of purified water as shown in the table and stir or shake to dissolve. Add a citric acid-trisodium citrate buffer solution of appropriate concentration and amount to the container, shake well, and slowly add an appropriate amount of polypeptide compound P016 (API). Shake well to dissolve. Then, adjust the pH to the target value shown in Table 2-12 by adding 0.1-0.5 M HCl or NaOH aqueous solution. Add purified water to 3 mL, mix well, and filter through a 0.22 μm PES membrane to obtain Compositions E1 to E14 containing the polypeptide compound P016 (API) of the present invention.

[0453] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain the test composition sample.

[0454] After standing, the clarity, color, purity and polymer content of the obtained composition samples were tested at different temperatures and for different time periods according to the aforementioned test methods, and the results were recorded in Tables 2-13 to 2-15 below.

[0455] In compositions E1 to E14, mannitol is used as an osmotic pressure regulator, citric acid-trisodium citrate, disodium hydrogen phosphate, or tromethamine (Tris) is used as a buffer, and glycine is used as a potential stabilizer.

[0456] Table 2-12 Combinations of different types and concentrations of buffers and different API concentrations at different pH values

[0457] Table 2-12 Combinations of different types and concentrations of buffers and different API concentrations at different pH values ​​(continued 1)

[0458] Table 2-12 Combinations of different types and concentrations of buffers and different API concentrations at different pH values ​​(continued 2)

[0459] Table 2-13 Effects of buffer type and concentration, API concentration, and pH on clarity and color

[0460] The results in Tables 2-13 above show that compositions E1-E10 and E13-E14 using citric acid-trisodium citrate (molar ratio 1:9) as a buffer and mannitol as an osmotic pressure regulator have a pH of 6.5-7.2 and contain 20-30 mg / mL of the polypeptide compound of the present invention. After storage at high temperatures (40°C, 60°C) for 5 days and 10 days, they remain clear and colorless.

[0461] Composition E11 using disodium hydrogen phosphate as a buffer gradually increased in turbidity and darkened in color after storage at 60° C. for 5 and 10 days, again indicating that citric acid-trisodium citrate is more suitable than disodium hydrogen phosphate as a buffer for the polypeptide compound of the present invention.

[0462] In compositions E3 and E4, the use or absence of glycine as a stabilizer had no significant effect on clarity and color stability.

[0463] Table 2-14 Effects of buffer type and concentration, API concentration, and pH value on purity

[0464] The results in Tables 2-14 above show that after 10 days at high temperature (40°C, 60°C), in compositions E11, E12, and E13 containing 30 mg / mL of the polypeptide compound of the present invention at pH 7.2, the improvement in purity stability by the buffer gradually deteriorated in the order of citric acid-trisodium citrate, tromethamine, and disodium hydrogen phosphate. When citric acid-trisodium citrate was used as the buffer, compositions E1-E10 and E13-E14 showed the best purity stability at high temperature (40°C, 60°C). Furthermore, when the total molar concentration of citric acid and trisodium citrate was in the range of 5-20 mM and the polypeptide compound of the present invention was 20-30 mg / mL, the purity stability of compositions E1-E10 and E13-E14 at high temperature (40°C, 60°C) was comparable. In compositions E3 and E4, the use and absence of glycine as a stabilizer had no significant effect on the purity stability of the compositions at high temperature (40°C, 60°C).

[0465] Table 2-15 Effects of buffer type and concentration, API concentration, and pH value on polymer content

[0466] The results in Tables 2-15 above show that after 10 days at high temperatures (40°C and 60°C), the polymer content in compositions E11, E12, and E13 at pH 7.2 and containing 30 mg / mL of the polypeptide compound of the present invention increased in the order of citric acid-trisodium citrate, tromethamine, and disodium hydrogen phosphate. When citric acid-trisodium citrate was used as the buffer, compositions E1-E10 and E13-E14 exhibited the best polymer content stability at high temperatures (40°C and 60°C). Furthermore, when the total molar concentration of citric acid-trisodium citrate was within the range of 5-20 mM and the polypeptide compound of the present invention was within the range of 20-30 mg / mL, the polymer content in the compositions increased minimally and comparable after 10 days at 40°C, with polymer contents of 0.10-0.13% in all compositions.

[0467] In combination with Tables 2-13, 2-14 and 2-15, in compositions E3 and E4, the use and non-use of glycine as a stabilizer had no significant effect on the solution clarity and color, purity, and polymer content of the compositions. The addition of glycine even resulted in a slight increase in the polymer content, indicating that glycine is not suitable as a stabilizer for the polypeptide compound of the present invention.

[0468] Composition E2 in Tables 2-13 and 2-14 and composition C3 in Tables 2-8 and 2-9 are compositions using 47 mg / mL mannitol, 10 mM buffer and 30 mg / mL API, pH 6.5. Composition E2 using 10 mM citric acid-trisodium citrate as a buffer shows improved clarity stability compared to composition C3 using only 10 mM trisodium citrate as a buffer, and the color and purity stability at high temperatures (40°C, 60°C) are comparable.

[0469] In summary, compositions E1-E10 and E13-E14 with different total molar concentrations of citric acid / trisodium citrate (5-20 mM), different API concentrations (20-30 mg / mL), and different pH values ​​(6.5-7.2) showed comparable clarity, color, API purity, and polymer content after storage at 40°C and 60°C for 10 days. These indicators exhibited excellent stability, indicating that the citric acid / trisodium citrate buffer was generally superior to the phosphate buffer and tromethamine buffer.

[0470] [Composition Example 6] Screening of Citric Acid-Trisodium Citrate Buffer Concentration, PO16 (API) Concentration, and pH Value—Citric Acid:Trisodium Citrate = 1:6.15 (1.05 mM:6.45 mM, molar ratio)

[0471] According to Tables 2-16 below, weigh appropriate amounts of mannitol, citric acid monohydrate, and trisodium citrate dihydrate into a beaker. Add an appropriate amount of purified water (approximately 80% of the total amount of purified water) and stir until dissolved. Slowly add an appropriate amount of polypeptide compound P016 (API) to the beaker while stirring until dissolved. Test the pH of the resulting mixture, then adjust to the target pH by adding 0.1M HCl aqueous solution or 0.1M NaOH aqueous solution to the beaker. Add purified water to the volume and mix thoroughly. Sterile filter through a 0.22μm PES membrane to obtain Compositions F1 to F12 containing the polypeptide compound P016 (API) of the present invention.

[0472] The solution was divided into 2 mL neutral borosilicate glass injection bottles (after sterilization), 1.0 mL / bottle, and the injection solution was fully plugged with a butyl rubber stopper partially covered with polytetrafluoroethylene membrane (after sterilization). The antibiotic was added and the cover was covered with an aluminum-plastic composite cap to obtain the composition sample to be tested.

[0473] The obtained composition samples were tested at different temperatures or under strong light (1800 lx, 28 μw / cm 2) After different time periods, the clarity, color, pH (micro pH meter), purity, and polymer content of the compositions of 4 mg / mL to 30 mg / mL API in the pH range of 6.5 to 7.4 were measured to investigate various indicators and their stability, which are recorded in Tables 2-17 to 2-20 below.

[0474] Unless otherwise specified, in compositions F1 to F12 of composition Example 6, the total molar concentration of the citric acid-trisodium citrate buffer is 7.5 mM, wherein the molar concentration of citric acid is 1.05 mM, the molar concentration of trisodium citrate is 6.45 mM, and the molar ratio of citric acid:trisodium citrate = 1.05:6.45 = 1:6.15.

[0475] Table 2-16 Combinations of different concentrations of citric acid-trisodium citrate buffer and different API concentrations at different pH values

[0476] Table 2-16 Combinations of different concentrations of citric acid-trisodium citrate buffer and different API concentrations at different pH values ​​(continued 1)

[0477] The results in Tables 2-16 above indicate that the initial osmotic pressures of compositions F1 to F12 all meet the requirement of near-physiological isotonicity, and are convenient for parenteral administration or injection.

[0478] Table 2-17 Effects of citric acid-trisodium citrate buffer concentration, API concentration, and pH on clarity and color

[0479] N / A: Because composition F2 is formulated identically to compositions F1 and F3, differing only in the concentration of P016, data were not collected for the time points marked with N / A. The same explanation applies to the other time points marked with N / A.

[0480] The results in Tables 2-17 above demonstrate that compositions F1 to F12 exhibit excellent solution clarity stability, remaining clear and colorless after exposure to high temperatures of 40°C for 10 and 30 days, 60°C for 10 days, and strong light exposure for 10 days. Compositions F1 and F3, F4 and F6, F7 and F9, and F10 and F12 containing 4 mg / mL or 30 mg / mL P016 all remained clear and colorless after exposure to low temperatures (2-8°C) for 30 days and room temperature (25°C) for 30 days.

[0481] Table 2-18 Effects of Citric Acid-Trisodium Citrate Buffer Concentration, API Concentration, and pH Value on pH Stability

[0482] The results in Table 2-18 above indicate that the pH of compositions F1 to F12 did not change by more than 0.14 after exposure to low temperature (2-8°C) for 30 days, room temperature (25°C) for 30 days, high temperature (40°C) for 10 days and 30 days, high temperature (60°C) for 10 days, and strong light for 10 days. In particular, as shown in Table 2-19 below, compositions F1 to F12 experienced a purity change of approximately 25% after exposure to high temperature (60°C) or strong light for 10 days. However, the pH of composition F12 did not change by more than 0.09, the pH of composition F5 did not change by more than 0.05, and the pH of the remaining ten compositions did not change by more than 0.03. This indicates that even after exposure to light for 10 days, a 7.5 mM total molar concentration of citric acid-trisodium citrate buffer (molar concentrations of citric acid and trisodium citrate are 1.05 mM and 6.45 mM, respectively) is sufficient to maintain pH stability of the compositions within the pH range of 6.5-7.4.

[0483] Table 2-19 Effects of the type and concentration of citric acid-trisodium citrate buffer, API concentration, and pH on API purity

[0484] The results in Tables 2-19 above indicate that the purity of compositions F1 to F12 of the polypeptide compound of the present invention was approximately 98.4%. After being placed at 60°C or under strong light for 10 days, the API purity of the 12 compositions decreased significantly, to 72.05-73.66% and 96.88-97.51%, respectively. After being placed at 40°C for 10 days and 30 days, the API purity of the 12 compositions decreased significantly, to 96.57-96.84% and 92.65-93.67%, respectively. After being placed at room temperature (25°C) for one month, the API purity of the compositions with API concentrations of 4 mg / mL and 30 mg / mL decreased significantly, to 97.65-98.02% (a decrease of less than 0.7%). No significant change in API purity was observed after being placed at low temperatures (2-8°C) for 30 days and 4 months. In general, the purity stability of the 12 compositions was comparable and highly temperature-dependent. Compositions F1 to F12 exhibited excellent purity stability at low temperatures of 2-8°C.

[0485] Table 2-20 Effect of citric acid-trisodium citrate buffer concentration, API concentration, and pH value on polymer content

[0486] The results in Tables 2-20 above show that the initial polymer content of compositions F1 to F12 was approximately 0.05%. After 10 days at 60°C, the polymer content increased to 0.66-0.78%. After 10 days of exposure to strong light, the content increased to 0.44-0.74%. After 1 month at 40°C, the content increased to 0.17-0.24%. After 30 days at room temperature (25°C) and low temperature (2-8°C), the polymer content of compositions F1 to F12 with API concentrations of 4 mg / mL to 30 mg / mL remained virtually unchanged at 0.02-0.06%. Overall, the polymer content stability of the 12 compositions was comparable, showing a high degree of temperature dependence. The polymer content of compositions F1 to F12 was stable at both room temperature (25°C) and low temperature (2-8°C).

[0487] In summary, a stability study was conducted on 12 compositions F1 to F12 (7.5 mM citric acid-trisodium citrate buffer, 4, 20, and 30 mg / mL API, pH 6.5, 6.8, 7.1, and 7.4) at 60°C, 40°C, strong light, 25°C room temperature, and 2-8°C low temperature for 1-4 months. The results showed that the stability of various indicators (clarity, color, pH, API purity, and polymer content) of the 12 compositions F1 to F12 at concentration levels of 4-30 mg / mL API and pH 6.5-7.4 were comparable. The compositions exhibited excellent stability in clarity, color, and pH at low temperature, room temperature, high temperature, and light. The stability of API purity and polymer content was highly temperature-dependent, with the best stability of API purity and polymer content at low temperature (2-8°C).

[0488] As shown in Tables 2-18 to 2-20, the clarity and color of compositions F1 to F12 remained unchanged under strong light, while the API purity and polymer content exhibited light sensitivity. Therefore, compositions containing the polypeptide compounds of the present invention should be packaged and stored in a dark environment to mitigate the risk of photodegradation.

[0489] 46 mg / mL mannitol is used as an osmotic pressure regulator, 7.5 mM citric acid-trisodium citrate buffer is used as a buffer, and 4-30 mg / mL of the polypeptide compound of the present invention is contained in the pH range of 6.5-7.4. The composition has excellent stability in various indicators (clarity, color, pH value, API purity, polymer content) at low temperature, room temperature, and high temperature, and has the best stability at low temperature (2-8°C).

[0490] [Composition Example 7]

[0491] [Preparation of Compositions G1, G1-a to G1-q]

[0492] According to Table 2-21-1 or 2-21-2 below, weigh 46.00g of mannitol, 1.90g of trisodium citrate dihydrate, and 0.22g of citric acid monohydrate, respectively, place in a container, add 800mL of water for injection, and stir evenly. Slowly add 0.5-1g or 1-30.00g of polypeptide compound P016 (API) while stirring. The pH value of the obtained mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the container to adjust the pH to 6.8-7.4, and water for injection was added to 1000 mL, and mixed to obtain a composition G1 or composition G1-a to G1-q having the following composition: comprising 0.5-1 mg / mL or 1-30 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and using 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM = 1:6.15) as a buffer, and a pH of 6.8-7.4.

[0493] The prepared liquid medicine can be packaged and sealed in 1) neutral borosilicate glass controlled injection bottles or 2) pre-filled syringes.

[0494] 1) Filter through a 0.22 μm PES membrane and dispense into 2 mL neutral borosilicate glass injection vials (after sterilization), 1.0 mL / vial. Add the injection solution and fully plug it with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). Add the antibiotic and cap it with an aluminum-plastic composite cap to obtain a sample of composition G1.

[0495] 2) Filter through a 0.22 μm PES membrane, dispense into prefilled syringes, seal with stoppers, and label with a volume of 0.5 mL to obtain compositions G1-a to G1-q. Similarly, compositions G1-a to G1-q were prepared according to Tables 2-21-3 to 2-21-6 below using the same method as described above, except that the raw materials were reduced in equal proportions.

[0496] Table 2-21-1 Composition of composition G1

[0497] Table 2-21-2 Compositions of compositions G1-a to G1-q (prefilled syringes, batch sizes can be scaled up or down proportionally)

[0498] Table 2-21-3 Stability results of composition G1-b (0.5mL: 0.5mg, 1mg / mL)

[0499] Note: In Table 2-21-3, the volume of the composition G1-b used for the test is 0.5 mL, the content of the polypeptide compound P016 is 0.5 mg, and the concentration of the polypeptide compound P016 in the composition is 1 mg / mL.

[0500] Table 2-21-4 Stability results of compositions G1-d (0.5 mL: 1 mg, 2 mg / mL), G1-h (0.5 mL: 2 mg, 4 mg / mL), G1-l (0.5 mL: 3 mg, 6 mg / mL), and G1-n (0.5 mL: 4 mg, 8 mg / mL)

[0501] Note: There is a measurement error of no more than 2.5% in the content of active ingredients in drugs.

[0502] Note: In Table 2-21-3, the volume of composition G1-d used for testing was 0.5 mL, the content of polypeptide compound P016 was 1 mg, and the concentration of polypeptide compound P016 in the composition was 2 mg / mL. The volume of composition G1-h used for testing was 0.5 mL, the content of polypeptide compound P016 was 2 mg, and the concentration of polypeptide compound P016 in the composition was 4 mg / mL. The volume of composition G1-l used for testing was 0.5 mL, the content of polypeptide compound P016 was 3 mg, and the concentration of polypeptide compound P016 in the composition was 6 mg / mL. The volume of composition G1-n used for testing was 0.5 mL, the content of polypeptide compound P016 was 4 mg, and the concentration of polypeptide compound P016 in the composition was 8 mg / mL.

[0503] Table 2-21-5 Composition G1-p stability results (0.5mL: 12mg, 24mg / mL)

[0504] Note: There is a measurement error of no more than 2.5% in the content of active ingredients in drugs.

[0505] Note: In Table 2-21-5, the volume of the composition G1-p used for the test is 0.5 mL, the content of the polypeptide compound P016 is 12 mg, and the concentration of the polypeptide compound P016 in the composition is 24 mg / mL.

[0506] Table 2-21-6 Stability results of composition G1-q (0.5mL: 15mg, 30mg / mL)

[0507] Note: In Table 2-21-6, the volume of the composition G1-q used for testing is 0.5 mL, the content of the polypeptide compound P016 is 15 mg, and the concentration of the polypeptide compound P016 in the composition is 30 mg / mL.

[0508] The results in Tables 2-21-3 to 2-21-6 above demonstrate that representative compositions at varying drug concentrations, for example, ranging from 1 mg / mL to 30 mg / mL, exhibit temperature-dependent stability. Furthermore, at 25°C, all compositions exhibited comparable stability trends under the related substance (total impurities) index. At 2-8°C, indicators such as properties, pH, solution clarity and color, polymer content, related substances, insoluble particulate matter, and visible foreign matter were stable, indicating that the compositions exhibited excellent stability at 2-8°C.

[0509] [Preparation of Composition G2]

[0510] According to Table 2-22 below, weigh 12.65g of mannitol, 0.52g of trisodium citrate dihydrate, and 0.06g of citric acid monohydrate, place in a beaker, add 220mL of water for injection, and stir thoroughly. Slowly add 1.1g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the resulting mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 7.2 (pH 6.8-7.4 was allowed), and water for injection was added to 275 mL, mixed, and filtered through a 0.22 μm PES membrane to obtain a composition G2 having the following composition: comprising 4 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and using 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM=1:6.15) as a buffer, and a pH of 7.2 (6.8-7.4 was allowed).

[0511] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.1 mL / bottle, for a total of 250 bottles. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization), and the antibiotic was added and capped with an aluminum-plastic composite cap to obtain a composition G2 sample.

[0512] Table 2-22 Composition of composition G2

[0513] [Preparation of Composition G3]

[0514] According to Table 2-23 below, weigh 12.65g of mannitol, 0.52g of trisodium citrate dihydrate, and 0.06g of citric acid monohydrate, place in a beaker, add 220mL of water for injection, and stir thoroughly. Slowly add 8.25g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the resulting mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 7.2 (pH 6.8-7.4 was allowed), and water for injection was added to the volume to 275 mL, mixed, and filtered through a 0.22 μm PES membrane to obtain a composition G3 having the following composition: comprising 30 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and using 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM=1:6.15) as a buffer, and a pH of 7.2 (6.8-7.4 was allowed).

[0515] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.1 mL / bottle, for a total of 250 bottles. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization), and the antibiotic was added and capped with an aluminum-plastic composite cap to obtain a composition G3 sample.

[0516] Table 2-23 Composition of composition G3

[0517] [Preparation of Composition G4]

[0518] According to Tables 2-24 below, weigh 46.00 g of mannitol, 1.985 g of trisodium citrate dihydrate, and 0.158 g of citric acid monohydrate, respectively, place in a beaker, add 800 mL of water for injection, and stir thoroughly. Slowly add 0.5-1 g or 1-30.00 g of polypeptide compound P016 (API) to the beaker while stirring. Test the pH of the resulting mixture, then adjust the pH to 6.8-7.4 using 0.1 M NaOH or 0.1 M HCl aqueous solution. The remaining water for injection was added to make the volume to 1000 mL, mixed, and filtered through a 0.22 μm PES membrane to obtain a composition G4 having the following composition: comprising 0.5-1 mg / mL or 1-30 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.75 mM:6.75 mM = 1:9) as a buffer, with a pH of 6.8-7.4.

[0519] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G4 sample.

[0520] Table 2-24 Composition of composition G4

[0521] [Preparation of Composition G5]

[0522] According to Table 2-25 below, weigh 46.00g of mannitol, 1.324g of trisodium citrate dihydrate, and 0.105g of citric acid monohydrate, respectively, place in a beaker, add 800mL of water for injection, and stir thoroughly. Slowly add 0.5-1g or 1-30.00g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the resulting mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 6.8-7.4, and the remaining water for injection was added to 1000 mL. The mixture was mixed and filtered through a 0.22 μm PES membrane to obtain a composition G5 having the following composition: comprising 0.5-1 mg / mL or 1-30 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 5.0 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.50 mM:4.50 mM = 1:9) as a buffer, and a pH of 6.8-7.4.

[0523] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G5 sample.

[0524] Table 2-25 Composition of composition G5

[0525] [Preparation of Composition G6]

[0526] According to Table 2-26 below, weigh 46.00g of mannitol, 1.90g of trisodium citrate dihydrate, and 0.22g of citric acid monohydrate, place in a beaker, add 800mL of water for injection, and stir thoroughly. Slowly add 1.00g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the obtained mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 6.8-7.4, and water for injection was added to 1000 mL, and mixed to obtain a composition G6 having the following composition: comprising 1.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM=1:6.15) as a buffer, and a pH of 6.8-7.4.

[0527] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G6 sample.

[0528] Table 2-26 Composition of composition G6

[0529] [Preparation of Composition G7]

[0530] According to Table 2-27 below, weigh 46.00 g of mannitol, 1.985 g of trisodium citrate dihydrate, and 0.158 g of citric acid monohydrate, place in a beaker, add 800 mL of water for injection, and stir thoroughly. Slowly add 1.00 g of polypeptide compound P016 (API) to the beaker while stirring. Test the pH of the resulting mixture, then adjust the pH to 6.8-7.4 using 0.1 M NaOH or 0.1 M HCl aqueous solution. In addition, the remaining water for injection was added to make the volume to 1000 mL and mixed to obtain a composition G7 having the following composition: comprising 1.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.75 mM: 6.75 mM = 1:9) as a buffer, and a pH of 6.8-7.4.

[0531] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G7 sample.

[0532] Table 2-27 Composition of composition G7

[0533] [Preparation of Composition G8-A and Composition G8-B]

[0534] According to Table 2-28 below, weigh 46.00g of mannitol, 1.90g of trisodium citrate dihydrate, and 0.22g of citric acid monohydrate, place in a container, add 800mL of water for injection, and stir evenly. Slowly add 30.00g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the obtained mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the container to adjust the pH to 6.8-7.4, and water for injection was added to 1000 mL, and mixed to obtain a composition G8-B having the following composition: comprising 30.0 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM=1:6.15) as a buffer, and a pH of 6.8-7.4.

[0535] Composition G8-A was prepared similarly to the above method, except that the amount of polypeptide compound P016 used was 4.00 g.

[0536] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain the composition G8-A or G8-B sample.

[0537] Table 2-28-1 Compositions of G8-A and G8-B (batch size enlarged and reduced)

[0538] Table 2-28-2 G8-A stability results (4 mg / mL)

[0539] Table 2-28-3 G8-B stability results (30 mg / mL)

[0540] The composition stability data shown in Tables 2-28-2 and 2-28-3 demonstrate that Compositions G8-A and G8-B exhibit temperature-dependent stability. Furthermore, after 12 months of long-term storage at 2-8°C, the composition parameters remained stable, indicating excellent stability. All parameters met the requirements for subcutaneous administration.

[0541] [Preparation of Composition G9]

[0542] According to Table 2-29 below, weigh 46.00 g of mannitol, 1.985 g of trisodium citrate dihydrate, and 0.158 g of citric acid monohydrate, place them in a beaker, add 800 mL of water for injection, and stir thoroughly. Slowly add 30.0 g of polypeptide compound P016 (API) to the beaker while stirring. Test the pH of the resulting mixture, then adjust the pH to 6.8-7.4 using 0.1 M NaOH or 0.1 M HCl aqueous solution. In addition, the remaining water for injection was added to make the volume to 1000 mL and mixed to obtain a composition G9 having the following composition: comprising 30.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 7.5 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.75 mM: 6.75 mM = 1:9) as a buffer, and a pH of 6.8-7.4.

[0543] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G9 sample.

[0544] Table 2-29 Composition of composition G9

[0545] [Preparation of Composition G10]

[0546] According to Table 2-30 below, weigh 46.00 g of mannitol, 1.324 g of trisodium citrate dihydrate, and 0.105 g of citric acid monohydrate, place in a beaker, add 800 mL of water for injection, and stir thoroughly. Slowly add 1.00 g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the resulting mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 6.8-7.4. The remaining water for injection was added to make the volume to 1000 mL, and mixed to obtain a composition G10 having the following composition: comprising 1.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and 5.0 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.50 mM:4.50 mM = 1:9) as a buffer, and a pH of 6.8-7.4.

[0547] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition G10 sample.

[0548] Table 2-30 Composition of composition G10

[0549] [Preparation of Composition G11]

[0550] According to Table 2-31 below, weigh 46.00 g of mannitol, 1.324 g of trisodium citrate dihydrate, and 0.105 g of citric acid monohydrate, place in a beaker, add 800 mL of water for injection, and stir thoroughly. Slowly add 30.0 g of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the obtained mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 6.8-7.4, and the remaining water for injection was added to 1000 mL, and mixed to obtain a composition G11 having the following composition: comprising 30.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and using 5.0 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 0.50 mM:4.50 mM=1:9) as a buffer, and a pH of 6.8-7.4.

[0551] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and the cover was covered with an aluminum-plastic composite cap to obtain a composition G11 sample.

[0552] Table 2-31 Composition of composition G11

[0553] The compositions G1-G11 of the present invention contain a combination of citrate and mannitol as excipients and have excellent solution clarity, color, purity, polymer content, and pH stability over time at high temperatures.

[0554] [Composition Example 8-Preservative Formulation]

[0555] [Preparation of Composition H1]

[0556] According to Table 2-32 below, 920 mg of mannitol, 37.94 mg of trisodium citrate dihydrate, and 4.41 mg of citric acid monohydrate were weighed and placed in a beaker. 16 mL of water for injection was added and stirred thoroughly. 600 mg of polypeptide compound P016 (API) was slowly added to the beaker with stirring. The pH of the resulting mixture was measured, and then the pH was adjusted to 7.2 using 0.1 M NaOH or 0.1 M HCl aqueous solution. The remaining water for injection was added to the beaker to make the volume 20 mL and mixed thoroughly. This yielded composition GH1 having the following composition: comprising 30.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutically active ingredient, 46 mg / mL of mannitol as an osmotic pressure regulator, and a 7.50 mM citric acid-trisodium citrate buffer (citric acid:trisodium citrate molar ratio of 1.05 mM:6.45 mM) as a buffer, with a pH of 7.2.

[0557] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain a composition H1 sample.

[0558] Table 2-32 Compositions of compositions H1 to H3 and comparative compositions H-C1 to H-C3

[0559] Note: The amount of excipients used is allowed to vary within 0.5%. For example, 4.41 mg of citric acid monohydrate can vary within the range of 4.378-4.422 mg.

[0560] [Comparative compositions H-C1, H-C2, H-C3]

[0561] As shown in Table 2-32 above, comparative compositions H-C1, H-C2, and H-C3 were prepared using the same method as H1. H-C1 differed only in that 37.94 mg of trisodium citrate dihydrate and 4.41 mg of citric acid monohydrate in H1 were replaced with 17.94 mg of sodium dihydrogen phosphate (7.5 mM); H-C2 differed only in that 920 mg of mannitol in H1 was replaced with 160 mg of sodium chloride; and H-C3 differed only in that 920 mg of mannitol in H1 was replaced with 560 mg of propylene glycol.

[0562] [Preparation of Composition H2 and Composition H3]

[0563] According to Table 2-32 above, weigh 920 mg of mannitol, 37.94 mg of trisodium citrate dihydrate, and 4.41 mg of citric acid monohydrate, respectively, place in a beaker, add 16 mL of water for injection, and stir thoroughly. Slowly add 600 mg of polypeptide compound P016 (API) to the beaker while stirring. The pH value of the obtained mixture was tested, and then 0.1M NaOH or 0.1M HCl aqueous solution was added to the beaker to adjust the pH to 7.2, and 60 mg of m-cresol or 110 mg of phenol was added while stirring. After stirring and dissolving, the pH was finely adjusted to 7.2, and the remaining water for injection was added to 20 mL, and mixed to obtain a composition H2 or H3 having the following composition: comprising 30.00 mg / mL of the polypeptide compound P016 (API) of the present invention as a pharmaceutical active ingredient, 46 mg / mL mannitol as an osmotic pressure regulator, 3.00 mg / mL of m-phenol or 5.5 mg of phenol as a preservative, and using 7.50 mM citric acid-trisodium citrate buffer (citric acid: trisodium citrate molar concentration ratio of 1.05 mM:6.45 mM) as a buffer, with a pH of 7.2.

[0564] The solution was filtered through a 0.22 μm PES membrane and dispensed into 2 mL neutral borosilicate glass tube injection bottles (after sterilization), 1.0 mL / bottle. The injection solution was fully plugged with a butyl rubber stopper partially covered with a polytetrafluoroethylene membrane (after sterilization). The antibiotics were added and capped with an aluminum-plastic composite cap to obtain the composition H2 or H3 sample.

[0565] Table 2-33 Clarity and color of different compositions

[0566] The results in Table 2-33 show that:

[0567] 1) Compared with the comparative compositions H-C1 (phosphate), H-C2 (sodium chloride), and H-C3 (propylene glycol), composition H1 (containing citrate and mannitol) still exhibited better solution clarity.

[0568] 2) Unexpectedly, the compositions H2 and H3 containing phenol or m-cresol as preservatives have smaller color depth changes at 40°C than the composition H1 without preservatives. The addition of preservatives improves the color stability of the compositions.

[0569] Table 2-34 Chromatographic purity of different compositions determined by HPLC-UV

[0570] Table 2-35 SEC-HPLC determination of polymers with different compositions

[0571] In summary, at high active P016 drug concentrations, the preservative-containing formulations H2 and H3 exhibited comparable polymer, related substance, and clarity stability profiles, as well as improved color and thermal stability, compared to the preservative-free formulation H1. Comparison of the inventive compositions H1 to H3 with the comparative compositions H-C1 to H-C3 further demonstrates that the excipient composition of the citrate buffer system and mannitol as an osmotic pressure regulator exhibits excellent physicochemical and thermal stability.

[0572] [Composition Example 9-Preservative Formulation]

[0573] 15mM Citrate Buffer: 1) 15mM Citric Acid: Weigh 0.3143g of citric acid monohydrate and dilute with water to 100mL. 2) 15mM Trisodium Citrate: Weigh 0.4414g of trisodium citrate dihydrate and dilute with water to 100mL. 3) Mix 10.5mL of 15mM citric acid with 64.5mL of 15mM trisodium citrate to prepare 15mM citrate buffer.

[0574] 55mg / 3mL phenol stock solution: Add an appropriate amount of phenol and dilute to volume with water.

[0575] 30mg / 3mL m-cresol stock solution: Add an appropriate amount of m-cresol and dilute with water to make up to volume.

[0576] Preparation of Compositions I1 to I12: According to Tables 2-36 below, add an appropriate amount of mannitol to each container. Add appropriate amounts of water for injection, citrate buffer, glycerol or propylene glycol, and sodium chloride as needed, and stir to mix. Then, weigh an appropriate amount of P016 polypeptide and slowly add it to the container. Shake well to dissolve to obtain a clear solution. Adjust the pH with 0.1M HCl or 0.1M NaOH. Add 3 mL of phenol stock solution or m-cresol stock solution dropwise as needed to adjust the pH to the target value. Make up to a final volume of 10 mL with water for injection and mix thoroughly. Filter through a 0.22 μm sterile polyethersulfone (PES) membrane and dispense into sterilized 2 mL injection vials, 1.0 mL per vial. Stopper with a sterilized, partially covered polytetrafluoroethylene membrane and butyl rubber stopper, and cap with an aluminum-plastic composite cap to obtain Compositions I1 to I12.

[0577] Table 2-36 Compositions of compositions I1 to I12

[0578] Table 2-37 Solution clarity and color of compositions I1 to I12

[0579] Table 2-38 Polymers of compositions I1 to I12 (%)

[0580] Table 2-39 Purity of compositions I1 to I12 (%)

[0581] The results in Tables 2-37 to 2-39 show that:

[0582] Solution clarity: In compositions I3 to I12 containing phenol or m-cresol as preservatives, despite containing propylene glycol, glycerol, sodium chloride, or a combination thereof as an osmotic pressure regulator, no increase in turbidity was observed at 25°C and 40°C for one month, or at 60°C for 10 days. This is different from preservative-free formulations, which are susceptible to increased turbidity when using propylene glycol or sodium chloride as an osmotic pressure regulator. It was unexpectedly discovered that the use of phenol or m-cresol as a preservative allows the composition to use a wider variety of osmotic pressure regulators (e.g., mannitol, propylene glycol, glycerol, sodium chloride, and a combination thereof) without compromising the physical stability of its solution properties / clarity.

[0583] Polymer (%): Compared to preservative-free compositions I1 and I2, preservative-containing compositions I3 to I12 exhibited comparable stability trends at 40°C, 25°C, or 2-8°C. The introduction of phenol or m-cresol preservatives, and osmotic pressure regulators, either alone or in combination, did not result in deterioration of polymer performance in compositions I3 to I12.

[0584] Purity (%): Compositions I1 to I12 all exhibited temperature-dependent stability. Compositions I3 to I12, using different combinations of preservatives (m-cresol or phenol) and osmotic pressure regulators (mannitol, propylene glycol, glycerol, sodium chloride), all exhibited similar temperature-dependent purity reduction.

[0585] Amino acid sequence:

Claims

1. A pharmaceutical composition, the pH of the pharmaceutical composition being from about 5.0 to about 9.0, and the pharmaceutical composition comprising from about 0.1 to about 50 mg / mL of a polypeptide compound of the following formula I: Y-Aib-E-G-T-αMePhe-T-S-D-Y-Aib-I-Aib-L-D-K-Q-A-Q-A-E-F-V-K 24 -W-L-L-K 28 -G-G-P-S-S-G-A-P-P-P-S-K Formula I (SEQ ID NO:12); Among them, Select the position of one K from the 16-bit, 24-bit, 28-bit, and 40-bit Ks of type I, and chemically modify it by conjugating ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z to the ε-amino group of the K side chain, where each a is independently selected from the integers 1, 2, 3, each b is independently selected from the integers 1, 2, 3, each c is independently selected from the integers 16, 18, 20, and where Z is independently selected from -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate / ester or sulfonate / ester, preferably -COOH; and the C-terminal amino acid is amidated to a C-terminal primary amide.

2. The pharmaceutical composition according to claim 1, wherein the polypeptide compound of formula I is Y-Aib-E-G-T-αMePhe-T-S-D-Y-Aib-I-Aib-L-D-K-Q-A-Q-A-E-F-V-K 24 -W-L-L-K 28 -G-G-P-S-S-G-A-P-P-P-S-K Formula I, wherein K at position 24 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P007, SEQ ID NO:2); or K at position 24 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 18 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P013, SEQ ID NO:4); or K at position 24 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P014, SEQ ID NO:5); or K at position 24 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P015, SEQ ID NO:6); or K at position 24 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl) 3-(γ-Glu) 1-CO-(CH2) 20 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P016, SEQ ID NO:7); or K at position 28 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P008, SEQ ID NO:3); or K at position 28 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P017, SEQ ID NO:8); or K at position 28 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P018, SEQ ID NO:9); or K at position 28 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 16 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P019, SEQ ID NO:10); or K at position 28 is chemically modified by conjugating the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H; and the C-terminal amino acid is amidated to a C-terminal primary amide (P020, SEQ ID NO:11).

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further contains an osmotic pressure regulator, a buffer, water, and optionally other excipients or carriers, and the other excipients or carriers include one or more of a pH regulator, a preservative, an antioxidant, and a stabilizer.

4. The pharmaceutical composition according to claim 3, wherein the buffer is selected from acetic acid, succinic acid, citric acid, phosphoric acid, gluconic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tromethamine (Tris), disodium hydrogen phosphate, trisodium citrate, disodium citrate, trisodium citrate dihydrate, citric acid - trisodium citrate, citric acid monohydrate - trisodium citrate dihydrate, disodium hydrogen phosphate - citric acid, sodium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium phosphate, disodium hydrogen phosphate - potassium dihydrogen phosphate, and combinations thereof.

5. The pharmaceutical composition according to claim 3 or 4, wherein based on the total volume of the aqueous solution, the molar concentration of the buffer is about 0.5 - about 50 mM.

6. The pharmaceutical composition according to any one of claims 3 - 5, wherein the osmotic pressure regulator is selected from sodium chloride, phosphate, citrate, borate, tartrate, lactose, trehalose, sucrose, glucose, mannitol, sorbitol, xylitol, L - glycine, L - histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, glycerol, 1,2 - propanediol, 1,3 - propanediol, 1,3 - butanediol, polyethylene glycol, or any combination thereof.

7. The pharmaceutical composition according to any one of claims 3 - 6, wherein based on the total volume of the aqueous solution, the content of the osmotic pressure regulator is about 1 - about 100 mg / mL.

8. The pharmaceutical composition according to any one of claims 3 - 7, wherein the pH regulator is an aqueous sodium hydroxide solution or hydrochloric acid.

9. The pharmaceutical composition according to any one of claims 3 - 8, wherein the preservative is selected from phenol, m - cresol, cresol, phenoxyethanol, chlorobutanol, benzyl alcohol, benzoate derivatives, and any combination thereof.

10. The pharmaceutical composition according to any one of claims 1 - 9, wherein the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition comprises: about 0.1 - about 50 mg / mL of the compound of formula I in claim 1, for example about 1 - about 40 mg / mL of compound P016 or P014 or P020, citric acid and trisodium citrate with a total molar concentration of about 0.5 - about 50 mM, preferably about 1.0 - 10 mM of citric acid - trisodium citrate, the molar ratio of citric acid to trisodium citrate being about 1:4 - 50, preferably about 1:4 - 20, 0 - about 50 mg / mL, preferably about 0.1 - about 50 mg / mL, more preferably about 1 - about 10 mg / mL, still more preferably about 2 - about 6 mg / mL of a preservative, An osmotic pressure regulator in an amount of about 1 to about 100 mg / mL, preferably mannitol, 1,2-propanediol, 1,3-propanediol, glycerol, sodium chloride or any combination thereof in an amount of about 1 to about 100 mg / mL, Optionally present aqueous sodium hydroxide solution or hydrochloric acid, and Water, Wherein the pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to about 8.8, more preferably about 6.5 to about 7.

4.

11. The pharmaceutical composition according to any one of claims 1-9, wherein the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, the pharmaceutical composition comprises: About 0.1 to about 50 mg / mL of the compound of formula I in claim 1, such as about 1 to about 40 mg / mL of compound P016 or P014 or P020, Citric acid and trisodium citrate with a total molar concentration of about 0.5 to about 50 mM, preferably about 1.0 to about 10 mM of citric acid-trisodium citrate, and the molar ratio of citric acid to trisodium citrate is about 1:4 to 50, preferably about 1:4 to 20, About 1 to about 100 mg / mL of mannitol, Optionally present aqueous sodium hydroxide or hydrochloric acid, Optionally present preservative, and Water, Wherein the pH value of the aqueous solution is about 5.0 to about 9.0, such as about 5.8 to about 9.0, preferably about 6.0 to about 8.8, more preferably about 6.5 to about 7.

4.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the osmotic pressure of the pharmaceutical composition is about 200 to about 500 mOsm / kg, preferably about 250 to about 350 mOsm / kg, more preferably about 260 to about 320 mOsm / kg, and the osmotic pressure is measured by an osmometer.

13. A kit, which comprises a container and the pharmaceutical composition according to any one of claims 1-12 contained in the container.

14. Use of the pharmaceutical composition according to any one of claims 1-12 for the preparation of a medicament for treating a disease, such as diabetes, obesity, diseases related to diabetes, and / or diseases related to obesity.

15. The use according to claim 14, wherein the unit dose of the pharmaceutical composition is 0.1 mg to about 0.5 mg or about 0.5 mg to about 50 mg.